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Sample records for activities gene expression

  1. Correspondence between resting state activity and brain gene expression

    PubMed Central

    Wang, Guang-Zhong; Belgard, T. Grant; Mao, Deng; Chen, Leslie; Berto, Stefano; Preuss, Todd M.; Lu, Hanzhang; Geschwind, Daniel H.; Konopka, Genevieve

    2015-01-01

    SUMMARY The relationship between functional brain activity and gene expression has not been fully explored in the human brain. Here, we identify significant correlations between gene expression in the brain and functional activity by comparing fractional Amplitude of Low Frequency Fluctuations (fALFF) from two independent human fMRI resting state datasets to regional cortical gene expression from a newly generated RNA-seq dataset and two additional gene expression datasets to obtain robust and reproducible correlations. We find significantly more genes correlated with fALFF than expected by chance, and identify specific genes correlated with the imaging signals in multiple expression datasets in the default mode network. Together, these data support a population-level relationship between regional steady state brain gene expression and resting state brain activity. PMID:26590343

  2. Carcinogen-induced trans activation of gene expression.

    PubMed Central

    Kleinberger, T; Flint, Y B; Blank, M; Etkin, S; Lavi, S

    1988-01-01

    We report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later. Images PMID:2835673

  3. Carcinogen-induced trans activation of gene expression

    SciTech Connect

    Kleinberger, T.; Flint, Y.B.; Blank, M.; Etkin, S.; Lavi, S.

    1988-03-01

    The authors report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later.

  4. Constitutive androstane receptor activation evokes the expression of glycolytic genes.

    PubMed

    Yarushkin, Andrei A; Kazantseva, Yuliya A; Prokopyeva, Elena A; Markova, Diana N; Pustylnyak, Yuliya A; Pustylnyak, Vladimir O

    2016-09-23

    It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in a mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation.

  5. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    EPA Science Inventory

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  6. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  7. Adaptation of muscle gene expression to changes in contractile activity

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Babij, P.; Thomason, D. B.; Wong, T. S.; Morrison, P. R.

    1987-01-01

    A review of the existing literature regarding the effects of different types of physical activities on the gene expression of adult skeletal muscles leads us to conclude that each type of exercise training program has, as a result, a different phenotype, which means that there are multiple mechanisms, each producing a unique phenotype. A portion of the facts which support this position is presented and interpreted here. [Abstract translated from the original French by NASA].

  8. Detecting microRNA activity from gene expression data

    PubMed Central

    2010-01-01

    Background MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. Results Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a database of miRNA target predictions from TargetScan, TargetScanS, PicTar4way PicTar5way, and miRanda and combining these data with gene expression levels from sets of microarrays, this method produces a ranked list of miRNAs associated with a specified split in samples. We applied this to three different microarray datasets, a papillary thyroid carcinoma dataset, an in-house dataset of lipopolysaccharide treated mouse macrophages, and a multi-tissue dataset. In each case we were able to identified miRNAs of biological importance. Conclusions We describe a technique to integrate gene expression data and miRNA target predictions from multiple sources. PMID:20482775

  9. Network activity-independent coordinated gene expression program for synapse assembly.

    PubMed

    Valor, Luis M; Charlesworth, Paul; Humphreys, Lawrence; Anderson, Chris N G; Grant, Seth G N

    2007-03-13

    Global biological datasets generated by genomics, transcriptomics, and proteomics provide new approaches to understanding the relationship between the genome and the synapse. Combined transcriptome analysis and multielectrode recordings of neuronal network activity were used in mouse embryonic primary neuronal cultures to examine synapse formation and activity-dependent gene regulation. Evidence for a coordinated gene expression program for assembly of synapses was observed in the expression of 642 genes encoding postsynaptic and plasticity proteins. This synaptogenesis gene expression program preceded protein expression of synapse markers and onset of spiking activity. Continued expression was followed by maturation of morphology and electrical neuronal networks, which was then followed by the expression of activity-dependent genes. Thus, two distinct sequentially active gene expression programs underlie the genomic programs of synapse function.

  10. Pharmacological and Genetic Modulation of REV-ERB Activity and Expression Affects Orexigenic Gene Expression

    PubMed Central

    Amador, Ariadna; Wang, Yongjun; Banerjee, Subhashis; Kameneka, Theodore M.; Solt, Laura A.; Burris, Thomas P.

    2016-01-01

    The nuclear receptors REV-ERBα and REV-ERBβ are transcription factors that play pivotal roles in the regulation of the circadian rhythm and various metabolic processes. The circadian rhythm is an endogenous mechanism, which generates entrainable biological changes that follow a 24-hour period. It regulates a number of physiological processes, including sleep/wakeful cycles and feeding behaviors. We recently demonstrated that REV-ERB-specific small molecules affect sleep and anxiety. The orexinergic system also plays a significant role in mammalian physiology and behavior, including the regulation of sleep and food intake. Importantly, orexin genes are expressed in a circadian manner. Given these overlaps in function and circadian expression, we wanted to determine whether the REV-ERBs might regulate orexin. We found that acute in vivo modulation of REV-ERB activity, with the REV-ERB-specific synthetic ligand SR9009, affects the circadian expression of orexinergic genes in mice. Long term dosing with SR9009 also suppresses orexinergic gene expression in mice. Finally, REV-ERBβ-deficient mice present with increased orexinergic transcripts. These data suggest that the REV-ERBs may be involved in the repression of orexinergic gene expression. PMID:26963516

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

    EPA Science Inventory

    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...

  12. Xenoestrogenic gene expression: structural features of active polycyclic aromatic hydrocarbons.

    PubMed

    Schultz, T Wayne; Sinks, Glendon D

    2002-04-01

    Estrogenicity was assessed using the Saccharomyces cerevisiae-based Lac-Z reporter assay and was reported as the logarithm of the inverse of the 50% molar beta-galactosidase activity (log[EC50(-1)]). In an effort to quantify the relationship between molecular structure of polycyclic aromatic hydrocarbons (PAHs) and estrogenic gene expression, a series of PAHs were evaluated. With noted exceptions, the results of these studies indicate that the initial two-dimensional structural warning for estrogenicity, the superpositioning of a hydroxylated aromatic system on the phenolic A-ring of 17-beta-estradiol, can be extended to the PAHs. This two-dimensional-alignment criterion correctly identified estrogenicity of 22 of the 29 PAHs evaluated. Moreover, the estrogenic potency of these compounds was directly related to the size of the hydrophobic backbone. The seven compounds classified incorrectly by this structural feature were either dihydroxylated naphthalenes or aromatic nitrogen-heterocyclic compounds; all such compounds were false positives. Results with dihydroxylated naphthalenes reveal derivatives that were nonestrogenic when superimposed on the phenolic A-ring of 17-beta-estradiol had the second hydroxyl group in the position of the C-ring or were catechol-like in structure. Structural alerts for nitrogen-heterocyclic compounds must take into account the position of the hydroxyl group and the in-ring nitrogen atom; compounds with the hydroxyl group and nitrogen atom involved with the same ring were observed to be nonactive.

  13. Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.

    PubMed Central

    De Vos, P; Lefebvre, A M; Miller, S G; Guerre-Millo, M; Wong, K; Saladin, R; Hamann, L G; Staels, B; Briggs, M R; Auwerx, J

    1996-01-01

    The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter. PMID:8770873

  14. Polyphenols from Chilean Propolis and Pinocembrin Reduce MMP-9 Gene Expression and Activity in Activated Macrophages

    PubMed Central

    Saavedra, Nicolás; Cuevas, Alejandro; Cavalcante, Marcela F.; Dörr, Felipe A.; Saavedra, Kathleen; Zambrano, Tomás; Abdalla, Dulcineia S. P.; Salazar, Luis A.

    2016-01-01

    Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measured in vitro using murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques. PMID:27119082

  15. Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities.

    PubMed

    Hesberg, Christine; Hänsch, Robert; Mendel, Ralf R; Bittner, Florian

    2004-04-02

    Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.

  16. A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus

    PubMed Central

    Sun, Ren; Lin, Su-Fang; Gradoville, Lyndle; Yuan, Yan; Zhu, Fanxiu; Miller, George

    1998-01-01

    Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases. PMID:9724796

  17. Growth enhancement and gene expression of Arabidopsis thaliana irradiated with active oxygen species

    NASA Astrophysics Data System (ADS)

    Watanabe, Satoshi; Ono, Reoto; Hayashi, Nobuya; Shiratani, Masaharu; Tashiro, Kosuke; Kuhara, Satoru; Inoue, Asami; Yasuda, Kaori; Hagiwara, Hiroko

    2016-07-01

    The characteristics of plant growth enhancement effect and the mechanism of the enhancement induced by plasma irradiation are investigated using various active species in plasma. Active oxygen species in oxygen plasma are effective for growth enhancement of plants. DNA microarray analysis of Arabidopsis thaliana indicates that the genes coding proteins that counter oxidative stresses by eliminating active oxygen species are expressed at significantly high levels. The size of plant cells increases owing to oxygen plasma irradiation. The increases in gene expression levels and cell size suggest that the increase in the expression level of the expansin protein is essential for plant growth enhancement phenomena.

  18. Gene activation properties of a mouse DNA sequence isolated by expression selection.

    PubMed Central

    von Hoyningen-Huene, V; Norbury, C; Griffiths, M; Fried, M

    1986-01-01

    The MES-1 element was previously isolated from restricted total mouse cellular DNA by "expression selection"--the ability to reactivate expression of a test gene devoid of its 5' enhancer sequences. Mes-1 has been tested in long-term transformation and short-term CAT expression assays. In both assays MES-1 is active independent of orientation and at a distance when placed 5' to the test gene. The element is active with heterologous promoters and functions efficiently in both rat and mouse cells. MES-1 activates expression by increasing transcription from the test gene's own start (cap) site. Thus the expression selection technique can be used for the isolation of DNA sequences with enhancer-like properties from total cellular DNA. Images PMID:3016657

  19. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

  20. Platelet-derived growth factor (PDGF) B-chain gene expression by activated blood monocytes precedes the expression of the PDGF A-chain gene

    SciTech Connect

    Martinet, Y.; Jaffe, H.A.; Yamauchi, K.; Betsholtz, C.; Westermark, B.; Heldin, C.H.; Crystal, R.G.

    1987-05-01

    When activated, normal human blood monocytes are known to express the c-sis proto-oncogene coding for PDGF B-chain. Since normal human platelet PDGF molecules are dimers of A and B chains and platelets and monocytes are derived from the same marrow precursors, activated blood monocytes were simultaneously evaluated for their expression of PDGF A and B chain genes. Human blood monocytes were purified by adherence, cultured with or without activation by lipopolysaccharide and poly(A)+ RNA evaluated using Northern analysis and /sup 32/P-labeled A-chain and B-chain (human c-sis) probes. Unstimulated blood monocytes did not express either A-chain or B-chain genes. In contrast, activated monocytes expressed a 4.2 kb mRNA B-chain transcript at 4 hr, but the B-chain mRNA levels declined significantly over the next 18 hr. In comparison, activated monocytes expressed very little A-chain mRNA at 4 hr, but at 12 hr 1.9, 2.3, and 2.8 kb transcripts were observed and persisted through 24 hr. Thus, activation of blood monocytes is followed by PDGF B-chain gene expression preceding PDGF A-chain gene expression, suggesting a difference in the regulation of the expression of the genes for these two chains by these cells.

  1. Regulation of Gene33 expression by insulin requires MEK-ERK activation.

    PubMed

    Keeton, Adam B; Xu, Jie; Franklin, J Lee; Messina, Joseph L

    2004-09-17

    Gene33 and its human homologue, mitogen inducible gene-6/receptor-associated late transducer (mig-6, RALT), is a 53-kDa soluble protein that was identified as a hepatic gene regulated by glucocorticoids and insulin. Its mRNA is expressed in numerous tissues in addition to the liver. Mitogen inducibility of Gene33 mRNA has been described in several experimental systems. Recent reports have suggested a role for Gene33 in inhibition of proliferation induced by factors that bind to members of the ErbB family of receptors. In the present work, we examine the regulation of Gene33 protein by insulin in hepatoma cells of rat (H4IIE) and human (HepG2/Hep3B) origin. Inhibition of MEK1 significantly inhibited extracellularly regulated kinase (ERK)1/2 activation and insulin-regulated Gene33 transcription and protein levels in H4IIE cells. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity alone did not significantly alter transcription of Gene33. In Hep3B and HepG2 cells, insulin did not significantly induce either ERK1/2 activation or Gene33 expression. This work suggests that the MEK-ERK, but not the phosphatidylinositol 3-kinase (PI3-K), pathway plays a direct role in insulin regulation of Gene33 transcription and protein expression.

  2. Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage.

    PubMed

    Thomas, David M; Francescutti-Verbeem, Dina M; Kuhn, Donald M

    2006-03-01

    Microglia are the resident antigen-presenting cells within the central nervous system (CNS), and they serve immune-like functions in protecting the brain against injury and invading pathogens. By contrast, activated microglia can secrete numerous reactants that damage neurons. The pathogenesis of various neurodegenerative diseases has been associated with microglial activation, but the signaling pathways that program a neuronally protective or destructive phenotype in microglia are not known. To increase the understanding of microglial activation, microarray analysis was used to profile the transcriptome of BV-2 microglial cells after activation. Microglia were activated by lipopolysaccharide, the HIV neurotoxic protein TAT, and dopamine quinone, each of which has been linked to dopamine neuronal damage. We identified 210 of 9882 genes whose expression was differentially regulated by all activators (116 increased and 94 decreased in expression). Gene ontology analysis assigned up-regulated genes to a number of specific biological processes and molecular functions, including immune response, inflammation, and cytokine/chemokine activity. Genes down-regulated in expression contribute to conditions that are permissive of microglial migration, lowered adhesion to matrix, lessened phagocytosis, and reduction in receptors that oppose chemotaxis and inflammation. These results elaborate a broad profile of microglial genes whose expression is altered by conditions associated with both neurodegenerative diseases and microglial activation.

  3. Process and genes for expression and overexpression of active [FeFe] hydrogenases

    DOEpatents

    Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

    2014-09-16

    A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

  4. Inhibiting AP-1 activity alters cocaine induced gene expression and potentiates sensitization

    PubMed Central

    Paletzki, Ronald F.; Myakishev, Max V.; Polesskaya, Oksana; Orosz, Andras; Hyman, Steven E.; Vinson, Charles

    2008-01-01

    We have expressed A-FOS, an inhibitor of AP-1 DNA binding, in adult mouse striatal neurons. We observe normal behavior including locomotion and exploratory activities. Following a single injection of cocaine, locomotion increased similarly in both the A-FOS expressing and littermate controls. However, following repeated injections of cocaine, the A-FOS expressing mice showed increased locomotion relative to littermate controls, an increase that persisted following a week of withdrawal and subsequent cocaine administration. These results indicate that AP-1 suppresses this behavioral responses to cocaine. We analyzed mRNA from the striatum before and 4 and 24 hours after a single cocaine injection in both A-FOS and control striata using Affymetrix microarrays (430 2.0 Array) to identify genes mis-regulated by A-FOS that may mediate the increased locomotor sensitization to cocaine. A-FOS expression did not change gene expression in the basal state or 4 hours following cocaine treatment relative to controls. However, 24 hours after an acute cocaine treatment, 84 genes were identified that were differentially expressed between the A-FOS and control mice. 56 gene are down regulated while 28 genes are up regulated including previously identified candidates for addiction including BDNF and Per1. Using a random sample of identified genes, quantitative PCR was used to verify the microarray studies. The chromosomal location of these 84 genes was compared to human genome scans of addiction to identify potential genes in humans that are involved in addiction. PMID:18355967

  5. BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks.

    PubMed

    Richiardi, Jonas; Altmann, Andre; Milazzo, Anna-Clare; Chang, Catie; Chakravarty, M Mallar; Banaschewski, Tobias; Barker, Gareth J; Bokde, Arun L W; Bromberg, Uli; Büchel, Christian; Conrod, Patricia; Fauth-Bühler, Mira; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny; Heinz, Andreas; Lemaître, Hervé; Mann, Karl F; Martinot, Jean-Luc; Nees, Frauke; Paus, Tomáš; Pausova, Zdenka; Rietschel, Marcella; Robbins, Trevor W; Smolka, Michael N; Spanagel, Rainer; Ströhle, Andreas; Schumann, Gunter; Hawrylycz, Mike; Poline, Jean-Baptiste; Greicius, Michael D

    2015-06-12

    During rest, brain activity is synchronized between different regions widely distributed throughout the brain, forming functional networks. However, the molecular mechanisms supporting functional connectivity remain undefined. We show that functional brain networks defined with resting-state functional magnetic resonance imaging can be recapitulated by using measures of correlated gene expression in a post mortem brain tissue data set. The set of 136 genes we identify is significantly enriched for ion channels. Polymorphisms in this set of genes significantly affect resting-state functional connectivity in a large sample of healthy adolescents. Expression levels of these genes are also significantly associated with axonal connectivity in the mouse. The results provide convergent, multimodal evidence that resting-state functional networks correlate with the orchestrated activity of dozens of genes linked to ion channel activity and synaptic function.

  6. SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression.

    PubMed

    Marathe, Himangi G; Mehta, Gaurav; Zhang, Xiaolu; Datar, Ila; Mehrotra, Aanchal; Yeung, Kam C; de la Serna, Ivana L

    2013-01-01

    SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to

  7. Modulation of activation-associated host cell gene expression by the apicomplexan parasite Theileria annulata

    PubMed Central

    Durrani, Zeeshan; Weir, William; Pillai, Sreerekha; Kinnaird, Jane; Shiels, Brian

    2012-01-01

    Summary Infection of bovine leucocytes by Theileria annulata results in establishment of transformed, infected cells. Infection of the host cell is known to promote constitutive activation of pro-inflammatory transcription factors that have the potential to be beneficial or detrimental. In this study we have compared the effect of LPS activation on uninfected bovine leucocytes (BL20 cells) and their Theileria-infected counterpart (TBL20). Gene expression profiles representing activated uninfected BL20 relative to TBL20 cells were also compared. The results show that while prolonged stimulation with LPS induces cell death and activation of NF-κB in BL20 cells, the viability of Theileria-infected cells was unaffected. Analysis of gene expression networks provided evidence that the parasite establishes tight control over pathways associated with cellular activation by modulating reception of extrinsic stimuli and by significantly altering the expression outcome of genes targeted by infection-activated transcription factors. Pathway analysis of the data set identified novel candidate genes involved in manipulation of cellular functions associated with the infected transformed cell. The data indicate that the T. annulata parasite can irreversibly reconfigure host cell gene expression networks associated with development of inflammatory disease and cancer to generate an outcome thatis beneficial to survival and propagation of the infected leucocyte. PMID:22533473

  8. Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis.

    PubMed

    Carnevali, O; Cionna, C; Tosti, L; Cerdà, J; Gioacchini, G

    2008-01-01

    The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.

  9. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    PubMed Central

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  10. Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator.

    PubMed Central

    Sheehan, B; Klarsfeld, A; Msadek, T; Cossart, P

    1995-01-01

    PrfA is a pleiotropic activator of virulence gene expression in the pathogenic bacterium Listeria monocytogenes. Several lines of evidence have suggested that a hierarchy of virulence gene activation by PrfA exists. This hypothesis was investigated by assessing the ability of PrfA to activate the expression of virulence gene fusions to lacZ in Bacillus subtilis. Expression of PrfA in this heterologous host was sufficient for activation of transcription at the hly, plcA, mpl, and actA promoters. Activation was most efficient at the divergently transcribed hly and plcA promoters. The putative PrfA binding site shared by these promoters is perfectly symmetrical and appears to represent the optimum sequence for target gene activation by PrfA. The activation of actA and mpl expression was considerably weaker and occurred more slowly than that observed at the hly and plcA promoters, suggesting that greater quantities of PrfA are required for productive interaction at these promoters. Interestingly, expression of an inlA-lacZ transcriptional fusion was very poorly activated by PrfA in B. subtilis, suggesting that other Listeria factors, in addition to PrfA, are required for PrfA-mediated activation at this promoter. Further support for the involvement of such factors was obtained by constructing and analyzing a prfA deletion mutant of L. monocytogenes. We observed that, in contrast to that of the other genes of the PrfA regulon, expression of inlA is only partially dependent on PrfA. PMID:7592422

  11. Intricate regulation of tyrosine hydroxylase activity and gene expression.

    PubMed

    Kumer, S C; Vrana, K E

    1996-08-01

    Tyrosine hydroxylase catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Therefore, the regulation of tyrosine hydroxylase enzyme number and intrinsic enzyme activity represents the central means for controlling the synthesis of these important biogenic amines. An intricate scheme has evolved whereby tyrosine hydroxylase activity is modulated by nearly every documented form of regulation. Beginning with the genomic DNA, evidence exists for the transcriptional regulation of tyrosine hydroxylase mRNA levels, alternative RNA processing, and the regulation of RNA stability. There is also experimental support for the role of both translational control and enzyme stability in establishing steady-state levels of active tyrosine hydroxylase protein. Finally, mechanisms have been proposed for feedback inhibition of the enzyme by catecholamine products, allosteric modulation of enzyme activity, and phosphorylation-dependent activation of the enzyme by various different kinase systems. Given the growing literature suggesting that different tissues regulate tyrosine hydroxylase mRNA levels and activity in different ways, regulatory mechanisms provide not only redundancy but also diversity in the control of catecholamine biosynthesis.

  12. DNA sequences that activate isocitrate lyase gene expression during late embryogenesis and during postgerminative growth.

    PubMed Central

    Zhang, J Z; Santes, C M; Engel, M L; Gasser, C S; Harada, J J

    1996-01-01

    We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants. PMID:8934622

  13. SNORD116 and SNORD115 change expression of multiple genes and modify each other's activity.

    PubMed

    Falaleeva, Marina; Surface, Justin; Shen, Manli; de la Grange, Pierre; Stamm, Stefan

    2015-11-10

    The loss of two gene clusters encoding small nucleolar RNAs, SNORD115 and SNORD116 contribute to Prader-Willi syndrome (PWS), the most common syndromic form of obesity in humans. SNORD115 and SNORD116 are considered to be orphan C/D box snoRNAs (SNORDs) as they do not target rRNAs or snRNAs. SNORD115 exhibits sequence complementarity towards the serotonin receptor 2C, but SNORD116 shows no extended complementarities to known RNAs. To identify molecular targets, we performed genome-wide array analysis after overexpressing SNORD115 and SNORD116 in HEK 293T cells, either alone or together. We found that SNORD116 changes the expression of over 200 genes. SNORD116 mainly changed mRNA expression levels. Surprisingly, we found that SNORD115 changes SNORD116's influence on gene expression. In similar experiments, we compared gene expression in post-mortem hypothalamus between individuals with PWS and aged-matched controls. The synopsis of these experiments resulted in 23 genes whose expression levels were influenced by SNORD116. Together our results indicate that SNORD115 and SNORD116 influence expression levels of multiple genes and modify each other activity.

  14. Gene expression profiling in Ishikawa cells: A fingerprint for estrogen active compounds

    SciTech Connect

    Boehme, Kathleen; Simon, Stephanie

    2009-04-01

    Several anthropogenous and naturally occurring substances, referred to as estrogen active compounds (EACs), are able to interfere with hormone and in particular estrogen receptor signaling. EACs can either cause adverse health effects in humans and wildlife populations or have beneficial effects on estrogen-dependent diseases. The aim of this study was to examine global gene expression profiles in estrogen receptor (ER)-proficient Ishikawa plus and ER-deficient Ishikawa minus endometrial cancer cells treated with selected well-known EACs (Diethylstilbestrol, Genistein, Zearalenone, Resveratrol, Bisphenol A and o,p'-DDT). We also investigated the effect of the pure antiestrogen ICI 182,780 (ICI) on the expression patterns caused by these compounds. Transcript levels were quantified 24 h after compound treatment using Illumina BeadChip Arrays. We identified 87 genes with similar expression changes in response to all EAC treatments in Ishikawa plus. ICI lowered the magnitude or reversed the expression of these genes, indicating ER dependent regulation. Apart from estrogenic gene regulation, Bisphenol A, o,p'-DDT, Zearalenone, Genistein and Resveratrol displayed similarities to ICI in their expression patterns, suggesting mixed estrogenic/antiestrogenic properties. In particular, the predominant antiestrogenic expression response of Resveratrol could be clearly distinguished from the other test compounds, indicating a distinct mechanism of action. Divergent gene expression patterns of the phytoestrogens, as well as weaker estrogenic gene expression regulation determined for the anthropogenous chemicals Bisphenol A and o,p'-DDT, warrants a careful assessment of potential detrimental and/or beneficial effects of EACs. The characteristic expression fingerprints and the identified subset of putative marker genes can be used for screening chemicals with an unknown mode of action and for predicting their potential to exert endocrine disrupting effects.

  15. Physical activity-associated gene expression signature in nonhuman primate motor cortex.

    PubMed

    Mitchell, Amanda C; Leak, Rehana K; Garbett, Krassimira; Zigmond, Michael J; Cameron, Judy L; Mirnics, Károly

    2012-03-01

    It has been established that weight gain and weight loss are heavily influenced by activity level. In this study, we hypothesized that the motor cortex exhibits a distinct physical activity-associated gene expression profile, which may underlie changes in weight associated with movement. Using DNA microarrays we profiled gene expression in the motor cortex of a group of 14 female rhesus monkeys (Macaca mulatta) with a wide range of stable physical activity levels. We found that neuronal growth factor signaling and nutrient sensing transcripts in the brain were highly correlated with physical activity. A follow-up of AKT3 expression changes (a gene at the apex of neuronal survival and nutrient sensing) revealed increased protein levels of total AKT, phosphorylated AKT, and forkhead box O3 (FOXO3), one of AKT's main downstream effectors. In addition, we successfully validated three other genes via quantitative polymerase chain reaction (qPCR) (cereblon (CRBN), origin recognition complex subunit 4-like, and pyruvate dehydrogenase 4 (PDK4)). We conclude that these genes are important in the physical activity-associated pathway in the motor cortex, and may be critical for physical activity-associated changes in body weight and neuroprotection.

  16. Gene-expression reversal of lncRNAs and associated mRNAs expression in active vs latent HIV infection

    PubMed Central

    Nair, Madhavan; Sagar, Vidya; Pilakka-Kanthikeel, Sudheesh

    2016-01-01

    Interplay between lncRNAs and mRNAs is rapidly emerging as a key epigenetic mechanism in controlling various cell functions. HIV can actively infect and/or can persist latently for years by manipulating host epigenetics; however, its molecular essence remains undiscovered in entirety. Here for the first time, we delineate the influence of HIV on global lncRNAs expression in monocytic cells lines. Our analysis revealed the expression modulation of nearly 1060 such lncRNAs which are associated with differentially expressed mRNAs in active and latent infection. This suggests a greater role of lncRNAs in regulating transcriptional and post-transcriptional gene expression during HIV infection. The differentially expressed mRNAs were involved in several different biological pathways where immunological networks were most enriched. Importantly, we discovered that HIV induces expression reversal of more than 150 lncRNAs between its active and latent infection. Also, hundreds of unique lncRNAs were identified in both infection conditions. The pathology specific “gene-expression reversal” and “on-and-off” switching of lncRNAs and associated mRNAs may lead to establish the relationship between active and HIV infection. PMID:27756902

  17. Conserved Overlapping Gene Arrangement, Restricted Expression, and Biochemical Activities of DNA Polymerase ν (POLN)*

    PubMed Central

    Takata, Kei-ichi; Tomida, Junya; Reh, Shelley; Swanhart, Lisa M.; Takata, Minoru; Hukriede, Neil A.; Wood, Richard D.

    2015-01-01

    DNA polymerase ν (POLN) is one of 16 DNA polymerases encoded in vertebrate genomes. It is important to determine its gene expression patterns, biological roles, and biochemical activities. By quantitative analysis of mRNA expression, we found that POLN from the zebrafish Danio rerio is expressed predominantly in testis. POLN is not detectably expressed in zebrafish embryos or in mouse embryonic stem cells. Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development. Analysis of transcripts revealed that vertebrate POLN has an unusual gene expression arrangement, sharing a first exon with HAUS3, the gene encoding augmin-like complex subunit 3. HAUS3 is broadly expressed in embryonic and adult tissues, in contrast to POLN. Differential expression of POLN and HAUS3 appears to arise by alternate splicing of transcripts in mammalian cells and zebrafish. When POLN was ectopically overexpressed in human cells, it specifically coimmunoprecipitated with the homologous recombination factors BRCA1 and FANCJ, but not with previously suggested interaction partners (HELQ and members of the Fanconi anemia core complex). Purified zebrafish POLN protein is capable of thymine glycol bypass and strand displacement, with activity dependent on a basic amino acid residue known to stabilize the primer-template. These properties are conserved with the human enzyme. Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase. PMID:26269593

  18. Coordinative modulation of human zinc transporter 2 gene expression through active and suppressive regulators.

    PubMed

    Lu, Yu-Ju; Liu, Ya-Chuan; Lin, Meng-Chieh; Chen, Yi-Ting; Lin, Lih-Yuan

    2015-04-01

    Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5' to 3') shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators.

  19. ERK signaling pathway regulates sleep duration through activity-induced gene expression during wakefulness.

    PubMed

    Mikhail, Cyril; Vaucher, Angélique; Jimenez, Sonia; Tafti, Mehdi

    2017-01-24

    Wakefulness is accompanied by experience-dependent synaptic plasticity and an increase in activity-regulated gene transcription. Wake-induced genes are certainly markers of neuronal activity and may also directly regulate the duration of and need for sleep. We stimulated murine cortical cultures with the neuromodulatory signals that are known to control wakefulness in the brain and found that norepinephrine alone or a mixture of these neuromodulators induced activity-regulated gene transcription. Pharmacological inhibition of the various signaling pathways involved in the regulation of gene expression indicated that the extracellular signal-regulated kinase (ERK) pathway is the principal one mediating the effects of waking neuromodulators on gene expression. In mice, ERK phosphorylation in the cortex increased and decreased with wakefulness and sleep. Whole-body or cortical neuron-specific deletion of Erk1 or Erk2 significantly increased the duration of wakefulness in mice, and pharmacological inhibition of ERK phosphorylation decreased sleep duration and increased the duration of wakefulness bouts. Thus, this signaling pathway, which is highly conserved from Drosophila to mammals, is a key pathway that links waking experience-induced neuronal gene expression to sleep duration and quality.

  20. Integrating Circadian Activity and Gene Expression Profiles to Predict Chronotoxicity of Drosophila suzukii Response to Insecticides

    PubMed Central

    Hamby, Kelly A.; Kwok, Rosanna S.; Zalom, Frank G.; Chiu, Joanna C.

    2013-01-01

    Native to Southeast Asia, Drosophila suzukii (Matsumura) is a recent invader that infests intact ripe and ripening fruit, leading to significant crop losses in the U.S., Canada, and Europe. Since current D. suzukii management strategies rely heavily on insecticide usage and insecticide detoxification gene expression is under circadian regulation in the closely related Drosophila melanogaster, we set out to determine if integrative analysis of daily activity patterns and detoxification gene expression can predict chronotoxicity of D. suzukii to insecticides. Locomotor assays were performed under conditions that approximate a typical summer or winter day in Watsonville, California, where D. suzukii was first detected in North America. As expected, daily activity patterns of D. suzukii appeared quite different between ‘summer’ and ‘winter’ conditions due to differences in photoperiod and temperature. In the ‘summer’, D. suzukii assumed a more bimodal activity pattern, with maximum activity occurring at dawn and dusk. In the ‘winter’, activity was unimodal and restricted to the warmest part of the circadian cycle. Expression analysis of six detoxification genes and acute contact bioassays were performed at multiple circadian times, but only in conditions approximating Watsonville summer, the cropping season, when most insecticide applications occur. Five of the genes tested exhibited rhythmic expression, with the majority showing peak expression at dawn (ZT0, 6am). We observed significant differences in the chronotoxicity of D. suzukii towards malathion, with highest susceptibility at ZT0 (6am), corresponding to peak expression of cytochrome P450s that may be involved in bioactivation of malathion. High activity levels were not found to correlate with high insecticide susceptibility as initially hypothesized. Chronobiology and chronotoxicity of D. suzukii provide valuable insights for monitoring and control efforts, because insect activity as well as

  1. Sensory experience and sensory activity regulate chemosensory receptor gene expression in Caenorhabditis elegans

    PubMed Central

    Peckol, Erin L.; Troemel, Emily R.; Bargmann, Cornelia I.

    2001-01-01

    Changes in the environment cause both short-term and long-term changes in an animal's behavior. Here we show that specific sensory experiences cause changes in chemosensory receptor gene expression that may alter sensory perception in the nematode Caenorhabditis elegans. Three predicted chemosensory receptor genes expressed in the ASI chemosensory neurons, srd-1, str-2, and str-3, are repressed by exposure to the dauer pheromone, a signal of crowding. Repression occurs at pheromone concentrations below those that induce formation of the alternative dauer larva stage, suggesting that exposure to pheromones can alter the chemosensory behaviors of non-dauer animals. In addition, ASI expression of srd-1, but not str-2 and str-3, is induced by sensory activity of the ASI neurons. Expression of two receptor genes is regulated by developmental entry into the dauer larva stage. srd-1 expression in ASI neurons is repressed in dauer larvae. str-2 expression in dauer animals is induced in the ASI neurons, but repressed in the AWC neurons. The ASI and AWC neurons remodel in the dauer stage, and these results suggest that their sensory specificity changes as well. We suggest that experience-dependent changes in chemosensory receptor gene expression may modify olfactory behaviors. PMID:11572964

  2. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  3. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M.; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher’s algorithm (p-value ≤ 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of

  4. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium.

    PubMed

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R; Aleksunes, Lauren M; Thomas, Russell S; Applegate, Dawn; Klaassen, Curtis D; Corton, J Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher's algorithm (p-value ≤ 10(-4))) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of

  5. Targeting c-Myc-activated genes with a correlation method: Detection of global changes in large gene expression network dynamics

    PubMed Central

    Remondini, D.; O'Connell, B.; Intrator, N.; Sedivy, J. M.; Neretti, N.; Castellani, G. C.; Cooper, L. N.

    2005-01-01

    This work studies the dynamics of a gene expression time series network. The network, which is obtained from the correlation of gene expressions, exhibits global dynamic properties that emerge after a cell state perturbation. The main features of this network appear to be more robust when compared with those obtained with a network obtained from a linear Markov model. In particular, the network properties strongly depend on the exact time sequence relationships between genes and are destroyed by random temporal data shuffling. We discuss in detail the problem of finding targets of the c-myc protooncogene, which encodes a transcriptional regulator whose inappropriate expression has been correlated with a wide array of malignancies. The data used for network construction are a time series of gene expression, collected by microarray analysis of a rat fibroblast cell line expressing a conditional Myc-estrogen receptor oncoprotein. We show that the correlation-based model can establish a clear relationship between network structure and the cascade of c-myc-activated genes. PMID:15867157

  6. Gene expression analysis during acute hepatitis C virus infection associates dendritic cell activation with viral clearance.

    PubMed

    Zabaleta, Aintzane; Riezu-Boj, Jose-Ignacio; Larrea, Esther; Villanueva, Lorea; Lasarte, Juan Jose; Guruceaga, Elizabeth; Fisicaro, Paola; Ezzikouri, Sayeh; Missale, Gabriele; Ferrari, Carlo; Benjelloun, Soumaya; Prieto, Jesús; Sarobe, Pablo

    2016-05-01

    Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T-cell responses. Since dendritic cells (DC) are essential in the activation of primary T-cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically-infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism-associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T-cell responses which lead to chronic HCV infection.

  7. Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli.

    PubMed Central

    Xie, W Q; Whitton, B A; Simon, J W; Jäger, K; Reed, D; Potts, M

    1989-01-01

    A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants. Images PMID:2536677

  8. Chromatin structure implicated in activation of HIV-1 gene expression by ultraviolet light

    SciTech Connect

    Valerie, K.; Rosenberg, M. )

    1990-08-01

    We have investigated the effects of different DNA-damaging agents on HIV-1 gene expression. We find that agents that produce bulky DNA lesions, similar to those induced by ultraviolet light (UV), all dramatically increase HIV-1 gene expression, whereas agents that produce primarily base damage and DNA breakage, such as ionizing radiation, have little or no effect. We show that these effects are independent of DNA synthesis per se and do not require DNA nucleotide excision repair. The drug novobiocin effectively prevents the UV activation process, consistent with the idea that a change in DNA chromatin structure may be required. We suggest that a transient decondensation of chromatin structure, an early step in DNA nucleotide excision repair but not in base excision repair, may be the triggering mechanism. The decondensation may allow the transcriptional machinery better access to the HIV-1 promoter region, thereby increasing gene expression.

  9. Differential activity of Drosophila Hox genes induces myosin expression and can maintain compartment boundaries.

    PubMed

    Curt, Jesús R; de Navas, Luis F; Sánchez-Herrero, Ernesto

    2013-01-01

    Compartments are units of cell lineage that subdivide territories with different developmental potential. In Drosophila, the wing and haltere discs are subdivided into anterior and posterior (A/P) compartments, which require the activity of Hedgehog, and into dorsal and ventral (D/V) compartments, needing Notch signaling. There is enrichment in actomyosin proteins at the compartment boundaries, suggesting a role for these proteins in their maintenance. Compartments also develop in the mouse hindbrain rhombomeres, which are characterized by the expression of different Hox genes, a group of genes specifying different structures along their main axis of bilaterians. We show here that the Drosophila Hox gene Ultrabithorax can maintain the A/P and D/V compartment boundaries when Hedgehog or Notch signaling is compromised, and that the interaction of cells with and without Ultrabithorax expression induces high levels of non-muscle myosin II. In the absence of Ultrabithorax there is occasional mixing of cells from different segments. We also show a similar role in cell segregation for the Abdominal-B Hox gene. Our results suggest that the juxtaposition of cells with different Hox gene expression leads to their sorting out, probably through the accumulation of non-muscle myosin II at the boundary of the different cell territories. The increase in myosin expression seems to be a general mechanism used by Hox genes or signaling pathways to maintain the segregation of different groups of cells.

  10. Synergistic and Antagonistic Interplay between Myostatin Gene Expression and Physical Activity Levels on Gene Expression Patterns in Triceps Brachii Muscles of C57/BL6 Mice

    PubMed Central

    Caetano-Anollés, Kelsey; Mishra, Sanjibita; Rodriguez-Zas, Sandra L.

    2015-01-01

    Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current

  11. SATB1 packages densely-looped, transciptionally-active chromatinfor coordinated expression of cytokine genes

    SciTech Connect

    Cai, Shutao; Lee, Charles C.; Kohwi-Shigematsu, Terumi

    2006-05-23

    SATB1 is an important regulator of nuclear architecture that anchors specialized DNA sequences onto its cage-like network and recruits chromatin remodeling/modifying factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4, and Il13 from the 200kb cytokine gene cluster region of mouse chromosome 11 during T-helper 2 (Th2)-cell activation. We show that upon cell activation, SATB1 is rapidly induced to form a unique transcriptionally-active chromatin structure that includes the cytokine gene region. Chromatin is folded into numerous small loops all anchored by SATB1, is histone H3 acetylated at lysine 9/14, and associated with Th2-specific factors, GATA3, STAT6, c-Maf, the chromatin-remodeling enzyme Brg-1, and RNA polymerase II across the 200kb region. Before activation, the chromatin displays some of these features, such as association with GATA3 and STAT6, but these were insufficient for cytokine gene expression. Using RNA interference (RNAi), we show that upon cell activation, SATB1 is not only required for chromatin folding into dense loops, but also for c-Maf induction and subsequently for Il4, Il5, and Il13 transcription. Our results show that SATB1 is an important determinant for chromatin architecture that constitutes a novel higher-order, transcriptionally-active chromatin structure upon Th2-cell activation.

  12. Maternal age effects on myometrial expression of contractile proteins, uterine gene expression, and contractile activity during labor in the rat

    PubMed Central

    Elmes, Matthew; Szyszka, Alexandra; Pauliat, Caroline; Clifford, Bethan; Daniel, Zoe; Cheng, Zhangrui; Wathes, Claire; McMullen, Sarah

    2015-01-01

    Advanced maternal age of first time pregnant mothers is associated with prolonged and dysfunctional labor and significant risk of emergency cesarean section. We investigated the influence of maternal age on myometrial contractility, expression of contractile associated proteins (CAPs), and global gene expression in the parturient uterus. Female Wistar rats either 8 (YOUNG n = 10) or 24 (OLDER n = 10) weeks old were fed laboratory chow, mated, and killed during parturition. Myometrial strips were dissected to determine contractile activity, cholesterol (CHOL) and triglycerides (TAG) content, protein expression of connexin-43 (GJA1), prostaglandin-endoperoxide synthase 2 (PTGS2), and caveolin 1 (CAV-1). Maternal plasma concentrations of prostaglandins PGE2, PGF2α, and progesterone were determined by RIA. Global gene expression in uterine samples was compared using Affymetrix Genechip Gene 2.0 ST arrays and Ingenuity Pathway analysis (IPA). Spontaneous contractility in myometrium exhibited by YOUNG rats was threefold greater than OLDER animals (P < 0.027) but maternal age had no significant effect on myometrial CAP expression, lipid profiles, or pregnancy-related hormones. OLDER myometrium increased contractile activity in response to PGF2α, phenylephrine, and carbachol, a response absent in YOUNG rats (all P < 0.002). Microarray analysis identified that maternal age affected expression of genes related to immune and inflammatory responses, lipid transport and metabolism, steroid metabolism, tissue remodeling, and smooth muscle contraction. In conclusion YOUNG laboring rat myometrium seems primed to contract maximally, whereas activity is blunted in OLDER animals and requires stimulation to meet contractile potential. Further work investigating maternal age effects on myometrial function is required with focus on lipid metabolism and inflammatory pathways. PMID:25876907

  13. Pyrophosphate Stimulates Differentiation, Matrix Gene Expression and Alkaline Phosphatase Activity in Osteoblasts

    PubMed Central

    Pujari-Palmer, Michael; Pujari-Palmer, Shiuli; Lu, Xi; Lind, Thomas; Melhus, Håkan; Engstrand, Thomas; Karlsson-Ott, Marjam; Engqvist, Hakan

    2016-01-01

    Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100μM and 0.1μM in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100μM) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100μM) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression. PMID:27701417

  14. Regulation of neuronal gene expression and survival by basal NMDA receptor activity: a role for histone deacetylase 4.

    PubMed

    Chen, Yelin; Wang, Yuanyuan; Modrusan, Zora; Sheng, Morgan; Kaminker, Joshua S

    2014-11-12

    Neuronal gene expression is modulated by activity via calcium-permeable receptors such as NMDA receptors (NMDARs). While gene expression changes downstream of evoked NMDAR activity have been well studied, much less is known about gene expression changes that occur under conditions of basal neuronal activity. In mouse dissociated hippocampal neuronal cultures, we found that a broad NMDAR antagonist, AP5, induced robust gene expression changes under basal activity, but subtype-specific antagonists did not. While some of the gene expression changes are also known to be downstream of stimulated NMDAR activity, others appear specific to basal NMDAR activity. The genes altered by AP5 treatment of basal cultures were enriched for pathways related to class IIa histone deacetylases (HDACs), apoptosis, and synapse-related signaling. Specifically, AP5 altered the expression of all three class IIa HDACs that are highly expressed in the brain, HDAC4, HDAC5, and HDAC9, and also induced nuclear accumulation of HDAC4. HDAC4 knockdown abolished a subset of the gene expression changes induced by AP5, and led to neuronal death under long-term tetrodotoxin or AP5 treatment in rat hippocampal organotypic slice cultures. These data suggest that basal, but not evoked, NMDAR activity regulates gene expression in part through HDAC4, and, that HDAC4 has neuroprotective functions under conditions of low NMDAR activity.

  15. MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation.

    PubMed

    Schmitt, Anja; Grondona, Paula; Maier, Tabea; Brändle, Marc; Schönfeld, Caroline; Jäger, Günter; Kosnopfel, Corinna; Eberle, Franziska C; Schittek, Birgit; Schulze-Osthoff, Klaus; Yazdi, Amir S; Hailfinger, Stephan

    2016-04-01

    The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.

  16. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  17. Diet quality determines lipase gene expression and lipase/esterase activity in Daphnia pulex

    PubMed Central

    Schwarzenberger, Anke; Wacker, Alexander

    2017-01-01

    ABSTRACT We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments. PMID:28069588

  18. Gene expression profiling of HGF/Met activation in neonatal mouse heart.

    PubMed

    Gatti, Stefano; Leo, Christian; Gallo, Simona; Sala, Valentina; Bucci, Enrico; Natale, Massimo; Cantarella, Daniela; Medico, Enzo; Crepaldi, Tiziana

    2013-06-01

    Hepatocyte Growth Factor (HGF) controls growth and differentiation in different cell types, including cardiac cells. However, its downstream effectors are poorly understood. To investigate the transcriptional targets of HGF, we analyzed the hearts of neonatal mice with cardiomyocyte-specific HGF overexpression with whole genome DNA microarrays. When comparing HGF expressing versus control hearts, we found a total of 249 transcripts with significant gene expression changes (210 upregulated and 39 downregulated). Gene Ontology (GO) annotation analysis revealed that the transcripts modulated by HGF were enriched for metabolic functions including: protein translation, vesicle-mediated transport, regulation of transcription, regulation of muscle development. Using an automated literature meta-analysis approach, we obtained a co-occurrence network oriented to the positive regulatory role of Myc and Notch1 in controlling some of the genes which are downstream to HGF. GO analysis of this network returned genes involved in the regulation of heart development. HGF positively controls MyocD, an activator of cardiac gene expression, and Hdac5, an inhibitor of cardiac growth. These results may unveil a new role of HGF in the modulation of signaling pathways implicated in the activation or repression of cardiomyogenesis.

  19. Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development.

    PubMed

    Louis, S A; Spiegelman, G B; Weeks, G

    1997-02-01

    It has been previously demonstrated that the expression of an activated rasD gene in wild-type Dictyostelium cells results in formation of aggregates with multitips, instead of the normal single tips, and a block in further development. In an attempt to better understand the role of activated RasD development, we examined cell-type-specific gene expression in a strain stably expressing high levels of RasD[G12T]. We found that the expression of prestalk cell-specific genes ecmA and tagB was markedly enhanced, whereas the expression of the prespore cell-specific gene cotC was reduced to very low levels. When the fate of cells in the multitipped aggregate was monitored with an ecmA/lacZ fusion, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When mixtures of the [G12T]rasD cells and Ax3 cells were induced to differentiate, chimeric pseudoplasmodia were not formed. Thus, although the [G12T]rasD transformant had a marked propensity to form prestalk cells, it could not supply the prestalk cell population when mixed with wild-type cells. Both stalk and spore cell formation occurred in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during multicellular development involved inhibitory cell interactions within the cell mass. Models for the possible role of rasD in development are discussed.

  20. Different Gene Expression and Activity Pattern of Antioxidant Enzymes in Bladder Cancer.

    PubMed

    Wieczorek, Edyta; Jablonowski, Zbigniew; Tomasik, Bartlomiej; Gromadzinska, Jolanta; Jablonska, Ewa; Konecki, Tomasz; Fendler, Wojciech; Sosnowski, Marek; Wasowicz, Wojciech; Reszka, Edyta

    2017-02-01

    The aim of this study was to evaluate the possible role in and contribution of antioxidant enzymes to bladder cancer (BC) etiology and recurrence after transurethral resection (TUR). We enrolled 40 patients with BC who underwent TUR and 100 sex- and age-matched healthy controls. The analysis was performed at diagnosis and recurrence, taking into account the time of recurrence. Gene expression of catalase (CAT), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (SOD2) was determined in peripheral blood leukocytes. The activity of glutathione peroxidase 3 (GPX3) was examined in plasma, and GPX1 and copper-zinc containing superoxide dismutase 1 (SOD1) in erythrocytes. SOD2 and GPX1 expression and GPX1 and SOD1 activity were significantly higher in patients at diagnosis of BC in comparison to controls. In patients who had recurrence earlier than 1 year from TUR, CAT and SOD2 expression was lower (at diagnosis p=0.024 and p=0.434, at recurrence p=0.022 and p=0.010), while the GPX1 and GPX3 activity was higher (at diagnosis p=0.242 and p=0.394, at recurrence p=0.019 and p=0.025) compared to patients with recurrence after 1 year from TUR. This study revealed that the gene expression and activity of the antioxidant enzymes are elevated in blood of patients with BC, although a low expression of CAT might contribute to the recurrence of BC, in early prognosis.

