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Sample records for upregulated cellular genes

  1. Kaposis Sarcoma-Associated Herpesvirus Interleukin-6 Modulates Endothelial Cell Movement by Upregulating Cellular Genes Involved in Migration

    PubMed Central

    Giffin, Louise; West, John A.

    2015-01-01

    ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of human Kaposis sarcoma, a tumor that arises from endothelial cells, as well as two B cell lymphoproliferative diseases, primary effusion lymphoma and multicentric Castlemans disease. KSHV utilizes a variety of mechanisms to evade host immune responses and promote cellular transformation and growth in order to persist for the life of the host. A viral homolog of human interleukin-6 (hIL-6) named viral interleukin-6 (vIL-6) is encoded by KSHV and expressed in KSHV-associated cancers. Similar to hIL-6, vIL-6 is secreted, but the majority of vIL-6 is retained within the endoplasmic reticulum, where it can initiate functional signaling through part of the interleukin-6 receptor complex. We sought to determine how intracellular vIL-6 modulates the host endothelial cell environment by analyzing vIL-6s impact on the endothelial cell transcriptome. vIL-6 significantly altered the expression of many cellular genes associated with cell migration. In particular, vIL-6 upregulated the host factor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) at the protein and message levels. CEACAM1 has been implicated in tumor invasion and metastasis and promotes migration and vascular remodeling in endothelial cells. We report that vIL-6 upregulates CEACAM1 by a STAT3-dependent mechanism and that CEACAM1 promotes vIL-6-mediated migration. Furthermore, latent and de novo KSHV infections of endothelial cells also induce CEACAM1 expression. Collectively, our data suggest that vIL-6 modulates endothelial cell migration by upregulating the expression of cellular factors, including CEACAM1. PMID:26646010

  2. Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication

    PubMed Central

    Mahller, YY; Sakthivel, B; Baird, WH; Aronow, BJ; Hsu, Y-H; Cripe, TP; Mehrian-Shai, R

    2010-01-01

    Oncolytic herpes simplex viruses (oHSVs) are promising anticancer therapeutics. We sought to characterize the functional genomic response of human cancer cells to oHSV infection using G207, an oHSV previously evaluated in a phase I trial. Five human malignant peripheral nerve sheath tumor cell lines, with differing sensitivity to oHSV, were infected with G207 for 6 h. Functional genomic analysis of virus-infected cells demonstrated large clusters of downregulated cellular mRNAs and smaller clusters of those upregulated, including 21 genes commonly upregulated in all five lines. Of these, 7 are known to be HSV-1 induced and 14 represent novel virus-regulated genes. Gene ontology analysis revealed that a majority of G207-upregulated genes are involved in Janus kinase/signal transducer and activator of transcription signaling, transcriptional regulation, nucleic acid metabolism, protein synthesis and apoptosis. Ingenuity networks highlighted nodes for AP-1 subunits and interferon signaling via STAT1, suppressor of cytokine signaling-1 (SOCS1), SOCS3 and RANTES. As biological confirmation, we found that virus-mediated upregulation of SOCS1 correlated with sensitivity to G207 and that depletion of SOCS1 impaired virus replication by >10-fold. Further characterization of roles provided by oHSV-induced cellular genes during virus replication may be utilized to predict oncolytic efficacy and to provide rational strategies for designing next-generation oncolytic viruses. PMID:18551144

  3. Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication.

    PubMed

    Mahller, Y Y; Sakthivel, B; Baird, W H; Aronow, B J; Hsu, Y-H; Cripe, T P; Mehrian-Shai, R

    2008-11-01

    Oncolytic herpes simplex viruses (oHSVs) are promising anticancer therapeutics. We sought to characterize the functional genomic response of human cancer cells to oHSV infection using G207, an oHSV previously evaluated in a phase I trial. Five human malignant peripheral nerve sheath tumor cell lines, with differing sensitivity to oHSV, were infected with G207 for 6 h. Functional genomic analysis of virus-infected cells demonstrated large clusters of downregulated cellular mRNAs and smaller clusters of those upregulated, including 21 genes commonly upregulated in all five lines. Of these, 7 are known to be HSV-1 induced and 14 represent novel virus-regulated genes. Gene ontology analysis revealed that a majority of G207-upregulated genes are involved in Janus kinase/signal transducer and activator of transcription signaling, transcriptional regulation, nucleic acid metabolism, protein synthesis and apoptosis. Ingenuity networks highlighted nodes for AP-1 subunits and interferon signaling via STAT1, suppressor of cytokine signaling-1 (SOCS1), SOCS3 and RANTES. As biological confirmation, we found that virus-mediated upregulation of SOCS1 correlated with sensitivity to G207 and that depletion of SOCS1 impaired virus replication by >10-fold. Further characterization of roles provided by oHSV-induced cellular genes during virus replication may be utilized to predict oncolytic efficacy and to provide rational strategies for designing next-generation oncolytic viruses. PMID:18551144

  4. Up-Regulation of leucocytes Genes Implicated in Telomere Dysfunction and Cellular Senescence Correlates with Depression and Anxiety Severity Scores

    PubMed Central

    Teyssier, Jean-Raymond; Chauvet-Gelinier, Jean-Christophe; Ragot, Sylviane; Bonin, Bernard