  1. Gene expression profiling of pituitary melanotrope cells during their physiological activation.

    PubMed

    Kuribara, Miyuki; van Bakel, Nick H M; Ramekers, Dyan; de Gouw, Daan; Neijts, Roel; Roubos, Eric W; Scheenen, Wim J J M; Martens, Gerard J M; Jenks, Bruce G

    2012-01-01

    The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.

  2. Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression

    PubMed Central

    Rivier-Cordey, Anne-Sophie; Caetano, Carlos; Fish, Richard J.; Kruithof, Egbert K. O.

    2016-01-01

    Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter. PMID:27973546

  3. Regulation of Drug Disposition Gene Expression in Pregnant Mice with Car Receptor Activation

    PubMed Central

    Bright, Amanda S.; Herrera-Garcia, Guadalupe; Moscovitz, Jamie E.; You, Dahea; Guo, Grace L.; Aleksunes, Lauren M.

    2016-01-01

    More than half of pregnant women use prescription medications in order to maintain both maternal and fetal health. The constitutive androstane receptor (Car) critically affects the disposition of chemicals by regulating the transcription of genes encoding metabolic enzymes and transporters. However, the effects of Car activation on chemical disposition during pregnancy are unclear. This study aims to determine the degree to which pregnancy alters the expression of drug metabolizing enzymes and transporters in response to the pharmacological activation of Car. To test this, pregnant C57BL/6 mice were administered IP doses of vehicle, or a potent Car agonist, TCPOBOP, on gestation days 14, 15 and 16. Hepatic mRNA and protein expression of Car target genes (phase I, II and transporters) were quantified on gestation day 17. Pregnancy-related changes, such as induction of Cyp2b10, Ugt1a1 and Sult1a1 and repression of Ugt1a6, Gsta1, Gsta2 and Mrp6, were observed. Interestingly, the induction of Cyp2b10, Gsta1, Gsta2 and Mrp2-4 mRNAs by TCPOBOP was attenuated in maternal livers suggesting that Car activation is impeded by the biochemical and/or physiological changes that occur during gestation. Taken together, these findings suggest that pregnancy and pharmacological activation of Car can differentially regulate the expression of drug metabolism and transport genes. PMID:27818994

  4. Cyclical strain modulates metalloprotease and matrix gene expression in human tenocytes via activation of TGFβ.

    PubMed

    Jones, Eleanor R; Jones, Gavin C; Legerlotz, Kirsten; Riley, Graham P

    2013-12-01

    Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1Hz for 48h. TGFβ1 and TGFβRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFβ protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFβ stimulation alone. We have also demonstrated that the inhibition of the TGFβ signalling pathway abrogated the strain induced changes in mRNA and that TGFβ activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFβ activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

  5. Passenger mutations and aberrant gene expression in congenic tissue plasminogen activator-deficient mouse strains

    PubMed Central

    Szabo, R.; Samson, A. L.; Lawrence, D. A.; Medcalf, R. L.; Bugge, T. H.

    2017-01-01

    Summary Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density SNP analysis, bioinformatics, and genome editing was used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat−/− mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel “passenger mutation”-free isogenic C57BL/6J-Plat−/− and FVB/NJ-Plat−/− mouse strains by introducing an 11 bp deletion in the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes. PMID:27079292

  6. Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD

    PubMed Central

    Colton, Carol A; Mott, Ryan T; Sharpe, Hayley; Xu, Qing; Van Nostrand, William E; Vitek, Michael P

    2006-01-01

    Background Microglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an "alternative" activation state. Methods To study alternative activation of microglia, we used quantitative RT-PCR to identify genes associated with alternative activation in microglia, including arginase I (AGI), mannose receptor (MRC1), found in inflammatory zone 1 (FIZZ1), and chitinase 3-like 3 (YM1). Results Our findings confirmed that treatment of microglia with anti-inflammatory cytokines such as IL-4 and IL-13 induces a gene profile typical of alternative activation similar to that previously observed in peripheral macrophages. We then used this gene expression profile to examine two mouse models of AD, the APPsw (Tg-2576) and Tg-SwDI, models for amyloid deposition and for cerebral amyloid angiopathy (CAA) respectively. AGI, MRC1 and YM1 mRNA levels were significantly increased in the Tg-2576 mouse brains compared to age-matched controls while TNFα and NOS2 mRNA levels, genes commonly associated with classical activation, increased or did not change, respectively. Only TNFα mRNA increased in the Tg-SwDI mouse brain. Alternative activation genes were also identified in brain samples from individuals with AD and were compared to age-matched control individuals. In AD brain, mRNAs for TNFα, AGI, MRC1 and the chitinase-3 like 1 and 2 genes (CHI3L1; CHI3L2) were significantly increased

  7. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    PubMed Central

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  8. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    SciTech Connect

    Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  9. VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression

    PubMed Central

    Pitzschke, Andrea; Djamei, Armin; Teige, Markus; Hirt, Heribert

    2009-01-01

    The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway. PMID:19820165

  10. Influence of redox-active compounds and PXR-activators on human MRP1 and MRP2 gene expression.

    PubMed

    Kauffmann, Hans Martin; Pfannschmidt, Sylvia; Zöller, Heike; Benz, Anke; Vorderstemann, Birgit; Webster, Jeanette I; Schrenk, Dieter

    2002-02-28

    In the present study, we investigated the inducibility of the drug conjugate transporter genes MRP1 and MRP2 by redox-active compounds such as tertiary butylated hydroquinone (tBHQ) and quercetin and by chemicals known to activate the pregnane X receptor (PXR) such as rifampicin and clotrimazol and by the metalloid compound arsenite. The human MRP2 gene was found to be inducible in HepG2 cells by rifampicin, clotrimazol, arsenite and tBHQ. As MRP1 expression is extremely low in HepG2 cells, its inducibility was studied in MCF-7 cells. However, only tBHQ and quercetin acted as inducers, but not the other compounds investigated. Reporter gene assays demonstrated that proximal promoter regions of the genes contribute to the induction by tBHQ, quercetin (MRP1) and clotrimazol (MRP2). However, the deletion of binding sites supposed to mediate the induction process (a PXR-binding element-like sequence for the clotrimazol effect and an ARE (antioxidative response element) for the tBHQ/quercetin effect) did not result in a significant decrease in the induction factor indicating that other parts of the promoter are probably involved in the induction process. In summary, expression of both genes can be up-regulated by redox-active compounds, while the other compounds tested induced only MRP2 but not MRP1 expression.

  11. AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

    PubMed

    Hubert, A; Husson, A; Chédeville, A; Lavoinne, A

    2000-09-22

    The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

  12. Tumor necrosis factor gene expression is mediated by protein kinase C following activation by ionizing radiation.

    SciTech Connect

    Hallahan, D. E.; Virudachalam, S.; Sherman, M. L.; Huberman, E.; Kufe, D. W.; Weichselbaum, R. R.; Univ. of Chicago; Dana-Farber Cancer Inst.; Univ. of Chicago

    1991-01-01

    Tumor necrosis factor (TNF) production following X-irradiation has been implicated in the biological response to ionizing radiation. Protein kinase C (PKC) is suggested to participate in TNF transcriptional induction and X-ray-mediated gene expression. We therefore studied radiation-mediated TNF expression in HL-60 cells with diminished PKC activity produced by either pretreatment with protein kinase inhibitors or prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Both treatments resulted in attenuation of radiation-mediated TNF induction. Consistent with these results, we found no detectable induction of TNF expression following X-irradiation in the HL-60 variant deficient in PKC-mediated signal transduction. The rapid activation of PKC following {gamma}-irradiation was established using an in vitro assay measuring phosphorylation of a PKC specific substrate. A 4.5-fold increase in PKC activity occurred 15 to 30 s following irradiation, which declined to baseline at 60 s. Two-dimensional gel electrophoresis of phosphoproteins extracted from irradiated cells demonstrated in vivo phosphorylation of the PKC specific substrate Mr 80,000 protein at 45 s following X-irradiation. These findings indicate that signal transduction via the PKC pathway is required for the induction of TNF gene expression by ionizing radiation.

  13. Temperature and water loss affect ADH activity and gene expression in grape berry during postharvest dehydration.

    PubMed

    Cirilli, Marco; Bellincontro, Andrea; De Santis, Diana; Botondi, Rinaldo; Colao, Maria Chiara; Muleo, Rosario; Mencarelli, Fabio

    2012-05-01

    Clusters of Aleatico wine grape were picked at 18°Brix and placed at 10, 20, or 30°C, 45% relative humidity (RH) and 1.5m/s of air flow to dehydrate the berries up to 40% of loss of initial fresh weight. Sampling was done at 0%, 10%, 20%, 30%, and 40% weight loss (wl). ADH (alcohol dehydrogenase) gene expression, enzyme activity, and related metabolites were analysed. At 10°C, acetaldehyde increased rapidly and then declined, while ethanol continued to rise. At 20°C, acetaldehyde and ethanol increased significantly with the same pattern and declined at 40%wl. At 30°C, acetaldehyde did not increase but ethanol increased rapidly already at 10%wl. At the latter temperature, a significant increase in acetic acid and ethyl acetate occurred, while at 10°C their values were low. At 30°C, the ADH activity (ethanol to acetaldehyde direction), increased rapidly but acetaldehyde did not rise because of its oxidation to acetic acid, which increased together with ethyl acetate. At 10°C, the ADH activity increased at 20%wl and continued to rise even at 40%wl, meaning that ethanol oxidation was delayed. At 20°C, the behaviour was intermediate to the other temperatures. The relative expression of the VvAdh2 gene was the highest at 10°C already at 10%wl in a synchrony with the ADH activity, indicating a rapid response likely due to low temperature. The expression subsequently declined. At 20 and 30°C, the expression was lower and increased slightly during dehydration in combination with the ADH activity. This imbalance between gene expression and ADH activity at 10°C, as well as the unexpected expression of the carotenoid cleavage dioxygenase 1 (CCD1) gene, opens the discussion on the stress sensitivity and transcription event during postharvest dehydration, and the importance of carefully monitoring temperature during dehydration.

  14. Modeling notch signaling in normal and neoplastic hematopoiesis: global gene expression profiling in response to activated notch expression.

    PubMed

    Ganapati, Uma; Tan, Hongying Tina; Lynch, Maureen; Dolezal, Milana; de Vos, Sven; Gasson, Judith C

    2007-08-01

    In normal hematopoiesis, proliferation is tightly linked to differentiation in ways that involve cell-cell interaction with stromal elements in the bone marrow stem cell niche. Numerous in vitro and in vivo studies strongly support a role for Notch signaling in the regulation of stem cell renewal and hematopoiesis. Not surprisingly, mutations in the Notch gene have been linked to a number of types of malignancies. To better define the function of Notch in both normal and neoplastic hematopoiesis, a tetracycline-inducible system regulating expression of a ligand-independent, constitutively active form of Notch1 was introduced into murine E14Tg2a embryonic stem cells. During coculture, OP9 stromal cells induce the embryonic stem cells to differentiate first to hemangioblasts and subsequently to hematopoietic stem cells. Our studies indicate that activation of Notch signaling in flk+ hemangioblasts dramatically reduces their survival and proliferative capacity and lowers the levels of hematopoietic stem cell markers CD34 and c-Kit and the myeloid marker CD11b. Global gene expression profiling of day 8 hematopoietic progenitors in the absence and presence of activated Notch yield candidate genes required for normal hematopoietic differentiation, as well as putative downstream targets of oncogenic forms of Notch including the noncanonical Wnts Wnt4 and 5A. Disclosure of potential conflicts of interest is found at the end of this article.

  15. Mitogen-activated protein kinase signaling controls basal and oncostatin M-mediated JUNB gene expression.

    PubMed

    Hicks, Mellissa J; Hu, Qiuping; Macrae, Erin; DeWille, James

    2015-05-01

    The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10A(met), SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10A(met) cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the "transcription initiating" form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

  16. The Light Wavelength Affects the Ontogeny of Clock Gene Expression and Activity Rhythms in Zebrafish Larvae.

    PubMed

    Di Rosa, Viviana; Frigato, Elena; López-Olmeda, José F; Sánchez-Vázquez, Francisco J; Bertolucci, Cristiano

    2015-01-01

    Light plays a key role in synchronizing rhythms and setting the phase of early development. However, to date, little is known about the impact of light wavelengths during the ontogeny of the molecular clock and the behavioural rhythmicity. The aim of this research was to determine the effect of light of different wavelengths (white, blue and red) on the onset of locomotor activity and clock gene (per1b, per2, clock1, bmal1 and dbp) expression rhythms. For this purpose, 4 groups of zebrafish embryo/larvae were raised from 0 to 7 days post-fertilization (dpf) under the following lighting conditions: three groups maintained under light:dark (LD) cycles with white (full visible spectrum, LDW), blue (LDB), or red light (LDR), and one group raised under constant darkness (DD). The results showed that lighting conditions influenced activity rhythms. Larvae were arrhythmic under DD, while under LD cycles they developed wavelength-dependent daily activity rhythms which appeared earlier under LDB (4 dpf) than under LDW or LDR (5 dpf). The results also revealed that development and lighting conditions influenced clock gene expression. While clock1 rhythmic expression appeared in all lighting conditions at 7 dpf, per1b, per2 and dbp showed daily variations already at 3 dpf. Curiously, bmal1 showed consistent rhythmic expression from embryonic stage (0 dpf). Summarizing, the data revealed that daily rhythms appeared earlier in the larvae reared under LDB than in those reared under LDW and LDR. These results emphasize the importance of lighting conditions and wavelengths during early development for the ontogeny of daily rhythms of gene expression and how these rhythms are reflected on the behavioural rhythmicity of zebrafish larvae.

  17. Hydrogen peroxide activates cell death and defense gene expression in birch.

    PubMed

    Pellinen, Riikka I; Korhonen, Minna-Sisko; Tauriainen, Airi A; Palva, E Tapio; Kangasjärvi, Jaakko

    2002-10-01

    The function of hydrogen peroxide (H(2)O(2)) as a signal molecule regulating gene expression and cell death induced by external stresses was studied in birch (Betula pendula). Ozone (O(3)), Pseudomonas syringae pv syringae (Pss), and wounding all induced cell death of various extents in birch leaves. This was temporally preceded and closely accompanied by H(2)O(2) accumulation at, and especially surrounding, the lesion sites. O(3) and Pss, along with an artificial H(2)O(2) producing system glucose (Glc)/Glc oxidase, elicited elevated mRNA levels corresponding to genes encoding reactive oxygen species detoxifying enzymes, Pal, Ypr10, and mitochondrial phosphate translocator 1. In addition to the regulation of gene expression, Glc/Glc oxidase also induced endogenous H(2)O(2) production in birch leaves, accompanied by cell death that resembled O(3) and Pss damage. Wound-induced gene expression differed from that induced by O(3) and Pss. Thus, it appears that at least two separate defense pathways can be activated in birch leaves by stress factors, even though the early H(2)O(2) accumulation response is common among them all.

  18. Controlling Hox gene expression and activity to build the vertebrate axial skeleton.

    PubMed

    Casaca, Ana; Santos, Ana Cristina; Mallo, Moisés

    2014-01-01

    It has long been known that Hox genes are central players in patterning the vertebrate axial skeleton. Extensive genetic studies in the mouse have revealed that the combinatorial activity of Hox genes along the anterior-posterior body axis specifies different vertebral identities. In addition, Hox genes were instrumental for the evolutionary diversification of the vertebrate body plan. In this review, we focus on fundamental questions regarding the intricate mechanisms controlling Hox gene activity. In particular, we discuss the functional relevance of the precise timing of Hox gene activation in the embryo. Moreover, we provide insight into the epigenetic regulatory mechanisms that are likely to control this process and are responsible for the maintenance of spatially restricted Hox expression domains throughout embryonic development. We also analyze how specific features of each Hox protein may contribute to the functional diversity of Hox family. Altogether, the work reviewed here further supports the notion that the Hox program is far more complex than initially assumed. Exciting new findings will surely emerge in the years ahead.

  19. Activation of GATA4 gene expression at the early stage of cardiac specification

    PubMed Central

    Yilbas, Ayse E.; Hamilton, Alison; Wang, Yingjian; Mach, Hymn; Lacroix, Natascha; Davis, Darryl R.; Chen, Jihong; Li, Qiao

    2014-01-01

    Currently, there are no effective treatments to directly repair damaged heart tissue after cardiac injury since existing therapies focus on rescuing or preserving reversibly damaged tissue. Cell-based therapies using cardiomyocytes generated from stem cells present a promising therapeutic approach to directly replace damaged myocardium with new healthy tissue. However, the molecular mechanisms underlying the commitment of stem cells into cardiomyocytes are not fully understood and will be critical to guide this new technology into the clinic. Since GATA4 is a critical regulator of cardiac differentiation, we examined the molecular basis underlying the early activation of GATA4 gene expression during cardiac differentiation of pluripotent stem cells. Our studies demonstrate the direct involvement of histone acetylation and transcriptional coactivator p300 in the regulation of GATA4 gene expression. More importantly, we show that histone acetyltransferase (HAT) activity is important for GATA4 gene expression with the use of curcumin, a HAT inhibitor. In addition, the widely used histone deacetylase inhibitor valproic acid enhances both histone acetylation and cardiac specification. PMID:24790981

  20. Activation of GATA4 gene expression at the early stage of cardiac specification

    NASA Astrophysics Data System (ADS)

    Yilbas, Ayse; Hamilton, Alison; Wang, Yingjian; Mach, Hymn; Lacroix, Natascha; Davis, Darryl; Chen, Jihong; LI, Qiao

    2014-03-01

    Currently, there are no effective treatments to directly repair damaged heart tissue after cardiac injury since existing therapies focus on rescuing or preserving reversibly damaged tissue. Cell-based therapies using cardiomyocytes generated from stem cells present a promising therapeutic approach to directly replace damaged myocardium with new healthy tissue. However, the molecular mechanisms underlying the commitment of stem cells into cardiomyocytes are not fully understood and will be critical to guide this new technology into the clinic. Since GATA4 is a critical regulator of cardiac differentiation, we examined the molecular basis underlying the early activation of GATA4 gene expression during cardiac differentiation of pluripotent stem cells. Our studies demonstrate the direct involvement of histone acetylation and transcriptional coactivator p300 in the regulation of GATA4 gene expression. More importantly, we show that histone acetyltransferase (HAT) activity is important for GATA4 gene expression with the use of curcumin, a HAT inhibitor. In addition, the widely used histone deacetylase inhibitor valproic acid enhances both histone acetylation and cardiac specification.

  1. Zebra fish myc family and max genes: differential expression and oncogenic activity throughout vertebrate evolution.

    PubMed Central

    Schreiber-Agus, N; Horner, J; Torres, R; Chiu, F C; DePinho, R A

    1993-01-01

    To gain insight into the role of Myc family oncoproteins and their associated protein Max in vertebrate growth and development, we sought to identify homologs in the zebra fish (Brachydanio rerio). A combination of a polymerase chain reaction-based cloning strategy and low-stringency hybridization screening allowed for the isolation of zebra fish c-, N-, and L-myc and max genes; subsequent structural characterization showed a high degree of conservation in regions that encode motifs of known functional significance. On the functional level, zebra fish Max, like its mammalian counterpart, served to suppress the transformation activity of mouse c-Myc in rat embryo fibroblasts. In addition, the zebra fish c-myc gene proved capable of cooperating with an activated H-ras to effect the malignant transformation of mammalian cells, albeit with diminished potency compared with mouse c-myc. With respect to their roles in normal developing tissues, the differential temporal and spatial patterns of steady-state mRNA expression observed for each zebra fish myc family member suggest unique functions for L-myc in early embryogenesis, for N-myc in establishment and growth of early organ systems, and for c-myc in increasingly differentiated tissues. Furthermore, significant alterations in the steady-state expression of zebra fish myc family genes concomitant with relatively constant max expression support the emerging model of regulation of Myc function in cellular growth and differentiation. Images PMID:8474440

  2. Targeting Activation of Specific NF-κB Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

    PubMed Central

    Djuric, Zdenka; Kashif, Muhammed; Fleming, Thomas; Muhammad, Sajjad; Piel, David; von Bauer, Rüdiger; Bea, Florian; Herzig, Stephan; Zeier, Martin; Pizzi, Marina; Isermann, Berend; Hecker, Markus; Schwaninger, Markus; Bierhaus, Angelika; Nawroth, Peter P

    2012-01-01

    Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein−/− (ApoE−/−) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease. PMID:23114885

  3. Riproximin's activity depends on gene expression and sensitizes PDAC cells to TRAIL.

    PubMed

    Adwan, Hassan; Murtaja, Ahmed; Kadhim Al-Taee, Khamael; Pervaiz, Asim; Hielscher, Thomas; Berger, Martin R

    2014-09-01

    Riproximin (Rpx) is a type II ribosome inactivating protein, which was investigated for its activity in pancreatic ductal adenocarcinoma (PDAC) in a panel of 17 human and rat PDAC cell lines and in rat pancreatic cancer liver metastasis. Cytotoxicity in response to Rpx was determined by MTT assay, apoptosis by flow cytometry and qRT-PCR for apoptosis related genes, and the modulation of the transcriptome was monitored by micro array analysis. The combination effect of Rpx and TRAIL was assessed by MTT assay. Rpx showed high but varying cytotoxicity in PDAC cells. Based on overall gene expression, the sensitivity of these cells was linked to genes involved in apoptosis. Furthermore, based on the affinity of Rpx for CEA, the expression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) genes was significantly related to Rpx's cytotoxicity in cells with CEACAM gene expression. Exposure of Suit2-007 cells to Rpx induced the mRNA expression of members of signaling pathways initiating from most death receptors, and down modulation of TRAIL. Apoptosis was increased as shown by FACS analysis. Combination of Rpx with TRAIL resulted in a synergistic cytotoxic effect in human Suit2-007 and rat ASML cells, as evidenced by a 6-fold lower tumor cell survival than expected from an additive combination effect. Treatment of BDX rats bearing intra-portally implanted Suit2-007 cells showed a highly significant anticancer effect and indicated an application of Rpx against pancreatic cancer metastasis to the liver. These data favor further evaluation of Rpx as anticancer agent in PDAC.

  4. Fur activates expression of the 2-oxoglutarate oxidoreductase genes (oorDABC) in Helicobacter pylori.

    PubMed

    Gilbreath, Jeremy J; West, Abby L; Pich, Oscar Q; Carpenter, Beth M; Michel, Sarah; Merrell, D Scott

    2012-12-01

    Helicobacter pylori is a highly successful pathogen that colonizes the gastric mucosa of ∼50% of the world's population. Within this colonization niche, the bacteria encounter large fluctuations in nutrient availability. As such, it is critical that this organism regulate expression of key metabolic enzymes so that they are present when environmental conditions are optimal for growth. One such enzyme is the 2-oxoglutarate (α-ketoglutarate) oxidoreductase (OOR), which catalyzes the conversion of α-ketoglutarate to succinyl coenzyme A (succinyl-CoA) and CO(2). Previous studies from our group suggested that the genes that encode the OOR are activated by iron-bound Fur (Fe-Fur); microarray analysis showed that expression of oorD, oorA, and oorC was altered in a fur mutant strain of H. pylori. The goal of the present work was to more thoroughly characterize expression of the oorDABC genes in H. pylori as well as to define the role of Fe-Fur in this process. Here we show that these four genes are cotranscribed as an operon and that expression of the operon is decreased in a fur mutant strain. Transcriptional start site mapping and promoter analysis revealed the presence of a canonical extended -10 element but a poorly conserved -35 element upstream of the +1. Additionally, we identified a conserved Fur binding sequence ∼130 bp upstream of the transcriptional start site. Transcriptional analysis using promoter fusions revealed that this binding sequence was required for Fe-Fur-mediated activation. Finally, fluorescence anisotropy assays indicate that Fe-Fur specifically bound this Fur box with a relatively high affinity (dissociation constant [K(d)] = 200 nM). These findings provide novel insight into the genetic regulation of a key metabolic enzyme and add to our understanding of the diverse roles Fur plays in gene regulation in H. pylori.

  5. Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity.

    PubMed

    Villamizar, Genis A Castillo; Nacke, Heiko; Daniel, Rolf

    2017-01-01

    The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

  6. Visualizing changes in circuit activity resulting from denervation and reinnervation using immediate early gene expression.

    PubMed

    Temple, Meredith D; Worley, Paul F; Steward, Oswald

    2003-04-01

    We describe a novel strategy to evaluate circuit function after brain injury that takes advantage of experience-dependent immediate early gene (IEG) expression. When normal rats undergo training or are exposed to a novel environment, there is a strong induction of IEG expression in forebrain regions, including the hippocampus. This gene induction identifies the neurons that are engaged during the experience. Here, we demonstrate that experience-dependent IEG induction is diminished after brain injury in young adult rats (120-200 gm), specifically after unilateral lesions of the entorhinal cortex (EC), and then recovers with a time course consistent with reinnervation. In situ hybridization techniques were used to assess the expression of the activity-regulated cytoskeleton-associated protein Arc at various times after the lesion (4, 8, 12, 16, or 30 d). One group of rats was allowed to explore a complex novel environment for 1 hr; control operated animals remained in their home cage. In unoperated animals, exposure to the novel environment induced Arc mRNA levels in most pyramidal neurons in CA1, in many pyramidal neurons in CA3, and in a small number of dentate granule cells. This characteristic pattern of induction was absent at early time points after unilateral EC lesions (4 and 8 d) but recovered progressively at later time points. The recovery of Arc expression occurred with approximately the same time course as the reinnervation of the dentate gyrus as a result of postlesion sprouting. These results document a novel approach for quantitatively assessing activity-regulated gene expression in polysynaptic circuits after trauma.

  7. Model-Based Characterization of Inflammatory Gene Expression Patterns of Activated Macrophages

    PubMed Central

    Ehlting, Christian; Thomas, Maria; Zanger, Ulrich M.; Sawodny, Oliver; Häussinger, Dieter; Bode, Johannes G.

    2016-01-01

    Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization. PMID:27464342

  8. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  9. Fine-tuning of nif and fix gene expression by upstream activator sequences in Bradyrhizobium japonicum.

    PubMed

    Gubler, M

    1989-02-01

    The significance of Bradyrhizobium japonicum upstream activator sequences (UASs) for differential NifA-mediated fix and nif gene expression was investigated by two means: (i) hybrid fixA- and fixB-lacZ fusions were constructed by transposing a nifH-UAS cartridge in front of their promoters; and (ii) B. japonicum mutants were generated carrying specific chromosomal deletions or UAS cartridge insertions within the fixA, fixB or nifH promoter-upstream regions. Expression of fixA was not affected, and expression of fixB decreased only to 42%, when the respective fixA and fixB promoter-upstream DNAs were deleted. This shows that in B. japonicum the NifA-dependent activation of at least the fixA promoter does not require the presence of a closely adjacent UAS. Deletion of the UASs in front of the nifH gene not only reduced the expression of nifH down to 2.5% but, surprisingly, also resulted in a reduction of the fixB mRNA level to less than 20%. This suggests that the nifH-UASs may exert a long-range effect on the expression of the 3-kb-distant fixBCX operon in nif cluster I or B. japonicum. Artificial transposition of the nifH-UASs in front of the fixA and fixB promoters strongly enhanced fixA and fixB expression.

  10. Changes in gene expression and catalase activity in Oryza sativa L. under abiotic stress.

    PubMed

    Vighi, I L; Benitez, L C; do Amaral, M N; Auler, P A; Moraes, G P; Rodrigues, G S; da Maia, L C; Pinto, L S; Braga, E J B

    2016-11-03

    Different rice (Oryza sativa L.) genotypes were subjected to high salinity and low temperature (150 mM NaCl and 13°C, respectively) for 0, 6, 24, 48, or 72 h. We evaluated the simultaneous expression of the genes OsCATA, OsCATB, and OsCATC, correlated gene expression with enzyme activity, and verified the regulation of these genes through identification of cis-elements in the promoter region. The hydrogen peroxide content increased in a tolerant genotype and decreased in a sensitive genotype under both stress conditions. Lipid peroxidation increased in the tolerant genotype when exposed to cold, and in the sensitive genotype when exposed to high salinity. Catalase activity significantly increased in both genotypes when subjected to 13°C. In the tolerant genotype, OsCATA and OsCATB were the most responsive to high salinity and cold, while in the sensitive genotype, OsCATA and OsCATC responded positively to saline stress, as did OsCATA and OsCATB to low temperature. Cis-element analysis identified different regulatory sequences in the catalase promoter region of each genotype. The sensitive genotype maintained a better balance between hydrogen oxyacid levels, catalase activity, and lipid peroxidation under low temperature than the resistant genotype. OsCATA and OsCATB were the most responsive in the salt-tolerant genotype to cold, OsCATA and OsCATC were the most responsive to saline stress, and OsCATA and OsCATB were the most responsive to chilling stress in the sensitive genotype. There were positive correlations between catalase activity and OsCATB expression in the tolerant genotype under saline stress and in the sensitive genotype under cold stress.

  11. Enhancement of glucocorticoid receptor-mediated gene expression by constitutively active heat shock factor 1.

    PubMed

    Jones, Thomas J; Li, Dapei; Wolf, Irene M; Wadekar, Subhagya A; Periyasamy, Sumudra; Sánchez, Edwin R

    2004-03-01

    To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorticoid receptor (GR) activity, we placed a constitutively active mutant of human HSF1 (hHSF1-E189) under the control of a doxycycline (DOX)-inducible vector. In mouse L929 cells, DOX-induced expression of hHSF1-E189 correlated with in vivo occupancy of the human heat shock protein 70 (hHsp70) promoter (chromatin-immunoprecipitation assay) and with increased activity under nonstress conditions at the hHsp70 promoter controlling expression of chloramphenicol acetyl transferase (CAT) (p2500-CAT). Comparison of hHSF1-E189 against stress-activated, endogenous HSF1 for DNA-binding, p2500-CAT, and Hsp70 protein expression activities showed the mutant factor to have lower, but clearly detectable, activities as compared with wild-type factor. Thus, the hHSF1-E189 mutant is capable of replicating these key functions of endogenous HSF1, albeit at reduced levels. To assess the involvement of hHSF1-E189 in GR activity, DOX-induced expression of hHSF1-E189 was performed in L929 cells expressing the minimal pGRE(2)E1B-CAT reporter. hHSF1-E189 protein expression in these cells was maximal at 24 h of DOX and remained constant up to 72 h. hHSF1-E189 expressed under these conditions was found both in the cytosolic and nuclear compartments, in a state capable of binding DNA. More importantly, GR activity at the pGRE(2)E1B-CAT promoter was found to increase after DOX-induced expression of hHSF1-E189. The potentiation of GR by hHSF1-E189 occurred at saturating concentrations of hormone and was dependent on at least 48 h of hHSF1-E189 up-regulation, suggesting that time was needed for an HSF1-induced factor to accumulate to a threshold level. Initial efforts to characterize how hHSF1-E189 controls GR signaling showed that it does not occur through alterations of GR protein levels or changes in GR hormone binding capacity. In summary, our observations provide the first molecular evidence for the

  12. Expression and Enzyme Activity Detection of a Sepiapterin Reductase Gene from Musca domestica Larva.

    PubMed

    Tang, Yan; Pei, Zhihua; Liu, Lei; Wang, Dongfang; Kong, Lingcong; Liu, Shuming; Jiang, Xiuyun; Gao, Yunhang; Ma, Hongxia

    2017-02-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of Vmax and Km of the MDSPR for SP were 6.83 μM/min and 23.48 μM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.

  13. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction.

    PubMed

    Rudenko, Andrii; Dawlaty, Meelad M; Seo, Jinsoo; Cheng, Albert W; Meng, Jia; Le, Thuc; Faull, Kym F; Jaenisch, Rudolf; Tsai, Li-Huei

    2013-09-18

    The ten-eleven translocation (Tet) family of methylcytosine dioxygenases catalyze oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and promote DNA demethylation. Despite the abundance of 5hmC and Tet proteins in the brain, little is known about the functions of the neuronal Tet enzymes. Here, we analyzed Tet1 knockout mice (Tet1KO) and found downregulation of multiple neuronal activity-regulated genes, including Npas4, c-Fos, and Arc. Furthermore, Tet1KO animals exhibited abnormal hippocampal long-term depression and impaired memory extinction. Analysis of the key regulatory gene, Npas4, indicated that its promoter region, containing multiple CpG dinucleotides, is hypermethylated in both naive Tet1KO mice and after extinction training. Such hypermethylation may account for the diminished expression of Npas4 itself and its downstream targets, impairing transcriptional programs underlying cognitive processes. In summary, we show that neuronal Tet1 regulates normal DNA methylation levels, expression of activity-regulated genes, synaptic plasticity, and memory extinction.

  14. The herbicide flumioxazin stimulates pathogenesis-related gene expression and enzyme activities in Vitis vinifera.

    PubMed

    Castro, Antonio Jesús; Saladin, Gäelle; Bézier, Annie; Mazeyrat-Gourbeyre, Florence; Baillieul, Fabienne; Clément, Christophe

    2008-11-01

    In this work, the capacity of the soil-applied herbicide flumioxazin (fmx) to trigger defence mechanisms was assessed using 6-week-old in vitro grown Vitis vinifera L. plantlets. Time-course studies demonstrated that the herbicide induced the expression of basic beta-1,3-glucanase (Vvglu), basic chitinase (Vvchit1b) and PR10 (VvPR10.3) genes encoding three pathogenesis-related (PR) proteins involved in grapevine defence against pathogens. Thus, all transcripts accumulated in grapevine tissues to reach maximum values after 24-72 h of herbicide exposure, except for VvPR10.3 gene expression, which was induced in roots and stems but not in leaves. Induction of PR genes was observed to a greater extent in roots and leaves, and its intensity diminished in the stems although still remained noteworthy. The activities of beta-1,3-glucanase and chitinase enzymes significantly increased in the whole plant after herbicide exposure and were still stimulated 21 days after the beginning of treatments. Similarly, the most remarkable effect occurred in roots. However, all enzyme activities tested were stimulated in the upper aerial tissues as well, indicating that fmx or a derived product acts systemically, likely via root uptake.

  15. Palmitate increases musclin gene expression through activation of PERK signaling pathway in C2C12 myotubes.

    PubMed

    Gu, Ning; Guo, Qian; Mao, Ke; Hu, Hailong; Jin, Sanli; Zhou, Ying; He, Hongjuan; Oh, Yuri; Liu, Chuanpeng; Wu, Qiong

    2015-11-20

    Musclin is a type of muscle-secreted cytokine and its increased gene expression induces insulin resistance in type 2 diabetes. However, the mechanism underlying increased musclin gene expression is currently unclear. Excessive saturated fatty acids (SFA) can activate the secretion of several muscle-secreted cytokines as well as endoplasmic reticulum (ER) stress pathway, thereby contributing to the development of type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the effect of palmitate, the most abundant SFA in the plasma, on the gene expression of musclin in C2C12 myotubes. Treatment of C2C12 myotubes with palmitate or tunicamycin significantly increased the expression of musclin as well as ER stress-related genes, but treatment with oleate did not. Pre-treatment of C2C12 myotubes with 4-phenyl butyrate suppressed the expression of ER stress-related genes, simultaneously, resulting in decreased expression of the musclin gene induced by palmitate or tunicamycin. These results indicate that ER stress is related to palmitate-induced musclin gene expression. Moreover, palmitate-induced musclin gene expression was significantly inhibited in C2C12 myotubes when PERK pathway signaling was suppressed by knockdown of the PERK gene or treatment with GSK2656157, a PERK autophosphorylation inhibitor. However, there was no difference in the palmitate-induced musclin gene expression when IRE1 and ATF6 signaling pathways were suppressed by knockdown of the IRE1 and ATF6 genes. These findings suggest that palmitate increases musclin gene expression via the activation of the PERK signaling pathway in C2C12 myotubes.

  16. Oscillation of p38 activity controls efficient pro-inflammatory gene expression

    PubMed Central

    Tomida, Taichiro; Takekawa, Mutsuhiro; Saito, Haruo

    2015-01-01

    The p38 MAP kinase signalling pathway controls inflammatory responses and is an important target of anti-inflammatory drugs. Although pro-inflammatory cytokines such as interleukin-1β (IL-1β) appear to induce only transient activation of p38 (over ∼60 min), longer cytokine exposure is necessary to induce p38-dependent effector genes. Here we study the dynamics of p38 activation in individual cells using a Förster resonance energy transfer (FRET)-based p38 activity reporter. We find that, after an initial burst of activity, p38 MAPK activity subsequently oscillates for more than 8 h under continuous IL-1β stimulation. However, as this oscillation is asynchronous, the measured p38 activity population average is only slightly higher than basal level. Mathematical modelling, which we have experimentally verified, indicates that the asynchronous oscillation of p38 is generated through a negative feedback loop involving the dual-specificity phosphatase MKP-1/DUSP1. We find that the oscillatory p38 activity is necessary for efficient expression of pro-inflammatory genes such as IL-6, IL-8 and COX-2. PMID:26399197

  17. Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

    PubMed

    Wagstaff, Carol; Leverentz, Michael K; Griffiths, Gareth; Thomas, Brian; Chanasut, Usawadee; Stead, Anthony D; Rogers, Hilary J

    2002-02-01

    The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

  18. Activity, expression and function of a second Drosophila protein kinase a catalytic subunit gene

    SciTech Connect

    Melendez, A.; Li, W.; Kalderon, D.

    1995-12-01

    The DC2 was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. 62 refs., 10 figs., 2 tabs.

  19. Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements.

    PubMed

    Ochi, Kozo; Tanaka, Yukinori; Tojo, Shigeo

    2014-02-01

    Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

  20. NF-kB activation and its downstream target genes expression after heavy ions exposure

    NASA Astrophysics Data System (ADS)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  1. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

    PubMed Central

    Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

  2. Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway

    PubMed Central

    Shen, Zhongliang; Liu, Yanfeng; Wang, Wei; Tao, Shuai; Cui, Xiaoxian; Liu, Jing

    2017-01-01

    The role of hepatocyte nuclear factor 1α (HNF1α) in the regulation of gene expression and replication of hepatitis B virus (HBV) is not fully understood. Previous reports have documented the induction of the expression of viral large surface protein (LHBs) by HNF1α through activating viral Sp1 promoter. Large amount of LHBs can block the secretion of hepatitis B surface antigen (HBsAg). Here we found that HNF1α overexpression inhibited HBV gene expression and replication in Huh7 cells, resulting in marked decreases in HBsAg, hepatitis B e antigen (HBeAg) and virion productions. In contrast, knockdown of endogenous HNF1α expression enhanced viral gene expression and replication. This HNF1α-mediated inhibition did not depend on LHBs. Instead, HNF1α promoted the expression of NF-κB p65 and slowed p65 protein degradation, leading to nuclear accumulation of p65 and activation of the NF-κB signaling, which in turn inhibited HBV gene expression and replication. The inhibitor of the NF-κB signaling, IκBα-SR, could abrogate this HNF1α-mediated inhibition. While the dimerization domain of HNF1α was dispensable for the induction of LHBs expression, all the domains of HNF1α was required for the inhibition of HBV gene expression. Our findings identify a novel role of HNF1α in the regulation of HBV gene expression and replication. PMID:28319127

  3. A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

    PubMed Central

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

  4. Discovering modulators of gene expression

    PubMed Central

    Babur, Özgün; Demir, Emek; Gönen, Mithat; Sander, Chris; Dogrusoz, Ugur

    2010-01-01

    Proteins that modulate the activity of transcription factors, often called modulators, play a critical role in creating tissue- and context-specific gene expression responses to the signals cells receive. GEM (Gene Expression Modulation) is a probabilistic framework that predicts modulators, their affected targets and mode of action by combining gene expression profiles, protein–protein interactions and transcription factor–target relationships. Using GEM, we correctly predicted a significant number of androgen receptor modulators and observed that most modulators can both act as co-activators and co-repressors for different target genes. PMID:20466809

  5. Disrupted Ultradian Activity Rhythms and Differential Expression of Several Clock Genes in Interleukin-6-Deficient Mice

    PubMed Central

    Monje, Francisco J.; Cicvaric, Ana; Acevedo Aguilar, Juan Pablo; Elbau, Immanuel; Horvath, Orsolya; Diao, Weifei; Glat, Micaela; Pollak, Daniela D.

    2017-01-01

    The characteristics of the cycles of activity and rest stand out among the most intensively investigated aspects of circadian rhythmicity in humans and experimental animals. Alterations in the circadian patterns of activity and rest are strongly linked to cognitive and emotional dysfunctions in severe mental illnesses such as Alzheimer’s disease (AD) and major depression (MDD). The proinflammatory cytokine interleukin 6 (IL-6) has been prominently associated with the pathogenesis of AD and MDD. However, the potential involvement of IL-6 in the modulation of the diurnal rhythms of activity and rest has not been investigated. Here, we set out to study the role of IL-6 in circadian rhythmicity through the characterization of patterns of behavioral locomotor activity in IL-6 knockout (IL-6 KO) mice and wild-type littermate controls. Deletion of IL-6 did not alter the length of the circadian period or the amount of locomotor activity under either light-entrained or free-running conditions. IL-6 KO mice also presented a normal phase shift in response to light exposure at night. However, the temporal architecture of the behavioral rhythmicity throughout the day, as characterized by the quantity of ultradian activity bouts, was significantly impaired under light-entrained and free-running conditions in IL-6 KO. Moreover, the assessment of clock gene expression in the hippocampus, a brain region involved in AD and depression, revealed altered levels of cry1, dec2, and rev-erb-beta in IL-6 KO mice. These data propose that IL-6 participates in the regulation of ultradian activity/rest rhythmicity and clock gene expression in the mammalian brain. Furthermore, we propose IL-6-dependent circadian misalignment as a common pathogenetic principle in some neurodegenerative and neuropsychiatric disorders. PMID:28382017

  6. Effects of Skilled Training on Sleep Slow Wave Activity and Cortical Gene Expression in the Rat

    PubMed Central

    Hanlon, Erin C.; Faraguna, Ugo; Vyazovskiy, Vladyslav V.; Tononi, Giulio; Cirelli, Chiara

    2009-01-01

    Study Objective: The best characterized marker of sleep homeostasis is the amount of slow wave activity (SWA, 0.5–4 Hz) during NREM sleep. SWA increases as a function of previous waking time and declines during sleep, but the underlying mechanisms remain unclear. We have suggested that SWA homeostasis is linked to synaptic potentiation associated with learning during wakefulness. Indeed, studies in rodents and humans found that SWA increases after manipulations that presumably enhance synaptic strength, but the evidence remains indirect. Here we trained rats in skilled reaching, a task known to elicit long-term potentiation in the trained motor cortex, and immediately after learning measured SWA and cortical protein levels of c-fos and Arc, 2 activity-dependent genes involved in motor learning. Design: Intracortical local field potential recordings and training on reaching task. Setting: Basic sleep research laboratory. Patients or Participants: Long Evans adult male rats. Interventions: N/A Measurements and Results: SWA increased post-training in the trained cortex (the frontal cortex contralateral to the limb used to learn the task), with smaller or no increase in other cortical areas. This increase was reversible within 1 hour, specific to NREM sleep, and positively correlated with changes in performance during the prior training session, suggesting that it reflects plasticity and not just motor activity. Fos and Arc levels were higher in the trained relative to untrained motor cortex immediately after training, but this asymmetry was no longer present after 1 hour of sleep. Conclusion: Learning to reach specifically affects gene expression in the trained motor cortex and, in the same area, increases sleep need as measured by a local change in SWA. Citation: Hanlon EC; Faraguna U; Vyazovskiy VV; Tononi G; Cirelli C. Effects of skilled training on sleep slow wave activity and cortical gene expression in the rat. SLEEP 2009;32(6):719-729. PMID:19544747

  7. Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester

    SciTech Connect

    Gius, D.; Laimins, L.A.

    1989-02-01

    Several viral trans-activators and a tumor promoter were examined for the ability to activate human papillomavirus type 18 (HPV-18) gene expression. A plasmid containing the HPV-18 noncoding region placed upstream of the chloramphenicol acetyltransferase reporter gene was cotransfected with different herpes simplex virus type 1 (HSV-1) genes into several cell lines. Both HSV-1 TIF and ICPO activated HPV-18 expression; however, activation by TIF was observed only in epithelial cells, while ICPO stimulated expression in a wide variety of cells. The element activated by both TIF and ICOP was mapped to a 229-base-pair fragment which also contains an HPV-18 epithelial cell-preferred enhancer. The inclusion of a papillomavirus E2 trans-activator with TIF and ICOP further increased HPV-18 expression. In contrast, the HSV-1 ICP4 and ICP27 genes, as well as the human T-cell lymphotropic virus type I and human immunodeficiency virus type 1 tat genes, were found to have no effect on HPV-18 expression. In transient assays, the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated HPV-18 expression. The region of HPV-18 activated by TPA was localized to a sequence which is homologous to other TPA-responsive elements.

  8. Gene Expression during the Generation and Activation of Mouse Neutrophils: Implication of Novel Functional and Regulatory Pathways

    PubMed Central

    Ericson, Jeffrey A.; Duffau, Pierre; Yasuda, Kei; Ortiz-Lopez, Adriana; Rothamel, Katherine; Rifkin, Ian R.; Monach, Paul A.

    2014-01-01

    As part of the Immunological Genome Project (ImmGen), gene expression was determined in unstimulated (circulating) mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes), thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1β, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the ImmGen regulatory

  9. Identification of Potential Anticancer Activities of Novel Ganoderma lucidum Extracts Using Gene Expression and Pathway Network Analysis.

    PubMed

    Kao, Chi H J; Bishop, Karen S; Xu, Yuanye; Han, Dug Yeo; Murray, Pamela M; Marlow, Gareth J; Ferguson, Lynnette R

    2016-01-01

    Ganoderma lucidum (lingzhi) has been used for the general promotion of health in Asia for many centuries. The common method of consumption is to boil lingzhi in water and then drink the liquid. In this study, we examined the potential anticancer activities of G. lucidum submerged in two commonly consumed forms of alcohol in East Asia: malt whiskey and rice wine. The anticancer effect of G. lucidum, using whiskey and rice wine-based extraction methods, has not been previously reported. The growth inhibition of G. lucidum whiskey and rice wine extracts on the prostate cancer cell lines, PC3 and DU145, was determined. Using Affymetrix gene expression assays, several biologically active pathways associated with the anticancer activities of G. lucidum extracts were identified. Using gene expression analysis (real-time polymerase chain reaction [RT-PCR]) and protein analysis (Western blotting), we confirmed the expression of key genes and their associated proteins that were initially identified with Affymetrix gene expression analysis.

  10. Gene expression profile in the activation of subperitoneal fibroblasts reflects prognosis of patients with colon cancer.

    PubMed

    Yokota, Mitsuru; Kojima, Motohiro; Higuchi, Youichi; Nishizawa, Yuji; Kobayashi, Akihiro; Ito, Masaaki; Saito, Norio; Ochiai, Atsushi

    2016-03-15

    Tumors can create a heterogenetic tumor microenvironment. We recently identified the pathologically unique cancer microenvironment formed by peritoneal invasion (CMPI), and revealed that subperitoneal fibroblasts (SPFs) within peritoneal tissue play a crucial role in tumor progression through their interaction with cancer cells. Therefore, the genes in SPFs altered by cancer stimulation may include some biologically important factors associated with patient prognosis. In this study, we aimed to identify new biomarkers using genes specifically upregulated in SPFs by cancer-cell-conditioned medium (CCCM) stimulation (SPFs CCCM response genes; SCR genes) in colon cancer (CC). We constructed two frameworks using SCR gene data: a publicly released microarray dataset, and validation cases with freshly frozen CC samples to identify genes related to short recurrence-free survival (RFS). In the first framework, we selected differentially expressed genes between the high and low SCR gene expression groups. In the second framework, genes significantly related to short RFS were selected by univariate analysis using all SCR genes, and multivariate analysis was performed to select robust genes associated with short RFS. We identified CTGF, CALD1, INHBA and TAGLN in the first framework, and PDLIM5, MAGI1, SPTBN1 and TAGLN in the second framework. Among these seven genes, high expression of three genes (CALD1, TAGLN and SPTBN1) showed a poor prognosis in our validation cases. In a public microarray dataset, SCR gene expression was associated with the expression of ECM component, EMT, and M2-macrophage associated genes, which was concordant with the pathological features of CMPI. Thus, we successfully identified new prognostic factors.

  11. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  12. Dynamic expression of the Slit-Robo GTPase activating protein genes during development of the murine nervous system.

    PubMed

    Bacon, Claire; Endris, Volker; Rappold, Gudrun

    2009-03-10

    We investigated the expression of the three known Slit-Robo GTPase activating protein (srGAP) genes in the developing murine nervous system using in situ hybridization. The three genes are expressed during embryonic and early postnatal development in the murine nervous system, showing a distinct pattern of expression in the olfactory system, the eye, forebrain and midbrain structures, the cerebellum, the spinal cord, and dorsal root ganglia, which we discuss in relation to Slit-Robo expression patterns and signaling pathways. We also report srGAP2 expression in zones of neuronal differentiation and srGAP3 in ventricular zones of neurogenesis in many different tissues of the central nervous system (CNS). Compared to srGAP2 and srGAP3, the onset of srGAP1 expression is later in most CNS tissues. We propose that these differences in expression point to functional differences between these three genes in the development of neural tissues.

  13. Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

    DOE PAGES

    Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; ...

    2016-06-08

    In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less

  14. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

    PubMed

    Book, Adam J; Lewin, Gina R; McDonald, Bradon R; Takasuka, Taichi E; Wendt-Pienkowski, Evelyn; Doering, Drew T; Suh, Steven; Raffa, Kenneth F; Fox, Brian G; Currie, Cameron R

    2016-06-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

  15. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression

    PubMed Central

    McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-01-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

  16. Soil type affects plant colonization, activity and catabolic gene expression of inoculated bacterial strains during phytoremediation of diesel.

    PubMed

    Afzal, Muhammad; Yousaf, Sohail; Reichenauer, Thomas G; Kuffner, Melanie; Sessitsch, Angela

    2011-02-28

    The combined use of plants and associated microorganisms has great potential for cleaning up soils contaminated with petroleum hydrocarbons. Apart from environmental conditions the physicochemical properties of the soil are the main factors influencing the survival and activity of an inoculated strain as well as the growth of plants. This study examined the effect of different soil types (sandy, loamy sand and loam) on the survival, gene abundance and catabolic gene expression of two inoculated strains (Pseudomonas sp. strain ITRI53 and Pantoea sp. strain BTRH79) in the rhizosphere and shoot interior of Italian ryegrass vegetated in diesel contaminated soils. High colonization, gene abundance and expression in loamy soils were observed. By contrast, low colonization, gene abundance and absence of gene expression in sandy soil were found. The highest levels of genes expression and hydrocarbon degradation were seen in loamy soil that had been inoculated with BTRH79 and were significantly higher compared to those in other soils. A positive correlation was observed between gene expression and hydrocarbon degradation indicating that catabolic gene expression is necessary for contaminant degradation. These results suggest that soil type influences the bacterial colonization and microbial activities and subsequently the efficiency of contaminant degradation.

  17. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

    PubMed Central

    Lux, Andreas; Salway, Fiona; Dressman, Holly K; Kröner-Lux, Gabriele; Hafner, Mathias; Day, Philip JR; Marchuk, Douglas A; Garland, John

    2006-01-01

    Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling. PMID:16594992

  18. PPAR{alpha} gene expression is up-regulated by LXR and PXR activators in the small intestine

    SciTech Connect

    Inoue, Jun; Satoh, Shin-ichi; Kita, Mariko; Nakahara, Mayuko; Hachimura, Satoshi; Miyata, Masaaki; Nishimaki-Mogami, Tomoko; Sato, Ryuichiro

    2008-07-11

    LXR, PXR, and PPAR{alpha} are members of a nuclear receptor family which regulate the expression of genes involved in lipid metabolism. Here, we show the administration of T0901317 stimulates PPAR{alpha} gene expression in the small intestine but not in the liver of both normal and FXR-null mice. The administration of LXR specific ligand GW3965, or PXR specific ligand PCN has the same effect, indicating that ligand-dependent activation of LXR and PXR, but not FXR, is responsible for the increased gene expression of PPAR{alpha} in the mouse small intestine.

  19. Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.

    PubMed

    Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam

    2014-09-03

    A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

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

    NASA Astrophysics Data System (ADS)

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

    1989-12-01

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

  1. Insulin Sensitizing Pharmacology of Thiazolidinediones Correlates with Mitochondrial Gene Expression rather than Activation of PPARγ

    PubMed Central

    Bolten, Charles W.; Blanner, Patrick M.; McDonald, William G.; Staten, Nicholas R.; Mazzarella, Richard A.; Arhancet, Graciela B.; Meier, Martin F.; Weiss, David J.; Sullivan, Patrick M.; Hromockyj, Alexander E.; Kletzien, Rolf F.; Colca, Jerry R.

    2007-01-01

    Insulin sensitizing thiazolidinediones (TZDs) are generally considered to work as agonists for the nuclear receptor peroxisome proliferative activated receptor-gamma (PPARγ). However, TZDs also have acute, non-genomic metabolic effects and it is unclear which actions are responsible for the beneficial pharmacology of these compounds. We have taken advantage of an analog, based on the metabolism of pioglitazone, which has much reduced ability to activate PPARγ. This analog (PNU-91325) was compared to rosiglitazone, the most potent PPARγ activator approved for human use, in a variety of studies both in vitro and in vivo. The data demonstrate that PNU-91325 is indeed much less effective than rosiglitazone at activating PPARγ both in vitro and in vivo. In contrast, both compounds bound similarly to a mitochondrial binding site and acutely activated PI-3 kinase-directed phosphorylation of AKT, an action that was not affected by elimination of PPARγ activation. The two compounds were then compared in vivo in both normal C57 mice and diabetic KKAy mice to determine whether their pharmacology correlated with biomarkers of PPARγ activation or with the expression of other gene transcripts. As expected from previous studies, both compounds improved insulin sensitivity in the diabetic mice, and this occurred in spite of the fact that there was little increase in expression of the classic PPARγ target biomarker adipocyte binding protein-2 (aP2) with PNU-91325 under these conditions. An examination of transcriptional profiling of key target tissues from mice treated for one week with both compounds demonstrated that the relative pharmacology of the two thiazolidinediones correlated best with an increased expression of an array of mitochondrial proteins and with expression of PPARγ coactivator 1-alpha (PGC1α), the master regulator of mitochondrial biogenesis. Thus, important pharmacology of the insulin sensitizing TZDs may involve acute actions, perhaps on the

  2. Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression.

    PubMed

    Chen, Xi-Lin; Dodd, Geraldine; Thomas, Suzanne; Zhang, Xiaolan; Wasserman, Martin A; Rovin, Brad H; Kunsch, Charles

    2006-05-01

    The antioxidant response element (ARE) is a transcriptional control element that mediates expression of a set of antioxidant proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that activates ARE-containing genes. In endothelial cells, the ARE-mediated genes are upregulated by atheroprotective laminar flow through a Nrf2-dependent mechanism. We tested the hypothesis that activation of ARE-regulated genes via adenovirus-mediated expression of Nrf2 may suppress redox-sensitive inflammatory gene expression. Expression of Nrf2 in human aortic endothelial cells (HAECs) resulted in a marked increase in ARE-driven transcriptional activity and protected HAECs from H2O2-mediated cytotoxicity. Nrf2 suppressed TNF-alpha-induced monocyte chemoattractant protein (MCP)-1 and VCAM-1 mRNA and protein expression in a dose-dependent manner and inhibited TNF-alpha-induced monocytic U937 cell adhesion to HAECs. Nrf2 also inhibited IL-1beta-induced MCP-1 gene expression in human mesangial cells. Expression of Nrf2 inhibited TNF-alpha-induced activation of p38 MAP kinase. Furthermore, expression of a constitutively active form of MKK6 (an upstream kinase for p38 MAP kinase) partially reversed Nrf2-mediated inhibition of VCAM-1 expression, suggesting that p38 MAP kinase, at least in part, mediates Nrf2's anti-inflammatory action. In contrast, Nrf2 did not inhibit TNF-alpha-induced NF-kappaB activation. These data identify the Nrf2/ARE pathway as an endogenous atheroprotective system for antioxidant protection and suppression of redox-sensitive inflammatory genes, suggesting that targeting the Nrf2/ARE pathway may represent a novel therapeutic approach for the treatment of inflammatory diseases such as atherosclerosis.

  3. KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes

    PubMed Central

    Solé, Laura; Vallejo-Gracia, Albert; Roig, Sara R.; Serrano-Albarrás, Antonio; Marruecos, Laura; Manils, Joan; Gómez, Diana; Soler, Concepció; Felipe, Antonio

    2013-01-01

    Voltage-dependent K+ (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1–5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1–5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response. PMID:23327879

  4. KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes.

    PubMed

    Solé, Laura; Vallejo-Gracia, Albert; Roig, Sara R; Serrano-Albarrás, Antonio; Marruecos, Laura; Manils, Joan; Gómez, Diana; Soler, Concepció; Felipe, Antonio

    2013-01-01

    Voltage-dependent K (+) (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1-5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1-5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response.

  5. Motor activity and gene expression in rats with neonatal 6-hydroxydopamine lesions.

    PubMed

    Masuo, Yoshinori; Ishido, Masami; Morita, Masatoshi; Oka, Syuichi; Niki, Etsuo

    2004-10-01

    A rat model of a hyperkinetic disorder was used to investigate the mechanisms underlying motor hyperactivity. Rats received an intracisternal injection of 6-hydroxydopamine on post-natal day 5. At 4 weeks of age, the animals showed significant motor hyperactivity during the dark phase, which was attenuated by methamphetamine injection. Gene expression profiling was carried out in the striatum and midbrain using a DNA macroarray. In the striatum at 4 weeks, there was increased gene expression of the NMDA receptor 1 and tachykinins, and decreased expression of a GABA transporter. At 8 weeks, expression of the NMDA receptor 1 in the striatum was attenuated, with enhanced expression of the glial glutamate/aspartate transporter. In the midbrain, a number of genes, including the GABA transporter gene, showed decreased expression at 4 weeks. At 8 weeks, gene expression was augmented for the dopamine transporter, D4 receptor, and several genes encoding peptides, such as tachykinins and their receptors. These results suggest that in the striatum the neurotransmitters glutamate, GABA and tachykinin may play crucial roles in motor hyperactivity during the juvenile period. Several classes of neurotransmitters, including dopamine and peptides, may be involved in compensatory mechanisms during early adulthood. These data may prompt further neurochemical investigations in hyperkinetic disorders.

  6. A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury.

    PubMed

    Guenther, Catherine A; Wang, Zhen; Li, Emma; Tran, Misha C; Logan, Catriona Y; Nusse, Roel; Pantalena-Filho, Luiz; Yang, George P; Kingsley, David M

    2015-08-01

    Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.

  7. Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

    PubMed Central

    McDonald, R A; Matthews, R P; Idzerda, R L; McKnight, G S

    1995-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types. Images Fig. 1 Fig. 3 PMID:7543684

  8. Dopamine quinones activate microglia and induce a neurotoxic gene expression profile: relationship to methamphetamine-induced nerve ending damage.

    PubMed

    Kuhn, Donald M; Francescutti-Verbeem, Dina M; Thomas, David M

    2006-08-01

    Methamphetamine (METH) intoxication leads to persistent damage of dopamine (DA) nerve endings of the striatum. Recently, we and others have suggested that the neurotoxicity associated with METH is mediated by extensive microglial activation. DA itself has been shown to play an obligatory role in METH neurotoxicity, possibly through the formation of quinone species. We show presently that DA-quinones (DAQ) cause a time-dependent activation of cultured microglial cells. Microarray analysis of the effects of DAQ on microglial gene expression revealed that 101 genes were significantly changed in expression, with 73 genes increasing and 28 genes decreasing in expression. Among those genes differentially regulated by DAQ were those often associated with neurotoxic conditions including inflammation, cytokines, chemokines, and prostaglandins. In addition, microglial genes associated with a neuronally protective phenotype were among those that were downregulated by DAQ. These results implicate DAQ as one species that could cause early activation of microglial cells in METH intoxication, manifested as an alteration in the expression of a broad biomarker panel of genes. These results also link oxidative stress, chemical alterations in DA to its quinone, and microglial activation as part of a cascade of glial-neuronal crosstalk that can amplify METH-induced neurotoxicity.

  9. Patterns of global gene expression in rat skeletal muscle during unloading and low-intensity ambulatory activity

    NASA Technical Reports Server (NTRS)

    Bey, Lionel; Akunuri, Nagabhavani; Zhao, Po; Hoffman, Eric P.; Hamilton, Deborah G.; Hamilton, Marc T.

    2003-01-01

    Physical inactivity and unloading lead to diverse skeletal muscle alterations. Our goal was to identify the genes in skeletal muscle whose expression is most sensitive to periods of unloading/reduced physical activity and that may be involved in triggering initial responses before phenotypic changes are evident. The ability of short periods of physical activity/loading as an effective countermeasure against changes in gene expression mediated by inactivity was also tested. Affymetrix microarrays were used to compare mRNA levels in the soleus muscle under three experimental treatments (n = 20-29 rats each): 12-h hindlimb unloading (HU), 12-h HU followed by 4 h of intermittent low-intensity ambulatory and postural activity (4-h reloading), and control (with ambulatory and postural activity). Using a combination of criteria, we identified a small set of genes (approximately 1% of 8,738 genes on the array or 4% of significant expressed genes) with the most reproducible and largest responses to altered activity. Analysis revealed a coordinated regulation of transcription for a large number of key signaling proteins and transcription factors involved in protein synthesis/degradation and energy metabolism. Most (21 of 25) of the gene expression changes that were downregulated during HU returned at least to control levels during the reloading. In surprising contrast, 27 of 38 of the genes upregulated during HU remained significantly above control, but most showed trends toward reversal. This introduces a new concept that, in general, genes that are upregulated during unloading/inactivity will be more resistant to periodic reloading than those genes that are downregulated. This study reveals genes that are the most sensitive to loading/activity in rat skeletal muscle and indicates new targets that may initiate muscle alterations during inactivity.

  10. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    SciTech Connect

    Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  11. Fasting activates the gene expression of UCP3 independent of genes necessary for lipid transport and oxidation in skeletal muscle.

    PubMed

    Tunstall, Rebecca J; Mehan, Kate A; Hargreaves, Mark; Spriet, Lawrence L; Cameron-Smith, David

    2002-06-07

    Fasting triggers a complex array of adaptive metabolic and hormonal responses including an augmentation in the capacity for mitochondrial fatty acid (FA) oxidation in skeletal muscle. This study hypothesized that this adaptive response is mediated by increased mRNA of key genes central to the regulation of fat oxidation in human skeletal muscle. Fasting dramatically increased UCP3 gene expression, by 5-fold at 15 h and 10-fold at 40 h. However the expression of key genes responsible for the uptake, transport, oxidation, and re-esterification of FA remained unchanged following 15 and 40 h of fasting. Likewise there was no change in the mRNA abundance of transcription factors. This suggests a unique role for UCP3 in the regulation of FA homeostasis during fasting as adaptation to 40 h of fasting does not require alterations in the expression of other genes necessary for lipid metabolism.

  12. Metabolic activity of Streptococcus mutans biofilms and gene expression during exposure to xylitol and sucrose.

    PubMed

    Decker, Eva-Maria; Klein, Christian; Schwindt, Dimitri; von Ohle, Christiane

    2014-12-01

    The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy (CLSM)-based analyses of the microbial vitality, respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride, CTC) and production of extracellular polysaccharides (EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However, xylitol exposure caused a difference in gene expression compared to the control. GtfC was upregulated only in the presence of xylitol. Under xylitol exposure, gtfB was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three. Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the

  13. Myoglobin-deficient mice activate a distinct cardiac gene expression program in response to isoproterenol-induced hypertrophy.

    PubMed

    Molojavyi, Andrei; Lindecke, Antje; Raupach, Annika; Moellendorf, Sarah; Köhrer, Karl; Gödecke, Axel

    2010-04-01

    Myoglobin knockout mice (myo-/-) adapt to the loss of myoglobin by the activation of a variety of compensatory mechanisms acting on the structural and functional level. To analyze to what extent myo-/- mice would tolerate cardiac stress we used the model of chronic isoproterenol application to induce cardiac hypertrophy in myo-/- mice and wild-type (WT) controls. After 14 days of isoproterenol infusion cardiac hypertrophy in WT and myo-/- mice reached a similar level. WT mice developed lung edema and left ventricular dilatation suggesting the development of heart failure. In contrast, myo-/- mice displayed conserved cardiac function and no signs of left ventricular dilatation. Analysis of the cardiac gene expression profiles using 40K mouse oligonucleotide arrays showed that isoproterenol affected the expression of 180 genes in WT but only 92 genes of myo-/- hearts. Only 40 of these genes were regulated in WT as well as in myo-/- hearts. In WT hearts a pronounced induction of genes of the extracellular matrix occurred suggesting a higher level of cardiac remodeling. myo-/- hearts showed altered transcription of genes involved in carbon metabolism, inhibition of apoptosis and muscular repair. Interestingly, a subset of genes that was altered in myo-/- mice already under basal conditions was differentially expressed in WT hearts under isoproterenol treatment. In summary, our data show a high capacity of myoglobin-deficient mice to adapt to catecholamine induced cardiac stress which is associated with activation of a distinct cardiac gene expression program.

  14. Perfluorooctane sulfonate (PFOS) affects hormone receptor activity, steroidogenesis, and expression of endocrine-related genes in vitro and in vivo.

    PubMed

    Du, Guizhen; Hu, Jialei; Huang, Hongyu; Qin, Yufeng; Han, Xiumei; Wu, Di; Song, Ling; Xia, Yankai; Wang, Xinru

    2013-02-01

    Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. We investigated the endocrine-disrupting effects of PFOS using a combination of in vitro and in vivo assays. Reporter gene assays were used to detect receptor-mediated (anti-)estrogenic, (anti-)androgenic, and (anti-)thyroid hormone activities. The effect of PFOS on steroidogenesis was assessed both at hormone levels in the supernatant and at expression levels of hormone-induced genes in the H295R cell. A zebrafish-based short-term screening method was developed to detect the effect of PFOS on endocrine function in vivo. The results indicate that PFOS can act as an estrogen receptor agonist and thyroid hormone receptor antagonist. Exposure to PFOS decreased supernatant testosterone (T), increased estradiol (E2) concentrations in H295R cell medium and altered the expression of several genes involved in steroidogenesis. In addition, PFOS increased early thyroid development gene (hhex and pax8) expression in a concentration-dependent manner, decreased steroidogenic enzyme gene (CYP17, CYP19a, CYP19b) expression, and changed the expression pattern of estrogen receptor production genes (esr1, esr2b) after 500 µg/L PFOS treatment in zebrafish embryos. These results indicate that PFOS has the ability to act as an endocrine disruptor both in vitro and in vivo by disrupting the function of nuclear hormone receptors, interfering with steroidogenesis, and altering the expression of endocrine-related genes in zebrafish embryo.

  15. A long non-coding RNA promotes full activation of adult gene expression in the chicken α-globin domain.

    PubMed

    Arriaga-Canon, Cristian; Fonseca-Guzmán, Yael; Valdes-Quezada, Christian; Arzate-Mejía, Rodrigo; Guerrero, Georgina; Recillas-Targa, Félix

    2014-01-01

    Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin remodelling activities. Their function in regulating gene expression switching during specific developmental stages is poorly understood. Here we describe a nuclear, non-coding transcript responsive for the stage-specific activation of the chicken adult α(D) globin gene. This non-coding transcript, named α-globin transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in late stages of chicken development, when active chromatin marks the adult α(D) gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific transcription. Furthermore, loss of function experiments showed that lncRNA-αGT is required for full activation of the α(D) adult gene and maintenance of transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an important part of the switching from embryonic to adult α-globin gene expression, and suggest a function of lncRNA-αGT in contributing to the maintenance of adult α-globin gene expression by promoting an active chromatin structure.

  16. Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

    PubMed Central

    2014-01-01

    Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

  17. Identification of active VQ motif-containing genes and the expression patterns under low nitrogen treatment in soybean.

    PubMed

    Wang, Xiaobo; Zhang, Haowei; Sun, Genlou; Jin, Yuan; Qiu, Lijuan

    2014-06-15

    Plant VQ motif-containing protein family plays crucial roles in plant growth, seed development, and defense responses in Arabidopsis. However, its function in soybean is still not well defined. We aim to identify the VQ gene family, and explore the genetic variation of active GmVQ genes in soybean and their expression patterns under low nitrogen stresses. A total of 74 VQ motif-containing genes were identified in soybean genome, and were clustered into five distinct subfamilies (GmVQI-V) with each gene having two or three copies except GmVQ55 (GmVQIV) with single copy. Fourteen genes with relatively high expression level, at least in one tissue, were defined as active GmVQ genes. Most of these active GmVQ genes specifically expressed in soybean pod shell (7/74), root (9/74) and/or nodule (10/74) respectively. Single nucleotide polymorphism (SNP) analysis in cultivated and wild soybeans revealed there were selected site(s) in GmVQ6, GmVQ7, GmVQ10, GmVQ26 and GmVQ61, which means that these genes have undergone artificial selection during soybean domestication. After low nitrogen treatment, enhanced expression of VQ genes was noticed in specific tissues, such as GmVQ53, GmVQ26, GmVQ58, GmVQ61, GmVQ70 and GmVQ6 in shoot, and GmVQ53, GmVQ58, GmVQ48 in root. On the contrary, suppressed expression of GmVQ57, GmVQ21 and GmVQ1 genes was noticed in root after the treatment. Duplicated copy of the active GmVQ genes showed similar expression pattern, suggesting that these genes might be complete copies. The results suggested that soybean VQ-motif containing genes may act as positive or negative regulators in soybean growth, development and nitrogen metabolism. Taken together, our results provided useful information for functional characterization of soybean GmVQ genes to unravel their biological roles.

  18. DNA demethylation in PD-1 gene promoter induced by 5-azacytidine activates PD-1 expression on Molt-4 cells.

    PubMed

    Zhang, Min; Xiao, Xin Q; Jiang, Yong F; Liang, Yun S; Peng, Min Y; Xu, Yun; Gong, Guo Z

    2011-01-01

    The expression of the programmed death 1 (PD-1) gene is an indicator of exhausted T-cells with decreased activation and function. It remains unknown, however, whether the methylation status of the PD-1 gene promoter is associated with PD-1 expression level. This study shows the changes of PD-1 expression levels and the demethylation status of the PD-1 promoter region in Molt-4 cells under different concentrations of 5-azacytidine (5-Zac). The result demonstrated that DNA demethylation at PD-1 promoter may contribute to PD-1 overexpression.

  19. Distinct promoter activation mechanisms modulate noise-driven HIV gene expression

    NASA Astrophysics Data System (ADS)

    Chavali, Arvind K.; Wong, Victor C.; Miller-Jensen, Kathryn

    2015-12-01

    Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance.

  20. Gene Expression and Histological Analysis of Activated Brown Adipocytes in Adipose Tissue.

    PubMed

    Lee, Yun-Hee

    2017-01-01

    With the rediscovery of brown adipose tissue in adult humans, identification and characterization of brown adipocytes have been topics of great interest in the field of adipose tissue research. In particular, identification of the molecular mechanisms that activate thermogenic adipocytes suggests promising targets for increasing energy expenditure and ultimately combatting obesity and obesity-related metabolic disease. Thus, the methodology for identifying brown adipocytes in vivo is important for the precise determination of the metabolic activity of brown adipose tissue and de novo brown adipogenesis in white adipose tissue. In addition, in vivo analysis of brown adipocytes in combination with lineage tracing is essential to investigate the cellular origins of brown adipocytes. This chapter first provides a brief overview of lineage tracing studies performed in the search for the cellular origins of brown adipocytes. The chapter then describes the immunohistochemistry methodology for identifying brown adipocytes in adipose tissue, including analyses in histologic tissue sections and whole mount tissue. Lastly, it discusses flow cytometric analysis of dissociated cells from adipose tissue, and isolation of live adipocytes for subsequent gene expression profiling using fluorescence-activated cell sorting.

  1. Distinct promoter activation mechanisms modulate noise-driven HIV gene expression

    PubMed Central

    Chavali, Arvind K.; Wong, Victor C.; Miller-Jensen, Kathryn

    2015-01-01

    Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance. PMID:26666681

  2. Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

    PubMed

    Sandoval, Angel; Fraisl, Peter; Arias-Barrau, Elsa; Dirusso, Concetta C; Singer, Diane; Sealls, Whitney; Black, Paul N

    2008-09-15

    These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The

  3. Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish

    PubMed Central

    Kung, Tiffany S.; Richardson, Jason R.; Cooper, Keith R.; White, Lori A.

    2015-01-01

    Pyrethroids are commonly used insecticides that are considered to pose little risk to human health. However, there is an increasing concern that children are more susceptible to the adverse effects of pesticides. We used the zebrafish model to test the hypothesis that developmental exposure to low doses of the pyrethroid deltamethrin results in persistent alterations in dopaminergic gene expression, neurochemistry, and locomotor activity. Zebrafish embryos were treated with deltamethrin (0.25–0.50 μg/l), at concentrations below the LOAEL, during the embryonic period [3–72 h postfertilization (hpf)], after which transferred to fresh water until the larval stage (2-weeks postfertilization). Deltamethrin exposure resulted in decreased transcript levels of the D1 dopamine (DA) receptor (drd1) and increased levels of tyrosine hydroxylase at 72 hpf. The reduction in drd1 transcripts persisted to the larval stage and was associated with decreased D2 dopamine receptor transcripts. Larval fish, exposed developmentally to deltamethrin, had increased levels of homovanillic acid, a DA metabolite. Since the DA system is involved in locomotor activity, we measured the swim activity of larval fish following a transition to darkness. Developmental exposure to deltamethrin significantly increased larval swim activity which was attenuated by concomitant knockdown of the DA transporter. Acute exposure to methylphenidate, a DA transporter inhibitor, increased swim activity in control larva, while reducing swim activity in larva developmentally exposed to deltamethrin. Developmental exposure to deltamethrin causes locomotor deficits in larval zebrafish, which is likely mediated by dopaminergic dysfunction. This highlights the need to understand the persistent effects of low-dose neurotoxicant exposure during development. PMID:25912032

  4. Calcitonin gene-related peptide receptor expression in alternatively activated monocytes/macrophages during irreversible pulpitis.

    PubMed

    Caviedes-Bucheli, Javier; Moreno, Gloria Cristina; López, María Paula; Bermeo-Noguera, Ana Milena; Pacheco-Rodríguez, Gloriana; Cuellar, Adriana; Muñoz, Hugo Roberto

    2008-08-01

    The purpose of this study was to quantify the percentage and the mean fluorescence intensity of viable alternatively activated monocytes/macrophages (AAMø) CD163+ positive for calcitonin gene-related peptide receptor (CGRPr) within the total AAMø population in human dental pulp. Pulp tissue samples were collected from teeth with a clinical diagnosis of irreversible pulpitis (n = 13), pulps with induced inflammation (n = 13), and normal pulps (n = 13). All samples were labeled to identify positive cells for CGRPr and CD163 using a flow cytometry assay. Results demonstrated that a high percentage of total viable AAMø CD163+ expressed CGRPr on their membranes (72.12% in healthy pulp, 62.20% in irreversible pulpitis, and 58.01% in induced pulpitis). Significant differences were found between mean AAMø CD163+ fluorescence for CGRPr according to pulp condition, being greater in irreversible pulpitis. It can be concluded that AAMø CD163+ are expressed during normal and inflammatory processes, supporting the hypothesis that they could exercise an anti-inflammatory action that could be controlled by CGRP signaling after its binding.

  5. YANA – a software tool for analyzing flux modes, gene-expression and enzyme activities

    PubMed Central

    Schwarz, Roland; Musch, Patrick; von Kamp, Axel; Engels, Bernd; Schirmer, Heiner; Schuster, Stefan; Dandekar, Thomas

    2005-01-01

    Background A number of algorithms for steady state analysis of metabolic networks have been developed over the years. Of these, Elementary Mode Analysis (EMA) has proven especially useful. Despite its low user-friendliness, METATOOL as a reliable high-performance implementation of the algorithm has been the instrument of choice up to now. As reported here, the analysis of metabolic networks has been improved by an editor and analyzer of metabolic flux modes. Analysis routines for expression levels and the most central, well connected metabolites and their metabolic connections are of particular interest. Results YANA features a platform-independent, dedicated toolbox for metabolic networks with a graphical user interface to calculate (integrating METATOOL), edit (including support for the SBML format), visualize, centralize, and compare elementary flux modes. Further, YANA calculates expected flux distributions for a given Elementary Mode (EM) activity pattern and vice versa. Moreover, a dissection algorithm, a centralization algorithm, and an average diameter routine can be used to simplify and analyze complex networks. Proteomics or gene expression data give a rough indication of some individual enzyme activities, whereas the complete flux distribution in the network is often not known. As such data are noisy, YANA features a fast evolutionary algorithm (EA) for the prediction of EM activities with minimum error, including alerts for inconsistent experimental data. We offer the possibility to include further known constraints (e.g. growth constraints) in the EA calculation process. The redox metabolism around glutathione reductase serves as an illustration example. All software and documentation are available for download at . Conclusion A graphical toolbox and an editor for METATOOL as well as a series of additional routines for metabolic network analyses constitute a new user-friendly software for such efforts. PMID:15929789

  6. Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays

    PubMed Central

    Xiong, Wu-Jun; Hu, Li-Juan; Jian, Yi-Cheng; Wang, Li-Jing; Jiang, Ming; Li, Wei; He, Yi

    2012-01-01

    AIM: To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions. METHODS: HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. Total RNA and mRNA of quiescent HSCs, and culture-activated HSCs were extracted, quantified and reversely transcripted into cDNA. The global gene expression profile was analyzed by microarray with Affymetrix rat genechip. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Microarray data were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi. The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl4)-induced fibrosis rat model was also analyzed with Western blotting. RESULTS: Of the 28 700 genes represented on this chip, 2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate. Of these, 1396 genes were upregulated, while 1170 genes were downregulated in culture-activated HSCs. These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms. The most enriched GO terms included response to wounding, wound healing, regulation of cell growth, vasculature development and actin cytoskeleton organization. KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs. Wnt5a was significantly increased in culture-activated HSCs as compared with quiescent HSCs. qRT-PCR validated the microarray data. Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation, downregulated expressions of

  7. Neighboring Genes Show Correlated Evolution in Gene Expression.

    PubMed

    Ghanbarian, Avazeh T; Hurst, Laurence D

    2015-07-01

    When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking.

  8. Nuclear Factor of Activated T Cells and Cytokines Gene Expression of the T Cells in AIDS Patients with Immune Reconstitution Inflammatory Syndrome during Highly Active Antiretroviral Therapy

    PubMed Central

    Chen, Heling; Xie, Yirui; Su, Junwei; Huang, Ying; Xu, Lijun; Yin, Michael; Zhou, Qihui

    2017-01-01

    Background. The etiology of immune reconstitution inflammatory syndrome (IRIS) in AIDS patients after the initiation of HAART remains unknown. Several researches indicated that the development of IRIS is associated with the production and variation of cytokines, whose gene expression are closely related to the Ca2+/CN-nuclear factor of activated T cells (NFAT) pathway. Methods. We studied the expression of NFAT isoforms and their major target cytokines genes in peripheral blood CD3+ T cells of subjects through fluorescence quantitative PCR and explored the expression changes of these genes before and after HAART. Results. After the initiation of HARRT, NFAT1, IL-6, and IL-8 gene expression showed a reversal trend in the CD3+ T cells of the IRIS group and changed from low expression before HARRT to high expression after HARRT. In particular, the relative gene expression of NFAT1 was markedly higher compared with the other three isoforms. The IRIS group also showed higher NFAT4, NFAT2, NFAT1, IL-1β, IL-10, IL-2, IL-18, and TNF-α gene expression than the non-IRIS group. Conclusion. This study suggested that high expression levels of IL-2, IL-6, IL-8, TNF-α, IL-1β, IL-10, IL-12, and IL-18 can predict the risk of IRIS. The increased expression of NFAT1 and NFAT4 may promote the expression of cytokines, such as IL-6, IL-8, and TNF-α, which may promote the occurrence of IRIS. PMID:28316373

  9. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

    PubMed Central

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

  10. Aryl Hydrocarbon Receptor Activation by TCDD Modulates Expression of Extracellular Matrix Remodeling Genes during Experimental Liver Fibrosis

    PubMed Central

    Lamb, Cheri L.; Cholico, Giovan N.; Perkins, Daniel E.; Fewkes, Michael T.; Oxford, Julia Thom; Lujan, Trevor J.; Morrill, Erica E.

    2016-01-01

    The aryl hydrocarbon receptor (AhR) is a soluble, ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence implicates the AhR in regulating extracellular matrix (ECM) homeostasis. We recently reported that TCDD increased necroinflammation and myofibroblast activation during liver injury elicited by carbon tetrachloride (CCl4). However, TCDD did not increase collagen deposition or exacerbate fibrosis in CCl4-treated mice, which raises the possibility that TCDD may enhance ECM turnover. The goal of this study was to determine how TCDD impacts ECM remodeling gene expression in the liver. Male C57BL/6 mice were treated for 8 weeks with 0.5 mL/kg CCl4, and TCDD (20 μg/kg) was administered during the last two weeks. Results indicate that TCDD increased mRNA levels of procollagen types I, III, IV, and VI and the collagen processing molecules HSP47 and lysyl oxidase. TCDD also increased gelatinase activity and mRNA levels of matrix metalloproteinase- (MMP-) 3, MMP-8, MMP-9, and MMP-13. Furthermore, TCDD modulated expression of genes in the plasminogen activator/plasmin system, which regulates MMP activation, and it also increased TIMP1 gene expression. These findings support the notion that AhR activation by TCDD dysregulates ECM remodeling gene expression and may facilitate ECM metabolism despite increased liver injury. PMID:27672655

  11. Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)

    EPA Science Inventory

    The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...

  12. Changes in expression of genes involved in apoptosis in activated human T-cells in response to modeled microgravity

    NASA Astrophysics Data System (ADS)

    Ward, Nancy E.; Pellis, Neal R.; Risin, Diana; Risin, Semyon A.; Liu, Wenbin

    2006-09-01

    Space flights result in remarkable effects on various physiological systems, including a decline in cellular immune functions. Previous studies have shown that exposure to microgravity, both true and modeled, can cause significant changes in numerous lymphocyte functions. The purpose of this study was to search for microgravity-sensitive genes, and specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. The experiments were performed on anti-CD3 and IL-2 activated human T cells. To model microgravity conditions we have utilized the NASA rotating wall vessel bioreactor. Control lymphocytes were cultured in static 1g conditions. To assess gene expression we used DNA microarray chip technology. We had shown that multiple genes (approximately 3-8% of tested genes) respond to microgravity conditions by 1.5 and more fold change in expression. There is a significant variability in the response. However, a certain reproducible pattern in gene response could be identified. Among the genes showing reproducible changes in expression in modeled microgravity, several genes involved in apoptosis as well as in immune response were identified. These are IL-7 receptor, Granzyme B, Beta-3-endonexin, Apo2 ligand and STAT1. Possible functional consequences of these changes are discussed.

  13. Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis.

    PubMed

    Primon-Barros, Muriel; Rigo, Graziela Vargas; Frasson, Amanda Piccoli; Santos, Odelta dos; Smiderle, Lisiane; Almeida, Silvana; Macedo, Alexandre José; Tasca, Tiana

    2015-11-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

  14. Virus infection and interferon can activate gene expression through a single synthetic element, but endogenous genes show distinct regulation.

    PubMed

    Raj, N B; Engelhardt, J; Au, W C; Levy, D E; Pitha, P M

    1989-10-05

    Virus inducible elements (IE) in promoters of mouse alpha-interferon and human beta 1-interferon genes contain multiple copies of the hexanucleotide sequence AGT-GAA or its variants which are also found in the interferon-stimulated response element of genes transcriptionally induced by interferon. We have examined the similarities between virus and interferon induction of gene expression and the role of AGTGAA and AAT-GAA hexamers in these responses. Hybrid plasmids were constructed by inserting the IE region, the alpha 4 promoter, or the multiple copies of AGTGAA or AAT-GAA 5' to the inactive-45 human immunodeficiency-chloramphenicol acetyltransferase hybrid gene, and their inducible expression was studied in a transient expression assay. In L-cells, multiple hexamers were efficiently induced both by infection with Newcastle disease virus and by interferon treatment; while the alpha 4 promoter and the IE inducible region were induced predominantly by virus rather than by interferon. In order to dissociate the effect of virus and endogenous interferon on the induction process, we examined the gene expression in Vero cells, which have undergone homozygous deletion of type 1 interferon genes, and in VNPT-159 cells, which were derived from Vero cells by insertion of an inducible human interferon beta 1 gene. The results show that while the alpha 4 promoter was efficiently induced only by virus in both cell types, the constructs containing shorter segments of the IE were induced by both virus and interferon in Vero cells. However, the inducibility by interferon was not detected in VNPT-159 cells, suggesting that the presence of endogenous interferon suppresses interferon-induced expression of hexanucleotide repeats and the short inducible region. In contrast, virus inducibility of endogenous interferon-stimulated genes, ISG-15 and ISG-54, was about 100-fold more efficient in VNPT-159 cells than in Vero cells, suggesting that this induction is largely mediated through

  15. Bortezomib and Arsenic Trioxide Activity on a Myelodysplastic Cell Line (P39): A Gene Expression Study

    PubMed Central

    Savlı, Hakan; Galimberti, Sara; Sünnetçi, Deniz; Canestraro, Martina; Palumbo, Giuseppe; Nagy, Balint; Raimondo, Francesco Di; Petrini, Mario

    2015-01-01

    Objective: We aimed to understand the molecular pathways affected by bortezomib and arsenic trioxide treatment on myelomonocytoid cell line P39. Materials and Methods: Oligonucleotide microarray platforms were used for gene expression and pathway analysis. Confirmation studies were performed using quantitative real time PCR. Results: Bortezomib treatment has shown upregulated DIABLO and NF-κBIB (a NF-κB inhibitor) and downregulated NF-κB1, NF-κB2, and BIRC1 gene expressions. Combination treatment of the two compounds showed gene expression deregulations in concordance by the results of single bortezomib treatment. Especially, P53 was a pathway more significantly modified and a gene network centralized around the beta estradiol gene. Beta estradiol, BRCA2, and FOXA1 genes were remarkable deregulations in our findings. Conclusion: Results support the suggestions about possible use of proteasome inhibitors in the treatment of high-risk myelodysplastic syndrome (MDS). NF-κB was observed as an important modulator in leukemic transformation of MDS. PMID:25913414

  16. Properties of a novel gene isolated from a Hodgkin's disease cell line that is expressed early during lymphoid cell activation

    SciTech Connect

    Bennett, J.S.; Tredway, T.L.; Dizikes, G.J.; Nawrocki, J.F. Hines Veterans Administration Hospital, IL )

    1994-03-01

    The authors have isolated a novel 667-bp cDNA clone, designated epag, from a Hodgkin's-disease cell line-derived library that is expressed in association with T cell activation and which is not related to any known gene family. By using reverse transcription/PCR, the authors have demonstrated that epag mRNA is expressed as early as 1 h after stimulation of normal PBMCs with anti-CD3. The levels of mRNA peaked by 4 h, and no expression was detectable by 12 h postactivation or in resting cells incubated in culture without activation. Expression of epag was also detected in PMA- and PHA-stimulated, but not in nonstimulated Jurkat cells, and overall its expression in transformed cell lines of hemopoietic origin is highly restricted. Sequence analysis of multiple independent cDNA clones showed that epag expressed in the Hodgkin's-disease cell line L428 is identical to the gene expressed in normal activated PBMC. Epag expression was detected by reverse transcription/PCR in RNA preparations made from various normal nonlymphoid tissues. Computer analysis of the sequence identified an open reading frame encoding a putative protein of 13.2 kDa initiating at a CUG translational codon. In vitro translation and Western blot analysis with anti-peptide serum supported this analysis. The authors hypothesize that epag functions as an early signal that helps mediate the activation of T cells. 63 refs., 11 figs.

  17. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression.

    PubMed Central

    Gambello, M J; Iglewski, B H

    1991-01-01

    We report the discovery of the lasR gene, which positively regulates elastase expression in Pseudomonas aeruginosa PAO1. The lasR gene was cloned by its ability to restore a positive elastase phenotype in strain PA103, a strain which possesses the elastase structural gene (lasB) but fails to synthesize the enzyme. Nucleotide sequence analysis revealed an open reading frame of 716 nucleotides encoding a protein of approximately 27 kDa. A labeled LasR protein of 27 kDa was detected in Escherichia coli by using a T7 RNA polymerase expression system. A chromosomal deletion mutant of the lasR gene was constructed in PAO1 by gene replacement. This mutant (PAO-R1) is devoid of elastolytic activity and elastase antigen. The deduced amino acid sequence of LasR is 27% homologous to the positive activator LuxR of Vibrio fischeri and the suspected activator 28K-UvrC of E. coli. Northern (RNA) analysis of total cellular RNA from PAO1, PAO-R1, and PAO-R1 containing the lasR gene on a multicopy plasmid (pMG1.7) revealed that a functional lasR gene is required for transcription of the elastase structural gene (lasB). Images PMID:1902216

  18. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver.

    PubMed

    Wortham, Matthew; Czerwinski, Maciej; He, Lin; Parkinson, Andrew; Wan, Yu-Jui Yvonne

    2007-09-01

    Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4 alpha (HNF4 alpha) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4 alpha expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4 alpha, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

  19. Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens

    PubMed Central

    Chen, Junfeng; Yang, Chingyuan; Tizioto, Polyana C.; Huang, Huan; Lee, Mi O. K.; Payne, Harold R.; Lawhon, Sara D.; Schroeder, Friedhelm; Taylor, Jeremy F.; Womack, James E.

    2016-01-01

    Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease. PMID:27409794

  20. Activation of mu opioid receptors in the striatum differentially augments methamphetamine-induced gene expression and enhances stereotypic behavior.

    PubMed

    Horner, Kristen A; Hebbard, John C; Logan, Anna S; Vanchipurakel, Golda A; Gilbert, Yamiece E

    2012-03-01

    Mu opioid receptors are densely expressed in the patch compartment of striatum and contribute to methamphetamine-induced patch-enhanced gene expression and stereotypy. To further elucidate the role of mu opioid receptor activation in these phenomena, we examined whether activation of mu opioid receptors would enhance methamphetamine-induced stereotypy and prodynorphin, c-fos, arc and zif/268 expression in the patch and/or matrix compartments of striatum, as well as the impact of mu opioid receptor activation on the relationship between patch-enhanced gene expression and stereotypy. Male Sprague-Dawley rats were intrastriatally infused with d-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO; 1 μg/μL), treated with methamphetamine (0.5 mg/kg) and killed at 45 min or 2 h later. DAMGO augmented methamphetamine-induced zif/268 mRNA expression in the patch and matrix compartments, while prodynorphin expression was increased in the dorsolateral patch compartment. DAMGO pre-treatment did not affect methamphetamine-induced arc and c-fos expression. DAMGO enhanced methamphetamine-induced stereotypy and resulted in greater patch versus matrix expression of prodynorphin in the dorsolateral striatum, leading to a negative correlation between the two. These findings indicate that mu opioid receptors contribute to methamphetamine-induced stereotypy, but can differentially influence the genomic responses to methamphetamine. These data also suggest that prodynorphin may offset the overstimulation of striatal neurons by methamphetamine.

  1. Optimal reference genes for qPCR in resting and activated human NK cells--Flow cytometric data correspond to qPCR gene expression analysis.

    PubMed

    Kaszubowska, Lucyna; Wierzbicki, Piotr Mieczysław; Karsznia, Sylwia; Damska, Marta; Ślebioda, Tomasz Jerzy; Foerster, Jerzy; Kmieć, Zbigniew

    2015-07-01

    Natural killer cells (NK cells) are cytotoxic lymphocytes critical to the innate immune system engaged in rapid response against tumor or virus infected cells. After activation NK cells acquire enhanced cytotoxicity and are capable of producing cytokines to stimulate other immune cells. Quantitative PCR (qPCR) is a method of choice for gene expression analysis but the usage of reliable reference genes for the normalization process is critical. Commonly used reference genes may vary in expression level between different experimental conditions providing wrong quantitative results of the studied genes' expression levels. Fourteen potential endogenous control genes were analyzed by qPCR method in NK-92 cell line that shows characteristics of human natural killer cells and is often used in studies on biology of NK lymphocytes. NK-92 cells were stimulated with IL-2 or TNF for 2, 24 or 72 h. Results were analyzed with RefFinder, a program which enables evaluation and screening of reference genes and integrates the currently available major computational programs (Genorm, Normfinder, BestKeeper and Delta Ct). The most stable gene in activated and non-activated NK cells was B2M, followed by IPO-8 and GAPDH and the least stable were HPRT1, PPIA and RPL32. The normalization process was performed on SOD2 gene and the results of qPCR experiments were confirmed by flow cytometry. The flow cytometric data corresponded to the results of qPCR gene expression analysis performed for the reference genes qualified by RefFinder as the most stable.

  2. The Caenorhabditis elegans NK-2 homeobox gene ceh-22 activates pharyngeal muscle gene expression in combination with pha-1 and is required for normal pharyngeal development.

    PubMed

    Okkema, P G; Ha, E; Haun, C; Chen, W; Fire, A

    1997-10-01

    Pharyngeal muscle development in the nematode Caenorhabditis elegans appears to share similarities with cardiac muscle development in other species. We have previously described CEH-22, an NK-2 class homeodomain transcription factor similar to Drosophila tinman and vertebrate Nkx2-5, which is expressed exclusively in the pharyngeal muscles. In vitro, CEH-22 binds the enhancer from myo-2, a pharyngeal muscle-specific myosin heavy chain gene. In this paper, we examine the role CEH-22 plays in pharyngeal muscle development and gene activation by (a) ectopically expressing ceh-22 in transgenic C. elegans and (b) examining the phenotype of a ceh-22 loss-of-function mutant. These experiments indicate that CEH-22 is an activator of myo-2 expression and that it is required for normal pharyngeal muscle development. However, ceh-22 is necessary for neither formation of the pharyngeal muscles, nor for myo-2 expression. Our data suggest parallel and potentially compensating pathways contribute to pharyngeal muscle differentiation. We also examine the relationship between ceh-22 and the pharyngeal organ-specific differentiation gene pha-1. Mutations in ceh-22 and pha-1 have strongly synergistic effects on pharyngeal muscle gene expression; in addition, a pha-1 mutation enhances the lethal phenotype caused by a mutation in ceh-22. Wild-type pha-1 is not required for the onset of ceh-22 expression but it appears necessary for maintained expression of ceh-22.

  3. Ethylene could influence ferric reductase, iron transporter, and H+-ATPase gene expression by affecting FER (or FER-like) gene activity.

    PubMed

    Lucena, Carlos; Waters, Brian M; Romera, F Javier; García, María José; Morales, María; Alcántara, Esteban; Pérez-Vicente, Rafael

    2006-01-01

    In previous works, it has been shown, by using ethylene inhibitors and precursors, that ethylene could participate in the regulation of the enhanced ferric reductase activity of Fe-deficient Strategy I plants. However, it was not known whether ethylene regulates the ferric reductase gene expression or other aspects related to this activity. This paper is a study of the effects of ethylene inhibitors and precursors on the expression of the genes encoding the ferric reductases and iron transporters of Arabidopsis thaliana (FRO2 and IRT1) and Lycopersicon esculentum (=Solanum lycopersicum) (FRO1 and IRT1) plants. The effects of ethylene inhibitors and precursors on the activity of the iron reductase and the iron transporter have been examined in parallel. Also studied were the effects of ethylene inhibitors and precursors on the expression of the H(+)-ATPase genes of cucumber (CsHA1 and CsHA2) and the transcription factor genes of tomato (LeFER) and Arabidopsis (AtFRU or AtFIT1, an LeFER homologue) that regulate ferric reductase, iron transporter, and H(+)-ATPse activity. The results obtained suggest that ethylene participates in the regulation of ferric reductase, the iron transporter, and H(+)-ATPase gene expression by affecting the FER (or FER-like) levels.