    2012-01-01

    Background Major depressive disorder (MDD) is frequently associated with chronic medical illness responsible of increased disability and mortality. Inflammation and oxidative stress are considered to be the major mediators of the allostatic load, and has been shown to correlate with telomere erosion in the leucocytes of MDD patients, leading to the model of accelerated aging. However, the significance of telomere length as an exclusive biomarker of aging has been questioned on both methodological and biological grounds. Furthermore, telomeres significantly shorten only in patients with long lasting MDD. Sensitive and dynamic functional biomarkers of aging would be clinically useful to evaluate the somatic impact of MDD. Methodology To address this issue we have measured in the blood leucocytes of MDD patients (N?=?17) and controls (N?=?16) the expression of two genes identified as robust biomarkers of human aging and telomere dysfunction: p16INK4a and STMN1. We have also quantified the transcripts of genes involved in the repair of oxidative DNA damage at telomeres (OGG1), telomere regulation and elongation (TERT), and in the response to biopsychological stress (FOS and DUSP1). Results The OGG1, p16INK4a, and STMN1 gene were significantly up-regulated (25 to 100%) in the leucocytes of MDD patients. Expression of p16INK4a and STMN1 was directly correlated with anxiety scores in the depression group, and that of p16INK4a, STMN and TERT with the depression and anxiety scores in the combined sample (MDD plus controls). Furthermore, we identified a unique correlative pattern of gene expression in the leucocytes of MDD subjects. Conclusions Expression of p16INK4 and STMN1 is a promising biomarker for future epidemiological assessment of the somatic impact of depressive and anxious symptoms, at both clinical and subclinical level in both depressive patients and general population. PMID:23185405

  5. Upregulating endogenous genes by an RNA-programmable artificial transactivator

    PubMed Central

    Fimiani, Cristina; Goina, Elisa; Mallamaci, Antonello

    2015-01-01

    To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action. PMID:26152305

  6. Upregulating endogenous genes by an RNA-programmable artificial transactivator.

    PubMed

    Fimiani, Cristina; Goina, Elisa; Mallamaci, Antonello

    2015-09-18

    To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action. PMID:26152305

  7. Nrf2 Protein Up-regulates Antiapoptotic Protein Bcl-2 and Prevents Cellular Apoptosis*

    PubMed Central

    Niture, Suryakant K.; Jaiswal, Anil K.

    2012-01-01

    Nuclear transcription factor Nrf2 regulates the expression and coordinated induction of a battery of genes encoding cytoprotective and drug transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival, and increased drug resistance. In this study, we investigated the role of Nrf2 in up-regulation of antiapoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Exposure of mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells to antioxidant tert-butylhydroquinone led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element between nucleotides −3148 and −3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/supershift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 antioxidant response element. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2, resulting in an increased Bcl-2 level and decreased etoposide- and UV/γ radiation-mediated DNA fragmentation. In addition, siRNA-mediated down-regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knockdown of Bcl-2 in Nrf2-activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and growth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance. PMID:22275372

  8. Acoustic trauma triggers upregulation of serotonin receptor genes.

    PubMed

    Smith, Adam R; Kwon, Jae Hyun; Navarro, Marco; Hurley, Laura M

    2014-09-01

    Hearing loss induces plasticity in excitatory and inhibitory neurotransmitter systems in auditory brain regions. Excitatory-inhibitory balance is also influenced by a range of neuromodulatory regulatory systems, but less is known about the effects of auditory damage on these networks. In this work, we studied the effects of acoustic trauma on neuromodulatory plasticity in the auditory midbrain of CBA/J mice. Quantitative PCR was used to measure the expression of serotonergic and GABAergic receptor genes in the inferior colliculus (IC) of mice that were unmanipulated, sham controls with no hearing loss, and experimental individuals with hearing loss induced by exposure to a 116dB, 10kHz pure tone for 3h. Acoustic trauma induced substantial hearing loss that was accompanied by selective upregulation of two serotonin receptor genes in the IC. The Htr1B receptor gene was upregulated tenfold following trauma relative to shams, while the Htr1A gene was upregulated threefold. In contrast, no plasticity in serotonin receptor gene expression was found in the hippocampus, a region also innervated by serotonergic projections. Analyses in the IC demonstrated that acoustic trauma also changed the coexpression of genes in relation to each other, leading to an overexpression of Htr1B compared to other genes. These data suggest that acoustic trauma induces serotonergic plasticity in the auditory system, and that this plasticity may involve comodulation of functionally-linked receptor genes. PMID:24997228

  9. Acoustic trauma triggers upregulation of serotonin receptor genes

    PubMed Central

    Smith, Adam R.; Kwon, Jae Hyun; Navarro, Marco; Hurley, Laura M.

    2014-01-01

    Hearing loss induces plasticity in excitatory and inhibitory neurotransmitter systems in auditory brain regions. Excitatory-inhibitory balance is also influenced by a range of neuromodulatory regulatory systems, but less is known about the effects of auditory damage on these networks. In this work, we studied the effects of acoustic trauma on neuromodulatory plasticity in the auditory midbrain of CBA/J mice. Quantitative PCR was used to measure the expression of serotonergic and GABAergic receptor genes in the inferior colliculus (IC) of mice that were unmanipulated, sham controls with no hearing loss, and experimental individuals with hearing loss induced by exposure to a 116 dB, 10 kHz pure tone for 3 hours. Acoustic trauma induced substantial hearing loss that was accompanied by selective upregulation of two serotonin receptor genes in the IC. The Htr1B receptor gene was upregulated tenfold following trauma relative to shams, while the Htr1A gene was upregulated threefold. In contrast, no plasticity in serotonin receptor gene expression was found in the hippocampus, a region also innervated by serotonergic projections. Analyses in the IC demonstrated that acoustic trauma also changed the coexpression of genes in relation to each other, leading to an overexpression of Htr1B compared to other genes.. These data suggest that acoustic trauma induces serotonergic plasticity in the auditory system, and that this plasticity may involve comodulation of functionally-linked receptor genes. PMID:24997228

  10. Sin3B expression is required for cellular senescence and is upregulated upon oncogenic stress

    PubMed Central

    Grandinetti, Kathryn B.; Jelinic, Petar; DiMauro, Teresa; Pellegrino, Jessica; Rodriguez, Ruben Fernandez; Finnerty, Patricia M.; Ruoff, Rachel; Bardeesy, Nabeel; Logan, Susan K.; David, Gregory

    2009-01-01

    Serial passage of primary mammalian cells or strong mitogenic signals induces a permanent exit from the cell cycle called senescence. A characteristic of senescent cells is the heterochromatinization of loci encoding pro-proliferative genes leading to their transcriptional silencing. Senescence is thought to represent a defense mechanism against uncontrolled proliferation and cancer. Consequently, genetic alterations that allow senescence bypass are associated with susceptibility to oncogenic transformation. We demonstrate that fibroblasts genetically inactivated for the chromatin associated Sin3B protein are refractory to replicative and oncogene-induced senescence. Conversely, overexpression of Sin3B triggers senescence and the formation of senescence associated heterochromatic foci. While Sin3B is strongly up-regulated upon oncogenic stress, decrease in expression of Sin3B is associated with tumor progression in vivo, suggesting that expression of Sin3B may represent a barrier against transformation. Together, these results underscore the contribution of senescence in tumor suppression and suggest that expression of chromatin modifiers is modulated at specific stages of cellular transformation. Consequently, these findings suggest that modulation of Sin3B-associated activities may represent new therapeutic opportunities for treatment of cancers. PMID:19654306

  11. The immediate early genes of human cytomegalovirus upregulate tumor necrosis factor-alpha gene expression.

    PubMed Central

    Geist, L J; Monick, M M; Stinski, M F; Hunninghake, G W

    1994-01-01

    Cytomegalovirus (CMV) is an important cause of disease in the immunocompromised patient and CMV infection is associated with predominantly mononuclear inflammatory response. Since products of the CMV immediate early (IE) gene region are potent trans-activators, we used the monocyte cell line THP-1 and a transient transfection assay to determine if these viral proteins upregulate expression of the TNF gene. The IE genes of CMV upregulated TNF gene activity as judged by increases in promoter activity, steady state mRNA, and protein production. The presence or absence of the 3' untranslated region of the TNF gene did not affect gene expression induced by the IE gene products. These studies suggest that activation of TNF gene expression by the CMV IE gene products may, in part, account for the inflammatory response associated with CMV infections. Images PMID:8113386

  12. Cytomegalovirus upregulates vascular endothelial growth factor and its second cellular kinase domain receptor in human fibroblasts.

    PubMed

    Heiske, Andreas; Roettger, Yvonne; Bacher, Michael

    2012-10-01

    Human cytomegalovirus (HCMV) activation and elevated levels of vascular endothelial growth factor (VEGF) have been found to be associated with transplant rejection. However, information is lacking about whether elevated levels of this multifunctional factor are directly due to viral activation, or if they derive from impurities within the culture supernatant of infected cell cultures. We used purified as well as unpurified viral stocks to infect human fibroblasts in vitro and applied PCR, Western blot, ELISA, and immunofluorescence staining to investigate the expression of VEGF and its receptors. Our data suggest that HCMV infection triggers an early and sustained induction of VEGF and kinase insert domain receptor (KDR) mRNAs, whereas transcript levels of FLT-1 remain unchanged by viral infection. Analysis of the extracellular VEGF and cellular KDR protein expression after infection with purified and unpurified HCMV strains AD169 and TOLEDO showed, in clear contrast to UV-inactivated virus preparations, an increased release of VEGF and KDR proteins. In addition, immunofluorescence revealed that HCMV infection was also accompanied by a profound increase in intracellular VEGF and KDR levels. These results confirm that active HCMV infection is required to induce VEGF and the most important VEGF receptor KDR, and that the upregulation of VEGF and KDR are a direct viral effect and not a secondary effect mediated by inflammatory cytokines within the supernatant. The HCMV-dependent upregulation of VEGF and KDR contributes to the theory that viral-induced immune mediators play a key role in transplant rejection. PMID:22985288

  13. Up-regulation of metallothionein gene expression in parkinsonian astrocytes.

    PubMed

    Michael, Gregory J; Esmailzadeh, Sharmin; Moran, Linda B; Christian, Lynne; Pearce, Ronald K B; Graeber, Manuel B

    2011-11-01

    The role of glial cells in Parkinson's disease (PD) is unclear. We have previously reported a striking up-regulation of DnaJB6 heat shock protein in PD substantia nigra astrocytes. Whole genome transcriptome analysis also indicated increased expression of metallothionein genes in substantia nigra and cortex of sporadic PD cases. Metallothioneins are metal-binding proteins in the CNS that are released by astrocytes and associated with neuroprotection. Metallothionein expression was investigated in 18 PD cases and 15 non-PD controls using quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridisation (ISH) and immunocytochemistry (ICC). We observed a strong increase in the expression of metallothioneins MT1E, MT1F, MT1G, MT1H, MT1M, MT1X and MT2A in both PD nigra and frontal cortex. Expression of LRP2 (megalin), the neuronal metallothionein receptor was also significantly increased. qRT-PCR confirmed metallothionein up-regulation. Astrocytes were found to be the main source of metallothioneins 1 and 2 based on ISH results, and this finding was confirmed by ICC. Our findings demonstrate metallothionein expression by reactive astrocytes in PD nigra and support a neuroprotective role for these cells. The traditional view that nigral astrocytes are non-reactive in PD is clearly incorrect. However, it is possible that astrocytes are themselves affected by the disease process which may explain their comparatively modest and previously overlooked response. PMID:21800131

  14. Decomposing gene expression into cellular processes.

    PubMed

    Segal, E; Battle, A; Koller, D

    2003-01-01

    We propose a probabilistic model for cellular processes, and an algorithm for discovering them from gene expression data. A process is associated with a set of genes that participate in it; unlike clustering techniques, our model allows genes to participate in multiple processes. Each process may be active to a different degree in each experiment. The expression measurement for gene g in array a is a sum, over all processes in which g participates, of the activity levels of these processes in array a. We describe an iterative procedure, based on the EM algorithm, for decomposing the expression matrix into a given number of processes. We present results on Yeast gene expression data, which indicate that our approach identifies real biological processes. PMID:12603020