  4. Heart failure alters matrix metalloproteinase gene expression and activity in rat skeletal muscle.

    PubMed

    Carvalho, Robson Francisco; Dariolli, Rafael; Justulin Junior, Luis Antonio; Sugizaki, Mário Mateus; Politi Okoshi, Marina; Cicogna, Antonio Carlos; Felisbino, Sérgio Luis; Dal Pai-Silva, Maeli

    2006-12-01

    Heart failure is associated with a skeletal muscle myopathy with cellular and extracellular alterations. The hypothesis of this investigation is that extracellular changes may be associated with enhanced mRNA expression and activity of matrix metalloproteinases (MMP). We examined MMP mRNA expression and MMP activity in Soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DIA) muscles of young Wistar rat with monocrotaline-induced heart failure. Rats injected with saline served as age-matched controls. MMP2 and MMP9 mRNA contents were determined by RT-PCR and MMP activity by electrophoresis in gelatin-containing polyacrylamide gels in the presence of SDS under non-reducing conditions. Heart failure increased MMP9 mRNA expression and activity in SOL, EDL and DIA and MMP2 mRNA expression in DIA. These results suggest that MMP changes may contribute to the skeletal muscle myopathy during heart failure.

  5. Effects of Neonatal Treatment With 6-Hydroxydopamine and Endocrine Disruptors on Motor Activity and Gene Expression in Rats

    PubMed Central

    Masuo, Yoshinori; Ishido, Masami; Morita, Masatoshi; Oka, Syuichi

    2004-01-01

    To investigate the mechanisms underlying motor hyperactivity, we performed intracisternal injection of 6-hydroxydopamine or endocrine disruptors in rats on postnatal day 5. 6-Hydroxydopamine (100 μg, 488 nmol) caused a significant increase in spontaneous motor activities at 4 weeks of age. Gene-expression profiling using a cDNA membrane array revealed alterations in several classes of gene at 8 weeks of age. In the midbrain, gene expression was enhanced in dopamine transporter 1; a platelet-derived growth factor receptor; dopamine receptor D4; galanin receptor 2; arginine vasopressin receptor 2; neuropeptide Y; tachykinin 2; and fibroblast growth factor 10. Expression was also enhanced in the glutamate/aspartate transporter gene in the striatum. Rats received an endocrine disruptor (87 nmol), such as bisphenol A, nonylphenol, p-octylphenol, or diethylhexylphthalate, which also caused motor hyperactivity at 4 weeks. The effects of bisphenol A on motor activity were dose-dependent from 0.87 to 87 nmol. The phenols caused a deficit in dopamine neurons, similarly to the deficit caused by 6-hydroxydopamine. Gene-expression profiles after treatment with endocrine disruptors showed variation and differed from those of 6- hydroxydopamine. The results suggest that neonatal treatment with environmental chemicals can generate an animal model of attention-deficit hyperactivity disorder, in which clinical symptoms are pervasive. PMID:15303306

  6. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.

    PubMed

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun; Kim, Young Woo; Cho, Il Je; Kim, Sang Chan; Shin, Sang Mi; Ki, Sung Hwan

    2014-01-15

    Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.

  7. Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

    PubMed

    Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

    2015-08-03

    Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

  8. PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression

    PubMed Central

    Thomas, Jean-Luc; Ravel-Chapuis, Aymeric; Valente, Carmen; Corda, Daniela; Méjat, Alexandre

    2015-01-01

    Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway. PMID:26416879

  9. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons.

    PubMed

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP-GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP-GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP-GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin-GPR54 signaling, which could be a potential pathway to suppress cell migration.

  10. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons

    PubMed Central

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S.

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP–GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP–GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP–GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin–GPR54 signaling, which could be a potential pathway to suppress cell migration. PMID:26973595

  11. Activation of class I major histocompatibility complex gene expression by hepatitis B virus.

    PubMed Central

    Zhou, D X; Taraboulos, A; Ou, J H; Yen, T S

    1990-01-01

    Normal hepatocytes express very few class I major histocompatibility complex (MHC I) molecules, but MHC I expression is elevated in hepatitis B virus (HBV) infection. We report here that hepatoblastoma cells with replicating HBV genomes express three- to fourfold-higher levels of MHC I protein and mRNA than do parent cells without HBV DNA. Transient transfection assays demonstrated that the HBV X protein trans activated transcription from an MHC I promoter and allowed identification of cis elements important for trans activation. Images PMID:2164611

  12. Primate Lentiviruses Modulate NF-κB Activity by Multiple Mechanisms to Fine-Tune Viral and Cellular Gene Expression

    PubMed Central

    Heusinger, Elena; Kirchhoff, Frank

    2017-01-01

    The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4+ T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4+ T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle. PMID:28261165

  13. Identification of Potential Anticancer Activities of Novel Ganoderma lucidum Extracts Using Gene Expression and Pathway Network Analysis

    PubMed Central

    Kao, Chi H.J.; Bishop, Karen S.; Xu, Yuanye; Han, Dug Yeo; Murray, Pamela M.; Marlow, Gareth J.; Ferguson, Lynnette R.

    2016-01-01

    Ganoderma lucidum (lingzhi) has been used for the general promotion of health in Asia for many centuries. The common method of consumption is to boil lingzhi in water and then drink the liquid. In this study, we examined the potential anticancer activities of G. lucidum submerged in two commonly consumed forms of alcohol in East Asia: malt whiskey and rice wine. The anticancer effect of G. lucidum, using whiskey and rice wine-based extraction methods, has not been previously reported. The growth inhibition of G. lucidum whiskey and rice wine extracts on the prostate cancer cell lines, PC3 and DU145, was determined. Using Affymetrix gene expression assays, several biologically active pathways associated with the anticancer activities of G. lucidum extracts were identified. Using gene expression analysis (real-time polymerase chain reaction [RT-PCR]) and protein analysis (Western blotting), we confirmed the expression of key genes and their associated proteins that were initially identified with Affymetrix gene expression analysis. PMID:27006591

  14. Hepatitis B Virus Induces Expression of Antioxidant Response Element-regulated Genes by Activation of Nrf2*

    PubMed Central

    Schaedler, Stephanie; Krause, Janis; Himmelsbach, Kiyoshi; Carvajal-Yepes, Monica; Lieder, Franziska; Klingel, Karin; Nassal, Michael; Weiss, Thomas S.; Werner, Sabine; Hildt, Eberhard

    2010-01-01

    The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Human hepatitis B virus (HBV) induces a strong activation of Nrf2/ARE-regulated genes in vitro and in vivo. This is triggered by the HBV-regulatory proteins (HBx and LHBs) via c-Raf and MEK. The Nrf2/ARE-mediated induction of cytoprotective genes by HBV results in a better protection of HBV-positive cells against oxidative damage as compared with control cells. Furthermore, there is a significantly increased expression of the Nrf2/ARE-regulated proteasomal subunit PSMB5 in HBV-positive cells that is associated with a decreased level of the immunoproteasome subunit PSMB5i. In accordance with this finding, HBV-positive cells display a higher constitutive proteasome activity and a decreased activity of the immunoproteasome as compared with control cells even after interferon α/γ treatment. The HBV-dependent induction of Nrf2/ARE-regulated genes might ensure survival of the infected cell, shape the immune response to HBV, and thereby promote establishment of the infection. PMID:20956535

  15. Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension

    NASA Astrophysics Data System (ADS)

    Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab

    2015-11-01

    To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P < 0.05). In the hypertensive chickens, superoxide dismutase activity was decreased (forebrain, midbrain, and hindbrain), while catalase activity was increased (forebrain and midbrain) ( P < 0.05). Glutathione peroxidase activity did not change. Relative gene expression of catalase and superoxide dismutases (1 and 2) was downregulated, while glutathione peroxidase was upregulated in the brain of the cold-induced pulmonary hypertensive chickens. Probably, these situations in the oxidant and antioxidant status of the brain especially hindbrain may change its function at cardiovascular center and sympathetic nervous system to exacerbate pulmonary hypertension.

  16. Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase.

    PubMed

    Nowak, Tomasz; Januszkiewicz, Danuta; Zawada, Mariola; Pernak, Monika; Lewandowski, Krzysztof; Rembowska, Jolanta; Nowicka, Karina; Mankowski, Przemyslaw; Nowak, Jerzy

    2006-08-01

    Reactivation of telomerase plays an important role in carcinogenesis. Malignant cells almost always possess high activity and expression of telomerase. The aim of this study was to see whether there is any relationship between telomerase activity and expression and hTERT and hTERC gene amplification in acute lymphoblastic leukemia (ALL) and non-lymphoblastic leukemia (ANLL) cells. In addition telomere length was tested in leukemic cells at the time of diagnosis and during remission. Expression of the three components of telomerase (hTERT, hTERC and TP1) as well as telomerase activity was found both in ALL and ANLL cells. Telomerase activity was diminished in patients in remission. The leukemic cells showed considerable heterogeneity of terminal restriction fragments, that is telomere length. ALL cells showed a variable pattern of telomere length in contrast to ANLL cells which produced a predominantly short telomere pattern. Telomere length in the lymphocytes of leukemia patients was shorter in remission as compared to the time of diagnosis. FISH analysis revealed amplification of hTERT and hTERC genes in ALL and ANLL cells. Quantitative analysis showed that leukemic cells possess higher number of hTERT and hTERC copies than the normal PBL. Our results suggest that the activation of telomerase in leukemic cells is connected with amplification of hTERT and hTERC genes. The high expression and activity of telomerase found in leukemic cells may be partially explained by amplified hTERT and hTERC genes. Amplification of the telomerase genes seems to be a common event in carcinogenesis and may play a role in telomerase reactivation leading to cell immortalization.

  17. Application of Gene Expression Trajectories Initiated from ErbB Receptor Activation Highlights the Dynamics of Divergent Promoter Usage.

    PubMed

    Carbajo, Daniel; Magi, Shigeyuki; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Arner, Erik; Forrest, Alistair R R; Carninci, Piero; Hayashizaki, Yoshihide; Daub, Carsten O; Okada-Hatakeyama, Mariko; Mar, Jessica C

    2015-01-01

    Understanding how cells use complex transcriptional programs to alter their fate in response to specific stimuli is an important question in biology. For the MCF-7 human breast cancer cell line, we applied gene expression trajectory models to identify the genes involved in driving cell fate transitions. We modified trajectory models to account for the scenario where cells were exposed to different stimuli, in this case epidermal growth factor and heregulin, to arrive at different cell fates, i.e. proliferation and differentiation respectively. Using genome-wide CAGE time series data collected from the FANTOM5 consortium, we identified the sets of promoters that were involved in the transition of MCF-7 cells to their specific fates versus those with expression changes that were generic to both stimuli. Of the 1,552 promoters identified, 1,091 had stimulus-specific expression while 461 promoters had generic expression profiles over the time course surveyed. Many of these stimulus-specific promoters mapped to key regulators of the ERK (extracellular signal-regulated kinases) signaling pathway such as FHL2 (four and a half LIM domains 2). We observed that in general, generic promoters peaked in their expression early on in the time course, while stimulus-specific promoters tended to show activation of their expression at a later stage. The genes that mapped to stimulus-specific promoters were enriched for pathways that control focal adhesion, p53 signaling and MAPK signaling while generic promoters were enriched for cell death, transcription and the cell cycle. We identified 162 genes that were controlled by an alternative promoter during the time course where a subset of 37 genes had separate promoters that were classified as stimulus-specific and generic. The results of our study highlighted the degree of complexity involved in regulating a cell fate transition where multiple promoters mapping to the same gene can demonstrate quite divergent expression profiles.

  18. Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

    NASA Astrophysics Data System (ADS)

    Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda

    2004-06-01

    Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

  19. Angelica Sinensis Polysaccharides Stimulated UDP-Sugar Synthase Genes through Promoting Gene Expression of IGF-1 and IGF1R in Chondrocytes: Promoting Anti-Osteoarthritic Activity

    PubMed Central

    Wen, Yinxian; Li, Jing; Tan, Yang; Qin, Jun; Xie, Xianfei; Wang, Linlong; Mei, Qibing; Wang, Hui; Magdalou, Jacques; Chen, Liaobin

    2014-01-01

    Background Osteoarthritis (OA) is a chronic joints disease characterized by progressive degeneration of articular cartilage due to the loss of cartilage matrix. Previously, we found, for the first time, that an acidic glycan from Angelica Sinensis Polysaccharides (APSs), namely the APS-3c, could protect rat cartilage from OA due to promoting glycosaminoglycan (GAG) synthesis in chondrocytes. In the present work, we tried to further the understanding of ASP-3c’s anti-OA activity. Methodology/Principal Findings Human primary chondrocytes were treated with APS-3c or/and recombinant human interleukin 1β (IL-1β). It turned out that APS-3c promoted synthesis of UDP-xylose and GAG, as well as the gene expression of UDP-sugar synthases (USSs), insulin like growth factor 1 (IGF1) and IGF1 receptor (IGF1R), and attenuated the degenerative phenotypes, suppressed biosynthesis of UDP-sugars and GAG, and inhibited the gene expression of USSs, IGF1 and IGF1R induced by IL-1β. Then, we induced a rat OA model with papain, and found that APS-3c also stimulated GAG synthesis and gene expression of USSs, IGF1 and IGF1R in vivo. Additionally, recombinant human IGF1 and IGF1R inhibitor NP-AEW541 were applied to figure out the correlation between stimulated gene expression of USSs, IGF1 and IGF1R induced by APS-3c. It tuned out that the promoted GAG synthesis and USSs gene expression induced by APS-3c was mediated by the stimulated IGF1 and IGF1R gene expression, but not through directly activation of IGF1R signaling pathway. Conclusions/Significances We demonstrated for the first time that APS-3c presented anti-OA activity through stimulating IGF-1 and IGF1R gene expression, but not directly activating the IGF1R signaling pathway, which consequently promoted UDP-sugars and GAG synthesis due to up-regulating gene expression of USSs. Our findings presented a better understanding of APS-3c’s anti-OA activity and suggested that APS-3c could potentially be a novel therapeutic agent

  20. Developmental alterations in CNS stress-related gene expression following postnatal immune activation.

    PubMed

    Amath, A; Foster, J A; Sidor, M M

    2012-09-18

    Early-life adversity is associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and increased susceptibility to later-life psychopathology. Specifically, there is mounting evidence suggesting that the immune system plays an important role in central nervous system (CNS) development and in the programing of behavior. The current study investigated how early-life immune challenge affects the development of CNS stress neurocircuitry by examining the gene expression profile of corticotropin-releasing hormone (CRH), CRH receptors, and the major corticosteroid receptors within the limbic-hypothalamic circuit of the developing rodent brain. Mice were administered a 0.05 mg/kg lipopolysaccharide (LPS) injection on postnatal day (P) 3 and 5 and gene expression was assessed using in situ hybridization from P14 to P28. Target genes investigated were CRH, CRH receptor-1 (CRHR1), CRH receptor-2, the mineralocorticoid receptor, and the glucocorticoid receptor (GR). Early LPS challenge resulted in a transient decrease in CRHR1 mRNA expression in the cornuammonis 1 (CA1) and CA3 regions of the hippocampus that were accompanied by increased hippocampal GR mRNA expression in the CA1 region between P14 and P21. This was followed by increased hypothalamic CRH expression in LPS-mice on P28. Our current findings suggest that early-life LPS challenge impacts the developmental trajectory of CNS stress neurocircuitry. These results lend insight into the molecular basis for the later development of stress-related behaviors as previously described in early immune challenge rodents.

  1. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

    SciTech Connect

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun; Kim, Young Woo; Cho, Il Je; Kim, Sang Chan; Shin, Sang Mi; Ki, Sung Hwan

    2014-01-15

    Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. - Highlights: • We investigated the effect of isorhamnetin on Nrf2 activation. • Isorhamnetin increased Nrf2

  2. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis.

    PubMed Central

    Virbasius, J V; Scarpulla, R C

    1994-01-01

    Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates transcription from the two divergent mitochondrial promoters and is likely the principal activator of mitochondrial gene expression in vertebrates. Here we establish that the proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implicated in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nucleotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro transcription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis. Images PMID:8108407

  3. TRANSLUCENT GREEN, an ERF family transcription factor, controls water balance in Arabidopsis by activating the expression of aquaporin genes.

    PubMed

    Zhu, Danling; Wu, Zhe; Cao, Guangyu; Li, Jigang; Wei, Jia; Tsuge, Tomohiko; Gu, Hongya; Aoyama, Takashi; Qu, Li-Jia

    2014-04-01

    Water is the most abundant molecule in almost all living organisms. Aquaporins are channel proteins that play critical roles in controlling the water content of cells. Here, we report the identification of an AP2/EREBP family transcription factor in Arabidopsis thaliana, TRANSLUCENT GREEN (TG), whose overexpression in transgenic plants gave enhanced drought tolerance and vitrified leaves. TG protein is localized in the nucleus, binds DRE and GCC elements in vitro, and acts as a transcriptional activator in yeast cells. Microarray analysis revealed a total of 330 genes regulated by TG, among which five genes encode aquaporins. A transient expression assay showed that TG directly binds to the promoters of three aquaporin genes, such as AtTIP1;1, AtTIP2;3, and AtPIP2;2, indicating that TG directly regulates the expression of these genes. Moreover, overexpression of AtTIP1;1 resulted in vitrified phenotypes in transgenic Arabidopsis plants, similar to those observed in TG overexpression lines. Water injection into wild-type leaves recapitulated the vitrified leaf phenotypes, which was reversed by cutting off the water supply from vascular bundles. Taken together, our data support that TG controls water balance in Arabidopsis through directly activating the expression of aquaporin genes.

  4. HoxBlinc RNA recruits Set1/MLL complexes to activate Hox gene expression patterns and mesoderm lineage development

    PubMed Central

    Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Nao; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

    2015-01-01

    Summary Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1+ mesoderm and then promotes hematopoietic differentiation through regulating hoxb gene pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated KD or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb gene expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1+ precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1+ precursors and differentiation of Flk1+ cells into hematopoietic lineages. PMID:26725110

  5. Using fluorescence activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.

    PubMed

    Schwarz, Jaclyn M

    2015-05-28

    The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

  6. Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

    PubMed Central

    Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall; Jørgensen, Louise Helskov; Dhawan, Jyotsna; Nielsen, Michael Friberg Bruun; Schrøder, Henrik Daa

    2016-01-01

    Objectives Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. Materials and Methods Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. Results and Conclusions We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts. PMID:27442119

  7. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation.

    PubMed

    Martinez, Emily M; Yoshida, Miya C; Candelario, Tara Lynne T; Hughes-Fulford, Millie

    2015-03-15

    Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (μg) conditions. Scientists on the ground use two models of simulated μg (sμg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true μg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true μg and sμg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sμg models provide an excellent test bed for scientists to develop baseline studies and augment true μg in spaceflight experiments. Ultimately, sμg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders.

  8. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation

    PubMed Central

    Martinez, Emily M.; Yoshida, Miya C.; Candelario, Tara Lynne T.

    2015-01-01

    Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (μg) conditions. Scientists on the ground use two models of simulated μg (sμg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true μg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true μg and sμg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sμg models provide an excellent test bed for scientists to develop baseline studies and augment true μg in spaceflight experiments. Ultimately, sμg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders. PMID:25568077

  9. Venom of Parasitoid Pteromalus puparum Impairs Host Humoral Antimicrobial Activity by Decreasing Host Cecropin and Lysozyme Gene Expression

    PubMed Central

    Fang, Qi; Wang, Bei-Bei; Ye, Xin-Hai; Wang, Fei; Ye, Gong-Yin

    2016-01-01

    Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Here, we report that Pteromalus puparum venom impairs the antimicrobial activity of its host Pieris rapae. Inhibition zone results showed that bead injection induced the antimicrobial activity of the host hemolymph but that venom inhibited it. The cDNAs encoding cecropin and lysozyme were screened. Relative quantitative PCR results indicated that all of the microorganisms and bead injections up-regulated the transcript levels of the two genes but that venom down-regulated them. At 8 h post bead challenge, there was a peak in the transcript level of the cecropin gene, whereas the peak of lysozyme gene occurred at 24 h. The transcripts levels of the two genes were higher in the granulocytes and fat body than in other tissues. RNA interference decreased the transcript levels of the two genes and the antimicrobial activity of the pupal hemolymph. Venom injections similarly silenced the expression of the two genes during the first 8 h post-treatment in time- and dose-dependent manners, after which the silence effects abated. Additionally, recombinant cecropin and lysozyme had no significant effect on the emergence rate of pupae that were parasitized by P. puparum females. These findings suggest one mechanism of impairing host antimicrobial activity by parasitoid venom. PMID:26907346

  10. Pyrroloquinoline quinone increases the expression and activity of Sirt1 and -3 genes in HepG2 cells.

    PubMed

    Zhang, Jian; Meruvu, Sunitha; Bedi, Yudhishtar Singh; Chau, Jason; Arguelles, Andrix; Rucker, Robert; Choudhury, Mahua

    2015-09-01

    Sirtuin (Sirt) 1 and Sirt 3 are nicotinamide adenine dinucleotide ((+))-dependent protein deacetylases that are important to a number of mitochondrial-related functions; thus, identification of sirtuin activators is important. Herein, we hypothesize that pyrroloquinoline quinone (PQQ) can act as a Sirt1/Sirt3 activator. In HepG2 cell cultures, PQQ increased the expression of Sirt1 and Sirt3 gene, protein, and activity levels (P < .05). We also observed a significant increase in nicotinamide phosphoribosyltransferase gene expression (as early as 18 hours) and increased NAD(+) activity at 24 hours. In addition, targets of Sirt1 and Sirt3 (peroxisome proliferator-activated receptor γ coactivator 1α, nuclear respiratory factor 1 and 2, and mitochondrial transcription factor A) were increased at 48 hours. This is the first report that demonstrates PQQ as an activator of Sirt1 and Sirt3 expression and activity, making it an attractive therapeutic agent for the treatment of metabolic diseases and for healthy aging. Based on our study and the available data in vivo, PQQ has the potential to serve as a therapeutic nutraceutical, when enhancing mitochondrial function.

  11. Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-{beta}

    SciTech Connect

    Zhao, Haige; Hiroi, Toyoko; Hansen, Baranda S.; Rade, Jeffrey J.

    2009-11-27

    Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A{sub 2}, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A{sub 2} synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-{kappa}{beta} signaling pathway.

  12. Altered activities of transcription factors and their related gene expression in cardiac tissues of diabetic rats.

    PubMed

    Nishio, Y; Kashiwagi, A; Taki, H; Shinozaki, K; Maeno, Y; Kojima, H; Maegawa, H; Haneda, M; Hidaka, H; Yasuda, H; Horiike, K; Kikkawa, R

    1998-08-01

    Gene regulation in the cardiovascular tissues of diabetic subjects has been reported to be altered. To examine abnormal activities in transcription factors as a possible cause of this altered gene regulation, we studied the activity of two redox-sensitive transcription factors--nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1)--and the change in the mRNA content of heme oxygenase-1, which is regulated by these transcription factors in the cardiac tissues of rats with streptozotocin-induced diabetes. Increased activity of NF-kappaB and AP-1 but not nuclear transcription-activating factor, as determined by an electrophoretic mobility shift assay, was found in the hearts of 4-week diabetic rats. Glycemic control by a subcutaneous injection of insulin prevented these diabetes-induced changes in transcription factor activity. In accordance with these changes, the mRNA content of heme oxygenase-1 was increased fourfold in 4-week diabetic rats and threefold in 24-week diabetic rats as compared with control rats (P < 0.01 and P < 0.05, respectively). Insulin treatment also consistently prevented changes in the mRNA content of heme oxygenase-1. The oral administration of an antioxidant, probucol, to these diabetic rats partially prevented the elevation of the activity of both NF-kappaB and AP-1, and normalized the mRNA content of heme oxygenase-1 without producing any change in the plasma glucose concentration. These results suggest that elevated oxidative stress is involved in the activation of the transcription factors NF-kappaB and AP-1 in the cardiac tissues of diabetic rats, and that these abnormal activities of transcription factors could be associated with the altered gene regulation observed in the cardiovascular tissues of diabetic rats.

  13. Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

    PubMed

    Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

    2016-12-01

    To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori.

  14. Gene expression and proteomic analysis of shoot apical meristem transition from dormancy to activation in Cunninghamia lanceolata (Lamb.) Hook

    PubMed Central

    Xu, Huimin; Cao, Dechang; Chen, Yanmei; Wei, Dongmei; Wang, Yanwei; Stevenson, Rebecca Ann; Zhu, Yingfang; Lin, Jinxing

    2016-01-01

    In contrast to annual plants, in perennial plants, the shoot apical meristem (SAM) can undergo seasonal transitions between dormancy and activity; understanding this transition is crucial for understanding growth in perennial plants. However, little is known about the molecular mechanisms of SAM development in trees. Here, light and transmission electron microscopy revealed that evident changes in starch granules, lipid bodies, and cell walls thickness of the SAM in C. lanceolata during the transition from dormancy to activation. HPLC-ESI-MS/MS analysis showed that levels of indole-3-acetic acid (IAA) increased and levels of abscisic acid (ABA) decreased from dormant to active stage. Examination of 20 genes and 132 differentially expressed proteins revealed that the expression of genes and proteins potentially involved in cell division and expansion significantly increased in the active stage, whereas those related to the abscisic acid insensitive 3(ABI3), the cytoskeleton and energy metabolism decreased in the dormant stage. These findings provide new insights into the complex mechanism of gene and protein expression and their relation to cytological and physiological changes of SAM in this coniferous species. PMID:26832850

  15. Age-Dependent Hepatic UDP-Glucuronosyltransferase Gene Expression and Activity in Children

    PubMed Central

    Neumann, Elizabeth; Mehboob, Huma; Ramírez, Jacqueline; Mirkov, Snezana; Zhang, Min; Liu, Wanqing

    2016-01-01

    UDP-glucuronosyltransferases (UGTs) are important phase II drug metabolism enzymes. The aim of this study was to explore the relationship between age and changes in mRNA expression and activity of major human hepatic UGTs, as well as to understand the potential regulatory mechanism underlying this relationship. Using previously generated data, we investigated age-dependent mRNA expression levels of 11 hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17) and 16 transcription factors (AHR, AR, CAR, ESR2, FXR, GCCR, HNF1a, HNF3a, HNF3b, HNF4a, PPARA, PPARG, PPARGC, PXR, SP1, and STAT3) in liver tissue of donors (n = 38) ranging from 0 to 25 years of age. We also examined the correlation between age and microsomal activities using 14 known UGT drug substrates in the liver samples (n = 19) of children donors. We found a statistically significant increase (nominal p < 0.05) in the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT2B7, and UGT2B17, as well as glucuronidation activities of serotonin, testosterone, and vorinostat during the first 25 years of life. Expression of estrogen receptor 1 and pregnane X receptor, two strong UGT transcriptional regulators, were significantly correlated with both age and UGT mRNA expression (p ≤ 0.05). These results suggest that both UGT expression and activity increase during childhood and adolescence, possibly driven in part by hormonal signaling. Our findings may help explain inter-patient variability in response to medications among children. PMID:27899892

  16. Rapid activation of MDR1 gene expression in human metastatic sarcoma after in vivo exposure to doxorubicin.

    PubMed

    Abolhoda, A; Wilson, A E; Ross, H; Danenberg, P V; Burt, M; Scotto, K W

    1999-11-01

    Overexpression of P-glycoprotein (Pgp), a multidrug transporter encoded by the MDR1 gene, is associated with chemoresistance in some human solid tumor malignancies. To date, analyses of MDR1 levels in solid tumors have examined constitutive increases in expression at relapse. In the present study, we have evaluated the acute induction of MDR1 gene expression in a solid human tumor as a function of time in response to in vivo exposure to chemotherapy. Five patients with unresectable sarcoma pulmonary metastases underwent isolated single lung perfusion with doxorubicin. Relative MDR1 gene expression was measured in metastatic tumor nodules and normal lung specimens after initiation of chemoperfusion. In four of five patients, a 3-15-fold (median, 6.8) increase in MDR1 RNA levels was detected in tumors at 50 min after administration of doxorubicin. In contrast, normal lung samples had very low levels of MDR1 RNA prior to perfusion, and no acute increases were observed after therapy. These findings demonstrate, for the first time, that MDR1 gene expression can be rapidly activated in human tumors after transient in vivo exposure to cytotoxic chemotherapy.

  17. Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3'-end-seq.

    PubMed

    Haenni, Simon; Ji, Zhe; Hoque, Mainul; Rust, Nigel; Sharpe, Helen; Eberhard, Ralf; Browne, Cathy; Hengartner, Michael O; Mellor, Jane; Tian, Bin; Furger, André

    2012-07-01

    Despite the many advantages of Caenorhabditis elegans, biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by fluorescence-activated nuclei sorting (FANS), followed by deep sequencing of linearly amplified 3'-end regions of transcripts (3'-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3'-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3'-end formation in the intestine.

  18. Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells.

    PubMed

    Arbon, Kate S; Christensen, Cody M; Harvey, Wendy A; Heggland, Sara J

    2012-02-01

    Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10μM CdCl(2) for 2-72h. We detected significant ERK activation in response to CdCl(2) and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl(2) and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl(2) exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl(2). Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity.

  19. Extensive Evolutionary Changes in Regulatory Element Activity during Human Origins Are Associated with Altered Gene Expression and Positive Selection

    PubMed Central

    Fedrigo, Olivier; Babbitt, Courtney C.; Wortham, Matthew; Tewari, Alok K.; London, Darin; Song, Lingyun; Lee, Bum-Kyu; Iyer, Vishwanath R.; Parker, Stephen C. J.; Margulies, Elliott H.; Wray, Gregory A.; Furey, Terrence S.; Crawford, Gregory E.

    2012-01-01

    Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species. PMID:22761590

  20. Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons

    PubMed Central

    Qiu, Jing; McQueen, Jamie; Bilican, Bilada; Dando, Owen; Magnani, Dario; Punovuori, Karolina; Selvaraj, Bhuvaneish T; Livesey, Matthew; Haghi, Ghazal; Heron, Samuel; Burr, Karen; Patani, Rickie; Rajan, Rinku; Sheppard, Olivia; Kind, Peter C; Simpson, T Ian; Tybulewicz, Victor LJ; Wyllie, David JA; Fisher, Elizabeth MC; Lowell, Sally; Chandran, Siddharthan; Hardingham, Giles E

    2016-01-01

    Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents. DOI: http://dx.doi.org/10.7554/eLife.20337.001 PMID:27692071

  1. Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1.

    PubMed

    Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H

    2017-04-01

    Since the discovery of μ-opioid receptor (MOR) gene two decades ago, various regulatory factors have been shown to interact with the MOR promoter and modulate transcript levels. However, the majority of early transcriptional studies on MOR gene have not addressed how intracellular signaling pathways mediate extracellular modulators. In this study, we demonstrate that MOR epigenetic regulation requires multiple coordinated signals converging at the MOR promoter, involving mitogen-activated protein kinase (MAPK) activation and mitogen- and stress-activated protein kinase 1 (MSK1)-ranges of intracellular signaling pathways similar to those activated by opioid agonists. Inhibiting p38 MAPK or extracellular signal-regulated kinase (ERK) 1/2 MAPK (upstream activators of MSK1) reduced MOR expression levels; accordingly, the functional role of MSK1, but not MSK2, was demonstrated using genetic approaches. However, for maximal MSK1 effect, an open chromatin configuration was required, because in vitro CpG methylation of the MOR promoter abolished MSK1 activity. Finally, endogenous MSK1 levels concomitantly increased to regulate MOR gene expression during neuronal differentiation of P19 cells, suggesting a conserved role of this kinase in the epigenic activation of MOR in neurons. Taken together, our findings indicate that the expression of MOR gene requires the activity of intracellular signaling pathways that have been implicated in the behavioral outcomes of opioid drugs, which suggests that an autoregulatory mechanism may function in opioid systems.

  2. The bacteriophage-derived transcriptional regulator, LscR, activates the expression of levansucrase genes in Pseudomonas syringae.

    PubMed

    Abdallah, Khaled; Hartman, Katharina; Pletzer, Daniel; Zhurina, Daria; Ullrich, Matthias S

    2016-12-01

    Synthesis of the exopolysaccharide levan occurs in the bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, when this bacterium encounters moderate to high concentrations of sucrose inside its host plant. The process is mediated by the temperature-dependent expression and secretion of two levansucrases, LscB and LscC. Previous studies showed the importance of a prophage-associated promoter element in driving the expression of levansucrase genes. Herein, heterologous screening for transcriptional activators revealed that the prophage-borne transcriptional regulator, LscR, from P. syringae mediates expression of levansucrase. A lscR-deficient mutant was generated and exhibited a levan-negative phenotype when grown on a sucrose-rich medium. This phenotype was confirmed by zymographic analysis and Western blots which demonstrated absence of levansucrase in the supernatant and total cell lysates. Transcriptional analysis showed a down-regulation of expression levels of levansucrase and glycosyl hydrolase genes in the lscR-deficient mutant. Ultimately, a direct binding of LscR to the promoter region of levansucrase was demonstrated using electrophoretic mobility shift assays allowing to conclude that a bacteriophage-derived regulator dictates expression of bacterial genes involved in in planta fitness.

  3. RNA-mediated gene activation

    PubMed Central

    Jiao, Alan L; Slack, Frank J

    2014-01-01

    The regulation of gene expression by non-coding RNAs (ncRNAs) has become a new paradigm in biology. RNA-mediated gene silencing pathways have been studied extensively, revealing diverse epigenetic and posttranscriptional mechanisms. In contrast, the roles of ncRNAs in activating gene expression remains poorly understood. In this review, we summarize the current knowledge of gene activation by small RNAs, long non-coding RNAs, and enhancer-derived RNAs, with an emphasis on epigenetic mechanisms. PMID:24185374

  4. Cystatin D Locates in the Nucleus at Sites of Active Transcription and Modulates Gene and Protein Expression*

    PubMed Central

    Ferrer-Mayorga, Gemma; Alvarez-Díaz, Silvia; Valle, Noelia; De Las Rivas, Javier; Mendes, Marta; Barderas, Rodrigo; Canals, Francesc; Tapia, Olga; Casal, J. Ignacio; Lafarga, Miguel; Muñoz, Alberto

    2015-01-01

    Cystatin D is an inhibitor of lysosomal and secreted cysteine proteases. Strikingly, cystatin D has been found to inhibit proliferation, migration, and invasion of colon carcinoma cells indicating tumor suppressor activity that is unrelated to protease inhibition. Here, we demonstrate that a proportion of cystatin D locates within the cell nucleus at specific transcriptionally active chromatin sites. Consistently, transcriptomic analysis show that cystatin D alters gene expression, including that of genes encoding transcription factors such as RUNX1, RUNX2, and MEF2C in HCT116 cells. In concordance with transcriptomic data, quantitative proteomic analysis identified 292 proteins differentially expressed in cystatin D-expressing cells involved in cell adhesion, cytoskeleton, and RNA synthesis and processing. Furthermore, using cytokine arrays we found that cystatin D reduces the secretion of several protumor cytokines such as fibroblast growth factor-4, CX3CL1/fractalkine, neurotrophin 4 oncostatin-M, pulmonary and activation-regulated chemokine/CCL18, and transforming growth factor B3. These results support an unanticipated role of cystatin D in the cell nucleus, controlling the transcription of specific genes involved in crucial cellular functions, which may mediate its protective action in colon cancer. PMID:26364852

  5. Stress gene (hsp70) sequences and quantitative expression in Milnesium tardigradum (Tardigrada) during active and cryptobiotic stages.

    PubMed

    Schill, Ralph O; Steinbrück, Günther H B; Köhler, Heinz-R

    2004-04-01

    The eutardigrade Milnesium tardigradum can undergo cryptobiosis, i.e. entry into a reversible ametabolic stage induced by dehydration, cooling and, probably, osmotic and anoxic stress. For the first time in tardigrades, we described partial sequences of three heat-shock protein (hsp70 family) genes and examined gene expression on the way from an active to a cryptobiotic and back to an active stage again. Results showed different patterns of gene expression in the hsp70 isoforms. All three isoforms seem to be true heat-shock proteins since transcription could be clearly enhanced by temperature elevation. Isoform 1 and, at a lower level, isoform 3 do not seem to have a specific function for cryptobiosis. By contrast, transcription of isoform 2 is significantly induced in the transitional stage between the active and the cryptobiotic stage, resulting in a comparatively high mRNA copy number also during cryptobiosis. This pattern of induction implies that isoform 2 is the most relevant hsp70 gene for M. tardigradum individuals entering the cryptobiotic stage.

  6. Lin28A binds active promoters and recruits Tet1 to regulate gene expression

    PubMed Central

    Zeng, Yaxue; Yao, Bing; Shin, Jaehoon; Lin, Li; Kim, Namshik; Song, Qifeng; Liu, Shuang; Su, Yijing; Guo, Junjie U.; Huang, Luoxiu; Wan, Jun; Wu, Hao; Qian, Jiang; Cheng, Xiaodong; Zhu, Heng; Ming, Guo-li; Jin, Peng; Song, Hongjun

    2015-01-01

    Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites, and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems. PMID:26711009

  7. Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.

    PubMed

    Parra, Loreto P; Espina, Giannina; Devia, Javier; Salazar, Oriana; Andrews, Barbara; Asenjo, Juan A

    2015-01-01

    Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C.

  8. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    SciTech Connect

    Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak; Ilgu, Muslum; Beasley, Jonathan; Bendickson, Lee; Mehanovic, Samir; Kraus, George A.; Nilsen-Hamilton, Marit

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  9. Partial Sleep Restriction Activates Immune Response-Related Gene Expression Pathways: Experimental and Epidemiological Studies in Humans

    PubMed Central

    Rantanen, Ville; Kronholm, Erkki; Surakka, Ida; van Leeuwen, Wessel M. A.; Lehto, Maili; Matikainen, Sampsa; Ripatti, Samuli; Härmä, Mikko; Sallinen, Mikael; Salomaa, Veikko; Jauhiainen, Matti; Alenius, Harri; Paunio, Tiina; Porkka-Heiskanen, Tarja

    2013-01-01

    Epidemiological studies have shown that short or insufficient sleep is associated with increased risk for metabolic diseases and mortality. To elucidate mechanisms behind this connection, we aimed to identify genes and pathways affected by experimentally induced, partial sleep restriction and to verify their connection to insufficient sleep at population level. The experimental design simulated sleep restriction during a working week: sleep of healthy men (N = 9) was restricted to 4 h/night for five nights. The control subjects (N = 4) spent 8 h/night in bed. Leukocyte RNA expression was analyzed at baseline, after sleep restriction, and after recovery using whole genome microarrays complemented with pathway and transcription factor analysis. Expression levels of the ten most up-regulated and ten most down-regulated transcripts were correlated with subjective assessment of insufficient sleep in a population cohort (N = 472). Experimental sleep restriction altered the expression of 117 genes. Eight of the 25 most up-regulated transcripts were related to immune function. Accordingly, fifteen of the 25 most up-regulated Gene Ontology pathways were also related to immune function, including those for B cell activation, interleukin 8 production, and NF-κB signaling (P<0.005). Of the ten most up-regulated genes, expression of STX16 correlated negatively with self-reported insufficient sleep in a population sample, while three other genes showed tendency for positive correlation. Of the ten most down-regulated genes, TBX21 and LGR6 correlated negatively and TGFBR3 positively with insufficient sleep. Partial sleep restriction affects the regulation of signaling pathways related to the immune system. Some of these changes appear to be long-lasting and may at least partly explain how prolonged sleep restriction can contribute to inflammation-associated pathological states, such as cardiometabolic diseases. PMID:24194869

  10. Epilepsy-causing sequence variations in SIK1 disrupt synaptic activity response gene expression and affect neuronal morphology.

    PubMed

    Pröschel, Christoph; Hansen, Jeanne N; Ali, Adil; Tuttle, Emily; Lacagnina, Michelle; Buscaglia, Georgia; Halterman, Marc W; Paciorkowski, Alex R

    2017-02-01

    SIK1 syndrome is a newly described developmental epilepsy disorder caused by heterozygous mutations in the salt-inducible kinase SIK1. To better understand the pathophysiology of SIK1 syndrome, we studied the effects of SIK1 pathogenic sequence variations in human neurons. Primary human fetal cortical neurons were transfected with a lentiviral vector to overexpress wild-type and mutant SIK1 protein. We evaluated the transcriptional activity of known downstream gene targets in neurons expressing mutant SIK1 compared with wild type. We then assayed neuronal morphology by measuring neurite length, number and branching. Truncating SIK1 sequence variations were associated with abnormal MEF2C transcriptional activity and decreased MEF2C protein levels. Epilepsy-causing SIK1 sequence variations were associated with significantly decreased expression of ARC (activity-regulated cytoskeletal-associated) and other synaptic activity response element genes. Assay of mRNA levels for other MEF2C target genes NR4A1 (Nur77) and NRG1, found significantly, decreased the expression of these genes as well. The missense p.(Pro287Thr) SIK1 sequence variation was associated with abnormal neuronal morphology, with significant decreases in mean neurite length, mean number of neurites and a significant increase in proximal branches compared with wild type. Epilepsy-causing SIK1 sequence variations resulted in abnormalities in the MEF2C-ARC pathway of neuronal development and synapse activity response. This work provides the first insights into the mechanisms of pathogenesis in SIK1 syndrome, and extends the ARX-MEF2C pathway in the pathogenesis of developmental epilepsy.

  11. Drosophila lysyl oxidases Dmloxl-1 and Dmloxl-2 are differentially expressed and the active DmLOXL-1 influences gene expression and development.

    PubMed

    Molnar, Janos; Ujfaludi, Zsuzsanna; Fong, Sheri F T; Bollinger, John A; Waro, Girma; Fogelgren, Ben; Dooley, David M; Mink, Matyas; Csiszar, Katalin

    2005-06-17

    Mammalian lysyl oxidase (LOX) is essential for the catalysis of lysyl-derived cross-links in fibrillar collagens and elastin in the extracellular matrix and has also been implicated in cell motility, differentiation, and tumor cell invasion. The active LOX has been shown to translocate to the nuclei of smooth muscle cells and regulate chromatin structure and transcription. It is difficult to interpret the role of the LOX protein as it is co-expressed with other members of the LOX amine oxidase family in most mammalian cells. To investigate the function of the LOX proteins, we have characterized the Drosophila lysyl oxidases Dmloxl-1 and Dmloxl-2. We present the gene, domain structure, and expression pattern of Dmloxl-1 and Dmloxl-2 during development. In early development, only Dmloxl-1 was expressed, which allowed functional studies. We have expressed Dmloxl-1 in S2 cells and determined that it is a catalytically active enzyme, inhibited by beta-amino-proprionitrile (BAPN), a specific LOX inhibitor. We localized DmLOXL-1 in the nuclei in embryos and in adult salivary gland cells in the nuclei, cytoplasm, and cell surface, using immunostaining and a DmLOXL-1 antibody. To address the biological function of Dmloxl-1, we raised larvae under BAPN inhibitory conditions and over-expressed Dmloxl-1 in transgenic Drosophila. DmLOXL-1 inhibition resulted in developmental delay and a shift in sex ratio; over-expression in the w(m4) variegating strain increased drosopterin production, demonstrating euchromatinization. Our previous data on the transcriptional down-regulation of seven ribosomal genes and the glue gene under inhibitory conditions and the current results collectively support a nuclear role for Dmloxl-1 in euchromatinization and gene regulation.

  12. A novel murrel Channa striatus mitochondrial manganese superoxide dismutase: gene silencing, SOD activity, superoxide anion production and expression.

    PubMed

    Arockiaraj, Jesu; Palanisamy, Rajesh; Bhatt, Prasanth; Kumaresan, Venkatesh; Gnanam, Annie J; Pasupuleti, Mukesh; Kasi, Marimuthu

    2014-12-01

    We have reported the molecular characterization including gene silencing, superoxide activity, superoxide anion production, gene expression and molecular characterization of a mitochondrial manganese superoxide dismutase (mMnSOD) from striped murrel Channa striatus (named as CsmMnSOD). The CsmMnSOD polypeptide contains 225 amino acids with a molecular weight of 25 kDa and a theoretical isoelectric point of 8.3. In the N-terminal region, CsmMnSOD carries a mitochondrial targeting sequence and a superoxide dismutases (SOD) Fe domain (28-109), and in C-terminal region, it carries another SOD Fe domain (114-220). The CsmMnSOD protein sequence shared significant similarity with its homolog of MnSOD from rock bream Oplegnathus fasciatus (96%). The phylogenetic analysis showed that the CsmMnSOD fell in the clade of fish mMnSOD group. The monomeric structure of CsmMnSOD possesses 9 α-helices (52.4%), 3 β-sheets (8.8%) and 38.8% random coils. The highest gene expression was noticed in liver, and its expression was inducted with fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infections. The gene silencing results show that the fish that received dsRNA exhibited significant (P < 0.05) changes in expression when compared to their non-injected and fish physiological saline-injected controls. The SOD activity shows that the activity increases with the spread of infection and decreases once the molecule controls the pathogen. The capacity of superoxide anion production was determined by calculating the granular blood cell count during infection in murrel. It shows that the infection influenced the superoxide radical production which plays a major role in killing the pathogens. Overall, this study indicated the defense potentiality of CsmMnSOD; however, further research is necessary to explore its capability at protein level.

  13. Blue light-mediated transcriptional activation and repression of gene expression in bacteria

    PubMed Central

    Jayaraman, Premkumar; Devarajan, Kavya; Chua, Tze Kwang; Zhang, Hanzhong; Gunawan, Erry; Poh, Chueh Loo

    2016-01-01

    Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes. PMID:27353329

  14. Regulation of vitellogenin gene expression in transgenic Caenorhabditis elegans: short sequences required for activation of the vit-2 promoter.

    PubMed Central

    MacMorris, M; Broverman, S; Greenspoon, S; Lea, K; Madej, C; Blumenthal, T; Spieth, J

    1992-01-01

    The Caenorhabditis elegans vitellogenin genes are subject to sex-, stage-, and tissue-specific regulation: they are expressed solely in the adult hermaphrodite intestine. Comparative sequence analysis of the DNA immediately upstream of these genes revealed the presence of two repeated heptameric elements, vit promoter element 1 (VPE1) and VPE2. VPE1 has the consensus sequence TGTCAAT, while VPE2, CTGATAA, shares the recognition sequence of the GATA family of transcription factors. We report here a functional analysis of the VPEs within the 5'-flanking region of the vit-2 gene using stable transgenic lines. The 247 upstream bp containing the VPEs was sufficient for high-level, regulated expression. Furthermore, none of the four deletion mutations or eight point mutations tested resulted in expression of the reporter gene in larvae, males, or inappropriate hermaphrodite tissues. Mutation of the VPE1 closest to the TATA box inactivated the promoter, in spite of the fact that four additional close matches to the VPE1 consensus sequence are present within the 5'-flanking 200 bp. Each of these upstream VPE1-like sequences could be mutated without loss of high-level transgene expression, suggesting that if these VPE1 sequences play a role in regulating vit-2, their effects are more subtle. A site-directed mutation in the overlapping VPE1 and VPE2 at -98 was sufficient to inactivate the promoter, indicating that one or both of these VPEs must be present for activation of vit-2 transcription. Similarly, a small perturbation of the VPE2 at -150 resulted in reduction of fp155 expression, while a more extensive mutation in this element eliminated expression. On the other hand, deletion of this VPE2 and all upstream DNA still permitted correctly regulated expression, although at a very low level, suggesting that this VPE2 performs an important role in activation of vit-2 expression but may not be absolutely required. The results, taken together, demonstrate that both VPE1 and

  15. Developmental Febrile Seizures Modulate Hippocampal Gene Expression of Hyperpolarization-Activated Channels in an Isoform- and Cell-Specific Manner

    PubMed Central

    Brewster, Amy; Bender, Roland A.; Chen, Yuncai; Dube, Celine; Eghbal-Ahmadi, Mariam; Baram, Tallie Z.