  15. The NS5A protein of hepatitis C virus transcriptionally upregulates the AGR3 gene expression.

    PubMed

    Chen, Ming; Gan, Xiang; Deng, Lin; Hotta, Hak

    2015-01-01

    The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) is a multifunctional protein involved in the HCV lifecycle and pathogenesis. The precise molecular mechanisms of NS5A-mediated pathogenesis still remain to be clarified. In this study, we performed cDNA microarray analysis on NS5A-expressing HEK293 cells and the non-expressing control to screen the possible cellular genes dysregulated by NS5A. Subsequent quantitative real time PCR (qRT-PCR) analysis on NS5A-expressing cells and the control confirmed that NS5A upregulated the anterior gradient homolog 3 (AGR3) mRNA expression. The domain III of NS5A was responsible for the activation of AGR3 gene expression. AGR3 mRNA expression levels were upregulated also in Huh7.5 cells harboring a full-genome HCV-1b RNA replicon (FGR) and in those infected with HCV-2a. Moreover, AGR3 promoter activity was activated in NS5A-expressing cells, FGR-harboring cells and HCV-infected cells. Taken together, our present results suggest that HCV NS5A transcriptionally activates the cancer-associated AGR3 gene. This may be a novel mechanism of HCV-mediated pathogenesis, especially hepatocarcinogenesis. PMID:25868611

  16. DNA stabilization by the upregulation of estrogen signaling in BRCA gene mutation carriers

    PubMed Central

    Suba, Zsuzsanna

    2015-01-01

    Currently available scientific evidence erroneously suggests that mutagenic weakness or loss of the BRCA1/2 genes may liberate the proliferative effects of estrogen signaling, which provokes DNA damage and genomic instability. Conversely, BRCA mutation seems to be an imbalanced defect, crudely inhibiting the upregulation of estrogen receptor expression and liganded transcriptional activity, whereas estrogen receptor-repressor functions become predominant. In BRCA-proficient cases, estrogen signaling orchestrates the activity of cell proliferation and differentiation with high safety, while upregulating the expression and DNA-stabilizing impact of BRCA genes. In turn, BRCA proteins promote estrogen signaling by proper estrogen synthesis via CYP19 gene regulation and by induction of the appropriate expression and transcriptional activity of estrogen receptors. In this exquisitely organized regulatory system, the dysfunction of each player may jeopardize genome stability and lead to severe chronic diseases, such as cancer development. Female organs, such as breast, endometrium, and ovary, exhibiting regular cyclic proliferative activity are particularly vulnerable in case of disturbances in either estrogen signaling or BRCA-mediated DNA repair. BRCA mutation carrier women may apparently be healthy or exhibit clinical signs of deficient estrogen signaling in spite of hyperestrogenism. Even women who enjoy sufficient compensatory DNA-defending activities are at risk of tumor development because many endogenous and environmental factors may jeopardize the mechanisms of extreme compensatory processes. Natural estrogens have numerous benefits in tumor prevention and therapy even in BRCA mutation carriers. There are no toxic effects even in sky-high doses and all physiologic cellular functions are strongly upregulated, while malignant tumor cells are recognized and killed in a Janus-faced manner. PMID:26028963

  17. Retinoblastoma protein promotes oxidative phosphorylation through upregulation of glycolytic genes in oncogene-induced senescent cells.

    PubMed

    Takebayashi, Shin-Ichiro; Tanaka, Hiroshi; Hino, Shinjiro; Nakatsu, Yuko; Igata, Tomoka; Sakamoto, Akihisa; Narita, Masashi; Nakao, Mitsuyoshi

    2015-08-01

    Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene-induced senescence (OIS). A combination of real-time metabolic monitoring, and metabolome and gene expression analyses showed that OIS-induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB-depleted or downstream glycolytic enzyme-depleted OIS cells, suggesting that RB-mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells. PMID:26009982

  18. In GERD patients, mucosal repair associated genes are upregulated in non-inflamed oesophageal epithelium

    PubMed Central

    De Vries, D R; Ter Linde, J J M; Van Herwaarden, M A; Schwartz, M P; Shephard, P; Geng, M M; Smout, A J P M; Samsom, M

    2009-01-01

    Abstract Previous studies addressing the effects of acid reflux and PPI therapy on gene expression in oesophageal epithelium concentrated on inflamed tissue. We aimed to determine changes in gene expression in non-inflamed oesophageal epithelium of GERD patients. Therefore, we included 20 GERD patients with pathological total 24-hr acid exposure of 6–12% and SAP ≥ 95%. Ten patients discontinued PPI treatment (PPI-), 10 took pantoprazole 40 mg bid (PPI+). Ten age/sex-matched healthy controls were recruited. Biopsies were taken from non-inflamed mucosa 6 cm and 16 cm proximal to the squamocolumnar junction (SCJ). Gene expression profiling of biopsies from 6 cm was performed on Human Genome U133 Plus 2.0 arrays (Affymetrix). Genes exhibiting a fold change >1.4 (t-test P-value < 1E– 4) were considered differentially expressed. Results were confirmed by real-time RT-PCR. In PPI- patients, 92 microarray probesets were deregulated. The majority of the corresponding genes were associated with cell–cell contacts, cytoskeletal reorganization and cellular motility, suggesting facilitation of a migratory phenotype. Genes encoding proteins with anti-apoptotic or anti-proliferative functions or stress-protective functions were also deregulated. No probesets were deregulated in PPI+ patients. QPCR analysis of 20 selected genes confirmed most of the deregulations in PPI- patients, and showed several deregulated genes in PPI+ patients as well. In the biopsies taken at 16 cm QPCR revealed no deregulations of the selected genes. We conclude that upon acid exposure, oesophageal epithelial cells activate a process globally known as epithelial restitution: up-regulation of anti-apoptotic, anti-oxidant and migration associated genes. Possibly this process helps maintaining barrier function. PMID:19413890

  19. Ginseng Berry Extract Prevents Atherogenesis via Anti-Inflammatory Action by Upregulating Phase II Gene Expression

    PubMed Central

    Kim, Chun-Ki; Cho, Dong Hui; Lee, Kyu-Sun; Lee, Dong-Keon; Park, Chan-Woong; Kim, Wan Gi; Lee, Sang Jun; Ha, Kwon-Soo; Goo Taeg, Oh; Kwon, Young-Guen; Kim, Young-Myeong

    2012-01-01

    Ginseng berry possesses higher ginsenoside content than its root, which has been traditionally used in herbal medicine for many human diseases, including atherosclerosis. We here examined the antiatherogenic effects of the Korean ginseng berry extract (KGBE) and investigated its underlying mechanism of action in vitro and in vivo. Administration of KGBE decreased atherosclerotic lesions, which was inversely correlated with the expression levels of phase II genes to include heme oxygenase-1 (HO-1) and glutamine-cysteine ligase (GCL). Furthermore, KGBE administration suppressed NF-?B-mediated expression of atherogenic inflammatory genes (TNF-?, IL-1?, iNOS, COX-2, ICAM-1, and VCAM-1), without altering serum cholesterol levels, in ApoE?/? mice fed a high fat-diet. Treatment with KGBE increased phase II gene expression and suppressed lipopolysaccharide-induced reactive oxygen species production, NF-?B activation, and inflammatory gene expression in primary macrophages. Importantly, these cellular events were blocked by selective inhibitors of HO-1 and GCL. In addition, these inhibitors reversed the suppressive effect of KGBE on TNF-?-mediated induction of ICAM-1 and VCAM-1, resulting in decreased interaction between endothelial cells and monocytes. These results suggest that KGBE ameliorates atherosclerosis by inhibiting NF-?B-mediated expression of atherogenic genes via upregulation of phase II enzymes and thus has therapeutic or preventive potential for atherosclerosis. PMID:23243449

  20. TNF-beta (lymphotoxin) strongly upregulates TNF-alpha gene expression in human peripheral blood lymphocytes.

    PubMed

    Owen-Schaub, L B; De Mars, M; Murphy, E C; Grimm, E A

    1990-01-01

    We investigated whether TNF-beta could induce or modulate TNF-alpha gene expression in human peripheral blood lymphocytes. For these experiments, lymphocytes were cultured in serum-free medium for 48 hours with human recombinant TNF-beta in the presence or absence of human recombinant IL-2. At the end of the culture period, total cellular RNA was isolated and probed for TNF-alpha mRNA using an S1 nuclease protection assay. TNF-beta alone was unable to induce lymphocyte TNF-alpha mRNA. However, when TNF-beta was used in combination with IL-2 an 8- to 12-fold increase in TNF-alpha mRNA compared to lymphocytes activated in IL-2 alone was observed. Secreted TNF-alpha was measured in the culture supernatants by TNF-alpha specific ELISA. Consistent with the results of mRNA analysis, TNF-beta alone did stimulate TNF-alpha secretion, but lymphocytes activated with TNF-beta/IL-2 demonstrated a 3- to 10-fold increase in secreted TNF-alpha over that induced by IL-2 alone. These experiments demonstrate that TNF-beta can participate in the upregulation of TNF-alpha gene expression in lymphocytes and suggest that cellular dysregulations involving autocrine TNF production may be exacerbated by this positive feedback pathway. PMID:1708843

  1. Ethanol Upregulates NMDA Receptor Subunit Gene Expression in Human Embryonic Stem Cell-Derived Cortical Neurons

    PubMed Central

    Gelernter, Joel; Park, In-Hyun; Zhang, Huiping

    2015-01-01

    Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as in vitro cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (ALDH2), anti-apoptosis (BCL2 and CCND2), neurotransmission (NMDA receptor subunit genes: GRIN1, GRIN2A, GRIN2B, and GRIN2D), calcium channel activity (ITPR2), or transcriptional repression (JARID2). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (GRIN1: 1.93-fold, P = 0.003; GRIN2A: 1.40-fold, P = 0.003; GRIN2B: 1.75-fold, P = 0.002; GRIN2D: 1.86-fold, P = 0.048; BCL2: 1.34-fold, P = 0.031), and the results of GRIN1, GRIN2A, and GRIN2B survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations. PMID:26266540

  2. DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

    PubMed Central

    Cao, Huimin; Wang, Li; Chen, Beibei; Zheng, Peng; He, Yi; Ding, Yubin; Deng, Yushuang; Lu, Xi; Guo, Xiuming; Zhang, Yuping; Li, Yu; Yu, Gang

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important transcription factor in the defense against oxidative stress. Cumulative evidence has shown that oxidative stress plays a key role in the pathogenesis of Alzheimer’s disease (AD). Previous animal and clinical studies had observed decreased expression of Nrf2 in AD. However, the underlying regulation mechanisms of Nrf2 in AD remain unclear. Here, we used the DNA methyltransferases (Dnmts) inhibitor 5-aza-2′-deoxycytidine (5-Aza) to test whether Nrf2 expression was regulated by methylation in N2a cells characterizing by expressing human Swedish mutant amyloid precursor protein (N2a/APPswe). We found 5-Aza treatment increased Nrf2 at both messenger RNA and protein levels via downregulating the expression of Dnmts and DNA demethylation. In addition, 5-Aza-mediated upregulation of Nrf2 expression was concomitant with increased nuclear translocation of Nrf2 and higher expression of Nrf2 downstream target gene NAD(P)H:quinone oxidoreductas (NQO1). Our study showed that DNA demethylation promoted the Nrf2 cell signaling pathway, which may enhance the antioxidant system against AD development. PMID:26779013

  3. Designer gene networks: Towards fundamental cellular control

    NASA Astrophysics Data System (ADS)

    Hasty, Jeff; Isaacs, Farren; Dolnik, Milos; McMillen, David; Collins, J. J.