    2012-01-01

    Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (IH). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network. PMID:12040066

  16. Developmental febrile seizures modulate hippocampal gene expression of hyperpolarization-activated channels in an isoform- and cell-specific manner.

    PubMed

    Brewster, Amy; Bender, Roland A; Chen, Yuncai; Dube, Celine; Eghbal-Ahmadi, Mariam; Baram, Tallie Z

    2002-06-01

    Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (I(H)). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.

  17. The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

    PubMed

    Hu, Y; Cooper, T G; Kohlhaw, G B

    1995-01-01

    The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

  18. HoxBlinc RNA Recruits Set1/MLL Complexes to Activate Hox Gene Expression Patterns and Mesoderm Lineage Development.

    PubMed

    Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Naohiro; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

    2016-01-05

    Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1(+) mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1(+) precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1(+) precursors and differentiation of Flk1(+) cells into hematopoietic lineages.

  19. The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.

    PubMed

    Ichiyama, Kenji; Chen, Tingting; Wang, Xiaohu; Yan, Xiaowei; Kim, Byung-Seok; Tanaka, Shinya; Ndiaye-Lobry, Delphine; Deng, Yuhua; Zou, Yanli; Zheng, Pan; Tian, Qiang; Aifantis, Iannis; Wei, Lai; Dong, Chen

    2015-04-21

    Epigenetic regulation of lineage-specific genes is important for the differentiation and function of T cells. Ten-eleven translocation (Tet) proteins catalyze 5-methylcytosine (5 mC) conversion to 5-hydroxymethylcytosine (5 hmC) to mediate DNA demethylation. However, the roles of Tet proteins in the immune response are unknown. Here, we characterized the genome-wide distribution of 5 hmC in CD4(+) T cells and found that 5 hmC marks putative regulatory elements in signature genes associated with effector cell differentiation. Moreover, Tet2 protein was recruited to 5 hmC-containing regions, dependent on lineage-specific transcription factors. Deletion of Tet2 in T cells decreased their cytokine expression, associated with reduced p300 recruitment. In vivo, Tet2 plays a critical role in the control of cytokine gene expression in autoimmune disease. Collectively, our findings suggest that Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.

  20. Regulation of gene expression by manipulating transcriptional repressor activity using a novel CoSRI technology.

    PubMed

    Xu, Yue; Li, Song Feng; Parish, Roger W

    2016-12-20

    Targeted gene manipulation is a central strategy for studying gene function and identifying related biological processes. However, a methodology for manipulating the regulatory motifs of transcription factors is lacking as these factors commonly possess multiple motifs (eg. repression and activation motifs) which collaborate with each other to regulate multiple biological processes. We describe a novel approach designated Conserved Sequence-guided Repressor Inhibition (CoSRI) that can specifically reduce or abolish the repressive activities of transcription factors in vivo. The technology was evaluated using the chimeric MYB80-EAR transcription factor and subsequently the endogenous WUS transcription factor. The technology was employed to develop a reversible male sterility system applicable to hybrid seed production. In order to determine the capacity of the technology to regulate the activity of endogenous transcription factors, the WUS repressor was chosen. The WUS repression motif could be inhibited in vivo and the transformed plants exhibited the wus-1 phenotype. Consequently, the technology can be used to manipulate the activities of transcriptional repressor motifs regulating beneficial traits in crop plants and other eukaryotic organisms. This article is protected by copyright. All rights reserved.

  1. Chromatin structure and gene expression changes associated with loss of MOP1 activity in Zea mays.

    PubMed

    Madzima, Thelma F; Huang, Ji; McGinnis, Karen M

    2014-07-01

    Though the mechanisms governing nuclear organization are not well understood, it is apparent that epigenetic modifications coordinately modulate chromatin organization as well as transcription. In maize, MEDIATOR OF PARAMUTATION1 (MOP1) is required for 24 nt siRNA-mediated epigenetic regulation and transcriptional gene silencing via a putative Pol IV- RdDM pathway. To elucidate the mechanisms of nuclear chromatin organization, we investigated the relationship between chromatin structure and transcription in response to loss of MOP1 function. We used a microarray based micrococcal nuclease sensitivity assay to identify genome-wide changes in chromatin structure in mop1-1 immature ears and observed an increase in chromatin accessibility at chromosome arms associated with loss of MOP1 function. Within the many genes misregulated in mop1 mutants, we identified one subset likely to be direct targets of epigenetic transcriptional silencing via Pol-IV RdDM. We found that target specificity for MOP1-mediated RdDM activity is governed by multiple signals that include accumulation of 24 nt siRNAs and the presence of specific classes of gene-proximal transposons, but neither of these attributes alone is sufficient to predict transcriptional misregulation in mop1-1 homozygous mutants. Our results suggest a role for MOP1 in regulation of higher-order chromatin organization where loss of MOP1 activity at a subset of loci triggers a broader cascade of transcriptional consequences and genome-wide changes in chromatin structure.

  2. Constitutive androstane receptor activation by 2,4,6-triphenyldioxane-1,3 suppresses the expression of the gluconeogenic genes.

    PubMed

    Kachaylo, Ekaterina M; Yarushkin, Andrei A; Pustylnyak, Vladimir O

    2012-03-15

    The constitutive androstane receptor (CAR, NR1I3) has a central role in detoxification processes, regulating the expression of a set of genes involved in metabolism. The dual role of NR1I3 as both a xenosensor and as a regulator of endogenous energy metabolism has recently been accepted. Here, we investigated the mechanism of transcriptional regulation of the glucose metabolising genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) by the cis isomer of 2,4,6-triphenyldioxane-1,3 (cisTPD), a highly effective NR1I3 activator in rat liver. It was shown that expression of the gluconeogenic genes PEPCK and G6Pase was repressed by cisTPD treatment under fasting conditions. Western-blot analysis demonstrated a clear reduction in the intensity of PEPCK and G6Pase immunobands from the livers of cisTPD-treated animals relative to bands from the livers of control animals. Chromatin immunoprecipitation assays demonstrated that cisTPD prevents the binding of FOXO1 to the insulin response sequences in the PEPCK and G6Pase gene promoters in rat liver. Moreover, cisTPD-activated NR1I3 inhibited NR2A1 (HNF-4) transactivation by competing with NR2A1 for binding to the NR2A1-binding element (DR1-site) in the gluconeogenic gene promoters. Thus, our results are consistent with the hypothesis that the cisTPD-activated NR1I3 participates in the regulation of the gluconeogenic genes PEPCK and G6Pase.

  3. Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

    PubMed Central

    González-Candelas, L; Aristoy, M C; Polaina, J; Flors, A

    1989-01-01

    DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar. Images PMID:2515802

  4. Methanobactin from Methylocystis sp. strain SB2 affects gene expression and methane monooxygenase activity in Methylosinus trichosporium OB3b.

    PubMed

    Farhan Ul-Haque, Muhammad; Kalidass, Bhagyalakshmi; Vorobev, Alexey; Baral, Bipin S; DiSpirito, Alan A; Semrau, Jeremy D

    2015-04-01

    Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl2, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace.

  5. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    NASA Astrophysics Data System (ADS)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  6. HUMAN PARAOXONASE-1 (PON1): GENE STRUCTURE AND EXPRESSION, PROMISCUOUS ACTIVITIES AND MULTIPLE PHYSIOLOGICAL ROLES

    PubMed Central

    Mackness, Mike; Mackness, Bharti

    2015-01-01

    Human PON1 is a HDL-associated lipolactonase capable of preventing LDL and cell membrane oxidation and is therefore considered to be atheroprotective. PON1 contributes to the antioxidative function of HDL and reductions in HDL-PON1 activity, prevalent in a wide variety of diseases with an inflammatory component, is believed to lead to dysfunctional HDL which can promote inflammation and atherosclerosis. However, PON1 is multifunctional and may contribute to other HDL functions such as in innate immunity, preventing infection by quorum sensing gram negative bacteria by destroying acyl lactone mediators of quorum sensing, and putative new roles in cancer development and the promotion of healthy ageing. In this review we explore the physiological roles of PON1 in disease development, as well as PON1 gene and protein structure, promiscuous activities and the roles of SNPs and ethnicity in determining PON1 activity. PMID:25965560

  7. A Novel TetR-Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression

    PubMed Central

    Schmidt, Sebastian; Berens, Christian; Klotzsche, Marcus

    2014-01-01

    Conditional regulation of gene expression is a powerful and indispensable method for analyzing gene function. The “Tet-On” system is a tool widely used for that purpose. Here, the transregulator rtTA mediates expression of a gene of interest after addition of the small molecule effector doxycycline. Although very effective in rapidly turning on gene expression, the system is hampered by the long half-life of doxycycline which makes shutting down gene expression rapidly very difficult to achieve. We isolated an rtTA-binding peptide by in vivo selection that acts as a doxycycline antagonist and leads to rapid and efficient shut down of rtTA-mediated reporter gene expression in a human cell line. This peptide represents the basis for novel effector molecules which complement the “Tet-system” by enabling the investigator to rapidly turn gene expression not just on at will, but now also off. PMID:24810590

  8. Gene expression profiling of microbial activities and interactions in sediments under haloclines of E. Mediterranean deep hypersaline anoxic basins.

    PubMed

    Edgcomb, Virginia P; Pachiadaki, Maria G; Mara, Paraskevi; Kormas, Konstantinos A; Leadbetter, Edward R; Bernhard, Joan M

    2016-11-01

    Deep-sea hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea are considered some of the most polyextreme habitats on Earth. In comparison to microbial activities occurring within the haloclines and brines of these unusual water column habitats near the Mediterranean seafloor, relatively little is known about microbial metabolic activities in the underlying sediments. In addition, it is not known whether activities are shaped by the unique chemistries of the different DHAB brines and whether evidence exists for active microbial eukaryotes in those sediments. Metatranscriptome analysis was applied to sediment samples collected using ROV Jason from underneath the haloclines of Urania, Discovery and L'Atalante DHABs and a control site. We report on expression of genes associated with sulfur and nitrogen cycling, putative osmolyte biosynthetic pathways and ion transporters, trace metal detoxification, selected eukaryotic activities (particularly of fungi), microbe-microbe interactions, and motility in sediments underlying the haloclines of three DHABs. Relative to our control sediment sample collected outside of Urania Basin, microbial communities (including eukaryotes) in the Urania and Discovery DHAB sediments showed upregulation of expressed genes associated with nitrogen transformations, osmolyte biosynthesis, heavy metals resistance and metabolism, eukaryotic organelle functions, and cell-cell interactions. Sediments underlying DHAB haloclines that have cumulative physico-chemical stressors within the limits of tolerance for microoorganisms can therefore be hotspots of activity in the deep Mediterranean Sea.

  9. Amygdala activation and GABAergic gene expression in hippocampal sub-regions at the interplay of stress and spatial learning

    PubMed Central

    Hadad-Ophir, Osnat; Albrecht, Anne; Stork, Oliver; Richter-Levin, Gal

    2014-01-01

    Molecular processes in GABAergic local circuit neurons critically contribute to information processing in the hippocampus and to stress-induced activation of the amygdala. In the current study, we determined expression changes in GABA-related factors induced in subregions of the dorsal hippocampus as well as in the BLA of rats 5 h after spatial learning in a Morris water maze (MWM), using laser microdissection and quantitative real-time PCR. Spatial learning resulted in highly selective pattern of changes in hippocampal subregions: gene expression levels of neuropeptide Y (NPY) were reduced in the hilus of the dentate gyrus (DG), whereas somatostatin (SST) was increased in the stratum oriens (SO) of CA3. The GABA-synthesizing enzymes GAD65 and GAD67 as well as the neuropeptide cholecystokinin (CCK) were reduced in SO of CA1. In the BLA, expression of GAD65 and GAD67 were reduced compared to a handled Control group. These expression patterns were further compared to alterations in a group of rats that have been exposed to the water maze but were not provided with an invisible escape platform. In this Water Exposure group, no expression changes were observed in any of the hippocampal subregions, but a differential regulation of all selected target genes was evident in the BLA. These findings suggest that expression changes of GABAergic factors in the hippocampus are associated with spatial learning, while additional stress effects modulate expression alterations in the BLA. Indeed, while in both experimental groups plasma corticosterone (CORT) levels were enhanced, only Water Exposure stress activated the basolateral amygdala (BLA), as indicated by increased levels of phosphorylated ERK 1/2. Altered GABAergic function in the BLA may thus contribute to memory consolidation in the hippocampus, in relation to levels of stress and emotionality associated with the experience. PMID:24478650

  10. Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

    PubMed Central

    van Zuylen, Wendy J.; Garceau, Valerie; Idris, Adi; Schroder, Kate; Irvine, Katharine M.; Lattin, Jane E.; Ovchinnikov, Dmitry A.; Perkins, Andrew C.; Cook, Andrew D.; Hamilton, John A.; Hertzog, Paul J.; Stacey, Katryn J.; Kellie, Stuart; Hume, David A.; Sweet, Matthew J.

    2011-01-01

    Background The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. Methodology/Principal Findings Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. Conclusions Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in

  11. Profiling gene expression to distinguish the likely active diazotrophs from a sea of genetic potential in marine sediments

    PubMed Central

    Brown, S M; Jenkins, B D

    2014-01-01

    Nitrogen (N) cycling microbial communities in marine sediments are extremely diverse, and it is unknown whether this diversity reflects extensive functional redundancy. Sedimentary denitrifiers remove significant amounts of N from the coastal ocean and diazotrophs are typically regarded as inconsequential. Recently, N fixation has been shown to be a potentially important source of N in estuarine and continental shelf sediments. Analysis of expressed genes for nitrite reductase (nirS) and a nitrogenase subunit (nifH) was used to identify the likely active denitrifiers and nitrogen fixers in surface sediments from different seasons in Narragansett Bay (Rhode Island, USA). The overall diversity of diazotrophs expressing nifH decreased along the estuarine gradient from the estuarine head to an offshore continental shelf site. Two groups of sequences related to anaerobic sulphur/iron reducers and sulphate reducers dominated libraries of expressed nifH genes. Quantitative polymerase chain reaction (qPCR) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) data shows the highest abundance of both groups at a mid bay site, and the highest nifH expression at the head of the estuary, regardless of season. Several potential environmental factors, including water temperature, oxygen concentration and metal contamination, may influence the abundance and nifH expression of these two bacterial groups. PMID:24447468

  12. Protein arginine methyltransferase 5 (Prmt5) promotes gene expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and its target genes during adipogenesis.

    PubMed

    LeBlanc, Scott E; Konda, Silvana; Wu, Qiong; Hu, Yu-Jie; Oslowski, Christine M; Sif, Saïd; Imbalzano, Anthony N

    2012-04-01

    Regulation of adipose tissue formation by adipogenic-regulatory proteins has long been a topic of interest given the ever-increasing health concerns of obesity and type 2 diabetes in the general population. Differentiation of precursor cells into adipocytes involves a complex network of cofactors that facilitate the functions of transcriptional regulators from the CCATT/enhancer binding protein, and the peroxisome proliferator-activated receptor (PPAR) families. Many of these cofactors are enzymes that modulate the structure of chromatin by altering histone-DNA contacts in an ATP-dependent manner or by posttranslationally modifying the histone proteins. Here we report that inhibition of protein arginine methyltransferase 5 (Prmt5) expression in multiple cell culture models for adipogenesis prevented the activation of adipogenic genes. In contrast, overexpression of Prmt5 enhanced adipogenic gene expression and differentiation. Chromatin immunoprecipitation experiments indicated that Prmt5 binds to and dimethylates histones at adipogenic promoters. Furthermore, the presence of Prmt5 promoted the binding of ATP-dependent chromatin-remodeling enzymes and was required for the binding of PPARγ2 at PPARγ2-regulated promoters. The data indicate that Prmt5 acts as a coactivator for the activation of adipogenic gene expression and promotes adipogenic differentiation.

  13. Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

    PubMed Central

    Bollmann, Franziska; Art, Julia; Henke, Jenny; Schrick, Katharina; Besche, Verena; Bros, Matthias; Li, Huige; Siuda, Daniel; Handler, Norbert; Bauer, Florian; Erker, Thomas; Behnke, Felix; Mönch, Bettina; Härdle, Lorena; Hoffmann, Markus; Chen, Ching-Yi; Förstermann, Ulrich; Dirsch, Verena M.; Werz, Oliver; Kleinert, Hartmut; Pautz, Andrea

    2014-01-01

    Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol. PMID:25352548

  14. Cystatin B-deficient mice have increased expression of apoptosis and glial activation genes

    SciTech Connect

    Lieuallen, Kimberly; Pennacchio, Len A.; Park, Morgan; Myers, Richard M.; Lennon, Gregory G.

    2001-07-05

    Loss-of-function mutations in the cystatin B (Cstb) gene cause a neurological disorder known as Unverricht Lundborg disease (EPM1) in human patients. Mice that lack Cstb provide a mammalian model for EPM1 by displaying progressive ataxia and myoclonic seizures. We analyzed RNAs from brains of Cstb-deficient mice by using modified differential display, oligonucleotide microarray hybridization and quantitative reverse transcriptase polymerase chain reaction to examine the molecular consequences of the lack of Cstb. We identified seven genes that have consistently increased transcript levels in neurological tissues from the knockout mice. These genes are cathepsin S, C1q B-chain of complement (C1qB), beta-2-microglobulin, glial fibrillary acidic protein (Gfap), apolipoprotein D, fibronectin 1 and metallothionein II, which are expected to be involved in increased proteolysis, apoptosis and glial activation. The molecular changes in Cstb-deficient mice are consistent with the pathology found in the mouse model and may provide clues towards the identification of therapeutic points of intervention for EPM1 patients.

  15. Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer.

    PubMed

    Wang, G; Qiu, J; Wang, R; Krause, A; Boyer, J L; Hackett, N R; Crystal, R G

    2010-08-01

    Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody (mAb) directed against an extracellular region of the human epidermal growth-factor receptor type 2 (HER2) protein. We hypothesized that a single adeno-associated virus (AAV)-mediated genetic delivery of an anti-HER2 antibody should be effective in mediating long-term production of anti-HER2 and in suppressing the growth of human tumors in a xenograft model in nude mice. The adeno-associated virus gene transfer vector AAVrh.10alphaHER2 was constructed based on a non-human primate AAV serotype rh.10 to express the complementary DNAs for the heavy and light chains of mAb 4D5, the murine precursor to trastuzumab. The data show that genetically transferred anti-HER2 selectively bound human HER2 protein and suppressed the proliferation of HER2(+) tumor cell lines. A single administration of AAVrh.10alphaHER2 provided long-term therapeutic levels of anti-HER2 antibody expression without inducing an anti-idiotype response, suppressed the growth of HER2(+) tumors and increased the survival of tumor bearing mice. In the context that trastuzumab therapy requires frequent and repeated administration, this strategy might be developed as an alternate platform for delivery of anti-HER2 therapy.

  16. Shifting boundaries of retinoic acid activity control hindbrain segmental gene expression.

    PubMed

    Sirbu, Ioan Ovidiu; Gresh, Lionel; Barra, Jacqueline; Duester, Gregg

    2005-06-01

    Retinoic acid (RA) generated by Raldh2 in paraxial mesoderm is required for specification of the posterior hindbrain, including restriction of Hoxb1 expression to presumptive rhombomere 4 (r4). Hoxb1 expression requires 3' and 5' RA response elements for widespread induction up to r4 and for r3/r5 repression, but RA has previously been detected only from r5-r8, and vHnf1 is required for repression of Hoxb1 posterior to r4 in zebrafish. We demonstrate in mouse embryos that an RA signal initially travels from the paraxial mesoderm to r3, forming a boundary next to the r2 expression domain of Cyp26a1 (which encodes an RA-degrading enzyme). After Hoxb1 induction, the RA boundary quickly shifts to r4/r5, coincident with induction of Cyp26c1 in r4. A functional role for Cyp26c1 in RA degradation was established through examination of RA-treated embryos. Analysis of Raldh2-/- and vHnf1-/- embryos supports a direct role for RA in Hoxb1 induction up to r4 and repression in r3/r5, as well as an indirect role for RA in Hoxb1 repression posterior to r4 via RA induction of vHnf1 up to the r4/r5 boundary. Our findings suggest that Raldh2 and Cyp26 generate shifting boundaries of RA activity, such that r3-r4 receives a short pulse of RA and r5-r8 receives a long pulse of RA. These two pulses of RA activity function to establish expression of Hoxb1 and vHnf1 on opposite sides of the r4/r5 boundary.

  17. Saccharomyces cerevisiae and metabolic activators: HXT3 gene expression and fructose/glucose discrepancy in sluggish fermentation conditions.

    PubMed

    Díaz-Hellín, Patricia; Naranjo, Victoria; Úbeda, Juan; Briones, Ana

    2016-12-01

    When exposed to mixtures of glucose and fructose, as occurs during the fermentation of grape juice into wine, Saccharomyces cerevisiae uses these sugars at different rates. Moreover, glucose and fructose are transported by the same hexose transporters (HXT), which present a greater affinity for glucose, so that late in fermentation, fructose becomes the predominant sugar. Only a few commercial fermentation activators are available to optimally solve the problems this entails. The aim of this study was to investigate the relation between HXT3 gene expression and fructose/glucose discrepancy in two different media inoculated with a commercial wine strain of S. cerevisiae in the presence of three metabolic activators. Fermentation kinetics, vitality and major metabolites were also measured. Rehydration with ergosterol improved the area under the curve and the growth rate (µ max ) in both studied media. Also, the fructose/glucose discrepancy values were improved with all activator treatments, highlighting rehydration in the presence of ascorbic acid. The yeast rehydration process was demonstrated to influence HXT3 expression under the studied conditions. Tetrahydrofolic acid treatment greatly influenced HXT3 gene expression, especially on the 12th day of the fermentation process. To a lesser extent, ergosterol and ascorbic acid also improved this parameter.

  18. Coal-burning endemic fluorosis is associated with reduced activity in antioxidative enzymes and Cu/Zn-SOD gene expression.

    PubMed

    Wang, Qi; Cui, Kang-ping; Xu, Yuan-yuan; Gao, Yan-ling; Zhao, Jing; Li, Da-sheng; Li, Xiao-lei; Huang, Hou-jin

    2014-02-01

    To study the effect of fluorine on the oxidative stress in coal-burning fluorosis, we investigated the environmental characteristics of coal-burning endemic fluorosis combined with fluorine content surveillance in air, water, food, briquette, and clay binder samples from Bijie region, Guizhou Province, southwest of China. The activities of antioxidant enzymes including copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and level of lipid peroxidation such as malondialdehyde (MDA) were measured in serum samples obtained from subjects residing in the Bijie region. Expression of the Cu/Zn-SOD gene was assessed by quantitative reverse transcriptase PCR (qRT-PCR). Our results showed that people suffering from endemic fluorosis (the high and low exposure groups) had much higher MDA level. Their antioxidant enzyme activities and Cu/Zn-SOD gene expression levels were lower when compared to healthy people (the control group). Fluorosis can decrease the activities of antioxidant enzymes, which was associated with exposure level of fluorine. Down-regulation of Cu/Zn-SOD expression may play an important role in the aggravation of oxidative stress in endemic fluorosis.

  19. Histone deacetylase inhibitors decrease Toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment

    PubMed Central

    Bode, Konrad A; Schroder, Kate; Hume, David A; Ravasi, Timothy; Heeg, Klaus; Sweet, Matthew J; Dalpke, Alexander H

    2007-01-01

    Post-translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibited induction of both interleukin-12 protein p40 (IL-12p40) mRNA and protein upon stimulation of Toll-like receptors (TLRs). Moreover, TLR-mediated up-regulation of costimulatory molecules was also inhibited. Up-regulation of tumour necrosis factor-α mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL-1β was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL-12p40 locus. Nevertheless, IL-12p40 chromatin remodelling, binding of Rel-A and IRF1 to the IL-12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor-κB and mitogen-activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment. PMID:17635610

  20. Activity-Dependent Plasticity and Gene Expression Modifications in the Adult CNS

    PubMed Central

    Carulli, Daniela; Foscarin, Simona; Rossi, Ferdinando

    2011-01-01

    Information processing, memory formation, or functional recovery after nervous system damage depend on the ability of neurons to modify their functional properties or their connections. At the cellular/molecular level, structural modifications of neural circuits are finely regulated by intrinsic neuronal properties and growth-regulatory cues in the extracellular milieu. Recently, it has become clear that stimuli coming from the external world, which comprise sensory inflow, motor activity, cognitive elaboration, or social interaction, not only provide the involved neurons with instructive information needed to shape connection patterns to sustain adaptive function, but also exert a powerful influence on intrinsic and extrinsic growth-related mechanisms, so to create permissive conditions for neuritic remodeling. Here, we present an overview of recent findings concerning the effects of experience on molecular mechanisms underlying CNS structural plasticity, both in physiological conditions and after damage, with particular focus on activity-dependent modulation of growth-regulatory genes and epigenetic modifications. PMID:22144945

  1. Effect of temperature and water activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium.

    PubMed

    Gallo, Antonia; Solfrizzo, Michele; Epifani, Filomena; Panzarini, Giuseppe; Perrone, Giancarlo

    2016-01-18

    Almonds are among the commodities at risk of aflatoxin contamination by Aspergillus flavus. Temperature and water activity are the two key determinants in pre and post-harvest environments influencing both the rate of fungal spoilage and aflatoxin production. Varying the combination of these parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it is fundamental to know which combinations can control or be conducive to aflatoxin contamination. Little information is available about the influence of these parameters on aflatoxin production on almonds. The objective of this study was to determine the influence of different combinations of temperature (20 °C, 28 °C, and 37 °C) and water activity (0.90, 0.93, 0.96, 0.99 aw) on growth, aflatoxin B1 (AFB1) production and expression of the two regulatory genes, aflR and aflS, and two structural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on an almond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1 production was obtained at 28 °C and 0.96 aw; no fungal growth and AFB1 production were observed at 20 °C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37 °C AFB1 production was 70-90% lower or completely suppressed, depending on aw. Reverse transcriptase quantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed at maximum (28 °C) and minimum (20 °C and 37 °C) AFB1 production. Conversely the two structural genes (aflD and aflO) were highly expressed only at maximum AFB1 production (28 °C and 0.96-0.99 aw). It seems that temperature acts as a key factor influencing aflatoxin production which is strictly correlated to the induction of expression of structural biosynthesis genes (aflD and aflO), but not to that of aflatoxin regulatory genes (aflR and aflS), whose functional products are most likely subordinated to other regulatory processes acting at post-translational level

  2. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue

    PubMed Central

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio

    2015-01-01

    Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males’ subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole. PMID:26587356

  3. Micronuclei in Cord Blood Lymphocytes and Associations with Biomarkers of Exposure to Carcinogens and Hormonally Active Factors, Gene Polymorphisms, and Gene Expression: The NewGeneris Cohort

    PubMed Central

    Merlo, Domenico Franco; Agramunt, Silvia; Anna, Lívia; Besselink, Harrie; Botsivali, Maria; Brady, Nigel J.; Ceppi, Marcello; Chatzi, Leda; Chen, Bowang; Decordier, Ilse; Farmer, Peter B.; Fleming, Sarah; Fontana, Vincenzo; Försti, Asta; Fthenou, Eleni; Gallo, Fabio; Georgiadis, Panagiotis; Gmuender, Hans; Godschalk, Roger W.; Granum, Berit; Hardie, Laura J.; Hemminki, Kari; Hochstenbach, Kevin; Knudsen, Lisbeth E.; Kogevinas, Manolis; Kovács, Katalin; Kyrtopoulos, Soterios A.; Løvik, Martinus; Nielsen, Jeanette K; Nygaard, Unni Cecilie; Pedersen, Marie; Rydberg, Per; Schoket, Bernadette; Segerbäck, Dan; Singh, Rajinder; Sunyer, Jordi; Törnqvist, Margareta; van Loveren, Henk; van Schooten, Frederik J.; Vande Loock, Kim; von Stedingk, Hans; Wright, John; Kirsch-Volders, Micheline; van Delft, Joost H.M.

    2013-01-01

    Background: Leukemia incidence has increased in recent decades among European children, suggesting that early-life environmental exposures play an important role in disease development. Objectives: We investigated the hypothesis that childhood susceptibility may increase as a result of in utero exposure to carcinogens and hormonally acting factors. Using cord blood samples from the NewGeneris cohort, we examined associations between a range of biomarkers of carcinogen exposure and hormonally acting factors with micronuclei (MN) frequency as a proxy measure of cancer risk. Associations with gene expression and genotype were also explored. Methods: DNA and protein adducts, gene expression profiles, circulating hormonally acting factors, and GWAS (genome-wide association study) data were investigated in relation to genomic damage measured by MN frequency in lymphocytes from 623 newborns enrolled between 2006 and 2010 across Europe. Results: Malondialdehyde DNA adducts (M1dG) were associated with increased MN frequency in binucleated lymphocytes (MNBN), and exposure to androgenic, estrogenic, and dioxin-like compounds was associated with MN frequency in mononucleated lymphocytes (MNMONO), although no monotonic exposure–outcome relationship was observed. Lower frequencies of MNBN were associated with a 1-unit increase expression of PDCD11, LATS2, TRIM13, CD28, SMC1A, IL7R, and NIPBL genes. Gene expression was significantly higher in association with the highest versus lowest category of bulky and M1dG–DNA adducts for five and six genes, respectively. Gene expression levels were significantly lower for 11 genes in association with the highest versus lowest category of plasma AR CALUX® (chemically activated luciferase expression for androgens) (8 genes), ERα CALUX® (for estrogens) (2 genes), and DR CALUX® (for dioxins). Several SNPs (single-nucleotide polymorphisms) on chromosome 11 near FOLH1 significantly modified associations between androgen activity and MNBN

  4. Gene expression and enzyme activities of the sodium pump during sea urchin development: implications for indices of physiological state.

    PubMed

    Marsh, A G; LeongPKK; Manahan, T

    2000-10-01

    The sodium pump consumes a large portion of the metabolic energy (40%) in sea urchin larvae. Understanding the developmental regulation of ion pumps is important for assessing the physiological state of embryos and larvae. We sequenced a partial cDNA clone (1769 bp) from the sea urchin Strongylocentrotus purpuratus and found it to contain the C-terminal portion of an open reading frame coding for 195 amino acids that exhibited high sequence similarity (89%) to invertebrate alpha-subunits of the Na+,K+-ATPase sodium pump. Northern blots using the 3' untranslated region of this cDNA specifically recognized a 4.6-kbp transcript under high stringency. During embryonic development, a rapid increase in levels of this mRNA transcript during gastrulation (25 h postfertilization) was paralleled by a concomitant increase in the total enzymatic activity of Na+,K+-ATPase. Expression of this subunit during gastrulation increased to a maximum at 36 h, followed by a rapid decline to trace levels by 60 h. The rate of removal of the transcript from the total RNA pool after 36 h closely followed a first-order exponential decay model (r2= 0.988), equivalent to a degradation rate of 7.8% h(-1). By 83 h, transcription of the alpha-subunit gene was low, yet sodium pump activity remained high. Molecular assays for the expression of this gene would underestimate sodium pump activities for assessing physiological state because of the temporal separation between maximal gene expression in a gastrula and maximal enzyme activities in the later larval stage. This finding illustrates the difficulty of using molecular probes for assessing the physiological state of invertebrate larvae.

  5. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    SciTech Connect

    Taylor, Cormac T.; Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T.; Ryan, Silke

    2014-05-16

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  6. Visible light exposure induces VEGF gene expression through activation of retinoic acid receptor-alpha in retinoblastoma Y79 cells.

    PubMed

    Akiyama, Hideo; Tanaka, Toru; Doi, Hiroshi; Kanai, Hiroyoshi; Maeno, Toshitaka; Itakura, Hirotaka; Iida, Tomohiro; Kimura, Yasutaka; Kishi, Shoji; Kurabayashi, Masahiko

    2005-04-01

    Neovascularization of the retina and choroids is the pathological hallmark of many retinopathies, but its molecular mechanisms remain unclear. Vascular endothelial growth factor (VEGF), which is induced by hypoxia or cytokines, plays a critical role in the abnormal growth of blood vessels. In this study, we report that visible light exposure induces VEGF gene expression in retinoblastoma Y79 cells. Fluorescent light exposure (700 lux, wavelength 400 approximately 740 nm) caused a significant increase in VEGF transcripts and protein levels. Such an induction seemed to be specific to certain cells, including photoreceptor cells, because light-induced VEGF expression was not observed in either nontransformed cells, such as retinal pigment epithelium cells, and bovine aortic endothelial cells or transformed cells, such as CV-1 and HepG2 cells. Pertussis toxin and guanosine 5'-[beta-thio]diphosphate, specific inhibitors for rhodopsin-associated G protein, blunted this induction. Progressive deletion and site-specific mutation analyses indicate that light stimulation increases VEGF promoter activity through G+C-rich sequence, which is proven by Sp1 binding sites by supershift assays. Electrophoretic mobility shift assays show that light stimulation increases Sp1 binding. Synthetic retinoic acid receptor-alpha (RARalpha) antagonist completely abrogated light-mediated increase in VEGF expression. Transfection of Y79 cells with dominant negative mutant of RARalpha significantly attenuated the light-mediated induction of VEGF promoter activity. In conclusion, our data indicate that light exposure increases VEGF expression through the mechanisms involving activation of Sp1 and RARalpha signaling in Y79 cells. This study provides new insight into the role of visible light in the transcription and induction of VEGF gene expression.

  7. Effects of temperature - heavy metal interactions, antioxidant enzyme activity and gene expression in wheat (Triticum aestivum L.) seedlings.

    PubMed

    Ergün, N; Özçubukçu, S; Kolukirik, M; Temizkan, Ö

    2014-12-01

    In this study, the effect of heat and chromium (Cr) heavy metal interactions on wheat seedlings (Triticum aestivum L. cv. Ç-1252 and Gun91) was investigated by measuring total chlorophyll and carotenoid levels, catalase (CAT) and ascorbate peroxidase (APX) antioxidant enzyme activities, and MYB73, ERF1 and TaSRG gene expression. Examination of pigment levels demonstrated a decrease in total chlorophyll in both species of wheat under combined heat and heavy metal stress, while the carotenoid levels showed a slight increase. APX activity increased in both species in response to heavy metal stress, but the increase in APX activity in the Gun91 seedlings was higher than that in the Ç-1252 seedlings. CAT activity increased in Gun91 seedlings but decreased in Ç-1252 seedlings. These results showed that Gun91 seedling had higher resistance to Cr and Cr + heat stresses than the Ç-1252 seedling. The quantitative molecular analyses implied that the higher resistance was related to the overexpression of TaMYB73, TaERF1 and TaSRG transcription factors. The increase in the expression levels of these transcription factors was profound under combined Cr and heat stress. This study suggests that TaMYB73, TaERF1 and TaSRG transcription factors regulate Cr and heat stress responsive genes in wheat.

  8. Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

    PubMed Central

    Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

    2017-01-01

    Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

  9. Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03.

    PubMed

    Ogino, Hiroyasu; Katou, Yoshikazu; Akagi, Rieko; Mimitsuka, Takashi; Hiroshima, Shinichi; Gemba, Yuichi; Doukyu, Noriyuki; Yasuda, Masahiro; Ishimi, Kosaku; Ishikawa, Haruo

    2007-11-01

    Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.

  10. Fosb gene products contribute to excitotoxic microglial activation by regulating the expression of complement C5a receptors in microglia

    PubMed Central

    Nomaru, Hiroko; Sakumi, Kunihiko; Katogi, Atsuhisa; Ohnishi, Yoshinori N; Kajitani, Kosuke; Tsuchimoto, Daisuke; Nestler, Eric J.; Nakabeppu, Yusaku

    2014-01-01

    The Fosb gene encodes subunits of the activator protein-1 transcription factor complex. Two mature mRNAs, Fosb and ΔFosb, encoding full-length FOSB and ΔFOSB proteins respectively, are formed by alternative splicing of Fosb mRNA. Fosb products are expressed in several brain regions. Moreover, Fosb-null mice exhibit depressive-like behaviors and adult-onset spontaneous epilepsy, demonstrating important roles in neurological and psychiatric disorders. Study of Fosb products has focused almost exclusively on neurons; their function in glial cells remains to be explored. In this study, we found that microglia express equivalent levels of Fosb and ΔFosb mRNAs to hippocampal neurons and, using microarray analysis, we identified six microglial genes whose expression is dependent on Fosb products. Of these genes, we focused on C5ar1 and C5ar2, which encode receptors for complement C5a. In isolated Fosb-null microglia, chemotactic responsiveness toward the truncated form of C5a was significantly lower than that in wild-type cells. Fosb-null mice were significantly resistant to kainate-induced seizures compared with wild-type mice. C5ar1 mRNA levels and C5aR1 immunoreactivity were increased in wild-type hippocampus 24 hours after kainate administration; however, such induction was significantly reduced in Fosb-null hippocampus. Furthermore, microglial activation after kainate administration was significantly diminished in Fosb-null hippocampus, as shown by significant reductions in CD68 immunoreactivity, morphological change and reduced levels of Il6 and Tnf mRNAs, although no change in the number of Iba-1-positive cells was observed. These findings demonstrate that, under excitotoxicity, Fosb products contribute to a neuroinflammatory response in the hippocampus through regulation of microglial C5ar1 and C5ar2 expression. PMID:24771617

  11. Peroxisome Proliferator-Activated Receptor-γGene Expression and Its Association with Oxidative Stress in Patients with Metabolic Syndrome

    PubMed Central

    Hatami, Mehdi; Saidijam, Massoud; Yadegarzari, Reza; Borzuei, Shiva; Soltanian, Alireza; Arian, Marzieh Safi

    2016-01-01

    Regulation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene plays an important role in controlling the metabolism of lipids and inflammatory processes. Therefore, it can be associated with the pathogenesis of metabolic syndrome (MetS). The purpose of this study was to determine the expression of this gene in peripheral blood mononuclear cells (PBMC) in patients with metabolic syndrome. Using real-time polymerase chain reaction (PCR), mRNA expression of PPAR-γ was found in PBMC from 37 subjects with MetS and 30 healthy controls. Serum levels of glucose and lipid profiles were measured. The total antioxidant capacity (TAC) was measured using the ferric reducing ability of plasma (FRAP) test. Malondialdehyde (MDA) was determined using a fluorimetric method. Total oxidant status (TOS) in serum was assayed according to oxidation of ferric to ferrous in the presence of methyl orange. Super oxide dismutase (SOD) activity was measured using a Randox kit. Expression of PPAR-γ gene was significantly increased in patients with MetS compared to the control subjects (p=0.002). There was no difference in serum levels of TAC, MDA and SOD between the two study groups, but a significant difference was observed in the TOS (p=0.03). Serum levels of triglycerides and glucose were significantly higher in subjects with MetS. According to the results of our study, an increase in the expression of PPAR-γ in subjects with MetS indicated a possible role of PPAR-γ in the pathogenesis of this disease. PMID:27689030

  12. Expression of the iron-activated nspA and secY genes in Neisseria meningitidis group B by Fur-dependent and -independent mechanisms.

    PubMed

    Shaik, Yazdani B; Grogan, Susan; Davey, Michael; Sebastian, Shite; Goswami, Sulip; Szmigielski, Borys; Genco, Caroline A

    2007-01-01

    Our whole-genome microarray studies of Neisseria meningitidis MC58 previously identified a set of 153 genes whose transcription was activated during growth in iron. In this study, Fur-mediated regulation of the iron-activated nspA gene was confirmed, whereas iron-activated regulation of the secY gene was demonstrated to be Fur independent. Analysis of the Fur binding sequences in the nspA gene and an additional iron-activated and Fur-regulated gene identified a hexameric (G/T)ATAAT unit in the operator regions of these genes similar to that observed in Fur- and iron-repressed genes. These studies indicate that the expression of the iron-activated nspA and secY genes in N. meningitidis occur by Fur-dependent and -independent mechanisms, respectively.

  13. Expression of the Iron-Activated nspA and secY Genes in Neisseria meningitidis Group B by Fur-Dependent and -Independent Mechanisms▿

    PubMed Central

    Shaik, Yazdani B.; Grogan, Susan; Davey, Michael; Sebastian, Shite; Goswami, Sulip; Szmigielski, Borys; Genco, Caroline A.

    2007-01-01

    Our whole-genome microarray studies of Neisseria meningitidis MC58 previously identified a set of 153 genes whose transcription was activated during growth in iron. In this study, Fur-mediated regulation of the iron-activated nspA gene was confirmed, whereas iron-activated regulation of the secY gene was demonstrated to be Fur independent. Analysis of the Fur binding sequences in the nspA gene and an additional iron-activated and Fur-regulated gene identified a hexameric (G/T)ATAAT unit in the operator regions of these genes similar to that observed in Fur- and iron-repressed genes. These studies indicate that the expression of the iron-activated nspA and secY genes in N. meningitidis occur by Fur-dependent and -independent mechanisms, respectively. PMID:17085550

  14. DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation.

    PubMed

    Liu, Haijing; Ling, Yun; Gong, Yilei; Sun, Ying; Hou, Lin; Zhang, Bo

    2007-06-01

    NAT10 (N-acetyltransferase 10) is a protein with histone acetylation activity and primarily identified to be involved in regulation of telomerase activity. The presented research shows its transcriptional activation by genotoxic agents and possible role in DNA damage. NAT10 mRNA could be markedly increased by using hydrogen peroxide (H2O2) or cisplatin in a dose- and time-dependent way, and the immunofluorescent staining revealed that the treatment of H2O2 or cisplatin induced focal accumulation of NAT10 protein in cellular nuclei. Both H2O2 and cisplatin could stimulate the transcriptional activity of the NAT10 promoter through the upstream sequences from -615 bp to +110 bp, with which some nuclear proteins interacted. Ectopic expression of NAT10 could enhance the number of survival cells in the presence of H2O2 or cisplatin. The above results suggested that NAT10 could be involved in DNA damage response and increased cellular resistance to genotoxicity.

  15. Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

    NASA Astrophysics Data System (ADS)

    Huang, Yong-Zhen; Zhang, Liang-Zhi; Lai, Xin-Sheng; Li, Ming-Xun; Sun, Yu-Jia; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-04-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

  16. Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

    PubMed Central

    Bevec, D; Klier, H; Holter, W; Tschachler, E; Valent, P; Lottspeich, F; Baumruker, T; Hauber, J

    1994-01-01

    The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation. Images PMID:7971969

  17. A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli.

    PubMed

    Wang, Liying; Zhang, Lihong; Liu, Zhanzhi; Liu, Zhangzhi; Zhao, Dehua; Liu, Xiaomeng; Zhang, Bo; Xie, Jianbo; Hong, Yuanyuan; Li, Pengfei; Chen, Sanfeng; Dixon, Ray; Li, Jilun

    2013-01-01

    Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ(70) (σ(A))-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.

  18. An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression

    PubMed Central

    Portnoy, Daniel A.

    2016-01-01

    Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5’ untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. PMID:27414028

  19. MALT1--a universal soldier: multiple strategies to ensure NF-κB activation and target gene expression.

    PubMed

    Afonina, Inna S; Elton, Lynn; Carpentier, Isabelle; Beyaert, Rudi

    2015-09-01

    The paracaspase MALT1 (mucosa associated lymphoid tissue lymphoma translocation gene 1) is an intracellular signaling protein that plays a key role in innate and adaptive immunity. It is essential for nuclear factor κB (NF-κB) activation and proinflammatory gene expression downstream of several cell surface receptors. MALT1 has been most studied in the context of T-cell receptor-induced NF-κB signaling, supporting T-cell activation and proliferation. In addition, MALT1 hyperactivation is associated with specific subtypes of B-cell lymphoma, where it controls tumor cell proliferation and survival. For a long time, MALT1 was believed to function solely as a scaffold protein, providing a platform for the assembly of other NF-κB signaling proteins. However, this view changed dramatically when MALT1 was found to have proteolytic activity that further fine-tunes signaling. MALT1 proteolytic activity is essential for T-cell activation and lymphomagenesis, suggesting that MALT1 is a promising therapeutic target for the treatment of autoimmune diseases and distinct lymphoma entities. However, interference with MALT1 activity may pose a dangerous threat to the normal functioning of the immune system and should be evaluated with great care. Here we discuss the current knowledge on the scaffold and protease functions of MALT1, including an overview of its substrates and the functional implications of their cleavage.

  20. Clobetasol and Halcinonide Act as Smoothened Agonists to Promote Myelin Gene Expression and RxRγ Receptor Activation

    PubMed Central

    De Nardis, Velia; Di Giandomenico, Daniele; Lucisano, Giuseppe; Scardapane, Marco; Poma, Anna; Ragnini-Wilson, Antonella

    2015-01-01

    One of the causes of permanent disability in chronic multiple sclerosis patients is the inability of oligodendrocyte progenitor cells (OPCs) to terminate their maturation program at lesions. To identify key regulators of myelin gene expression acting at the last stages of OPC maturation we developed a drug repositioning strategy based on the mouse immortalized oligodendrocyte (OL) cell line Oli-neu brought to the premyelination stage by stably expressing a key factor regulating the last stages of OL maturation. The Prestwick Chemical Library® of 1,200 FDA-approved compound(s) was repositioned at three dosages based on the induction of Myelin Basic Protein (MBP) expression. Drug hits were further validated using dosage-dependent reproducibility tests and biochemical assays. The glucocorticoid class of compounds was the most highly represented and we found that they can be divided in three groups according to their efficacy on MBP up-regulation. Since target identification is crucial before bringing compounds to the clinic, we searched for common targets of the primary screen hits based on their known chemical-target interactomes, and the pathways predicted by top ranking compounds were validated using specific inhibitors. Two of the top ranking compounds, Halcinonide and Clobetasol, act as Smoothened (Smo) agonists to up-regulate myelin gene expression in the Oli-neuM cell line. Further, RxRγ activation is required for MBP expression upon Halcinonide and Clobetasol treatment. These data indicate Clobetasol and Halcinonide as potential promyelinating drugs and also provide a mechanistic understanding of their mode of action in the pathway leading to myelination in OPCs. Furthermore, our classification of glucocorticoids with respect to MBP expression provides important novel insights into their effects in the CNS and a rational criteria for their choice in combinatorial therapies in de-myelinating diseases. PMID:26658258

  1. Clobetasol and Halcinonide Act as Smoothened Agonists to Promote Myelin Gene Expression and RxRγ Receptor Activation.

    PubMed

    Porcu, Giampiero; Serone, Eliseo; De Nardis, Velia; Di Giandomenico, Daniele; Lucisano, Giuseppe; Scardapane, Marco; Poma, Anna; Ragnini-Wilson, Antonella

    2015-01-01

    One of the causes of permanent disability in chronic multiple sclerosis patients is the inability of oligodendrocyte progenitor cells (OPCs) to terminate their maturation program at lesions. To identify key regulators of myelin gene expression acting at the last stages of OPC maturation we developed a drug repositioning strategy based on the mouse immortalized oligodendrocyte (OL) cell line Oli-neu brought to the premyelination stage by stably expressing a key factor regulating the last stages of OL maturation. The Prestwick Chemical Library of 1,200 FDA-approved compound(s) was repositioned at three dosages based on the induction of Myelin Basic Protein (MBP) expression. Drug hits were further validated using dosage-dependent reproducibility tests and biochemical assays. The glucocorticoid class of compounds was the most highly represented and we found that they can be divided in three groups according to their efficacy on MBP up-regulation. Since target identification is crucial before bringing compounds to the clinic, we searched for common targets of the primary screen hits based on their known chemical-target interactomes, and the pathways predicted by top ranking compounds were validated using specific inhibitors. Two of the top ranking compounds, Halcinonide and Clobetasol, act as Smoothened (Smo) agonists to up-regulate myelin gene expression in the Oli-neuM cell line. Further, RxRγ activation is required for MBP expression upon Halcinonide and Clobetasol treatment. These data indicate Clobetasol and Halcinonide as potential promyelinating drugs and also provide a mechanistic understanding of their mode of action in the pathway leading to myelination in OPCs. Furthermore, our classification of glucocorticoids with respect to MBP expression provides important novel insights into their effects in the CNS and a rational criteria for their choice in combinatorial therapies in de-myelinating diseases.