    2001-03-01

    The engineered control of cellular function through the design of synthetic genetic networks is becoming plausible. Here we show how a naturally occurring network can be used as a parts list for artificial network design, and how model formulation leads to computational and analytical approaches relevant to nonlinear dynamics and statistical physics. We first review the relevant work on synthetic gene networks, highlighting the important experimental findings with regard to genetic switches and oscillators. We then present the derivation of a deterministic model describing the temporal evolution of the concentration of protein in a single-gene network. Bistability in the steady-state protein concentration arises naturally as a consequence of autoregulatory feedback, and we focus on the hysteretic properties of the protein concentration as a function of the degradation rate. We then formulate the effect of an external noise source which interacts with the protein degradation rate. We demonstrate the utility of such a formulation by constructing a protein switch, whereby external noise pulses are used to switch the protein concentration between two values. Following the lead of earlier work, we show how the addition of a second network component can be used to construct a relaxation oscillator, whereby the system is driven around the hysteresis loop. We highlight the frequency dependence on the tunable parameter values, and discuss design plausibility. We emphasize how the model equations can be used to develop design criteria for robust oscillations, and illustrate this point with parameter plots illuminating the oscillatory regions for given parameter values. We then turn to the utilization of an intrinsic cellular process as a means of controlling the oscillations. We consider a network design which exhibits self-sustained oscillations, and discuss the driving of the oscillator in the context of synchronization. Then, as a second design, we consider a synthetic network with parameter values near, but outside, the oscillatory boundary. In this case, we show how resonance can lead to the induction of oscillations and amplification of a cellular signal. Finally, we construct a toggle switch from positive regulatory elements, and compare the switching properties for this network with those of a network constructed using negative regulation. Our results demonstrate the utility of model analysis in the construction of synthetic gene regulatory networks.

  4. Proteomic and Transcriptomic Analyses Reveal Genes Upregulated by cis-Dichloroethene in Polaromonas sp. Strain JS666?

    PubMed Central

    Jennings, Laura K.; Chartrand, Michelle M. G.; Lacrampe-Couloume, Georges; Lollar, Barbara Sherwood; Spain, Jim C.; Gossett, James M.

    2009-01-01

    Polaromonas sp. strain JS666 is the only bacterial isolate capable of using cis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathione S-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from ?17.4 to ?22.4. Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C=C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666. PMID:19363075

  5. Downregulation of lysyl oxidase and upregulation of cellular thiols in rat fetal lung fibroblasts treated with cigarette smoke condensate.

    PubMed

    Chen, Li-Jun; Zhao, Yinzhi; Gao, Song; Chou, Iih-Nan; Toselli, Paul; Stone, Phillip; Li, Wande

    2005-02-01

    Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as gamma-GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema. PMID:15509664

  6. Identification of a conserved set of upregulated genes in mouse skeletal muscle hypertrophy and regrowth

    PubMed Central

    Chaillou, Thomas; Jackson, Janna R.; England, Jonathan H.; Kirby, Tyler J.; Richards-White, Jena; Esser, Karyn A.; Dupont-Versteegden, Esther E.

    2014-01-01

    The purpose of this study was to compare the gene expression profile of mouse skeletal muscle undergoing two forms of growth (hypertrophy and regrowth) with the goal of identifying a conserved set of differentially expressed genes. Expression profiling by microarray was performed on the plantaris muscle subjected to 1, 3, 5, 7, 10, and 14 days of hypertrophy or regrowth following 2 wk of hind-limb suspension. We identified 97 differentially expressed genes (?2-fold increase or ?50% decrease compared with control muscle) that were conserved during the two forms of muscle growth. The vast majority (?90%) of the differentially expressed genes was upregulated and occurred at a single time point (64 out of 86 genes), which most often was on the first day of the time course. Microarray analysis from the conserved upregulated genes showed a set of genes related to contractile apparatus and stress response at day 1, including three genes involved in mechanotransduction and four genes encoding heat shock proteins. Our analysis further identified three cell cycle-related genes at day and several genes associated with extracellular matrix (ECM) at both days 3 and 10. In conclusion, we have identified a core set of genes commonly upregulated in two forms of muscle growth that could play a role in the maintenance of sarcomere stability, ECM remodeling, cell proliferation, fast-to-slow fiber type transition, and the regulation of skeletal muscle growth. These findings suggest conserved regulatory mechanisms involved in the adaptation of skeletal muscle to increased mechanical loading. PMID:25554798

  7. Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing

    PubMed Central

    Huber, Evamaria; Vlasny, Daniela; Jeckel, Sonja; Stubenrauch, Frank; Iftner, Thomas

    2004-01-01

    To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 ? and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin ?, which are involved in cell motility and tissue invasion, as well as casein kinase 1 ?, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin ? in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays. PMID:15220421

  8. Thyroid hormones upregulate apolipoprotein E gene expression in astrocytes.

    PubMed

    Roman, Corina; Fuior, Elena V; Trusca, Violeta G; Kardassis, Dimitris; Simionescu, Maya; Gafencu, Anca V