  2. A WRKY Transcription Factor Recruits the SYG1-Like Protein SHB1 to Activate Gene Expression and Seed Cavity Enlargement

    PubMed Central

    Kang, Xiaojun; Li, Wei; Zhou, Yun; Ni, Min

    2013-01-01

    Seed development in Arabidopsis and in many dicots involves an early proliferation of the endosperm to form a large embryo sac or seed cavity close to the size of the mature seed, followed by a second phase during which the embryo grows and replaces the endosperm. SHORT HYPOCOTYL UNDER BLUE1 (SHB1) is a member of the SYG1 protein family in fungi, Caenorhabditis elegans, flies, and mammals. SHB1 gain-of-function enhances endosperm proliferation, increases seed size, and up-regulates the expression of the WRKY transcription factor gene MINISEED3 (MINI3) and the LRR receptor kinase gene HAIKU2 (IKU2). Mutations in either IKU2 or MINI3 retard endosperm proliferation and reduce seed size. However, the molecular mechanisms underlying the establishment of the seed cavity and hence the seed size remain largely unknown. Here, we show that the expression of MINI3 and IKU2 is repressed before fertilization and after 4 days after pollination (DAP), but is activated by SHB1 from 2 to 4 DAP prior to the formation of the seed cavity. SHB1 associates with their promoters but without a recognizable DNA binding motif, and this association is abolished in mini3 mutant. MINI3 binds to W-boxes in, and recruits SHB1 to, its own and IKU2 promoters. Interestingly, SHB1, but not MINI3, activates transcription of pMINI3::GUS or pIKU2::GUS. We reveal a critical developmental switch through the activation of MINI3 expression by SHB1. The recruitment of SHB1 by MINI3 to its own and IKU2 promoters represents a novel two-step amplification to counter the low expression level of IKU2, which is a trigger for endosperm proliferation and seed cavity enlargement. PMID:23505389

  3. Signal transducer and activator of transcription (STAT)-3 regulates microRNA gene expression in chronic lymphocytic leukemia cells

    PubMed Central

    2013-01-01

    Backgrounds Approximately 1,000 microRNAs (miRs) are present in the human genome; however, little is known about the regulation of miR transcription. Because miR levels are deregulated in chronic lymphocytic leukemia (CLL) and signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL, we sought to determine whether STAT3 affects the transcription of miR genes in CLL cells. Methods We used publically available data from the ENCODE project to identify putative STAT3 binding sites in the promoters of miR genes. Then we transfected CLL cells with STAT3-shRNA or with an empty vector, and to determine which miRs are differentially expressed, we used a miR microarray approach followed by validation of the microarray results for 6 miRs using quantitative real-time polymerase chain reaction (qRT-PCR). Results We identified putative STAT3 binding sites in 160 promoter regions of 200 miRs, including miR-21, miR-29, and miR-155, whose levels have been reported to be upregulated in CLL. Levels of 72 miRs were downregulated (n = 63) or upregulated (n = 9). qRT-PCR confirmed the array data in 5 of 6 miRs. Conclusions The presence of activated STAT3 has a profound effect on miR expression in CLL cells. PMID:23725032

  4. Phytol directly activates peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and regulates gene expression involved in lipid metabolism in PPAR{alpha}-expressing HepG2 hepatocytes

    SciTech Connect

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo . E-mail: fat@kais.kyoto-u.ac.jp

    2005-11-18

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

  5. Does FACS perturb gene expression?

    PubMed

    Richardson, Graham M; Lannigan, Joanne; Macara, Ian G

    2015-02-01

    Fluorescence activated cell sorting is the technique most commonly used to separate primary mammary epithelial sub-populations. Many studies incorporate this technique before analyzing gene expression within specific cellular lineages. However, to our knowledge, no one has examined the effects of fluorescence activated cell sorting (FACS) separation on short-term transcriptional profiles. In this study, we isolated a heterogeneous mixture of cells from the mouse mammary gland. To determine the effects of the isolation and separation process on gene expression, we harvested RNA from the cells before enzymatic digestion, following enzymatic digestion, and following a mock FACS sort where the entire cohort of cells was retained. A strict protocol was followed to minimize disruption to the cells, and to ensure that no subpopulations were enriched or lost. Microarray analysis demonstrated that FACS causes minimal disruptions to gene expression patterns, but prior steps in the mammary cell isolation process are followed by upregulation of 18 miRNA's and rapid decreases in their predicted target transcripts. © 2015 International Society for Advancement of Cytometry.

  6. Quantitative perturbation-based analysis of gene expression predicts enhancer activity in early Drosophila embryo

    PubMed Central

    Sayal, Rupinder; Dresch, Jacqueline M; Pushel, Irina; Taylor, Benjamin R; Arnosti, David N

    2016-01-01

    Enhancers constitute one of the major components of regulatory machinery of metazoans. Although several genome-wide studies have focused on finding and locating enhancers in the genomes, the fundamental principles governing their internal architecture and cis-regulatory grammar remain elusive. Here, we describe an extensive, quantitative perturbation analysis targeting the dorsal-ventral patterning gene regulatory network (GRN) controlled by Drosophila NF-κB homolog Dorsal. To understand transcription factor interactions on enhancers, we employed an ensemble of mathematical models, testing effects of cooperativity, repression, and factor potency. Models trained on the dataset correctly predict activity of evolutionarily divergent regulatory regions, providing insights into spatial relationships between repressor and activator binding sites. Importantly, the collective predictions of sets of models were effective at novel enhancer identification and characterization. Our study demonstrates how experimental dataset and modeling can be effectively combined to provide quantitative insights into cis-regulatory information on a genome-wide scale. DOI: http://dx.doi.org/10.7554/eLife.08445.001 PMID:27152947

  7. [Neuronal plasticity and gene expression].

    PubMed

    Sokolova, O O; Shtark, M B; Lisachev, P D

    2010-01-01

    Neuronal plasticity--a fundamental feature of brain--provides adequate interactions with dynamic environment. One of the most deeply investigated forms of the neuronal plasticity is a long-term potentiation (LTP)--a phenomenon underlying learning and memory. Signal paths activated during LTP converge into the nuclear of the neuron, giving rise to launch of the molecular-genetic programs, which mediate structural and functional remodeling of synapses. In the review data concerning involvement of multilevel gene expression into plastic change under neuronal activation are summarized.

  8. Cytokinins act synergistically with salicylic acid to activate defense gene expression in rice.

    PubMed

    Jiang, Chang-Jie; Shimono, Masaki; Sugano, Shoji; Kojima, Mikiko; Liu, Xinqiong; Inoue, Haruhiko; Sakakibara, Hitoshi; Takatsuji, Hiroshi

    2013-03-01

    Hormone crosstalk is pivotal in plant-pathogen interactions. Here, we report on the accumulation of cytokinins (CK) in rice seedlings after infection of blast fungus Magnaporthe oryzae and its potential significance in rice-M. oryzae interaction. Blast infection to rice seedlings increased levels of N(6)-(Δ(2)-isopentenyl) adenine (iP), iP riboside (iPR), and iPR 5'-phosphates (iPRP) in leaf blades. Consistent with this, CK signaling was activated around the infection sites, as shown by histochemical staining for β-glucuronidase activity driven by a CK-responsive OsRR6 promoter. Diverse CK species were also detected in the hyphae (mycelium), conidia, and culture filtrates of blast fungus, indicating that M. oryzae is capable of production as well as hyphal secretion of CK. Co-treatment of leaf blades with CK and salicylic acid (SA), but not with either one alone, markedly induced pathogenesis-related genes OsPR1b and probenazole-induced protein 1 (PBZ1). These effects were diminished by RNAi-knockdown of OsNPR1 or WRKY45, the key regulators of the SA signaling pathway in rice, indicating that the effects of CK depend on these two regulators. Taken together, our data imply a coevolutionary rice-M. oryzae interaction, wherein M. oryzae probably elevates rice CK levels for its own benefits such as nutrient translocation. Rice plants, on the other hand, sense it as an infection signal and activate defense reactions through the synergistic action with SA.

  9. Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells

    PubMed Central

    1990-01-01

    In canine thyrocytes in primary culture, our previous studies have identified three mitogenic agents and pathways: thyrotropin (TSH) acting through cyclic AMP (cAMP), EGF and its receptor tyrosine protein kinase, and the phorbol esters that stimulate protein kinase C. TSH enhances, while EGF and phorbol esters inhibit, the expression of differentiation. Given that growth and differentiation expression are often considered as mutually exclusive activities of the cells, it was conceivable that the differentiating action of TSH was restricted to noncycling (Go) cells, while the inhibition of the differentiation expression by EGF and phorbol esters only concerned proliferating cells. Therefore, the capacity to express the thyroglobulin (Tg) gene, the most prominent marker of differentiation in thyrocytes, was studied in proliferative cells (with insulin) and in quiescent cells (without insulin). Using cRNA in situ hybridization, we observed that TSH (and, to a lesser extent, insulin and insulin-like growth factor I) restored or maintained the expression of the Tg gene. Without these hormones, the Tg mRNA content became undetectable in most of the cells. EGF and 12-0-tetradecanoyl phorbol-13-acetate (TPA) inhibited the Tg mRNA accumulation induced by TSH (and/or insulin). Most of the cells (up to 90%) responded to both TSH and EGF. Nevertheless, the range of individual response was quite variable. The effects of TSH and EGF on differentiation expression were not dependent on insulin and can therefore be dissociated from their mitogenic effects. Cell cycling did not affect the induction of Tg gene. Indeed, the same cell distribution of Tg mRNA content was observed in quiescent cells stimulated by TSH alone, or in cells approximately 50% of which had performed one mitotic cycle in response to TSH + insulin. Moreover, after proliferation in "dedifferentiating" conditions (EGF + serum + insulin), thyrocytes had acquired a fusiform fibroblast-like morphology, and responded

  10. 4-Hydroxynonenal differentially regulates adiponectin gene expression and secretion via activating PPARγ and accelerating ubiquitin–proteasome degradation

    PubMed Central

    Wanga, Zhigang; Dou, Xiaobing; Gu, Dongfang; Shen, Chen; Yao, Tong; Nguyen, Van; Braunschweig, Carol; Song, Zhenyuan

    2011-01-01

    Although well-established, the underlying mechanisms involved in obesity-related plasma adiponectin decline remain elusive. Oxidative stress is associated with obesity and insulin resistance and considered to contribute to the progression toward obesity-related metabolic disorders. In this study, we investigated the effects of 4-hydroxynonenal (4-HNE), the most abundant lipid peroxidation end product, on adiponectin production and its potential implication in obesity-related adiponectin decrease. Long-term high-fat diet feeding led to obesity in mouse, accompanied by decreased plasma adiponectin and increased adipose tissue 4-HNE content. Exposure of adipocytes to exogenous 4-HNE resulted in decreased adiponectin secretion in a dose-dependent manner, which was consistent with significantly decreased intracellular adiponectin protein abundance. In contrast, adiponectin gene expression was significantly elevated by 4-HNE treatment, which was concomitant with increased peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression and transactivity. The effect was abolished by T0070907, a PPAR-γ antagonist, suggesting that PPAR-γ activation plays a critical role in this process. To gain insight into mechanisms involved in adiponectin protein decrease, we examined the effects of 4-HNE on adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that 4-HNE exposure accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that 4-HNE exposure increased ubiquitinated adiponectin protein levels. These data altogether indicated that 4-HNE enhanced adiponectin protein degradation via ubiquitin–proteasome system. Finally, we demonstrated that supplementation of HF diet with betaine, an antioxidant and methyl donor, alleviated high-fat-induced adipose tissue 4-HNE increase and attenuated plasma adiponectin decline. Taken together, our findings suggest that the lipid

  11. Insulin continues to induce plasminogen activator inhibitor 1 gene expression in insulin-resistant mice and adipocytes.

    PubMed Central

    Samad, F.; Pandey, M.; Bell, P. A.; Loskutoff, D. J.

    2000-01-01

    BACKGROUND: Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. MATERIALS AND METHODS: We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. CONCLUSIONS: These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease. PMID:11055587

  12. High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain

    PubMed Central

    Bravim, Fernanda; Lippman, Soyeon I.; da Silva, Lucas F.; Souza, Diego T.; Fernandes, A. Alberto R.; Masuda, Claudio A.; Broach, James R.

    2016-01-01

    High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries. PMID:22915193

  13. Methylphenidate regulates activity regulated cytoskeletal associated but not brain-derived neurotrophic factor gene expression in the developing rat striatum.

    PubMed

    Chase, T; Carrey, N; Soo, E; Wilkinson, M

    2007-02-09

    Methylphenidate (MPH) is a psychostimulant drug used to treat attention deficit hyperactivity disorder in children. To explore the central effects of chronic MPH, we investigated the expression of an effector immediate early gene, activity regulated cytoskeletal associated (arc), and the neurotrophin, brain-derived neurotrophic factor (bdnf) in the brain of immature and adult rats following repeated MPH. Prepubertal (postnatal day (PD) 25-38) and adult (PD 53-66) male rats were injected once daily for: a) 14 days with saline or MPH (2 or 10 mg/kg; s.c.) or b) 13 days with saline followed by a single dose of MPH (2 or 10 mg/kg; s.c.). To determine possible long-term effects of MPH, prepubertal rats were allowed a drug-free period of 4 weeks following the 14 days of treatment, and then were given a challenge dose of MPH. We demonstrated, for the first time, that an acute injection of MPH increased levels of activity-regulated cytoskeletal protein (ARC) and arc mRNA in the prepubertal rat striatum and cingulate/frontal cortex. This response was significantly attenuated by chronic MPH. The desensitization in arc expression observed in prepubertal rats persisted in the adult striatum following a later MPH challenge. In contrast to these data we observed little effect of MPH on bdnf expression. We also developed an effective, non-stressful technique to treat freely moving immature rats with oral MPH. Consistent with the results described above, we observed that oral MPH (7.5 and 10 mg/kg) also increased arc expression in the prepubertal rat striatum. However, unlike the effects of injected MPH, repeated oral MPH (7.5 mg/kg) did not alter the normal arc response. This result raises the important possibility that oral doses of MPH that reproduce clinically relevant blood levels of MPH may not down-regulate gene expression, at least in the short term (14 days). We confirmed, using mass spectrometry, that the oral doses of MPH used in our experiments yielded blood levels

  14. Modular Utilization of Distal cis-Regulatory Elements Controls Ifng Gene Expression in T Cells Activated by Distinct Stimuli

    PubMed Central

    Balasubramani, Anand; Shibata, Yoichiro; Crawford, Gregory E.; Baldwin, Albert S.; Hatton, Robin D.; Weaver, Casey T.

    2010-01-01

    SUMMARY Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three transacting factors – NF-κB, STAT4 and T-bet – with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and co-recruitment of STAT4. STAT4 and NF-κB therefore cooperate at multiple cis elements to enable NF-κB–dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in Th1 and Tc1 effector cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals. PMID:20643337

  15. Combining modelling and experimental approaches to explain how calcium signatures are decoded by calmodulin-binding transcription activators (CAMTAs) to produce specific gene expression responses.

    PubMed

    Liu, Junli; Whalley, Helen J; Knight, Marc R

    2015-10-01

    Experimental data show that Arabidopsis thaliana is able to decode different calcium signatures to produce specific gene expression responses. It is also known that calmodulin-binding transcription activators (CAMTAs) have calmodulin (CaM)-binding domains. Therefore, the gene expression responses regulated by CAMTAs respond to calcium signals. However, little is known about how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses. A dynamic model of Ca(2+) -CaM-CAMTA binding and gene expression responses is developed following thermodynamic and kinetic principles. The model is parameterized using experimental data. Then it is used to analyse how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses. Modelling analysis reveals that: calcium signals in the form of cytosolic calcium concentration elevations are nonlinearly amplified by binding of Ca(2+) , CaM and CAMTAs; amplification of Ca(2+) signals enables calcium signatures to be decoded to give specific CAMTA-regulated gene expression responses; gene expression responses to a calcium signature depend upon its history and accumulate all the information during the lifetime of the calcium signature. Information flow from calcium signatures to CAMTA-regulated gene expression responses has been established by combining experimental data with mathematical modelling.

  16. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue.

    PubMed

    Rubio, F Javier; Li, Xuan; Liu, Qing-Rong; Cimbro, Raffaello; Hope, Bruce T

    2016-08-27

    The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 ºC for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

  17. Jasmonic Acid Modulates the Physio-Biochemical Attributes, Antioxidant Enzyme Activity, and Gene Expression in Glycine max under Nickel Toxicity

    PubMed Central

    Sirhindi, Geetika; Mir, Mudaser Ahmad; Abd-Allah, Elsayed Fathi; Ahmad, Parvaiz; Gucel, Salih

    2016-01-01

    In present study, we evaluated the effects of Jasmonic acid (JA) on physio-biochemical attributes, antioxidant enzyme activity, and gene expression in soybean (Glycine max L.) plants subjected to nickel (Ni) stress. Ni stress decreases the shoot and root length and chlorophyll content by 37.23, 38.31, and 39.21%, respectively, over the control. However, application of JA was found to improve the chlorophyll content and length of shoot and root of Ni-fed seedlings. Plants supplemented with JA restores the chlorophyll fluorescence, which was disturbed by Ni stress. The present study demonstrated increase in proline, glycinebetaine, total protein, and total soluble sugar (TSS) by 33.09, 51.26, 22.58, and 49.15%, respectively, under Ni toxicity over the control. Addition of JA to Ni stressed plants further enhanced the above parameters. Ni stress increases hydrogen peroxide (H2O2) by 68.49%, lipid peroxidation (MDA) by 50.57% and NADPH oxidase by 50.92% over the control. Supplementation of JA minimizes the accumulation of H2O2, MDA, and NADPH oxidase, which helps in stabilization of biomolecules. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) increases by 40.04, 28.22, 48.53, and 56.79%, respectively, over the control in Ni treated seedlings and further enhancement in the antioxidant activity was observed by the application of JA. Ni treated soybean seedlings showed increase in expression of Fe-SOD by 77.62, CAT by 15.25, POD by 58.33, and APX by 80.58% over the control. Nevertheless, application of JA further enhanced the expression of the above genes in the present study. Our results signified that Ni stress caused negative impacts on soybean seedlings, but, co-application of JA facilitate the seedlings to combat the detrimental effects of Ni through enhanced osmolytes, activity of antioxidant enzymes and gene expression. PMID:27242811

  18. Expression of Human NSAID Activated Gene 1 in Mice Leads to Altered Mammary Gland Differentiation and Impaired Lactation

    PubMed Central

    Binder, April K.; Kosak, Justin P.; Janhardhan, Kyathanahalli S.; Moser, Glenda; Eling, Thomas E.; Korach, Kenneth S.

    2016-01-01

    Transgenic mice expressing human non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) have less adipose tissue, improved insulin sensitivity, lower insulin levels and are resistant to dietary induced obesity. The hNAG-1 expressing mice are more metabolically active with a higher energy expenditure. This study investigates female reproduction in the hNAG-1 transgenic mice and finds the female mice are fertile but have reduced pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 expressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells on lactation day 2. Pups nursing from hNAG-1 expressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25 ng/mL) from hNAG-1 dams. The WT dams had no detectable hNAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development. To examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Microarray analysis revealed that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. Furthermore, the expression of Cidea/CIDEA that has been shown to regulate milk lipid secretion in the mammary gland was reduced in hNAG-1 mammary glands. This study suggests that expression of hNAG-1 in mice leads to impaired lactation and reduces pup survival due to

  19. In Vivo Biochemical and Gene Expression Analyses of the Antioxidant Activities and Hypocholesterolaemic Properties of Tamarindus indica Fruit Pulp Extract

    PubMed Central

    Lim, Chor Yin; Mat Junit, Sarni; Abdulla, Mahmood Ameen; Abdul Aziz, Azlina

    2013-01-01

    Background Tamarindus indica (T. indica) is a medicinal plant with many biological activities including anti-diabetic, hypolipidaemic and anti-bacterial activities. A recent study demonstrated the hypolipidaemic effect of T. indica fruit pulp in hamsters. However, the biochemical and molecular mechanisms responsible for these effects have not been fully elucidated. Hence, the aims of this study were to evaluate the antioxidant activities and potential hypocholesterolaemic properties of T. indica, using in vitro and in vivo approaches. Methodology/Principal Findings The in vitro study demonstrated that T. indica fruit pulp had significant amount of phenolic (244.9±10.1 mg GAE/extract) and flavonoid (93.9±2.6 mg RE/g extract) content and possessed antioxidant activities. In the in vivo study, hamsters fed with high-cholesterol diet for ten weeks showed elevated serum triglyceride, total cholesterol, HDL-C and LDL-C levels. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters significantly lowered serum triglyceride, total cholesterol and LDL-C levels but had no effect on the HDL-C level. The lipid-lowering effect was accompanied with significant increase in the expression of Apo A1, Abcg5 and LDL receptor genes and significant decrease in the expression of HMG-CoA reductase and Mtp genes. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters also protected against oxidative damage by increasing hepatic antioxidant enzymes, antioxidant activities and preventing hepatic lipid peroxidation. Conclusion/Significance It is postulated that tamarind fruit pulp exerts its hypocholesterolaemic effect by increasing cholesterol efflux, enhancing LDL-C uptake and clearance, suppressing triglyceride accumulation and inhibiting cholesterol biosynthesis. T. indica fruit pulp has potential antioxidative effects and is potentially protective against diet-induced hypercholesterolaemia. PMID:23894592

  20. Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down.

    PubMed

    Hogg, K; Robinson, W P; Beristain, A G

    2014-07-01

    Increasingly, placental DNA methylation is assessed as a factor in pregnancy-related complications, yet the transcriptional impact of such findings is not always clear. Using a proliferative in vitro placental model, the effect of DNA methylation loss on gene activation was evaluated at a number of genes selected for being differentially methylated in pre-eclampsia-associated placentae in vivo. We aimed to determine whether reduced DNA methylation at specific loci was associated with transcriptional changes at the corresponding gene, thus providing mechanistic underpinnings for previous clinical findings and to assess the degree of transcriptional response amongst our candidate genes. BeWo and JEG3 choriocarcinoma cells were exposed to 1 μM 5-Aza-2'-deoxycytidine (5-Aza-CdR) or vehicle control for 48 h, and re-plated and cultured for a further 72 h in normal media before cells were harvested for RNA and DNA. Bisulphite pyrosequencing confirmed that DNA methylation was reduced by ∼30-50% points at the selected loci studied in both cell lines. Gene activation, measured by qRT-PCR, was highly variable and transcript specific, indicating differential sensitivity to DNA methylation. Most notably, loss of DNA methylation at the leptin (LEP) promoter corresponded to a 200-fold and 40-fold increase in LEP expression in BeWo and JEG3 cells, respectively (P < 0.01). Transcripts of steroidogenic pathway enzymes CYP11A1 and HSD3B1 were up-regulated ∼40-fold in response to 5-Aza-CdR exposure in BeWo cells (P < 0.01). Other transcripts, including aromatase (CYP19), HSD11B2, inhibin (INHBA) and glucocorticoid receptor (NR3C1) were more moderately, although significantly, affected by loss of associated DNA methylation. These data present a mixed effect of DNA methylation changes at selected loci supporting cautionary interpretation of DNA methylation results in the absence of functional data.

  1. Glutathione S-transferase (GST) family in barley: identification of members, enzyme activity, and gene expression pattern.

    PubMed

    Rezaei, Mohammad Kazem; Shobbar, Zahra-Sadat; Shahbazi, Maryam; Abedini, Raha; Zare, Sajjad

    2013-09-15

    Barley (Hordeum vulgare) is one of the most important cereals in many developing countries where drought stress considerably diminishes agricultural production. Glutathione S-transferases (GSTs EC 2.5.1.18) are multifunctional enzymes which play a crucial role in cellular detoxification and oxidative stress tolerance. In this study, 84 GST genes were identified in barley by a comprehensive in silico approach. Sequence alignment and phylogenetic analysis grouped these HvGST proteins in eight classes. The largest numbers of the HvGST genes (50) were included in the Tau class followed by 21 genes in Phi, five in Zeta, two in DHAR, two in EF1G, two in Lambda, and one each in TCHQD and Theta classes. Phylogenetic analysis of the putative GSTs from Arabidopsis, rice, and barley indicated that major functional diversification within the GST family predated the monocot/dicot divergence. However, intra-specious duplication seems to be common. Expression patterns of five GST genes from Phi and Tau classes were investigated in three barley genotypes (Yusof [drought-tolerant], Moroc9-75 [drought-sensitive], and HS1 [wild ecotype]) under control and drought-stressed conditions, during the vegetative stage. All investigated genes were up-regulated significantly under drought stress and/or showed a higher level of transcripts in the tolerant cultivar. Additionally, GST enzyme activity was superior in Yusof and induced in the extreme-drought-treated leaves, while it was not changed in Moroc9-75 under drought conditions. Moreover, the lowest and highest levels of lipid peroxidation were observed in the Yusof and Moroc9-75 cultivars, respectively. Based on the achieved results, detoxification and antioxidant activity of GSTs might be considered an important factor in the drought tolerance of barley genotypes for further investigations.

  2. The dose dependence of glucocorticoid-inducible gene expression results from changes in the number of transcriptionally active templates.

    PubMed Central

    Ko, M S; Nakauchi, H; Takahashi, N

    1990-01-01

    Glucocorticoid hormones induce the transcription of genes having glucocorticoid response elements in a dose dependent manner. To determine whether this dose dependence represents a response of individual templates or of the mass of templates, we introduced a bacterial beta-galactosidase gene linked to the glucocorticoid-inducible enhancer/promoter of the mouse mammary tumor virus (MTV) into Ltk- cells and obtained stable transformants containing a single or a few templates per cell. Visual inspection and flow cytometry analysis by enzyme histochemistry assay for beta-galactosidase revealed that individual cells showed very heterogeneous beta-galactosidase activity after 48 h induction with dexamethasone. When the glucocorticoid concentration was increased, an increasing cell population producing beta-galactosidase was observed. These phenomena were probably not due to heterogeneity of template copy number or to a predetermined cellular state among individual cells, since cells forming a single small colony gave similar results. This was also supported by data showing that recloned cells retained both their responsiveness to the glucocorticoid hormone and their digestion pattern in Southern blotting analyses. These results indicate that the dose dependent increase of glucocorticoid-inducible gene expression is caused by an increase in the number of transcriptionally active templates. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 8. PMID:2167833

  3. Constitutive gene expression in monocytes from chronic HIV-1 infection overlaps with acute Toll-like receptor induced monocyte activation profiles.

    PubMed

    Gekonge, Bethsebah; Giri, Malavika S; Kossenkov, Andrew V; Nebozyhn, Michael; Yousef, Malik; Mounzer, Karam; Showe, Louise; Montaner, Luis J

    2012-01-01

    Elevated TLR expression/signalling in monocyte/macrophages has been shown to mediate systemic immune activation, a hallmark of progressive HIV-1 infection. Here we show, via differential gene expression comparisons, the presence of a constitutive in vivo TLR-like gene activation signature in steady-state circulating monocytes from chronically HIV-1 infected subjects. The TLR2-like gene signature was defined as an 82 gene subset of the 376 genes constitutively modulated in in vivo HIV-1 monocytes, based on their overlap with de novo TLR2-induced genes in uninfected subjects' monocytes following acute ex vivo stimulation with Staphylococcus Aureus Cowan (SAC). Additional comparison of in vivo gene networks with available datasets from acute TLR activations in M/M expanded the overlap to 151-gene concordance among the 376 differential genes with emphasis on ERK/MAPK, TNF/IL6 (NFκB) and p53 gene networks. TLR2 stimulation of monocytes from HIV-1 infected subjects resulted in further upregulation of inflammatory genes indicative of a sustained transcriptional potential upon stimulation. In summary, our data support the presence of a sustained TLR-like gene activation profile in circulating monocyte from steady-state viremia in HIV-1 infected subjects.

  4. Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields

    PubMed Central

    Moffett, John; Fray, Linley M; Kubat, Nicole J

    2012-01-01

    Background Pulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways. Methods and Results Using cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types. Conclusion These findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting. PMID:23055776

  5. Physicochemical properties of starches and expression and activity of starch biosynthesis-related genes in sweet potatoes.

    PubMed

    Lai, Yung C; Wang, Shu Y; Gao, Huan Y; Nguyen, Khiem M; Nguyen, Chinh H; Shih, Ming C; Lin, Kuan H

    2016-05-15

    The functional properties of starches from six sweet potato varieties containing various starch components and structures were studied in an attempt to identify starch sources for industrial uses. Tainan 18 (TNN18) with high-amylose (AM) starch exhibited high setback and breakdown viscosities, high water solubility at 85°C but low swelling volume at 65°C, and high hardness and adhesiveness; in contrast, the low-AM starch of Tainung 31 (TNG31) had opposite characteristics. Seven genes related to starch biosynthesis were tested, and GBSS, SS, SBEII, ISA, and AGPase were highly expressed in TNN18 and TNG31; however, transcript levels in DBE and SBE were extremely low. GBSS and SS activity reflected the abundance of GBSS and SS mRNA in TNG31 and TNN18, and expression of AGPase, GBSS, SS, and SBE in TNN18 substantially increased content of AM. The expression and activity of DBE had a significant effect on TNG31 with increased AP content.

  6. Expression of plasminogen activator-related genes in the adipose tissue of lactating dairy sheep in the early post-weaning period.

    PubMed

    Theodorou, G; Lampidonis, A D; Laliotis, G P; Bizelis, I; Politis, I

    2012-06-01

    There is growing evidence that plasminogen activator inhibitor type 1 (PAI-1) is expressed in adipose tissue and its expression is implicated in inflammation that accompanies obesity-associated diseases. The physiological role of other genes implicated in the plasminogen-activating cascade such as urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitor type 2 (PAI-2) in ovine adipose tissue remains unknown. The objective of this study was to examine the changes in the expression of four plasminogen activator (PA)-related genes during the early post-weaning period in dairy ewes. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating dairy ewes of the Chios breed at weeks 1, 2 and 4 after weaning. Results indicated that expression of all PA-related genes was detected in most of the samples examined. Greatest expression of u-PAR corresponded to highest (week 1), while greatest expression of PAI-2 corresponded to lowest (week 4) rate of lipolysis, as indicated by the expression of hormone-sensitive lipase, in the ovine adipose tissue. There were no significant differences in the expression of the other two PA-related genes (u-PA, PAI-1) throughout the experimental period. Plasminogen activator-related genes are not expressed in a coordinated manner in the adipose tissue of lactating dairy sheep in the early post-weaning period. In conclusion, adipose tissue mobilization is correlated with highest expression of u-PAR and lowest expression of PAI-2.

  7. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    PubMed

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  8. Characterization of the third SERK gene in pineapple (Ananas comosus) and analysis of its expression and autophosphorylation activity in vitro.

    PubMed

    Ma, Jun; He, Yehua; Hu, Zhongyi; Xu, Wentian; Xia, Jingxian; Guo, Cuihong; Lin, Shunquan; Chen, Chengjie; Wu, Chenghou; Zhang, Junli

    2014-09-01

    Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20-80 fold more than in anther but similar to that of non-embryogenic callus, which was 20-50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity.

  9. Gold nanoprobe-based method for sensing activated leukocyte cell adhesion molecule (ALCAM) gene expression, as a breast cancer biomarker.

    PubMed

    Eskandari, Leila; Akbarzadeh, Abolfazl; Zarghami, Nosratollah; Rahmati-Yamchi, Mohammad

    2017-03-01

    In breast cancer, a proper biomarker for the assessment of metastasis and poor prognosis is the RNA of activated leukocyte cell adhesion molecule (ALCAM) gene, which is expressed at high levels in breast tumor. We applied DNA-functionalized gold nanoparticles as the target-specific probes, for detecting specific sequences of DNA or RNA. At high MgCL2 concentrations, nanoprobes aggregate in the absence of the complementary DNA sequence and alteration in the solution color is detectable by evaluating the localized surface plasmon resonance (LSPR). But in the presence of complementary DNA, nanoprobes hybridize to the complementary sequence; therefore, no aggregation takes place, and no color change is observed. We designed a gold nanoprobe-based method that promptly detects the ALCAM gene expression in a low reaction volume with high sensitivity and specificity. This method is simple, fast, selective, and quantitative and can be done with small concentrations of the target (fmol/μL). Limit of detection of the method corresponded to 300 fmol/μL of synthetic ALCAM target.

  10. Hepatic expression of inflammatory genes and microRNAs in pigs with high "cholesteryl ester transfer protein" (CETP) activity.

    PubMed

    Cirera, Susanna; Tørsleff, Benedicte C Juul; Ritz, Christian; Fredholm, Merete; Heegaard, Peter M H; Skovgaard, Kerstin

    2016-10-01

    Human obesity and obesity-related diseases (ORD) are growing health problems worldwide and represent a major public health challenge. Most of these diseases are complex conditions, influenced by many genes (including microRNAs) and environmental factors. Many metabolic perturbations are associated with obesity; e.g., low levels of high-density lipoproteins (HDL) are high risk factors of cardiovascular events. A number of genetic, lifestyle, and environmental factors have been shown to contribute to the lowering of HDL-cholesterol. One of these factors is cholesteryl ester transfer protein (CETP) promoting the redistribution of cholesteryl esters, triglycerides, and phospholipids between plasma proteins. Moreover, obesity and ORD are often linked with chronic low-grade inflammation leading to insulin resistance and endothelial and microvascular dysfunctions. The aim of this study was to detect differences in the hepatic expression of genes involved in low-grade inflammation and of obesity- and cholesterol-related microRNAs in two mixed breed populations of pigs (Yorkshire-Göttingen minipig, YM and Duroc-Göttingen minipig, DM) including males and females, with extreme phenotypes for CETP activity levels (designated as CETP-high and CETP-low, respectively). Furthermore, breed and gender differences were also investigated. We found significant difference (P < 0.05) in hepatic expression levels of several mRNAs and microRNAs between the CETP-high and -low groups (C5, IL1RN, IL18, and miR-223-5p); between the two mixed breeds (IL1RAP and miR-140-5p); and between gender (APOA1, IL1RN, and FBLN1). Furthermore, when taking breed into account we show that the transcriptional levels of TNF, miR20a, miR33b, and miR130a differed between the two CETP groups. We conclude that increased CETP activity is accompanied by a modest differential hepatic expression of several microRNAs and inflammatory-related genes. Furthermore, our study demonstrates that when modeling the analysis

  11. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes

    SciTech Connect

    Smetanina, Mariya A.; Pakharukova, Mariya Y.; Kurinna, Svitlana M.; Dong, Bingning; Hernandez, Juan P.; Moore, David D.; Merkulova, Tatyana I.

    2011-08-15

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car{sup -/-}) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car{sup -/-} livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. - Highlights: > The azo dye and mouse carcinogen OAT is a very effective mCAR activator. > OAT increases mCAR transactivation in a dose-dependent manner. > OAT CAR-dependently increases the expression of a specific subset of CAR target genes. > OAT induces an unexpectedly deferred, but CAR-dependent hepatocyte proliferation.

  12. Influence of low glycolytic activities in gcr1 and gcr2 mutants on the expression of other metabolic pathway genes in Saccharomyces cerevisiae.

    PubMed

    Sasaki, Hiromi; Uemura, Hiroshi

    2005-01-30

    A complex of the transcription factors Gcr1p and Gcr2p coordinately regulates the expression of glycolytic genes in Saccharomyces cerevisiae. To understand the effects of gcr mutations on other metabolic pathways, genome-wide gene expression profiles in gcr1 and gcr2 mutants were examined. The biggest effects of gcr1 and gcr2 mutations were observed on the glycolytic genes and the expressions of most of the glycolytic genes were substantially decreased compared to those in the wild-type strain in both glucose and glycerol+lactate growth conditions. On the other hand, the expressions of genes encoding the TCA cycle and respiration were increased in gcr mutants when the cells were grown in glucose. RT-PCR analyses revealed that the expression of SIP4 and HAP5, which are known to affect the expression of some of the gluconeogenic, TCA cycle and respiratory genes, were also increased under this condition. The growth of gcr mutants on glucose was impaired by adding respiration inhibitor antimycin A, whereas the growth of the wild-type strain was not. The conversion of glucose to biomass was higher and, to the contrary, ethanol yield was lower in the gcr2 mutant compared to those in the wild-type strain. These results suggest the possibility that the gcr mutants, in which glycolytic activities are low, changed their metabolic patterns under glucose growth condition to enhance the expression of TCA cycle and respiratory genes to produce more energy.

  13. Method of controlling gene expression

    DOEpatents

    Peters, Norman K.; Frost, John W.; Long, Sharon R.

    1991-12-03

    A method of controlling expression of a DNA segment under the control of a nod gene promoter which comprises administering to a host containing a nod gene promoter an amount sufficient to control expression of the DNA segment of a compound of the formula: ##STR1## in which each R is independently H or OH, is described.

  14. The flow of gene expression.

    PubMed

    Misteli, Tom

    2004-03-01

    Gene expression is a highly interconnected multistep process. A recent meeting in Iguazu Falls, Argentina, highlighted the need to uncover both the molecular details of each single step as well as the mechanisms of coordination among processes in order to fully understand the expression of genes.

  15. Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression.

    PubMed

    Ribeiro, Regislane P; Portela, Antonia M L R; Silva, Anderson W B; Costa, José J N; Passos, José R S; Cunha, Ellen V; Souza, Glaucinete B; Saraiva, Márcia V A; Donato, Mariana A M; Peixoto, Christina A; van den Hurk, Robert; Silva, José R V

    2015-08-01

    This study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml - Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

  16. Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars

    PubMed Central

    Gharsallah, Charfeddine; Fakhfakh, Hatem; Grubb, Douglas; Gorsane, Faten

    2016-01-01

    Salinity is a constraint limiting plant growth and productivity of crops throughout the world. Understanding the mechanism underlying plant response to salinity provides new insights into the improvement of salt tolerance-crops of importance. In the present study, we report on the responses of twenty cultivars of tomato. We have clustered genotypes into scale classes according to their response to increased NaCl levels. Three local tomato genotypes, representative of different saline scale classes, were selected for further investigation. During early (0 h, 6 h and 12 h) and later (7 days) stages of the response to salt treatment, ion concentrations (Na+, K+  and Ca2+), proline content, enzyme activities (catalase, ascorbate peroxidase and guiacol peroxidase) were recorded. qPCR analysis of candidate genes WRKY (8, 31and 39), ERF (9, 16 and 80), LeNHX (1, 3 and 4) and HKT (class I) were performed. A high K+, Ca2 +and proline accumulation as well as a decrease of Na+  concentration-mediated salt tolerance. Concomitant with a pattern of high-antioxidant enzyme activities, tolerant genotypes also displayed differential patterns of gene expression during the response to salt stress. PMID:27543452

  17. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes.

    PubMed

    Smetanina, Mariya A; Pakharukova, Mariya Y; Kurinna, Svitlana M; Dong, Bingning; Hernandez, Juan P; Moore, David D; Merkulova, Tatyana I

    2011-08-15

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car(-/-)) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car(-/-) livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor.

  18. Astrocyte activation in the anterior cingulate cortex and altered glutamatergic gene expression during paclitaxel-induced neuropathic pain in mice

    PubMed Central

    2015-01-01

    Spinal astrocyte activation contributes to the pathogenesis of paclitaxel-induced neuropathic pain (PINP) in animal models. We examined glial fibrillary acidic protein (GFAP; an astrocyte marker) immunoreactivity and gene expression of GFAP, glutamate transporters and receptor subunits by real time PCR in the anterior cingulate cortex (ACC) at 7 days post first administration of paclitaxel, a time point when mice had developed thermal hyperalgesia. The ACC, an area in the brain involved in pain perception and modulation, was chosen because changes in this area might contribute to the pathophysiology of PINP. GFAP transcripts levels were elevated by more than fivefold and GFAP immunoreactivity increased in the ACC of paclitaxel-treated mice. The 6 glutamate transporters (GLAST, GLT-1 EAAC1, EAAT4, VGLUT-1 and VGLUT-2) quantified were not significantly altered by paclitaxel treatment. Of the 12 ionotropic glutamate receptor subunits transcripts analysed 6 (GLuA1, GLuA3, GLuK2, GLuK3, GLuK5 and GLuN1) were significantly up-regulated, whereas GLuA2, GLuK1, GLuK4, GLuN2A and GLuN2B were not significantly altered and GLuA4 was lowly expressed. Amongst the 8 metabotropic receptor subunits analysed only mGLuR8 was significantly elevated. In conclusion, during PINP there is astrocyte activation, with no change in glutamate transporter expression and differential up-regulation of glutamate receptor subunits in the ACC. Thus, targeting astrocyte activation and the glutamatergic system might be another therapeutic avenue for management of PINP. PMID:26528412

  19. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-05

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  20. Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells.

    PubMed

    Chen, H C; Chou, C K; Lee, S D; Wang, J C; Yeh, S F

    1995-05-01

    We have examined the antiviral activity of the crude extract prepared from the root of Saussurea lappa Clarks, a Chinese medicinal herb which is widely used for many illnesses including cancer. Two active components, costunolide and dehydrocostus lactone, were identified which show strong suppressive effect on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells, but have little effect on the viability of the cells. Both costunolide and dehydrocostus lactone suppress the HBsAg production by Hep3B cells in a dose-dependent manner with IC50s of 1.0 and 2.0 microM, respectively. Northern blotting analysis shows that the suppression of HBsAg gene expression by both costunolide and dehydrocostus lactone were mainly at the mRNA level. Furthermore, the suppressive effect of costunolide and dehydrocostus lactone on HBsAg and hepatitis B e antigen (HBeAg), a marker for hepatitis B viral genome replication in human liver cells, was also observed in another human hepatoma cell line HepA2 which was derived from HepG2 cells by transfecting a tandemly repeat hepatitis B virus (HBV) DNA. Similarly, the mRNA of HBsAg in HepA2 cells was also suppressed by these two compounds. Our findings suggest that costunolide and dehydrocostus lactone may have potential to develop as specific anti-HBV drugs in the future.

  1. [Effects of light intensity on associated enzyme activity and gene expression during callus formation of Vitis vinifera].

    PubMed

    Liu, Rong; Yang, Guowei; Wu, Yueyan; Rao, Huiyun; Li, Xuefu; Li, Meiqin; Qian, Pingxian

    2015-08-01

    We analyzed the best light intensity for callus induction and maintenance in Vitis vinifera and explored the mechanism of grape callus browning. Tender stem segments of grape cultivar "gold finger" were used to study the effects of different light intensities (0, 500, 1 000, 1 500, 2 000, 2 500, 3 000 and 4 000 Lx) on the induction rate, browning rate and associated enzyme activity and gene expression during Vitis vinifera callus formation. The callus induction rate under 0, 500, 1 000 and 1 500 Lx was more than 92%, significantly higher than in other treatments (P < 0.05). A lower browning rate and better callus growth were also observed during subculture under 1 000 and 1 500 Lx treatments. We found that chlorogenic acid, caffeic acid, p-hydroxybenzoic acid and coumaric acid contents were correlated with the browning rate of callus, among which chlorogenic acid content was positively correlated with the browning rate (P < 0.05). Peroxidase (POD) and polyphenol oxidase (PPO) activities were negatively correlated with the browning rate of callus (P < 0.01). The POD, PPO and phenylalanine ammonialyase (PAL) expression levels were positively correlated with the browning rate at P < 0.05 or P < 0.01. An appropriate light intensity for the tissue culture of Vitis vinifera was 1 000-1 500 Lx, higher or lower light intensities significantly impaired normal callus growth.

  2. Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

    SciTech Connect

    Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Suh, Steven; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-06-08

    In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

  3. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

    PubMed Central

    Olinto, S.C.F.; Adrião, M.G.; Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T.

    2012-01-01

    The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression. PMID:22641416

  4. Osmostress-induced gene expression--a model to understand how stress-activated protein kinases (SAPKs) regulate transcription.

    PubMed

    de Nadal, Eulàlia; Posas, Francesc

    2015-09-01

    Adaptation is essential for maximizing cell survival and for cell fitness in response to sudden changes in the environment. Several aspects of cell physiology change during adaptation. Major changes in gene expression are associated with cell exposure to environmental changes, and several aspects of mRNA biogenesis appear to be targeted by signaling pathways upon stress. Exhaustive reviews have been written regarding adaptation to stress and regulation of gene expression. In this review, using osmostress in yeast as a prototypical case study, we highlight those aspects of regulation of gene induction that are general to various environmental stresses as well as mechanistic aspects that are potentially conserved from yeast to mammals.

  5. HIV-1 Negative Female Sex Workers Sustain High Cervical IFNε, Low Immune Activation and Low Expression of HIV-1 Required Host Genes

    PubMed Central

    Abdulhaqq, Shaheed A.; Zorrilla, Carmen; Kang, Guobin; Yin, Xiangfan; Tamayo, Vivian; Seaton, Kelly E.; Joseph, Jocelin; Garced, Sheyla; Tomaras, Georgia D.; Linn, Kristin A.; Foulkes, Andrea S.; Azzoni, Livio; VerMilyea, Matthew; Coutifaris, Christos; Kossenkov, Andrew V.; Showe, Louise; Kraiselburd, Edmundo N.; Li, Qingsheng; Montaner, Luis J.

    2015-01-01

    Sex workers within high HIV endemic areas are often a target population where anti-HIV prophylactic strategies are tested. We hypothesize that in women with high levels of genital exposure to semen changes in cervicovaginal mucosal and/or systemic immune activation will contribute to a decreased susceptibility to HIV-1 infection. To address this question, we assessed sexual activity, immune activation status (in peripheral blood), as well as cellular infiltrates and gene expression in ectocervical mucosa biopsies in female sex workers [FSW] (n=50), as compared to control women [CG] (n=32). FSW had low to absent HIV-1 specific immune responses with significantly lower CD38 expression on circulating CD4+ or CD8+ T-Cells (both: p<0.001) together with lower cervical gene expression of genes associated with leukocyte homing and chemotaxis. FSW also had increased levels of Interferon-ε gene and protein expression in the cervical epithelium together with reduced expression of genes associated with HIV-1 integration and replication. A correlative relationship between semen exposure and elevated type-1 IFN expression in FSW was also established. Overall, our data suggest that long-term condomless sex work can result in multiple changes within the cervicovaginal compartment that would contribute to sustaining a lower susceptibility for HIV-1 infection in absence of HIV-specific responses. PMID:26555708

  6. Nucleosome repositioning underlies dynamic gene expression.

    PubMed

    Nocetti, Nicolas; Whitehouse, Iestyn

    2016-03-15

    Nucleosome repositioning at gene promoters is a fundamental aspect of the regulation of gene expression. However, the extent to which nucleosome repositioning is used within eukaryotic genomes is poorly understood. Here we report a comprehensive analysis of nucleosome positions as budding yeast transit through an ultradian cycle in which expression of >50% of all genes is highly synchronized. We present evidence of extensive nucleosome repositioning at thousands of gene promoters as genes are activated and repressed. During activation, nucleosomes are relocated to allow sites of general transcription factor binding and transcription initiation to become accessible. The extent of nucleosome shifting is closely related to the dynamic range of gene transcription and generally related to DNA sequence properties and use of the coactivators TFIID or SAGA. However, dynamic gene expression is not limited to SAGA-regulated promoters and is an inherent feature of most genes. While nucleosome repositioning occurs pervasively, we found that a class of genes required for growth experience acute nucleosome shifting as cells enter the cell cycle. Significantly, our data identify that the ATP-dependent chromatin-remodeling enzyme Snf2 plays a fundamental role in nucleosome repositioning and the expression of growth genes. We also reveal that nucleosome organization changes extensively in concert with phases of the cell cycle, with large, regularly spaced nucleosome arrays being established in mitosis. Collectively, our data and analysis provide a framework for understanding nucleosome dynamics in relation to fundamental DNA-dependent transactions.

  7. Relationship between male sterility and β-1,3-glucanase activity and callose deposition-related gene expression in wheat (Triticum aestivum L.).

    PubMed

    Liu, H Z; Zhang, G S; Zhu, W W; Ba, Q S; Niu, N; Wang, J W; Ma, S C; Wang, J S

    2015-01-26

    In previous studies, we first isolated one different protein β-1,3-glucanase using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from normal wheat (Triticum aestivum L.) and chemical hybridization agent-induced male sterility (CIMS) wheat. In this experiment, β-1,3-glucanase activity and the expression of a callose deposition-related gene, UDP-glucose phosphorylase (UGPase), were determinate in normal, CIMS, and genetic male sterility (GS) wheat. β-1,3-glucanase activity was significantly different between the fertile and sterile lines during callose synthesis and degradation, but there was no difference between CIMS and GS wheat. The UGPase gene of callose deposition was highly expressed in the meiophase and sharply decreased in the tetrad stage. However, the expression of the UGPase gene was significantly different between the fertile and sterile lines. These data indicated that β-1,3-glucanase activity and the expression of the UGPase gene play important roles in the male sterility of wheat. Consequently, pollen mother cells (PMCs) might degenerate at the early meiosis stage, and differences in UGPase gene expression and β-1,3-glucanase activity might eventually result in complete pollen collapse. In addition, the critical period of anther abortion might be the meiosis stage to the tetrad stage rather than what we previously thought, the mononuclear period.

  8. Thyroid hormones directly activate the expression of the human and mouse uncoupling protein-3 genes through a thyroid response element in the proximal promoter region

    PubMed Central

    2004-01-01

    The transcription of the human UCP3 (uncoupling protein-3) gene in skeletal muscle is tightly regulated by metabolic signals related to fatty acid availability. However, changes in thyroid status also modulate UCP3 gene expression, albeit by unknown mechanisms. We created transgenic mice bearing the entire human UCP3 gene to investigate the effect of thyroid hormones on human UCP3 gene expression. Treatment of human UCP3 transgenic mice with thyroid hormones induced the expression of the human gene in skeletal muscle. In addition, transient transfection experiments demonstrate that thyroid hormones activate the transcription of the human UCP3 gene promoter when MyoD and the TR (thyroid hormone receptor) were co-transfected. The action of thyroid hormones on UCP3 gene transcription is mediated by the binding of the TR to a proximal region in the UCP3 gene promoter that contains a direct repeat structure. An intact DNA sequence of this site is required for thyroid hormone responsiveness and TR binding. Chromatin immunoprecipitation assays revealed that the TR binds this element in vivo. The murine Ucp3 gene promoter was also dependent on MyoD and responsive to thyroid hormone in transient transfection assays. However, it was much less sensitive to thyroid hormone than the human UCP3 promoter. In summary, UCP3 gene transcription is activated by thyroid hormone treatment in vivo, and this activation is mediated by a TRE (thyroid hormone response element) in the proximal promoter region. Such regulation suggests a link between UCP3 gene expression and the effects of thyroid hormone on mitochondrial function in skeletal muscle. PMID:15496137

  9. Fluorescent activated cell sorting (FACS) combined with gene expression microarrays for transcription enrichment profiling of zebrafish lateral line cells.

    PubMed

    Gallardo, Viviana E; Behra, Martine

    2013-08-15

    Transgenic lines carrying fluorescent reporter genes like GFP have been of great value in the elucidation of developmental features and physiological processes in various animal models, including zebrafish. The lateral line (LL), which is a fish specific superficial sensory organ, is an emerging organ model for studying complex cellular processes in the context of the whole living animal. Cell migration, mechanosensory cell development/differentiation and regeneration are some examples. This sensory system is made of superficial and sparse small sensory patches called neuromasts, with less than 50 cells in any given patch. The paucity of cells is a real problem in any effort to characterize those cells at the transcriptional level. We describe here a method which we applied to efficiently separate subpopulation of cells of the LL, using two distinct stable transgenic zebrafish lines, Tg(cldnb:gfp) and Tg(tnks1bp1:EGFP). In both cases, the GFP positive (GFP+) cells were separated from the remainder of the animal by using a Fluorescent Activated Cell Sorter (FACS). The transcripts of the GFP+ cells were subsequently analyzed on gene expression microarrays. The combination of FACS and microarrays is an efficient method to establish a transcriptional signature for discrete cell populations which would otherwise be masked in whole animal preparation.

  10. Ultraviolet Radiation-Elicited Enhancement of Isoflavonoid Accumulation, Biosynthetic Gene Expression, and Antioxidant Activity in Astragalus membranaceus Hairy Root Cultures.

    PubMed

    Jiao, Jiao; Gai, Qing-Yan; Wang, Wei; Luo, Meng; Gu, Cheng-Bo; Fu, Yu-Jie; Ma, Wei

    2015-09-23

    In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting isoflavonoid accumulation. The optimum enhancement for isoflavonoid production was achieved in 34-day-old AMHRCs elicited by 86.4 kJ/m(2) of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g dry weight (DW), which was 2.29-fold higher relative to control (232.93 ± 3.08 μg/g DW). UV-B up-regulated the transcriptional expressions of all investigated genes involved in isoflavonoid biosynthetic pathway. PAL and C4H were found to be two potential key genes that controlled isoflavonoid biosynthesis. Moreover, a significant increase was noted in antioxidant activity of extracts from UV-B-elicited AMHRCs (IC50 values = 0.85 and 1.08 mg/mL) in comparison with control (1.38 and 1.71 mg/mL). Overall, this study offered a feasible elicitation strategy to enhance isoflavonoid accumulation in AMHRCs and also provided a basis for metabolic engineering of isoflavonoid biosynthesis in the future.

  11. Sodium-pump gene-expression, protein abundance and enzyme activity in isolated nephron segments of the aging rat kidney

    PubMed Central

    Scherzer, Pnina; Gal-Moscovici, Anca; Sheikh-Hamad, David; Popovtzer, Mordecai M

    2015-01-01

    Aging is associated with alteration in renal tubular functions, including sodium handling and concentrating ability. Na-K-ATPase plays a key role in driving tubular transport, and we hypothesized that decreased concentrating ability of the aging kidney is due in part to downregulation of Na-K-ATPase. In this study, we evaluated Na and K balance, aldosterone levels, and Na-K-ATPase gene expression, protein abundance, and activity in aging rat kidney. Na-K-ATPase activity (assayed microfluorometrically), mRNA (RT-PCR), and protein abundance (immunoblotting) were quantitated in the following isolated nephron segments: PCT, PST, MTAL, DCT, and CCD from 2, 8, 15, and 24 month-old-rats. In the course of aging, creatinine clearance decreased from 0.48 ± 0.02 mL/min/100 g BW to 0.28 ± 0.06 (P < 0.001) and aldosterone decreased from 23.6 ± 0.8 ng/dL to 13.2 ± 0.6 (P < 0.001). Serum Na+ and K+ increased by 4.0% and 22.5%, respectively. Na-K-ATPase activity, mRNA, and protein abundance of the α1 subunit displayed similar trends in all assayed segments; increasing in PCT and PST; decreasing in MTAL and DCT; increasing in CCD: in PCT they increased by 40%, 75%, and 250%, respectively; while in PST they increased by 80%, 50%, and 100%, respectively (P < 0.001). In MTAL they declined by 36%, 24%, and 34%, respectively, and in DCT by 38%, 59%, and 60%, respectively (P < 0.001). They were higher in CCD by 110%, 115%, and 246%, respectively (P < 0.001). Rats maintained Na/K balance; however with a steady state elevated serum K+. These results reveal quantitative changes in axial distribution of Na-K-ATPase at the level of gene expression, protein abundance, and activity in the nephrons of aging animals and may explain, in part, the pathophysiology of the senescent kidney. PMID:26056060

  12. Genetic associations for activated partial thromboplastin time and prothrombin time, their gene expression profiles, and risk of coronary artery disease.

    PubMed

    Tang, Weihong; Schwienbacher, Christine; Lopez, Lorna M; Ben-Shlomo, Yoav; Oudot-Mellakh, Tiphaine; Johnson, Andrew D; Samani, Nilesh J; Basu, Saonli; Gögele, Martin; Davies, Gail; Lowe, Gordon D O; Tregouet, David-Alexandre; Tan, Adrian; Pankow, James S; Tenesa, Albert; Levy, Daniel; Volpato, Claudia B; Rumley, Ann; Gow, Alan J; Minelli, Cosetta; Yarnell, John W G; Porteous, David J; Starr, John M; Gallacher, John; Boerwinkle, Eric; Visscher, Peter M; Pramstaller, Peter P; Cushman, Mary; Emilsson, Valur; Plump, Andrew S; Matijevic, Nena; Morange, Pierre-Emmanuel; Deary, Ian J; Hicks, Andrew A; Folsom, Aaron R

    2012-07-13

    Activated partial thromboplastin time (aPTT) and prothrombin time (PT) are clinical tests commonly used to screen for coagulation-factor deficiencies. One genome-wide association study (GWAS) has been reported previously for aPTT, but no GWAS has been reported for PT. We conducted a GWAS and meta-analysis to identify genetic loci for aPTT and PT. The GWAS for aPTT was conducted in 9,240 individuals of European ancestry from the Atherosclerosis Risk in Communities (ARIC) study, and the GWAS for PT was conducted in 2,583 participants from the Genetic Study of Three Population Microisolates in South Tyrol (MICROS) and the Lothian Birth Cohorts (LBC) of 1921 and 1936. Replication was assessed in 1,041 to 3,467 individuals. For aPTT, previously reported associations with KNG1, HRG, F11, F12, and ABO were confirmed. A second independent association in ABO was identified and replicated (rs8176704, p = 4.26 × 10(-24)). Pooling the ARIC and replication data yielded two additional loci in F5 (rs6028, p = 3.22 × 10(-9)) and AGBL1 (rs2469184, p = 3.61 × 10(-8)). For PT, significant associations were identified and confirmed in F7 (rs561241, p = 3.71 × 10(-56)) and PROCR/EDEM2 (rs2295888, p = 5.25 × 10(-13)). Assessment of existing gene expression and coronary artery disease (CAD) databases identified associations of five of the GWAS loci with altered gene expression and two with CAD. In summary, eight genetic loci that account for ∼29% of the variance in aPTT and two loci that account for ∼14% of the variance in PT were detected and supported by functional data.

  13. Genetic Associations for Activated Partial Thromboplastin Time and Prothrombin Time, their Gene Expression Profiles, and Risk of Coronary Artery Disease

    PubMed Central

    Tang, Weihong; Schwienbacher, Christine; Lopez, Lorna M.; Ben-Shlomo, Yoav; Oudot-Mellakh, Tiphaine; Johnson, Andrew D.; Samani, Nilesh J.; Basu, Saonli; Gögele, Martin; Davies, Gail; Lowe, Gordon D.O.; Tregouet, David-Alexandre; Tan, Adrian; Pankow, James S.; Tenesa, Albert; Levy, Daniel; Volpato, Claudia B.; Rumley, Ann; Gow, Alan J.; Minelli, Cosetta; Yarnell, John W.G.; Porteous, David J.; Starr, John M.; Gallacher, John; Boerwinkle, Eric; Visscher, Peter M.; Pramstaller, Peter P.; Cushman, Mary; Emilsson, Valur; Plump, Andrew S.; Matijevic, Nena; Morange, Pierre-Emmanuel; Deary, Ian J.; Hicks, Andrew A.; Folsom, Aaron R.

    2012-01-01

    Activated partial thromboplastin time (aPTT) and prothrombin time (PT) are clinical tests commonly used to screen for coagulation-factor deficiencies. One genome-wide association study (GWAS) has been reported previously for aPTT, but no GWAS has been reported for PT. We conducted a GWAS and meta-analysis to identify genetic loci for aPTT and PT. The GWAS for aPTT was conducted in 9,240 individuals of European ancestry from the Atherosclerosis Risk in Communities (ARIC) study, and the GWAS for PT was conducted in 2,583 participants from the Genetic Study of Three Population Microisolates in South Tyrol (MICROS) and the Lothian Birth Cohorts (LBC) of 1921 and 1936. Replication was assessed in 1,041 to 3,467 individuals. For aPTT, previously reported associations with KNG1, HRG, F11, F12, and ABO were confirmed. A second independent association in ABO was identified and replicated (rs8176704, p = 4.26 × 10−24). Pooling the ARIC and replication data yielded two additional loci in F5 (rs6028, p = 3.22 × 10−9) and AGBL1 (rs2469184, p = 3.61 × 10−8). For PT, significant associations were identified and confirmed in F7 (rs561241, p = 3.71 × 10−56) and PROCR/EDEM2 (rs2295888, p = 5.25 × 10−13). Assessment of existing gene expression and coronary artery disease (CAD) databases identified associations of five of the GWAS loci with altered gene expression and two with CAD. In summary, eight genetic loci that account for ∼29% of the variance in aPTT and two loci that account for ∼14% of the variance in PT were detected and supported by functional data. PMID:22703881

  14. Suppression of activity-regulated cytoskeleton-associated gene expression in the dorsal striatum attenuates extinction of cocaine-seeking.

    PubMed

    Hearing, Matthew C; Schwendt, Marek; McGinty, Jacqueline F

    2011-07-01

    The caudate putamen (CPu) has been implicated in habit learning and neuroadaptive changes that mediate the compulsive nature of drug-seeking following chronic cocaine self-administration. Re-exposure to an operant chamber previously associated with cocaine, but not yoked-saline, increases activity-regulated cytoskeleton-associated (Arc) gene mRNA expression within the dorsolateral (dl) CPu following prolonged abstinence. In this study, we tested the hypothesis that antisense gene knockdown of Arc within the dlCPu would alter cocaine-seeking. Initial studies showed that a single infusion of Arc antisense oligodeoxynucleotide (ODN) into the dlCPu significantly attenuated the induction of Arc mRNA and Arc protein by a single cocaine exposure (20 mg/kg i.p.) compared to scrambled-ODN-infused controls. In cocaine self-administering rats, infusion of Arc antisense ODN into the dlCPu 3 h prior to a test of context-driven drug-seeking significantly attenuated Arc protein induction, but failed to alter responding during testing, suggesting striatal Arc does not facilitate context-induced drug-seeking following prolonged abstinence. However, Arc antisense ODN infusion blunted the decrease in responding during subsequent 1-h extinction tests 24 and 48 h later. Following re-exposure to a cocaine-paired context, surface expression of the AMPA-type glutamate receptor GluR1 was significantly reduced whereas GluR2 was significantly increased in the dlCPu, independent of Arc antisense ODN infusion. Together, these findings indicate an important role for Arc in neuroadaptations within brain regions responsible for drug-seeking after abstinence and direct attention to changes occurring within striatal circuitry that are necessary to break down the habitual behaviour that leads to relapse.

  15. The Correlation of the Presence and Expression Levels of cry Genes with the Insecticidal Activities against Plutella xylostella for Bacillus thuringiensis Strains

    PubMed Central

    Chen, Ming-Lun; Chen, Pin-Hsin; Pang, Jen-Chieh; Lin, Chia-Wei; Hwang, Chin-Fa; Tsen, Hau-Yang

    2014-01-01

    The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella. PMID:25153253

  16. The correlation of the presence and expression levels of cry genes with the insecticidal activities against Plutella xylostella for Bacillus thuringiensis strains.

    PubMed

    Chen, Ming-Lun; Chen, Pin-Hsin; Pang, Jen-Chieh; Lin, Chia-Wei; Hwang, Chin-Fa; Tsen, Hau-Yang

    2014-08-19

    The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella.

  17. Human Lacrimal Gland Gene Expression

    PubMed Central

    Aakalu, Vinay Kumar; Parameswaran, Sowmya; Maienschein-Cline, Mark; Bahroos, Neil; Shah, Dhara; Ali, Marwan; Krishnakumar, Subramanian

    2017-01-01

    Background The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development. Methods We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium. Results The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described. Conclusions Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas. PMID:28081151

  18. Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion

    PubMed Central

    Brombacher, Eva; Baratto, Andrea; Dorel, Corinne; Landini, Paolo

    2006-01-01

    Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene. PMID:16513732

  19. Gene expression regulation by the Curli activator CsgD protein: modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion.

    PubMed

    Brombacher, Eva; Baratto, Andrea; Dorel, Corinne; Landini, Paolo

    2006-03-01

    Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene.

  20. Activity-dependent gene expression in honey bee mushroom bodies in response to orientation flight

    PubMed Central

    Lutz, Claudia C.; Robinson, Gene E.

    2013-01-01

    SUMMARY The natural history of adult worker honey bees (Apis mellifera) provides an opportunity to study the molecular basis of learning in an ecological context. Foragers must learn to navigate between the hive and floral locations that may be up to miles away. Young pre-foragers prepare for this task by performing orientation flights near the hive, during which they begin to learn navigational cues such as the appearance of the hive, the position of landmarks, and the movement of the sun. Despite well-described spatial learning and navigation behavior, there is currently limited information on the neural basis of insect spatial learning. We found that Egr, an insect homolog of Egr-1, is rapidly and transiently upregulated in the mushroom bodies in response to orientation. This result is the first example of an Egr-1 homolog acting as a learning-related immediate-early gene in an insect and also demonstrates that honey bee orientation uses a molecular mechanism that is known to be involved in many other forms of learning. This transcriptional response occurred both in naïve bees and in foragers induced to re-orient. Further experiments suggest that visual environmental novelty, rather than exercise or memorization of specific visual cues, acts as the stimulus for Egr upregulation. Our results implicate the mushroom bodies in spatial learning and emphasize the deep conservation of Egr-related pathways in experience-dependent plasticity. PMID:23678099

  1. Identification and expression profiling analysis of calmodulin-binding transcription activator genes in maize (Zea mays L.) under abiotic and biotic stresses.

    PubMed

    Yue, Runqing; Lu, Caixia; Sun, Tao; Peng, Tingting; Han, Xiaohua; Qi, Jianshuang; Yan, Shufeng; Tie, Shuanggui

    2015-01-01

    The calmodulin-binding transcription activators (CAMTA) play critical roles in plant growth and responses to environmental stimuli. However, how CAMTAs function in responses to abiotic and biotic stresses in maize (Zea mays L.) is largely unknown. In this study, we first identified all the CAMTA homologous genes in the whole genome of maize. The results showed that nine ZmCAMTA genes showed highly diversified gene structures and tissue-specific expression patterns. Many ZmCAMTA genes displayed high expression levels in the roots. We then surveyed the distribution of stress-related cis-regulatory elements in the -1.5 kb promoter regions of ZmCAMTA genes. Notably, a large number of stress-related elements present in the promoter regions of some ZmCAMTA genes, indicating a genetic basis of stress expression regulation of these genes. Quantitative real-time PCR was used to test the expression of ZmCAMTA genes under several abiotic stresses (drought, salt, and cold), various stress-related hormones [abscisic acid, auxin, salicylic acid (SA), and jasmonic acid] and biotic stress [rice black-streaked dwarf virus (RBSDV) infection]. Furthermore, the expression pattern of ZmCAMTA genes under RBSDV infection was analyzed to investigate their potential roles in responses of different maize cultivated varieties to RBSDV. The expression of most ZmCAMTA genes responded to both abiotic and biotic stresses. The data will help us to understand the roles of CAMTA-mediated Ca(2+) signaling in maize tolerance to environmental stresses.

  2. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    PubMed

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  3. Superoxide dismutase activity and gene expression levels in Saudi women with recurrent miscarriage

    PubMed Central

    GHNEIM, HAZEM K.; AL-SHEIKH, YAZEED A.; ALSHEBLY, MASHAEL M.; ABOUL-SOUD, MOURAD A. M.

    2016-01-01

    The antioxidant activities of superoxide dismutase 1 (SOD1) and SOD2, as well as the levels of the oxidant superoxide anion (SOA) and the micronutrients zinc (Zn), copper (Cu) and manganese (Mn), were assayed in plasma, whole blood and placental tissue of non-pregnant (NP), healthy pregnant (HP) women and recurrent miscarriage (RM) patients. The results showed that SOD1 and SOD2 activities and the levels of Zn, Cu and Mn in plasma and whole blood of HP women were slightly, but significantly lower, and even more significantly decreased in RM patients compared to those observed in NP women (P<0.05 and P<0.0001, respectively). Additionally, whereas plasma SOD1 and SOD2 activities and Zn, Cu and Mn levels were significantly lower in RM patients, those of whole blood and placental tissue were significantly lower when compared to HP women (P<0.001 and P<0.0001, respectively). Concurrently, there were consistent increases of equal magnitude and statistical significance in SOA levels in all the assayed samples as identified by a comparison between the subjects. The findings thus supported oxidative metabolism and excessive reactive oxygen species generation. The resultant oxidative stress, identified in whole blood and placental tissues of RM patients, may have been a primary cause of RM. Dietary supplementation of Zn, Cu and Mn may be beneficial to these patients pre- and post-conception. PMID:26821085

  4. RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level.

    PubMed

    Lai, Yi-Chyi; Peng, Hwei-Ling; Chang, Hwan-You

    2003-02-01

    The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease.

  5. Activation of defense against Phytophthora infestans in potato by down-regulation of syntaxin gene expression.

    PubMed

    Eschen-Lippold, Lennart; Landgraf, Ramona; Smolka, Ulrike; Schulze, Sebastian; Heilmann, Mareike; Heilmann, Ingo; Hause, Gerd; Rosahl, Sabine

    2012-03-01

    The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed. Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane-localized SYNTAXIN-RELATED 1 (StSYR1) and SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively. Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1-RNAi plants, but not StSNAP33-RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans-infected StSYR1-RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines. The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1-RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose-containing papillae.

  6. Changes in phenylalanine ammonia-lyase activity and gene expression during storage of asparagus spears.

    PubMed

    Bhowmik, Pankaj K; Matsui, Toshiyuki

    2005-01-01

    A cDNA clone coding phenylalanine ammonia-lyase (PAL) was isolated from a cDNA library prepared from asparagus spears (Asparagus officinalis L. cv. Welcome) using the reverse transcription-polymerase chain reaction (RT-PCR). The partial cDNA clone encoded an mRNA of 527 bp and the derived amino acid sequence was found highly homologous to PAL from rice, maize and barley. Northern blot analysis showed an increase of pAS-PAL mRNA until 24 h at 20 degrees C, which coincided well with PAL activity and fiber development, suggesting that the increase is a response to the wounding associated with harvest.

  7. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Thomas, Russell S.; Applegate, Dawn; Rosen, Mitch; Abbott, Barbara; Lau, Christopher; Guo, Grace; Aleksunes, Lauren M.; Klaassen, Curtis; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents increases liver cancer incidence, whereas suppression of PPARα activity leads to hepatocellular steatosis. Analytical approaches were developed to identify biosets (i.e., gene expression differences between two conditions) in a genomic database in which PPARα activity was altered. A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice after exposure to three structurally diverse PPARα activators (WY-14,643, fenofibrate and perfluorohexane sulfonate). A fold-change rank-based test (Running Fisher’s test (p-value ≤ 10-4)) was used to evaluate the similarity between the PPARα signature and a test set of 48 and 31 biosets positive or negative, respectively for PPARα activation; the test resulted in a balanced accuracy of 98%. The signature was then used to identify factors that activate or suppress PPARα in an annotated mouse liver/primary hepatocyte gene expression compendium of ~1850 biosets. In addition to the expected activation of PPARα by fibrate drugs, di(2-ethylhexyl) phthalate, and perfluorinated compounds, PPARα was activated by benzofuran, galactosamine, and TCDD and suppressed by hepatotoxins acetaminophen, lipopolysaccharide, silicon dioxide nanoparticles, and trovafloxacin. Additional factors that activate (fasting, caloric restriction) or suppress (infections) PPARα were also identified. This study 1) developed methods useful for future screening of environmental chemicals, 2) identified chemicals that activate or suppress PPARα, and 3) identified factors including diets and infections that modulate PPARα activity and would be hypothesized to affect chemical-induced PPAR

  8. The BCL11A transcription factor directly activates RAG gene expression and V(D)J recombination.

    PubMed

    Lee, Baeck-seung; Dekker, Joseph D; Lee, Bum-kyu; Iyer, Vishwanath R; Sleckman, Barry P; Shaffer, Arthur L; Ippolito, Gregory C; Tucker, Philip W

    2013-05-01

    Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a(lox/lox) deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.

  9. Egg yolks inhibit activation of NF-κB and expression of its target genes in adipocytes after partial delipidation

    PubMed Central

    Shen, Qiwen; Riedl, Ken M.; Cole, Rachel M.; Lehman, Christopher; Xu, Lu; Alder, Hansjuerg; Belury, Martha A.; Schwartz, Steven J.; Ziouzenkova, Ouliana

    2015-01-01

    How composition of egg yolk (EY) influences NF-κB, a key transcription pathway in inflammation, remains unclear. We performed partial delipidation of EY that removed 20–30% of cholesterol and triglycerides. The resulting polar and non-polar fractions were termed EY-P and EY-NP. NF-κB activation in response to EY from different suppliers and their fractions was examined in 3T3-L1 adipocytes using a NF-κB response element reporter assay and by analyzing expression of 248 inflammatory genes. Although EY-P and EY contained similar level of vitamins, carotenoids, and fatty acids, only delipidated EY-P fraction suppressed NF-κB via down-regulation of toll like receptor-2 and up-regulation of inhibitory toll interacting protein (Tollip) and lymphocyte antigen 96 (Ly96). Our data suggest that anti-inflammatory activity of lutein and retinol were blunted by non-polar lipids in EY likely via crosstalk between SREBP and NF-κB pathways in adipocytes. Thus, moderate delipidation may improve their beneficial properties of regular eggs. PMID:25620076

  10. T-2 toxin inhibits gene expression and activity of key steroidogenesis enzymes in mouse Leydig cells.

    PubMed

    Yang, Jian Ying; Zhang, Yong Fa; Meng, Xiang Ping; Li, Yuan Xiao; Ma, Kai Wang; Bai, Xue Fei

    2015-08-01

    T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of the testosterone secretion in the primary Leydig cells derived from the mouse testis. The previous study demonstrated the effects of T-2 toxin through direct decrease of the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis. In this study, we further examined the direct biological effects of T-2 toxin on steroidogenesis production, primarily in Leydig cells of mice. Mature mouse Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining. To examine T-2 toxin-induced testosterone secretion decrease, we measured the transcription levels of 3 key steroidogenic enzymes and 5 enzyme activities including 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD in T-2 toxin/human chorionicgonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7) M, 10(-8) M and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with the decreases in the levels of transcription of 3β-HSD-1, P450scc, and StAR (P<0.05) and also in enzyme activities of 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD (P<0.05).

  11. IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells

    PubMed Central

    Mao, Yumeng; van Hoef, Vincent; Zhang, Xiaonan; Wennerberg, Erik; Lorent, Julie; Witt, Kristina; Masvidal, Laia; Liang, Shuo; Murray, Shannon; Larsson, Ola; Kiessling, Rolf

    2016-01-01

    Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15–induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15–stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy. PMID:27465917

  12. Chemically regulated gene expression in plants.

    PubMed

    Padidam, Malla

    2003-04-01

    Chemically inducible systems that activate or inactivate gene expression have many potential applications in the determination of gene function and in plant biotechnology. The precise timing and control of gene expression are important aspects of chemically inducible systems. Several systems have been developed and used to analyze gene function, marker-free plant transformation, site-specific DNA excision, activation tagging, conditional genetic complementation, and restoration of male fertility. Chemicals that are used to regulate transgene expression include the antibiotic tetracycline, the steroids dexamethasone and estradiol, copper, ethanol, the inducer of pathogen-related proteins benzothiadiazol, herbicide safeners, and the insecticide methoxyfenozide. Systems that are suitable for field application are particularly useful for experimental systems and have potential applications in biotechnology.

  13. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  14. Two hAT transposon genes were transferred from Brassicaceae to broomrapes and are actively expressed in some recipients

    PubMed Central

    Sun, Ting; Renner, Susanne S.; Xu, Yuxing; Qin, Yan; Wu, Jianqiang; Sun, Guiling

    2016-01-01

    A growing body of evidence is pointing to an important role of horizontal gene transfer (HGT) in the evolution of higher plants. However, reports of HGTs of transposable elements (TEs) in plants are still scarce, and only one case is known of a class II transposon horizontally transferred between grasses. To investigate possible TE transfers in dicots, we performed transcriptome screening in the obligate root parasite Phelipanche aegyptiaca (Orobanchaceae), data-mining in the draft genome assemblies of four other Orobanchaceae, gene cloning, gene annotation in species with genomic information, and a molecular phylogenetic analysis. We discovered that the broomrape genera Phelipanche and Orobanche acquired two related nuclear genes (christened BO transposase genes), a new group of the hAT superfamily of class II transposons, from Asian Sisymbrieae or a closely related tribe of Brassicaceae, by HGT. The collinearity of the flanking genes, lack of a classic border structure, and low expression levels suggest that BO transposase genes cannot transpose in Brassicaceae, whereas they are highly expressed in P. aegyptiaca. PMID:27452947

  15. The time course of activity-regulated cytoskeletal (ARC) gene and protein expression in the whisker-barrel circuit using two paradigms of whisker stimulation.

    PubMed

    Khodadad, Aida; Adelson, P David; Lifshitz, Jonathan; Thomas, Theresa Currier

    2015-05-01

    Immediate early genes have previously demonstrated a rapid increase in gene expression after various behavioral paradigms. The main focus of this article is to identify a molecular marker of circuit activation after manual whisker stimulation or exploration of a novel environment. To this end, we investigated the dynamics of ARC transcription in adult male rats during whisker somatosensation throughout the whisker barrel circuit. At various time points, tissue was biopsied from the ventral posterior medial nucleus (VPM) of the thalamus, primary somatosensory barrel field (S1BF) cortex and hippocampus for quantification using real-time PCR and western blot. Our results show that there were no significant differences in ARC gene or protein expression in the VPM after both types of stimulation. However, manual whisker stimulation resulted in increased ARC gene expression at 15, 30, 60 and 300 min in the S1BF, and 15 min in the hippocampus (p<0.05). Also, exploration of a novel environment resulted in increased ARC mRNA expression at 15 and 30 min in the S1BF and at 15 min in the hippocampus (p<0.05). The type of stimulation (manual versus exploration of a novel environment) influenced the magnitude of ARC gene expression in the S1BF (p<0.05). These data are the first to demonstrate that ARC is a specific, quantifiable and input dependent molecular marker of circuit activation which can serve to quantify the impact of brain injury and subsequent rehabilitation on whisker sensation.

  16. The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake

    PubMed Central

    Salter, Rebecca C.; Foka, Pelagia; Davies, Thomas S.; Gallagher, Hayley; Michael, Daryn R.; Ashlin, Tim G.; Ramji, Dipak P.

    2016-01-01

    The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis. PMID:27687241

  17. The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake.

    PubMed

    Salter, Rebecca C; Foka, Pelagia; Davies, Thomas S; Gallagher, Hayley; Michael, Daryn R; Ashlin, Tim G; Ramji, Dipak P

    2016-09-30

    The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis.

  18. Monoallelic Gene Expression in Mammals.

    PubMed

    Chess, Andrew

    2016-11-23

    Monoallelic expression not due to cis-regulatory sequence polymorphism poses an intriguing problem in epigenetics because it requires the unequal treatment of two segments of DNA that are present in the same nucleus and that can indeed have absolutely identical sequences. Here, I focus on a few recent developments in the field of monoallelic expression that are of particular interest and raise interesting questions for future work. One development is regarding analyses of imprinted genes, in which recent work suggests the possibility that intriguing networks of imprinted genes exist and are important for genetic and physiological studies. Another issue that has been raised in recent years by a number of publications is the question of how skewed allelic expression should be for it to be designated as monoallelic expression and, further, what methods are appropriate or inappropriate for analyzing genomic data to examine allele-specific expression. Perhaps the most exciting recent development in mammalian monoallelic expression is a clever and carefully executed analysis of genetic diversity of autosomal genes subject to random monoallelic expression (RMAE), which provides compelling evidence for distinct evolutionary forces acting on random monoallelically expressed genes.

  19. Tuning noise in gene expression.

    PubMed

    Tyagi, Sanjay

    2015-05-05

    The relative contribution of promoter architecture and the associated chromatin environment in regulating gene expression noise has remained elusive. In their recent work, Arkin, Schaffer and colleagues (Dey et al, 2015) show that mean expression and noise for a given promoter at different genomic loci are uncorrelated and influenced by the local chromatin environment.

  20. Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: their expression during embryonic organogenesis of the chicken.

    PubMed

    Uchikawa, M; Kamachi, Y; Kondoh, H

    1999-06-01

    Group B Sox genes, Sox1, -2 and -3 are known to activate crystallin genes and to be involved in differentiation of lens and neural tissues. Screening of chicken genomic sequences for more Group B Sox genes identified two additional genes, Sox14 and Sox21. Proteins encoded by Sox14 and Sox21 genes are similar to each other but distinct from those coded by Sox1-3 (subgroup B1) except for the HMG domain and Group B homology immediately C-proximal of the HMG domain. C-terminal domains of SOX21 and SOX14 proteins function as strong and weak repression domains, respectively, when linked to the GAL4 DNA binding domain. These SOX proteins strongly (SOX21) or moderately (SOX14) inhibited activation of delta1-crystallin DC5 enhancer by SOX1 or SOX2, establishing that Sox14 and Sox21 are repressing subgroup (B2) of Group B Sox genes. This provides the first evidence for the occurrence of repressor SOX proteins. Activating (B1) and repressing (B2) subgroups of Group B Sox genes display interesting overlaps of expression domains in developing tissues (e.g. optic tectum, spinal cord, inner ear, alimentary tract, branchial arches). Within each subgroup, most expression domains of Sox1 and -3 are included in those of Sox2 (e.g. CNS, PNS, inner ear), while co-expression of Sox14 and Sox21 occurs in highly restricted sites of the CNS, with the likely temporal order of Sox21 preceding Sox14 (e.g. interneurons of the spinal cord). These expression patterns suggest that target genes of Group B SOX proteins are finely regulated by the counterbalance of activating and repressing SOX proteins.

  1. Lipopolysaccharide derived from the digestive tract activates inflammatory gene expression and inhibits casein synthesis in the mammary glands of lactating dairy cows

    PubMed Central

    Zhang, Kai; Chang, Guangjun; Xu, Tianle; Xu, Lei; Guo, Junfei; Jin, Di; Shen, Xiangzhen

    2016-01-01

    To meet the nutrition requirements of lactation, dairy cows are usually fed a high concentrate diet (HC). However, high-grain feeding causes subacute ruminal acidosis (SARA), a metabolic disorder that causes milk protein depression. This study aimed to investigate the effect of lipopolysaccharide (LPS) released in the rumen on inflammatory gene expression and casein synthesis in mammary glands of lactating dairy cows fed a HC diet. We found that milk protein was significantly decreased in the HC group after 15 weeks of feeding. Overall, LPS concentrations in the rumen fluid, lacteal artery and vein were increased in the HC group. Transcriptome microarray was used to evaluate alterations in the signaling pathway in mammary glands. Signaling pathways involved in inflammatory responses were activated, whereas those involved in protein synthesis were inhibited in the HC group. mRNA expression involved in inflammatory responses, including that of TLR4, NF-кB and pro-inflammatory genes, was increased in the HC group, while αs1-casein (CSN1S1), β-casein (CSN2), mTOR and S6K gene expression were decreased. Moreover, protein expression was consistent with the corresponding gene expression. After feeding with an HC diet, LPS derived from the rumen increased inflammatory gene expression and inhibited casein synthesis in the mammary glands of lactating dairy cows fed a HC diet. PMID:26893357

  2. Histone Deacetylase Activity Represses Gamma Interferon-Inducible HLA-DR Gene Expression following the Establishment of a DNase I-Hypersensitive Chromatin Conformation

    PubMed Central

    Osborne, Aaron; Zhang, Hongquan; Yang, Wen-Ming; Seto, Edward; Blanck, George

    2001-01-01

    Expression of the retinoblastoma tumor suppressor protein (Rb) is required for gamma interferon (IFN-γ)-inducible major histocompatibility complex class II gene expression and transcriptionally productive HLA-DRA promoter occupancy in several human tumor cell lines. Treatment of these Rb-defective tumor cell lines with histone deacetylase (HDAC) inhibitors rescued IFN-γ-inducible HLA-DRA and -DRB mRNA and cell surface protein expression, demonstrating repression of these genes by endogenous cellular HDAC activity. Additionally, Rb-defective, transcriptionally incompetent tumor cells retained the HLA-DRA promoter DNase I-hypersensitive site. Thus, HDAC-mediated repression of the HLA-DRA promoter occurs following the establishment of an apparent nucleosome-free promoter region and before transcriptionally productive occupancy of the promoter by the required transactivators. Repression of HLA-DRA promoter activation by HDAC activity likely involves a YY1 binding element located in the first exon of the HLA-DRA gene. Chromatin immunoprecipitation experiments localized YY1 to the HLA-DRA gene in Rb-defective tumor cells. Additionally, mutation of the YY1 binding site prevented repression of the promoter by HDAC1 and partially prevented activation of the promoter by trichostatin A. Mutation of the octamer element also significantly reduced the ability of HDAC1 to confer repression of inducible HLA-DRA promoter activation. Treatment of Rb-defective tumor cells with HDAC inhibitors greatly reduced the DNA binding activity of Oct-1, a repressor of inducible HLA-DRA promoter activation. These findings represent the first evidence that HDAC activity can repress IFN-γ-inducible HLA class II gene expression and also demonstrate that HDAC activity can contribute to promoter repression following the establishment of a DNase I-hypersensitive chromatin conformation. PMID:11533238

  3. Gene expression homeostasis and chromosome architecture

    PubMed Central

    Seshasayee, Aswin Sai Narain

    2014-01-01

    In rapidly growing populations of bacterial cells, including those of the model organism Escherichia coli, genes essential for growth - such as those involved in protein synthesis - are expressed at high levels; this is in contrast to many horizontally-acquired genes, which are maintained at low transcriptional levels.1 This balance in gene expression states between 2 distinct classes of genes is established by a galaxy of transcriptional regulators, including the so-called nucleoid associated proteins (NAP) that contribute to shaping the chromosome.2 Besides these active players in gene regulation, it is not too far-fetched to anticipate that genome organization in terms of how genes are arranged on the chromosome,3 which is the result of long-drawn transactions among genome rearrangement processes and selection, and the manner in which it is structured inside the cell, plays a role in establishing this balance. A recent study from our group has contributed to the literature investigating the interplay between global transcriptional regulators and genome organization in establishing gene expression homeostasis.4 In particular, we address a triangle of functional interactions among genome organization, gene expression homeostasis and horizontal gene transfer. PMID:25997086

  4. Glucocorticoids activate Epstein Barr Virus lytic replication through the upregulation of immediate early BZLF1 gene expression

    PubMed Central

    Yang, Eric V.; Webster Marketon, Jeanette I.; Chen, Min; Lo, Kwok Wai; Kim, Seung-jae; Glaser, Ronald

    2010-01-01

    Psychological stress-associated immune dysregulation has been shown to disrupt the steady state expression and reactivate latent herpes viruses. One such virus is the Epstein Barr virus (EBV), which is associated with several human malignancies. EBV infects >90% of people living in North America and persists for life in latently infected cells. Although several studies have shown that glucocorticoids (GCs) can directly induce reactivation of the latent virus, the mechanism of stress hormone involvement in the control of EBV gene expression is not well understood. In this study, we tested the hypothesis that GCs can induce the latent EBV genome to lytically replicate through the induction of the EBV immediate early gene BZLF1 which encodes the lytic transactivator protein ZEBRA. We show a dose-dependent upregulation of BZLF1 mRNA expression by hydrocortisone (HC) and dexamethasone (Dex) in Daudi cells, an EBV genome positive Burkitt’s lymphoma cell line, and Dex-induction of the early gene products BLLF3 (encoding for the EBV dUTPase) and BALF5 (encoding for the EBV DNA polymerase). We show that Daudi cells express glucocorticoid receptors (GR) that mediate Dex-dependent upregulation of BZLF1 mRNA levels. This effect was inhibited by both the glucocorticoid receptor antagonist RU486 and by cycloheximide. The results suggest that GCs, in addition to inducing stress-related immune dysregulation, can mediate latent EBV reactivation through the induction of the BZLF1 gene. PMID:20466055

  5. Cloning and expression of lipoxygenase genes and enzyme activity in ripening persimmon fruit in response to GA and ABA treatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two genes of the lipoxygenase (LOX) family, DkLox1 and DkLox3 (GenBank accession No. JF436951 and JF436950), were cloned from persimmon fruit (Diospyros kaki L. ‘Fuping Jianshi’). Sequence analysis indicated that they belong to the 9-LOX sub-group. Heterologous expression of DkLox1 in E. coli produc...

  6. The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen.

    PubMed

    Boutaud, A; Castellino, F J

    1993-06-01

    A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees

  7. Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway.

    PubMed

    Hsu, Shih-Che; Huang, Shih-Ming; Chen, Ann; Sun, Chiao-Yin; Lin, Shih-Hua; Chen, Jin-Shuen; Liu, Shu-Ting; Hsu, Yu-Juei

    2014-08-01

    The Klotho gene functions as an aging suppressor gene. Evidence from animal models suggests that induction of Klotho expression may be a potential treatment for age-associated diseases. However, the molecular mechanism involved in regulating renal Klotho gene expression remains unclear. In this study, we determined that resveratrol, a natural polyphenol, induced renal Klotho expression both in vivo and in vitro. In the mouse kidney, resveratrol administration markedly increased both Klotho mRNA and protein expression. In resveratrol-treated NRK-52E cells, increased Klotho expression was accompanied by the upregulation and nuclear translocation of activating transcription factor 3 (ATF3) and c-Jun. ATF3 or c-Jun overexpression enhanced the transcriptional activation of Klotho. Conversely, resveratrol-induced Klotho expression was attenuated in the presence of dominant-negative ATF3 or c-Jun. Coimmunoprecipitation and a chromatin immunoprecipitation assay revealed that ATF3 physically interacted with c-Jun and that the ATF3/c-Jun complex directly bound to the Klotho promoter through ATF3- and AP-1-binding elements. c-Jun cotransfection augmented the effects of ATF3 on Klotho transcription in vitro. Although Sirtuin 1 mRNA expression was induced by resveratrol and involved in regulating Klotho mRNA expression, it was not the primary cause for the aforementioned ATF3/c-Jun pathway. In summary, resveratrol enhances the renal expression of the anti-aging Klotho gene, and the transcriptional factors ATF3 and c-Jun functionally interact and coordinately regulate the resveratrol-mediated transcriptional activation of Klotho.

  8. Light-responsive control of bacterial gene expression: precise triggering of the lac promoter activity using photocaged IPTG.

    PubMed

    Binder, Dennis; Grünberger, Alexander; Loeschcke, Anita; Probst, Christopher; Bier, Claus; Pietruszka, Jörg; Wiechert, Wolfgang; Kohlheyer, Dietrich; Jaeger, Karl-Erich; Drepper, Thomas

    2014-08-01

    Light can be used to control numerous cellular processes including protein function and interaction as well as gene expression in a non-invasive fashion and with unprecedented spatiotemporal resolution. However, for chemical phototriggers tight, gradual, and homogeneous light response has never been attained in living cells. Here, we report on a light-responsive bacterial T7 RNA polymerase expression system based on a photocaged derivative of the inducer molecule isopropyl-β-d-thiogalactopyranoside (IPTG). We have comparatively analyzed different Escherichia coli lac promoter-regulated expression systems in batch and microfluidic single-cell cultivation. The lacY-deficient E. coli strain Tuner(DE3) harboring additional plasmid-born copies of the lacI gene exhibited a sensitive and defined response to increasing IPTG concentrations. Photocaged IPTG served as a synthetic photo-switch to convert the E. coli system into an optogenetic expression module allowing for precise and gradual light-triggering of gene expression as demonstrated at the single cell level.

  9. A soluble factor from Trypanosoma cruzi inhibits transforming growth factor-ß-induced MAP kinase activation and gene expression in dermal fibroblasts.

    PubMed

    Mott, G Adam; Costales, Jaime A; Burleigh, Barbara A

    2011-01-01

    The protozoan parasite Trypanosoma cruzi, which causes human Chagas' disease, exerts a variety of effects on host extracellular matrix (ECM) including proteolytic degradation of collagens and dampening of ECM gene expression. Exposure of primary human dermal fibroblasts to live infective T. cruzi trypomastigotes or their shed/secreted products results in a rapid down-regulation of the fibrogenic genes collagenIα1, fibronectin and connective tissue growth factor (CTGF/CCN2). Here we demonstrate the ability of a secreted/released T. cruzi factor to antagonize ctgf/ccn2 expression in dermal fibroblasts in response to TGF-ß, lysophosphatidic acid or serum, where agonist-induced phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2, p38 and JNK was also inhibited. Global analysis of gene expression in dermal fibroblasts identified a discrete subset of TGF-ß-inducible genes involved in cell proliferation, wound repair, and immune regulation that are inhibited by T. cruzi secreted/released factors, where the genes exhibiting the highest sensitivity to T. cruzi are known to be regulated by MAP kinase-activated transcription factors. Consistent with this observation, the Ets-family transcription factor binding site in the proximal promoter region of the ctgf/ccn2 gene (-91 bp to -84 bp) was shown to be required for T. cruzi-mediated down-regulation of ctgf/ccn2 reporter expression. The cumulative data suggest a model in which T. cruzi-derived molecules secreted/released early in the infective process dampen MAP kinase signaling and the activation of transcription factors that regulate expression of fibroblast genes involved in wound repair and tissue remodelling, including ctgf/ccn2. These findings have broader implications for local modulation of ECM synthesis/remodelling by T. cruzi during the early establishment of infection in the mammalian host and highlight the potential for pathogen-derived molecules to be exploited as tools to modulate the

  10. Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells

    PubMed Central

    Vajravelu, Bathri N.; Moktar, Afsoon; Cao, Pengxiao; Moore, Joseph B.; Bolli, Roberto

    2017-01-01

    Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy. PMID:28355297

  11. Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells.

    PubMed

    Al-Maqtari, Tareq; Hong, Kyung U; Vajravelu, Bathri N; Moktar, Afsoon; Cao, Pengxiao; Moore, Joseph B; Bolli, Roberto

    2017-01-01

    Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.

  12. Specific expression of optically active reporter gene in arginine vasopressin-secreting neurosecretory cells in the hypothalamic-neurohypophyseal system.

    PubMed

    Ueta, Y; Fujihara, H; Dayanithi, G; Kawata, M; Murphy, D

    2008-06-01

    The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca(2+) imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion.

  13. Differential inhibition of activity, activation and gene expression of MMP-9 in THP-1 cells by azithromycin and minocycline versus bortezomib: A comparative study

    PubMed Central

    Knoops, Sofie; Aldinucci Buzzo, João L.; Boon, Lise; Martens, Erik; Opdenakker, Ghislain; Kolaczkowska, Elzbieta

    2017-01-01

    Gelatinase B or matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) is increased in inflammatory processes and cancer, and is associated with disease progression. In part, this is due to MMP-9-mediated degradation of extracellular matrix, facilitating influx of leukocytes into inflamed tissues and invasion or metastasis of cancer cells. MMP-9 is produced as proMMP-9 and its propeptide is subsequently removed by other proteases to generate proteolytically active MMP-9. The significance of MMP-9 in pathologies triggered the development of specific inhibitors of this protease. However, clinical trials with synthetic inhibitors of MMPs in the fight against cancer were disappointing. Reports on active compounds which inhibit MMP-9 should be carefully examined in this regard. In a considerable set of recent publications, two antibiotics (minocycline and azythromycin) and the proteasome inhibitor bortezomib, used in cancers, were reported to inhibit MMP-9 at different stages of its expression, activation or activity. The current study was undertaken to compare and to verify the impact of these compounds on MMP-9. With exception of minocycline at high concentrations (>100 μM), the compounds did not affect processing of proMMP-9 into MMP-9, nor did they affect direct MMP-9 gelatinolytic activity. In contrast, azithromycin specifically reduced MMP-9 mRNA and protein levels without affecting NF-κB in endotoxin-challenged monocytic THP-1 cells. Bortezomib, although being highly toxic, had no MMP-9-specific effects but significantly upregulated cyclooxygenase-2 (COX-2) activity and PGE2 levels. Overall, our study clarified that azithromycin decreased the levels of MMP-9 by reduction of gene and protein expression while minocycline inhibits proteolytic activity at high concentrations. PMID:28369077

  14. Heparanase-mediated Loss of Nuclear Syndecan-1 Enhances Histone Acetyltransferase (HAT) Activity to Promote Expression of Genes That Drive an Aggressive Tumor Phenotype*

    PubMed Central

    Purushothaman, Anurag; Hurst, Douglas R.; Pisano, Claudio; Mizumoto, Shuji; Sugahara, Kazuyuki; Sanderson, Ralph D.