    Apolipoprotein E (apoE), a protein mainly involved in lipid metabolism, is associated with several neurodegenerative disorders including Alzheimer's disease. Despite numerous attempts to elucidate apoE gene regulation in the brain, the exact mechanism is still uncovered. The mechanism of apoE gene regulation in the brain involves the proximal promoter and multienhancers ME.1 and ME.2, which evolved by gene duplication. Herein we questioned whether thyroid hormones and their nuclear receptors have a role in apoE gene regulation in astrocytes. Our data showed that thyroid hormones increase apoE gene expression in HTB14 astrocytes in a dose-dependent manner. This effect can be intermediated by the thyroid receptor β (TRβ) which is expressed in these cells. In the presence of triiodothyronine (T3) and 9-cis retinoic acid, in astrocytes transfected to overexpress TRβ and retinoid X receptor α (RXRα), apoE promoter was indirectly activated through the interaction with ME.2. To determine the location of TRβ/RXRα binding site on ME.2, we performed DNA pull down assays and found that TRβ/RXRα complex bound to the region 341-488 of ME.2. This result was confirmed by transient transfection experiments in which a series of 5'- and 3'-deletion mutants of ME.2 were used. These data support the existence of a biologically active TRβ binding site starting at 409 in ME.2. In conclusion, our data revealed that ligand-activated TRβ/RXRα heterodimers bind with high efficiency on tissue-specific distal regulatory element ME.2 and thus modulate apoE gene expression in the brain. PMID:26519880

  9. Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B

    SciTech Connect

    Hamm, Rebecca; Zeino, Maen; Frewert, Simon; Efferth, Thomas

    2014-11-15

    Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H{sup +}-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes, is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation.

  10. Lapatinib enhances trastuzumab-mediated antibody-dependent cellular cytotoxicity via upregulation of HER2 in malignant mesothelioma cells

    PubMed Central

    OKITA, RIKI; SHIMIZU, KATSUHIKO; NOJIMA, YUJI; YUKAWA, TAKURO; MAEDA, AI; SAISHO, SHINSUKE; NAKATA, MASAO

    2015-01-01

    EGFR/HER2 are frequently expressed in MPM tissues, however, no studies have shown the clinical benefit of using EGFR/HER2-targeting drugs in patients with malignant pleural mesothelioma (MPM). It was reported that the tyrosine kinase inhibitor (TKI) lapatinib enhanced trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) in HER2-positive breast cancer, suggesting that this combination is a promising strategy for MPM treatment. The aim of the present study was to explore the possibility of a TKI combined with trastuzumab to enhance ADCC in MPM cells. Five MPM cell lines were used to test the effects of TKIs targeting EGFR (gefitinib, afatinib and lapatinib) on cell proliferation and the expression of the HER family receptor. The combined effects of TKI with trastuzumab on ADCC were evaluated using the LDH release assay. Additionally, MPM cells were isolated from patients and evaluated for lapatinib-induced upregulation of HER family receptors and trastuzumab- or cetuximab-mediated ADCC. In MPM cell lines, HER2 expression was upregulated by lapatinib, downregulated by afatinib and unaffected by gefitinib. As expected, more trastuzumab bound to MPM cells pretreated with lapatinib than untreated cells, resulting in the enhancement of trastuzumab-mediated ADCC in MPM cells. In patient-derived MPM cells, both HER2 and EGFR were upregulated by lapatinib, resulting in the enhancement of both trastuzumab- and cetuximab-mediated ADCC. Of the three TKIs, only lapatinib enhanced trastuzumab-mediated ADCC via the upregulation of HER2 expression in MPM cells, suggesting that sequential combination of lapatinib and trastuzumab may be a promising strategy for MPM treatment. PMID:26503698

  11. HPV16 E2 could act as down-regulator in cellular genes implicated in apoptosis, proliferation and cell differentiation

    PubMed Central

    2011-01-01

    Background Human Papillomavirus (HPV) E2 plays several important roles in the viral cycle, including the transcriptional regulation of the oncogenes E6 and E7, the regulation of the viral genome replication by its association with E1 helicase and participates in the viral genome segregation during mitosis by its association with the cellular protein Brd4. It has been shown that E2 protein can regulate negative or positively the activity of several cellular promoters, although the precise mechanism of this regulation is uncertain. In this work we constructed a recombinant adenoviral vector to overexpress HPV16 E2 and evaluated the global pattern of biological processes regulated by E2 using microarrays expression analysis. Results The gene expression profile was strongly modified in cells expressing HPV16 E2, finding 1048 down-regulated genes, and 581 up-regulated. The main cellular pathway modified was WNT since we found 28 genes down-regulated and 15 up-regulated. Interestingly, this pathway is a convergence point for regulating the expression of genes involved in several cellular processes, including apoptosis, proliferation and cell differentiation; MYCN, JAG1 and MAPK13 genes were selected to validate by RT-qPCR the microarray data as these genes in an altered level of expression, modify very important cellular processes. Additionally, we found that a large number of genes from pathways such as PDGF, angiogenesis and cytokines and chemokines mediated inflammation, were also modified in their expression. Conclusions Our results demonstrate that HPV16 E2 has regulatory effects on cellular gene expression in HPV negative cells, independent of the other HPV proteins, and the gene profile observed indicates that these effects could be mediated by interactions with cellular proteins. The cellular processes affected suggest that E2 expression leads to the cells in to a convenient environment for a replicative cycle of the virus. PMID:21599968

  12. Accumulated SET protein up-regulates and interacts with hnRNPK, increasing its binding to nucleic acids, the Bcl-xS repression, and cellular proliferation

    SciTech Connect

    Almeida, Luciana O.; Garcia, Cristiana B.; Matos-Silva, Flavia A.; Curti, Carlos; Leopoldino, Andria M.

    2014-02-28

    Highlights: hnRNPK is a new target of SET. SET regulates hnRNPK. SET and hnRNPK accumulation promotes tumorigenesis. SET accumulation is a potential model to study genes regulated by SET-hnRNPK. - Abstract: SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SEThnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.