    2011-01-01

    Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype. PMID:21757697

  15. Benzaldehyde Schiff bases regulation to the metabolism, hemolysis, and virulence genes expression in vitro and their structure-microbicidal activity relationship.

    PubMed

    Xia, Lei; Xia, Yu-Fen; Huang, Li-Rong; Xiao, Xiao; Lou, Hua-Yong; Liu, Tang-Jingjun; Pan, Wei-Dong; Luo, Heng

    2015-06-05

    There is an urgent need to develop new antibacterial agents because of multidrug resistance by bacteria and fungi. Schiff bases (aldehyde or ketone-like compounds) exhibit intense antibacterial characteristics, and are therefore, promising candidates as antibacterial agents. To investigate the mechanism of action of newly designed benzaldehyde Schiff bases, a series of high-yielding benzaldehyde Schiff bases were synthesized, and their structures were determined by NMR and MS spectra data. The structure-microbicidal activity relationship of derivatives was investigated, and the antibacterial mechanisms were investigated by gene assays for the expression of functional genes in vitro using Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The active compounds were selective for certain active groups. The polar substitution of the R2 group of the amino acids in the Schiff bases, affected the antibacterial activity against E. coli and S. aureus; specific active group at the R3 or R4 groups of the acylhydrazone Schiff bases could improve their inhibitory activity against these three tested organisms. The antibacterial mechanism of the active benzaldehyde Schiff bases appeared to regulate the expression of metabolism-associated genes in E. coli, hemolysis-associated genes in B. subtilis, and key virulence genes in S. aureus. Some benzaldehyde Schiff bases were bactericidal to all the three strains and appeared to regulate gene expression associated with metabolism, hemolysis, and virulence, in vitro. The newly designed benzaldehyde Schiff bases possessed unique antibacterial activity and might be potentially useful for prophylactic or therapeutic intervention of bacterial infections.

  16. NonO binds to the CpG island of oct4 promoter and functions as a transcriptional activator of oct4 gene expression.

    PubMed

    Park, Yoojin; Lee, Ja-Myong; Hwang, Min-Young; Son, Gi-hoon; Geum, Dongho

    2013-01-01

    We investigated the relationship between oct4 gene expression patterns and CpG sites methylation profiles during ES cell differentiation into neurons, and identified relevant binding factor. The oct4 gene expression level gradually declined as ES cell differentiation progressed, and the CpG sites in the oct4 proximal enhancer (PE) and promoter regions were methylated in concert with ES cell differentiation. An electro-mobility shift assay (EMSA) showed that putative proteins bind to CpG sites in the oct4 PE/promoter. We purified CpG binding proteins with DNAbinding purification method, and NonO was identified by liquid chromatography-mass spectrometry. EMSA with specific competitors revealed that NonO specifically binds to the conserved CCGGTGAC sequence in the oct4 promoter. Methylation at a specific cytosine residue (CC* GGTGAC) reduced the binding affinity of NonO for the recognition sequence. Chromatin immunoprecipitation analysis confirmed that NonO binds to the unmethylated oct4 promoter. There were no changes in the NonO mRNA and protein levels between ES cells and differentiated cells. The transcriptional role of NonO in oct4 gene expression was evaluated by luciferase assays and knockdown experiments. The luciferase activity significantly increased threefold when the NonO expression vector was cotransfected with the NonO recognition sequence, indicating that NonO has a transcription activator effect on oct4 gene expression. In accordance with this effect, when NonO expression was inhibited by siRNA treatment, oct4 expression was also significantly reduced. In summary, we purified NonO, a novel protein that binds to the CpG island of oct4 promoter, and positively regulates oct4 gene expression in ES cells.

  17. Differential expression of the ras gene family in mice.

    PubMed Central

    Leon, J; Guerrero, I; Pellicer, A

    1987-01-01

    We compared the expression of the ras gene family (H-ras, K-ras, and N-ras) in adult mouse tissues and during development. We found substantial variations in expression among different organs and in the amounts of the different transcripts originating from each gene, especially for the N-ras gene. The expression patterns were consistent with the reported preferential tissue activation of ras genes and suggested different cellular functions for each of the ras genes. Images PMID:3600635

  18. Heat Stress and Lipopolysaccharide Stimulation of Chicken Macrophage-Like Cell Line Activates Expression of Distinct Sets of Genes

    PubMed Central

    Slawinska, Anna; Hsieh, John C.; Schmidt, Carl J.; Lamont, Susan J.

    2016-01-01

    Acute heat stress requires immediate adjustment of the stressed individual to sudden changes of ambient temperatures. Chickens are particularly sensitive to heat stress due to development of insufficient physiological mechanisms to mitigate its effects. One of the symptoms of heat stress is endotoxemia that results from release of the lipopolysaccharide (LPS) from the guts. Heat-related cytotoxicity is mitigated by the innate immune system, which is comprised mostly of phagocytic cells such as monocytes and macrophages. The objective of this study was to analyze the molecular responses of the chicken macrophage-like HD11 cell line to combined heat stress and lipopolysaccharide treatment in vitro. The cells were heat-stressed and then allowed a temperature-recovery period, during which the gene expression was investigated. LPS was added to the cells to mimic the heat-stress-related endotoxemia. Semi high-throughput gene expression analysis was used to study a gene panel comprised of heat shock proteins, stress-related genes, signaling molecules and immune response genes. HD11 cell line responded to heat stress with increased mRNA abundance of the HSP25, HSPA2 and HSPH1 chaperones as well as DNAJA4 and DNAJB6 co-chaperones. The anti-apoptotic gene BAG3 was also highly up-regulated, providing evidence that the cells expressed pro-survival processes. The immune response of the HD11 cell line to LPS in the heat stress environment (up-regulation of CCL4, CCL5, IL1B, IL8 and iNOS) was higher than in thermoneutral conditions. However, the peak in the transcriptional regulation of the immune genes was after two hours of temperature-recovery. Therefore, we propose the potential influence of the extracellular heat shock proteins not only in mitigating effects of abiotic stress but also in triggering the higher level of the immune responses. Finally, use of correlation networks for the data analysis aided in discovering subtle differences in the gene expression (i.e. the role

  19. C-terminal binding protein (CtBP) activates the expression of E-box clock genes with CLOCK/CYCLE in Drosophila.

    PubMed

    Itoh, Taichi Q; Matsumoto, Akira; Tanimura, Teiichi

    2013-01-01

    In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC) is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes"). Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP), a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per), vrille (vri), and PAR domain protein 1ε (Pdp1ε). Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.

  20. Prevalence of interferon type I signature in CD14 monocytes of patients with Sjögren's syndrome and association with disease activity and BAFF gene expression

    PubMed Central

    Brkic, Zana; Maria, Naomi I; van Helden-Meeuwsen, Cornelia G; van de Merwe, Joop P; van Daele, Paul L; Dalm, Virgil A; Wildenberg, Manon E; Beumer, Wouter; Drexhage, Hemmo A; Versnel, Marjan A

    2013-01-01

    Objective To determine the prevalence of upregulation of interferon (IFN) type I inducible genes, the so called ‘IFN type I signature’, in CD14 monocytes in 69 patients with primary Sjögren's syndrome (pSS) and 44 healthy controls (HC) and correlate it with disease manifestations and expression of B cell activating factor (BAFF). Methods Expression of IFI44L, IFI44, IFIT3, LY6E and MX1 was measured using real time quantitative PCR in monocytes. Expression values were used to calculate IFN type I scores for each subject. pSS patients positive for the IFN type I signature (IFN score≥10) and patients negative for the signature (IFN score<10) were then compared for clinical disease manifestations and BAFF expression. A bioassay using a monocytic cell line was performed to study whether BAFF mRNA expression was inducible by IFN type I activity in serum of patients with pSS. Results An IFN type I signature was present in 55% of patients with pSS compared with 4.5% of HC. Patients with the IFN type I signature showed: (a) higher EULAR Sjögren's Syndrome Disease Activity Index scores; higher anti-Ro52, anti-Ro60 and anti-La autoantibodies; higher rheumatoid factor; higher serum IgG; lower C3, lower absolute lymphocyte and neutrophil counts; (b)higher BAFF gene expression in monocytes. In addition, serum of signature-positive patients induced BAFF gene expression in monocytes. Conclusions The monocyte IFN type I signature identifies a subgroup of patients with pSS with a higher clinical disease activity together with higher BAFF mRNA expression. Such patients might benefit from treatment blocking IFN type I production or activity. PMID:22736090

  1. Differences in muscle and adipose tissue gene expression and cardio-metabolic risk factors in the members of physical activity discordant twin pairs.

    PubMed

    Leskinen, Tuija; Rinnankoski-Tuikka, Rita; Rintala, Mirva; Seppänen-Laakso, Tuulikki; Pöllänen, Eija; Alen, Markku; Sipilä, Sarianna; Kaprio, Jaakko; Kovanen, Vuokko; Rahkila, Paavo; Oresic, Matej; Kainulainen, Heikki; Kujala, Urho M

    2010-09-16

    High physical activity/aerobic fitness predicts low morbidity and mortality. Our aim was to identify the most up-regulated gene sets related to long-term physical activity vs. inactivity in skeletal muscle and adipose tissues and to obtain further information about their link with cardio-metabolic risk factors. We studied ten same-sex twin pairs (age range 50-74 years) who had been discordant for leisure-time physical activity for 30 years. The examinations included biopsies from m. vastus lateralis and abdominal subcutaneous adipose tissue. RNA was analyzed with the genome-wide Illumina Human WG-6 v3.0 Expression BeadChip. For pathway analysis we used Gene Set Enrichment Analysis utilizing active vs. inactive co-twin gene expression ratios. Our findings showed that among the physically active members of twin pairs, as compared to their inactive co-twins, gene expression in the muscle tissue samples was chronically up-regulated for the central pathways related to energy metabolism, including oxidative phosphorylation, lipid metabolism and supportive metabolic pathways. Up-regulation of these pathways was associated in particular with aerobic fitness and high HDL cholesterol levels. In fat tissue we found physical activity-associated increases in the expression of polyunsaturated fatty acid metabolism and branched-chain amino acid degradation gene sets both of which associated with decreased 'high-risk' ectopic body fat and plasma glucose levels. Consistent with other findings, plasma lipidomics analysis showed up-regulation of the triacylglycerols containing the polyunsaturated fatty acids. Our findings identified skeletal muscle and fat tissue pathways which are associated with the long-term physical activity and reduced cardio-metabolic disease risk, including increased aerobic fitness. In particular, improved skeletal muscle oxidative energy and lipid metabolism as well as changes in adipocyte function and redistribution of body fat are associated with reduced

  2. Concomitant Interferon Alpha Stimulation and TLR3 Activation Induces Neuronal Expression of Depression-Related Genes That Are Elevated in the Brain of Suicidal Persons

    PubMed Central

    Trippler, Martin; Lutterbeck, Melanie; Liu, Zijian J.; Truebner, Kurt; Bajanowski, Thomas; Gerken, Guido; Hermann, Dirk M.; Schlaak, Joerg F.

    2013-01-01

    We have previously identified 15 genes that are associated with the development of severe depressive side effects during the standard therapy with interferon alpha and ribavirin in the peripheral blood of hepatitis C virus infected patients. An enhanced expression of these genes was also found in the blood of psychiatric patients suffering severe depressive episode. Herein, we demonstrate that the same depression-related interferon-inducible genes (DRIIs) are also upregulated in post-mortem brains of suicidal individuals. Using cultured mouse hippocampal and prefrontal neurons we show that costimulation with murine IFN (mIFN) and the TLR3 agonist poly(I:C) promotes the expression of the described DRIIs, at the same time inducing pro-inflammatory cytokine expression through Stat1 and Stat3 activation, promoting neuronal apoptosis. Consequently, the upregulation of selective DRIIs, production of inflammatory cytokines and inhibition of neuronal plasticity may be involved in the pathogenesis of IFN-associated depression. PMID:24391741

  3. Anti-Eimeria activity of berberine and identification of associated gene expression changes in the mouse jejunum infected with Eimeria papillata.

    PubMed

    Dkhil, Mohamed A; Metwaly, Mahmoud S; Al-Quraishy, Saleh; Sherif, Nour E; Delic, Denis; Al Omar, Suliman Y; Wunderlich, Frank

    2015-04-01

    Plant-based natural products are promising sources for identifying novel agents with potential anti-Eimeria activity. This study explores possible effects of berberine on Eimeria papillata infections in the jejunum of male Swiss albino mice. Berberine chloride, when daily administered to mice during infection, impairs intracellular development and multiplication of E. papillata, evidenced as 60% reduction of maximal fecal output of oocysts on day 5 p.i. Concomitantly, berberine attenuates the inflammatory response, evidenced as decreased messenger RNA (mRNA) expression of IL-1β, IL-6, TNFα, IFNγ, and iNOS, as well as the oxidative stress response, evidenced as impaired increase in malondialdehyde, nitrate, and H2O2 and as prevented decrease in glutathione and catalase activity. Berberine also alters gene expression in the infected jejunum. On day 5 p.i., mRNA expression of 29 genes with annotated functions is more than 10-fold upregulated and that of 14 genes downregulated. Berberine downregulates the genes Xaf1, Itgb3bp, and Faim3 involved in apoptotic processes and upregulates genes involved in innate immune responses, as e.g., Colec11, Saa2, Klra8, Clec1b, and Crtam, especially the genes Cpa3, Fcer1a, and Mcpt1, Mcpt2, and Mcpt4 involved in mast cell activity. Additionally, 18 noncoding lincRNA species are differentially expressed more than 10-fold under berberine. Our data suggest that berberine induces hosts to exert anti-Eimeria activity by attenuating the inflammatory and oxidative stress response, by impairing apoptotic processes, and by activating local innate immune responses and epigenetic mechanisms in the host jejunum. Berberine has the potential as an anti-Eimeria food additive in animal farming.

  4. Chronic diclofenac exposure affects gill integrity and pituitary gene expression and displays estrogenic activity in nile tilapia (Oreochromis niloticus).

    PubMed

    Gröner, Frederike; Höhne, Christin; Kleiner, Wibke; Kloas, Werner

    2017-01-01

    Oreochromis niloticus has been exposed to diclofenac (DCF), a nonsteroidal anti-inflammatory drug prevalent in the aquatic environment, for 80 days post-hatch (dph). Concentrations ranged from environmentally relevant (0.1 μg L(-1) and 1 μg L(-1) DCF) up to 100-fold thereof. Population relevant endpoints (hatching, survival, growth) as well as gill histopathology were analyzed. On this level of examination only gills exhibited mild to moderate alterations. On the contrary, biomarkers associated with reproduction were affected due to DCF exposure, indicating the potential to affect sexual differentiation and gametogenesis by acting as an estrogenic endocrine disrupting compound (EDC) in tilapia. Vitellogenin (VTG) gene expression was significantly induced at 1 μg L(-1) DCF. In order to find an explanation, gene expression patterns of key enzymes of the biotransformation phases I, II, and III have been analyzed. It seems very likely that the detoxification metabolism is induced in a dose dependent manner at higher concentrations of DCF leading to the expression pattern of VTG mRNA. Our results suggest that DCF at environmentally relevant concentrations adversely affects O. niloticus gill histopathology and pituitary gene expression, and has the potential to act as an estrogenic EDC. The sensitivity of various endpoints, however, differs and therefore these endpoints should be linked.

  5. ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway

    PubMed Central

    1996-01-01

    AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Since some RTKs act through Ras and phosphatidylinositol 3-kinase (PI3K), we investigated the role of these pathways in ARIA signaling. Expression of activated Ras or Raf mimicked ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocked the effect of ARIA. ARIA rapidly activated erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulated association of PI3K with erbB3, expression of an activated PI3K led to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolished the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways. PMID:8707830

  6. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

    PubMed Central

    Liu, Zhiqin; Shi, Lanping; Liu, Yanyan; Tang, Qian; Shen, Lei; Yang, Sheng; Cai, Jinsen; Yu, Huanxin; Wang, Rongzhang; Wen, Jiayu; Lin, Youquan; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; He, Shuilin

    2015-01-01

    The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper. PMID:26442088

  7. Differential Gene Expression in Glaucoma

    PubMed Central

    Jakobs, Tatjana C.

    2014-01-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell–matrix interactions and adhesion, the cell cycle, and the endothelin system. PMID:24985133

  8. Differential gene expression in glaucoma.

    PubMed

    Jakobs, Tatjana C

    2014-07-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell-matrix interactions and adhesion, the cell cycle, and the endothelin system.

  9. Resemblance and Dissemblance of Arabidopsis Type II Peroxiredoxins: Similar Sequences for Divergent Gene Expression, Protein Localization, and Activity1

    PubMed Central

    Bréhélin, Claire; Meyer, Etienne H.; de Souris, Jean-Paul; Bonnard, Géraldine; Meyer, Yves

    2003-01-01

    The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified. PMID:12913160

  10. The Rb/E2F pathway and Ras activation regulate RecQ helicase gene expression.

    PubMed

    Liu, Yongqing; El-Naggar, Shahenda; Clem, Brian; Chesney, Jason; Dean, Douglas C

    2008-06-01

    Disruption of the Rb (retinoblastoma protein)/E2F cell-cycle pathway and Ras activation are two of the most frequent events in cancer, and both of these mutations place oncogenic stress on cells to increase DNA replication. In the present study, we demonstrate that these mutations have an additive effect on induction of members of the RecQ DNA helicase family. RecQ activity is important for genomic stability, initiation of DNA replication and telomere maintenance, and mutation of the BLM (Bloom's syndrome gene), WRN (Werner's syndrome gene) or RECQL4 (Rothmund-Thomson syndrome gene) family members leads to premature aging syndromes characterized by genetic instability and telomere loss. RecQ family members are frequently overexpressed in cancers, and overexpression of BLM has been shown to cause telomere elongation. Concomitant with induction of RecQ genes in response to Rb family mutation and Ras activation, we show an increase in the number of telomeric repeats. We suggest that this induction of RecQ genes in response to common oncogenic mutations may explain the up-regulation of the genes seen in cancers, and it may provide a means for transformed cells to respond to an increased demand for DNA replication.

  11. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  12. Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain

    PubMed Central

    Fleiss, Bobbi; Kawano, Go; Ezzati, Mojgan; Rocha-Ferreira, Eridan; Hristova, Mariya; Bennett, Kate; Fierens, Igor; Burnett, Ryan; Chaban, Badr; Alonso-Alconada, Daniel; Oliver-Taylor, Aaron; Tachsidis, Ilias; Rostami, Jamshid; Gressens, Pierre; Sanders, Robert D.

    2016-01-01

    Exposure of the brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex, incompletely understood and may be sexually dimorphic, but include developmentally inappropriate apoptosis, inflammation and a disruption to cognitively salient gene expression. We investigated the effects of a 6h isoflurane exposure on cell death, microglial activation and gene expression in the male neonatal piglet brain. Piglets (n = 6) were randomised to: (i) naive controls or (ii) 6h isoflurane. Cell death (TUNEL and caspase-3) and microglial activation were recorded in 7 brain regions. Changes in gene expression (microarray and qPCR) were assessed in the cingulate cortex. Electroencephalography (EEG) was recorded throughout. Isoflurane anesthesia induced significant increases in cell death in the cingulate and insular cortices, caudate nucleus, thalamus, putamen, internal capsule, periventricular white matter and hippocampus. Dying cells included both neurons and oligodendrocytes. Significantly, microglial activation was observed in the insula, pyriform, hippocampus, internal capsule, caudate and thalamus. Isoflurane induced significant disruption to the expression of 79 gene transcripts, of these 26 are important for the control of transcription and 23 are important for the mediation of neural plasticity, memory formation and recall. Our observations confirm that isoflurane increases apoptosis and inflammatory responses in the neonatal piglet brain but also suggests novel additional mechanisms by which isoflurane may induce adverse neural and cognitive development by disrupting the expression of genes mediating activity dependent development of neural circuits, the predictive adaptive responses of the brain, memory formation and recall. PMID:27898690

  13. Zipf's Law in Gene Expression

    NASA Astrophysics Data System (ADS)

    Furusawa, Chikara; Kaneko, Kunihiko

    2003-02-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1; i.e., they obey Zipf’s law. Furthermore, by simulations of a simple model with an intracellular reaction network, we found that Zipf’s law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  14. Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Shieh, Jia-Ching; Cheng, Yu-Che; Su, Mao-Chang; Moore, Michael; Choo, Yen; Klug, Aaron

    2007-01-01

    Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity. PMID:17710146

  15. Under the influence of the active deodorant ingredient 4-hydroxy-3-methoxybenzyl alcohol, the skin bacterium Corynebacterium jeikeium moderately responds with differential gene expression.

    PubMed

    Brune, Iris; Becker, Anke; Paarmann, Daniel; Albersmeier, Andreas; Kalinowski, Jörn; Pühler, Alfred; Tauch, Andreas

    2006-12-15

    A 70mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Corynebacterium jeikeium, a skin bacterium that is predominantly present in the human axilla and involved in axillary odor formation. Oligonucleotides representing 100% of the predicted coding regions of the C. jeikeium K411 genome were designed and spotted in quadruplicate onto epoxy-coated glass slides. The quality of the printed microarray was demonstrated by co-hybridization with fluorescently labeled cDNA probes obtained from exponentially growing C. jeikeium cultures. Accordingly, genes detected with different intensities resulting in log(2) transformed ratios greater than 0.8 or smaller than -0.8 can be regarded as differentially expressed with a confidence level greater than 99%. In an application example, we measured global changes of gene expression during growth of C. jeikeium in the presence of different concentrations of the deodorant component 4-hydroxy-3-methoxybenzyl alcohol that is active in preventing body odor formation. Global expression profiling revealed that low concentrations of 4-hydroxy-3-methoxybenzyl alcohol (0.5 and 2.5mg/ml) had almost no detectable effect on the transcriptome of C. jeikeium. A slightly higher concentration of 4-hydroxy-3-methoxybenzyl alcohol (5mg/ml) resulted in differential expression of 95 genes, 86 of which showed an enhanced expression when compared to a control culture. Besides many genes encoding proteins that apparently participate in transcription and translation, the drug resistance determinant cmx and the predicted virulence factors sapA and sapD showed significantly enhanced expression levels. Differential expression of relevant genes was validated by real-time reverse transcription PCR assays.

  16. Control of gene expression in trypanosomes.

    PubMed Central

    Vanhamme, L; Pays, E

    1995-01-01

    Trypanosomes are protozoan agents of major parasitic diseases such as Chagas' disease in South America and sleeping sickness of humans and nagana disease of cattle in Africa. They are transmitted to mammalian hosts by specific insect vectors. Their life cycle consists of a succession of differentiation and growth phases requiring regulated gene expression to adapt to the changing extracellular environment. Typical of such stage-specific expression is that of the major surface antigens of Trypanosoma brucei, procyclin in the procyclic (insect) form and the variant surface glycoprotein (VSG) in the bloodstream (mammalian) form. In trypanosomes, the regulation of gene expression is effected mainly at posttranscriptional levels, since primary transcription of most of the genes occurs in long polycistronic units and is constitutive. The transcripts are processed by transsplicing and polyadenylation under the influence of intergenic polypyrimidine tracts. These events show some developmental regulation. Untranslated sequences of the mRNAs seem to play a prominent role in the stage-specific control of individual gene expression, through a modulation of mRNA abundance. The VSG and procyclin transcription units exhibit particular features that are probably related to the need for a high level of expression. The promoters and RNA polymerase driving the expression of these units resemble those of the ribosomal genes. Their mutually exclusive expression is ensured by controls operating at several levels, including RNA elongation. Antigenic variation in the bloodstream is achieved through DNA rearrangements or alternative activation of the telomeric VSG gene expression sites. Recent discoveries, such as the existence of a novel nucleotide in telomeric DNA and the generation of point mutations in VSG genes, have shed new light on the mechanisms and consequences of antigenic variation. PMID:7603410

  17. Pathogen-associated molecular patterns activate expression of genes involved in cell proliferation, immunity and detoxification in the amebocyte-producing organ of the snail Biomphalaria glabrata

    PubMed Central

    Zhang, Si-Ming; Loker, Eric S.; Sullivan, John T.

    2017-01-01

    The anterior pericardial wall of the snail Biomphalaria glabrata has been identified as a site of hemocyte production, hence has been named the amebocyte-producing organ (APO). A number of studies have shown that exogenous abiotic and biotic substances, including pathogen associated molecular patterns (PAMPs), are able to stimulate APO mitotic activity and/or enlarge its size, implying a role for the APO in innate immunity. The molecular mechanisms underlying such responses have not yet been explored, in part due to the difficulty in obtaining sufficient APO tissue for gene expression studies. By using a modified RNA extraction technique and microarray technology, we investigated transcriptomic responses of APOs dissected from snails at 24 hours post-injection with two bacterial PAMPs, lipopolysaccharide (LPS) and peptidoglycan (PGN), or with fucoidan (FCN), which may mimic fucosyl-rich glycan PAMPs on sporocysts of Schistosoma mansoni. Based upon the number of genes differentially expressed, LPS exhibited the strongest activity, relative to saline-injected controls. A concurrent activation of genes involved in cell proliferation, immune response and detoxification metabolism was observed. A gene encoding checkpoint 1 kinase, a key regulator of mitosis, was highly expressed after stimulation by LPS. Also, seven different aminoacyl-tRNA synthetases that play an essential role in protein synthesis were found to be highly expressed. In addition to stimulating genes involved in cell proliferation, the injected substances, especially LPS, also induced expression of a number of immune-related genes including arginase, peptidoglycan recognition protein short form, tumor necrosis factor receptor, ficolin, calmodulin, bacterial permeability increasing proteins and E3 ubiquitin-protein ligase. Importantly, significant up-regulation was observed in four GiMAP (GTPase of immunity-associated protein) genes, a result which provides the first evidence suggesting an immune role of

  18. Isolation of 10 differentially expressed cDNAs in p53-induced apoptosis: activation of the vertebrate homologue of the drosophila seven in absentia gene.

    PubMed Central

    Amson, R B; Nemani, M; Roperch, J P; Israeli, D; Bougueleret, L; Le Gall, I; Medhioub, M; Linares-Cruz, G; Lethrosne, F; Pasturaud, P; Piouffre, L; Prieur, S; Susini, L; Alvaro, V; Millasseau, P; Guidicelli, C; Bui, H; Massart, C; Cazes, L; Dufour, F; Bruzzoni-Giovanelli, H; Owadi, H; Hennion, C; Charpak, G; Telerman, A

    1996-01-01

    We report the isolation of 10 differentially expressed cDNAs in the process of apoptosis induced by the p53 tamor suppressor. As a global analytical method, we performed a differential display of mRNA between mouse M1 myeloid leukemia cells and derived clone LTR6 cells, which contain a stably transfected temperature-sensitive mutant of p53. At 32 degrees C wild-type p53 function is activated in LTR6 cells, resulting in programmed cell death. Eight genes are activated (TSAP; tumor suppressor activated pathway), and two are inhibited (TSIP, tumor suppressor inhibited pathway) in their expression. None of the 10 sequences has hitherto been recognized as part of the p53 signaling pathway. Three TSAPs are homologous to known genes. TSAP1 corresponds to phospholipase C beta 4. TSAP2 has a conserved domain homologous to a multiple endocrine neoplasia I (ZFM1) candidate gene. TSAP3 is the mouse homologue of the Drosophila seven in absentia gene. These data provide novel molecules involved in the pathway of wild-type p53 activation. They establish a functional link between a homologue of a conserved developmental Drosophila gene and signal transduction in tumor suppression leading to programmed cell death. Images Fig. 2 Fig. 3 PMID:8632996

  19. Neutral Lipids and Peroxisome Proliferator-Activated Receptor-γ Control Pulmonary Gene Expression and Inflammation-Triggered Pathogenesis in Lysosomal Acid Lipase Knockout Mice

    PubMed Central

    Lian, Xuemei; Yan, Cong; Qin, Yulin; Knox, Lana; Li, Tingyu; Du, Hong

    2005-01-01

    The functional roles of neutral lipids in the lung are poorly understood. However, blocking cholesteryl ester and triglyceride metabolism in lysosomal acid lipase gene knockout mice (lal−/−) results in severe pathogenic phenotypes in the lung, including massive neutrophil infiltration, foamy macrophage accumulation, unwanted cell growth, and emphysema. To elucidate the mechanism underlining these pathologies, we performed Affymetrix GeneChip microarray analysis of 1-, 3-, and 6-month-old mice and identified aberrant gene expression that progressed with age. Among changed genes, matrix metalloproteinase (MMP)-12, apoptosis inhibitor 6 (Api-6), erythroblast transformation-specific domain (Ets) transcription factor family member Spi-C, and oncogene MafB were increased 100-, 70-, 40-, and 10-fold, respectively, in lal−/− lungs versus the wild-type lungs. The pathogenic increases of these molecules occurred primarily in alveolar type II epithelial cells. Transcriptional activities of the MMP-12 and Api-6 promoters were stimulated by Spi-C or MafB in respiratory epithelial cells. Treatment with 9-hydroxyoctadecanoic acids and ciglitazone significantly rescued lal−/− pulmonary inflammation and aberrant gene expression. In addition, both compounds as well as peroxisome proliferator-activated receptor gamma inhibited MMP-12 and Api-6 promoter activities. These data suggest that inflammation-triggered cell growth and emphysema during lysosomal acid lipase deficiency are partially caused by peroxisome proliferator-activated receptor-γ inactivation. PMID:16127159

  20. Expression of mosquito active toxin genes by a Colombian native strain of the gram-negative bacterium Asticcacaulis excentricus.

    PubMed

    Romero, M; Gil, F M; Orduz, S

    2001-02-01

    Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.

  1. Two short sequences in OsNAR2.1 promoter are necessary for fully activating the nitrate induced gene expression in rice roots

    PubMed Central

    Liu, Xiaoqin; Feng, Huimin; Huang, Daimin; Song, Miaoquan; Fan, Xiaorong; Xu, Guohua

    2015-01-01

    Nitrate is an essential nitrogen source and serves as a signal to control growth and gene expression in plants. In rice, OsNAR2.1 is an essential partner of multiple OsNRT2 nitrate transporters for nitrate uptake over low and high concentration range. Previously, we have reported that −311 bp upstream fragment from the translational start site in the promoter of OsNAR2.1 gene is the nitrate responsive region. To identify the cis-acting DNA elements necessary for nitrate induced gene expression, we detected the expression of beta-glucuronidase (GUS) reporter in the transgenic rice driven by the OsNAR2.1 promoter with different lengths and site mutations of the 311 bp region. We found that −129 to −1 bp region is necessary for the nitrate-induced full activation of OsNAR2.1. Besides, the site mutations showed that the 20 bp fragment between −191 and −172 bp contains an enhancer binding site necessary to fully drive the OsNAR2.1 expression. Part of the 20 bp fragment is commonly presented in the sequences of different promoters of both the nitrate induced NAR2 genes and nitrite reductase NIR1 genes from various higher plants. These findings thus reveal the presence of conserved cis-acting element for mediating nitrate responses in plants. PMID:26150107

  2. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    SciTech Connect

    Pittius, C.W.; Hennighausen, L.; Lee, E.; Westphal, H.; Nicols, E.; Vitale, J.; Gordon, K. )

    1988-08-01

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

  3. An RT-qPCR approach to study the expression of genes responsible for sugar assimilation during rehydration of active dry yeast.

    PubMed

    Vaudano, Enrico; Costantini, Antonella; Noti, Olta; Garcia-Moruno, Emilia

    2010-09-01

    A short reactivation period in aqueous media is required for active dry yeast (ADY) to be utilised in winemaking. Rehydration restores the active metabolic conditions necessary for good fermentative and competitive abilities. We used a reverse transcription-quantitative PCR (RT-qPCR) method with relative quantification to investigate the expression of seven hexose transporter genes (HXT1-7) and one invertase-encoding gene (SUC2) during ADY rehydration in water with or without sucrose. For this, seven candidate reference genes were evaluated, and the three most stably expressed genes were selected and used for mRNA normalisation. The results show that, during the rehydration in the presence of sucrose, yeast cells are able to immediately hydrolyse this sugar into glucose and fructose as soon as they are introduced in the medium. Subsequently, differential glucose/fructose uptake occurs, which is mediated by hexose transporters. At the transcriptomic level, there is a strong induction of the high-affinity transporters, HXT2 and HXT4, and the low-affinity transporters, HXT3 and HXT1, when ADY is rehydrated with sucrose, while HXT5 and HXT6/7 are expressed at high levels with a moderate tendency to decrease. In water, the HXT2 gene was the only one of the transporter genes studied that showed significant variations. These results suggest that during rehydration, expression is not simply regulated by the affinity to hexose but is also controlled by other mechanisms that allow the cell to bypass glucose control. Moreover, the expression of SUC2 showed little variation in media with sucrose, suggesting that other invertases and/or posttranscriptional controls exist.

  4. The regulation of Jmjd3 upon the expression of NF-κB downstream inflammatory genes in LPS activated vascular endothelial cells.

    PubMed

    Yu, Shaoqing; Chen, Xia; Xiu, Min; He, Feng; Xing, Juanjuan; Min, Dinghong; Guo, Fei

    2017-02-09

    Inflammatory mediators and adhesion molecules have been implicated in a variety of diseases including atherosclerosis. As both the mediator-releasing and targeted cells, vascular endothelial cells play key role in pathological processes. NF-κB signaling regulates a cluster of inflammatory factors in LPS-activated vascular endothelial cells but the underlying mechanisms remain largely unknown. Here, we investigated the epigenetic regulation of LPS upon the expression of inflammatory mediators and adhesion molecules. We found that LPS treatment promoted jmjd3 expression, enhanced Jmjd3 nuclear accumulation in human vascular endothelial cells. In addition, LPS enhanced the demethylation of H3K27me3, a specific substrate of Jmjd3. LPS treatment recruited Jmjd3 and NF-κB to the promoter region of target genes, suggesting Jmjd3 synergizes with NF-κB to activate the expression of target genes. We further found that Jmjd3 attenuated the methylation status in promoter region of target genes, culminating in target gene expression. Our findings unveil epigenetic regulations of LPS upon NF-κB pathway and identify Jmjd3 as a critical modulator of NF-κB pathway and potential therapeutic target for NF-κB related diseases including atherosclerosis.

  5. Gene cloning, characterization and expression and enzymatic activities related to trehalose metabolism during diapause of the onion maggot Delia antiqua (Diptera: Anthomyiidae).

    PubMed

    Guo, Qiang; Hao, You-Jin; Li, Yuan; Zhang, Yu-Juan; Ren, Shuang; Si, Feng-Ling; Chen, Bin

    2015-07-01

    Trehalose represents the main hemolymph sugar in many insects, and it functions in energy metabolism and protection in extreme environmental conditions. To gain an insight into trehalose functions in Delia antiqua diapausing pupae, genes encoding trehalose-6-phosphate synthase (TPS), trehalose-6-phosphatase (TPP) and trehalase (TRE) were identified and characterized. Analysis of the deduced amino acid sequences indicated that these genes were highly similar to each homolog from Diptera insects. Gene expressions and their enzyme activities were also investigated. The differential expressions of TPS and TPP shared very similar trends for summer and winter diapausing pupae. Their enzyme activities were consistent with the gene expressions. Trehalose concentrations in summer- and winter-diapausing pupae were lower at the initial phase (4.37-5.09μg/mg) but increased gradually and peaked in the maintenance phase (10.59-14.36μg/mg); the concentrations then declined in the quiescence phase. We speculated that a higher trehalose content during the maintenance stage may contribute to protein and/or biological membrane stabilization in winter or to desiccation resistance in the summertime. Diapause termination requires a decrease in the trehalose concentration to promote pupal-adult development. The glucose content also varied during the diapausing processes. Our results provide an overview of the differential expression levels of trehalose metabolic enzymes, confirming the important roles of trehalose in diapausing pupae of the onion maggot. Further work remains to explore its actual functions.

  6. The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes.

    PubMed Central

    Laurent, B C; Treitel, M A; Carlson, M

    1990-01-01

    The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose- and phosphate-regulated genes and appears to function in transcription. We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein. The N-terminal third of the protein is extremely rich in glutamine and proline. Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential. Evidence that SNF5 affects expression of the cell type-specific genes MF alpha 1 and BAR1 at the RNA level extends the known range of SNF5 function. SNF5 is apparently required for expression of a wide variety of differently regulated genes. A bifunctional SNF5-beta-galactosidase fusion protein was localized in the nucleus by immunofluorescence. No DNA-binding activity was detected for SNF5. A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator. Images PMID:2233708

  7. Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.

    PubMed

    Li, J; Gao, E; Mendelson, C R

    1998-02-20

    Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form

  8. 'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings.

    PubMed

    Saini, Geetanjali; Meskauskiene, Rasa; Pijacka, Wioletta; Roszak, Pawel; Sjögren, Lars L E; Clarke, Adrian K; Straus, Marco; Apel, Klaus

    2011-03-01

    Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.

  9. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

    PubMed Central

    Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

  10. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

    PubMed

    Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

  11. Wounding induces changes in tuber polyamine content, polyamine metabolic gene expression, and enzyme activity during closing layer formation and initiation of wound periderm formation.

    PubMed

    Lulai, Edward C; Neubauer, Jonathan D; Olson, Linda L; Suttle, Jeffrey C

    2015-03-15

    Tuber wound-healing processes are complex, and the associated regulation and modulation of these processes are poorly understood. Polyamines (PA) are involved in modulating a variety of responses to biotic and abiotic plant stresses and have been suggested to be involved in tuber wound responses. However, the time course of wound-induced changes in tuber PA content, activity of key biosynthetic enzymes and associated gene expression has not been determined and coordinated with major wound-healing processes. The objective of this study was to determine these wound-induced changes and their coordination with wound-healing processes. Wounding induced increases in putrescine (Put) and spermidine (Spd), but had only minor effects on spermine (Spm) content during the 168 h time course which encompassed the initiation and completion of the closing layer formation, and the initiation of cell division and wound periderm formation. As determinants of the first committed step in PA biosynthesis, arginine and ornithine decarboxylase (ADC and ODC, respectively) activities were below levels of detectability in resting tubers and expression of genes encoding these two enzymes was low. Within 6h of wounding, increases in the in vitro activities of ADC and ODC and expression of their cognate genes were observed. Expression of a gene encoding S-adenosylmethionine decarboxylase, required for Spd and Spm biosynthesis, was also increased 6h after wounding and remained elevated throughout the time course. Expression of a polyamine catabolic gene, encoding polyamine oxidase, was down-regulated after wounding. Results indicated a rapid wound-induced increase in PA biosynthesis during closing layer formation and the time of nuclei entry and exit from S-phase. PA content remained elevated as wound-induced cells became meristematic and initiated formation of the wound periderm suggesting sustained involvement in wound-healing.

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

    PubMed

    DeSmet, Marsha L; Fleet, James C

    2017-01-16

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

  13. Diosgenin inhibits osteoclastogenesis, invasion, and proliferation through the downregulation of Akt, I kappa B kinase activation and NF-kappa B-regulated gene expression.

    PubMed

    Shishodia, S; Aggarwal, B B

    2006-03-09

    Diosgenin, a steroidal saponin present in fenugreek (Trigonella foenum graecum) and other plants, has been shown to suppress inflammation, inhibit proliferation, and induce apoptosis in a variety of tumor cells, but through a mechanism that is poorly understood. In the present study, we report that diosgenin inhibits receptor-activated nuclear factor-kappaB ligand-induced osteoclastogenesis, suppresses tumor necrosis factor (TNF)-induced invasion, and blocks the proliferation of tumor cells, all activities known to be regulated by NF-kappaB. Diosgenin suppressed TNF-induced NF-kappaB activation as determined by DNA binding, activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation through inhibition of Akt activation. NF-kappaB-dependent reporter gene expression was also abrogated by diosgenin. TNF-induced expression of NF-kappaB-regulated gene products involved in cell proliferation (cyclin D1, COX-2, c-myc), antiapoptosis (IAP1, Bcl-2, Bcl-X(L), Bfl-1/A1, TRAF1 and cFLIP), and invasion (MMP-9) were also downregulated by the saponin. Diosgenin also potentiated the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results suggest that diosgenin suppresses proliferation, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression and enhances apoptosis induced by cytokines and chemotherapeutic agents.

  14. Keratin gene expression profiles after digit amputation in C57BL/6 vs. regenerative MRL mice imply an early regenerative keratinocyte activated-like state

    PubMed Central

    Cheng, Chia-Ho; Leferovich, John; Zhang, Xiang-Ming; Bedelbaeva, Khamilia; Gourevitch, Dmitri; Hatcher, Cathy J.; Basson, Craig T.; Heber-Katz, Ellen

    2013-01-01

    Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre- and postamputation at the midsecond phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding. PMID:23512742

  15. X chromosome regulation of autosomal gene expression in bovine blastocysts.

    PubMed

    Itoh, Yuichiro; Arnold, Arthur P

    2014-10-01

    Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.

  16. Intestinotrophic Glucagon-Like Peptide-2 (GLP-2) Activates Intestinal Gene Expression and Growth Factor-Dependent Pathways Independent of the Vasoactive Intestinal Peptide Gene in Mice

    PubMed Central

    Yusta, Bernardo; Holland, Dianne; Waschek, James A.

    2012-01-01

    The enteroendocrine and enteric nervous systems convey signals through an overlapping network of regulatory peptides that act either as circulating hormones or as localized neurotransmitters within the gastrointestinal tract. Because recent studies invoke an important role for vasoactive intestinal peptide (VIP) as a downstream mediator of glucagon-like peptide-2 (GLP-2) action in the gut, we examined the importance of the VIP-GLP-2 interaction through analysis of Vip−/− mice. Unexpectedly, we detected abnormal villous architecture, expansion of the crypt compartment, increased crypt cell proliferation, enhanced Igf1 and Kgf gene expression, and reduced expression of Paneth cell products in the Vip−/− small bowel. These abnormalities were not reproduced by antagonizing VIP action in wild-type mice, and VIP administration did not reverse the intestinal phenotype of Vip−/− mice. Exogenous administration of GLP-2 induced the expression of ErbB ligands and immediate-early genes to similar levels in Vip+/+ vs. Vip−/− mice. Moreover, GLP-2 significantly increased crypt cell proliferation and small bowel growth to comparable levels in Vip+/+ vs. Vip−/− mice. Unexpectedly, exogenous GLP-2 administration had no therapeutic effect in mice with dextran sulfate-induced colitis; the severity of colonic injury and weight loss was modestly reduced in female but not male Vip−/− mice. Taken together, these findings extend our understanding of the complex intestinal phenotype arising from loss of the Vip gene. Furthermore, although VIP action may be important for the antiinflammatory actions of GLP-2, the Vip gene is not required for induction of a gene expression program linked to small bowel growth after enhancement of GLP-2 receptor signaling. PMID:22535770

  17. Expression of urokinase-type plasminogen activator, stromelysin 1, stromelysin 3, and matrilysin genes in lung carcinomas.

    PubMed Central

    Bolon, I.; Devouassoux, M.; Robert, C.; Moro, D.; Brambilla, C.; Brambilla, E.

    1997-01-01

    We have previously shown that the extracellular-matrix-degrading enzymes, urokinase-type plasminogen activator (u-PA), stromelysin 1, stromelysin 3, and matrilysin, may play an important role in the transition from lung preneoplasia to invasive carcinoma. Using in situ hybridization and immunohistochemistry, we analyzed serial frozen sections for the expression of these enzymes in 89 lung carcinomas including 25 neuroendocrine (NE) carcinomas (10 small-cell lung carcinomas, 7 large-cell NE carcinomas, 1 atypical, and 7 typical carcinoids) and 64 non-small-cell, non-NE carcinomas (29 squamous and 7 basaloid carcinomas, 23 adenocarcinomas, and 5 large-cell carcinomas). Proteases, except matrilysin, were more often expressed in stromal than cancer cells. In non-small-cell, non-NE carcinomas, stromal co-expression of u-PA and stromelysin 3 was seen in 80 to 90% of the tumors and was highly correlated (P < 0.0001). Stromal u-PA and stromelysin 3 expression was linked to tumor size (P = 0.01 and 0.03, respectively) and lymph node involvement (P = 0.001 and 0.02, respectively). Epithelial expression of u-PA was correlated to tumor size (P = 0.04). Epithelial expression of stromelysin 3 predominated in squamous and basaloid carcinomas (P = 0.0005) and was inversely correlated to squamous differentiation (P = 0.018). Epithelial expression of matrilysin predominated in adenocarcinomas and large-cell carcinomas (P = 0.07). In NE carcinomas including small-cell lung carcinomas, stromal expression of u-PA was correlated to lymph node metastasis (P = 0.017). Epithelial expression of all enzymes were significantly less frequent in NE than in non-NE tumors. We conclude that 1) epithelial expression of matrix proteases in lung cancer is linked to cell phenotype (NE versus non-NE, squamous versus glandular) and 2) their stromal, rather than epithelial, expression influences local metastasis. Images Figure 1 PMID:9137088

  18. An extensive network of coupling among gene expression machines.

    PubMed

    Maniatis, Tom; Reed, Robin

    2002-04-04

    Gene expression in eukaryotes requires several multi-component cellular machines. Each machine carries out a separate step in the gene expression pathway, which includes transcription, several pre-messenger RNA processing steps and the export of mature mRNA to the cytoplasm. Recent studies lead to the view that, in contrast to a simple linear assembly line, a complex and extensively coupled network has evolved to coordinate the activities of the gene expression machines. The extensive coupling is consistent with a model in which the machines are tethered to each other to form 'gene expression factories' that maximize the efficiency and specificity of each step in gene expression.

  19. p53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer

    PubMed Central

    Zhang, Liang; Yang, Weiping; Zhu, Xiao; Wei, Changyuan

    2016-01-01

    p125 is one of four subunits of human DNA polymerases – DNA Pol δ as well as one of p53 target protein encoded by POLD1. However, the function and significance of p125 and the role that p53 plays in regulating p125 expression are not fully understood in breast cancer. Tissue sections of human breast cancer obtained from 70 patients whose median age was 47.6 years (range: 38–69 years) with stage II–III breast cancer were studied with normal breast tissue from the same patients and two human breast cell lines (MCF-7 and MCF-10A). p53 expression levels were reduced, while p125 protein expression was increased in human breast cancer tissues and cell line detected by Western blot and quantitative reverse transcriptase-polymerase chain reaction. The methylation level of the POLD1 gene promoter was greater in breast cancer tissues and cells when compared with normal tissues and cells. In MCF-7 cell model, p53 overexpression caused a decrease in the level of p125 protein, while the methylation level of the p125 gene promoter was also inhibited by p53 overexpression. To further investigate the regulating mechanism of p53 on p125 expression, our study focused on DNA methyltransferase 1 (DNMT1) and transcription factor Sp1. Both DNMT1 and Sp1 protein expression were reduced when p53 was overexpressed in MCF-7 cells. The Sp1 binding site appears to be important for DNMT1 gene transcription; Sp1 and p53 can bind together, which means that DNMT1 gene expression may be downregulated by p53 through binding to Sp1. Because DNMT1 methylation level of the p125 gene promoter can affect p125 gene transcription, we propose that p53 may indirectly regulate p125 gene promoter expression through the control of DNMT1 gene transcription. In conclusion, the data from this preliminary study have shown that p53 inhibits the methylation of p125 gene promoter by downregulating the activities of Sp1 and DNMT1 in breast cancer. PMID:27022290