  13. Molecular crowding shapes gene expression in synthetic cellular nanosystems

    NASA Astrophysics Data System (ADS)

    Tan, Cheemeng; Saurabh, Saumya; Bruchez, Marcel P.; Schwartz, Russell; Leduc, Philip

    2013-08-01

    The integration of synthetic and cell-free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution-based chemistry, where only the concentrations of reacting species modulate gene expression rates. However, it is known that macromolecular crowding, a key feature in natural cells, can dramatically influence biochemical kinetics via volume exclusion effects, which reduce diffusion rates and enhance binding rates of macromolecules. Here, we demonstrate that macromolecular crowding can increase the robustness of gene expression by integrating synthetic cellular components of biological circuits and artificial cellular nanosystems. Furthermore, we reveal how ubiquitous cellular modules, including genetic components, a negative feedback loop and the size of the crowding molecules can fine-tune gene circuit response to molecular crowding. By bridging a key gap between artificial and living cells, our work has implications for efficient and robust control of both synthetic and natural cellular circuits.

  14. Expression patterns and action analysis of genes associated with physiological responses during rat liver regeneration: Cellular immune response

    PubMed Central

    Zhang, Lian-Xing; Zhao, Li-Feng; Zhang, An-Shi; Chen, Xiao-Guang; Xu, Cun-Shuan

    2006-01-01

    AIM: To study the cellular immune response during rat liver regeneration (LR) at a transcriptional level. METHODS: Genes associated with the cellular immune response were obtained by collecting the data from databases and retrieving articles. Gene expression changes during LR were detected by rat genome 230 2.0 array. RESULTS: A total of 127 genes were found to be associated with LR. The number of initially and totally expressing genes in the initial phase of LR [0.5-4 h after partial hepatectomy (PH)], transition from G0-G1 (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and structure-function reconstruction (66-168 h after PH) was 54, 11, 34, 3 and 54, 49, 70, 49 respectively, illustrating that the associated genes were mainly triggered at the initiation of LR, and worked at different phases. According to their expression similarity, these genes were classified into 41 up-regulated, 21 predominantly up-regulated, 41 down-regulated, 14 predominantly down-regulated, 10 similarly up-regulated and down-regulated genes, respectively. The total up- and down-regulated expression times were 419 and 274, respectively, demonstrating that the expression of most genes was increased while the expression of a small number of genes was decreased. Their time relevance was classified into 14 groups, showing that the cellular physiological and biochemical activities were staggered during LR. According to the gene expression patterns, they were classified into 21 types, showing the activities were diverse and complicated during LR. CONCLUSION: Antigen processing and presentation are enhanced mainly in the forepart, prophase and anaphase of LR. T-cell activation and antigen elimination are enhanced mainly in the forepart and prophase of LR. A total of 127 genes associated with LR play an important role in cellular immunity. PMID:17167843

  15. Gene up-regulation in response to predator kairomones in the water flea, Daphnia pulex

    PubMed Central

    2010-01-01

    Background Numerous cases of predator-induced polyphenisms, in which alternate phenotypes are produced in response to extrinsic stimuli, have been reported in aquatic taxa to date. The genus Daphnia (Branchiopoda, Cladocera) provides a model experimental system for the study of the developmental mechanisms and evolutionary processes associated with predator-induced polyphenisms. In D. pulex, juveniles form neckteeth in response to predatory kairomones released by Chaoborus larvae (Insecta, Diptera). Results Previous studies suggest that the timing of the sensitivity to kairomones in D. pulex can generally be divided into the embryonic and postembryonic developmental periods. We therefore examined which of the genes in the embryonic and first-instar juvenile stages exhibit different expression levels in the presence or absence of predator kairomones. Employing a candidate gene approach and identifying differentially-expressed genes revealed that the morphogenetic factors, Hox3, extradenticle and escargot, were up-regulated by kairomones in the postembryonic stage and may potentially be responsible for defense morph formation. In addition, the juvenile hormone pathway genes, JHAMT and Met, and the insulin signaling pathway genes, InR and IRS-1, were up-regulated in the first-instar stage. It is well known that these hormonal pathways are involved in physiological regulation following morphogenesis in many insect species. During the embryonic stage when morphotypes were determined, one of the novel genes identified by differential display was up-regulated, suggesting that this gene may be related to morphotype determination. Biological functions of the up-regulated genes are discussed in the context of defense morph formation. Conclusions It is suggested that, following the reception of kairomone signals, the identified genes are involved in a series of defensive phenotypic alterations and the production of a defensive phenotype. PMID:20433737

  16. Upregulated Annexin A1 promotes cellular invasion in triple-negative breast cancer.

    PubMed

    Okano, Maiko; Kumamoto, Kensuke; Saito, Motonobu; Onozawa, Hisashi; Saito, Katsuharu; Abe, Noriko; Ohtake, Tohru; Takenoshita, Seiichi

    2015-03-01

    Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein, involved in anti-inflammatory effects, regulation of cellular differentiation, proliferation and apoptosis. While many studies have investigated the ANXA1 expression in various tumor types, the role of ANXA1 is not fully understood. Therefore, in the present study, we evaluated the ANXA1 expression in 211 breast cancer patients and compared the levels with clinicopathological factors. ANXA1 was positively expressed in 31 (14.7%) of the 211 cases in our cohort, and these positive cases were associated with triple-negative breast cancer (TNBC) (P=0.007) and venous invasion (P=0.028). The in vitro cell experiment found that the MDA-MB-231 cell line, which is a TNBC cell line, highly expressed ANXA1. Using this cell line, the functional role of ANXA1 in breast cancer was revealed and the knockdown of ANXA1 by specific siRNA demonstrated a significant reduction in cellular invasion. Further experiments indicated that ANXA1 was induced by hypoxia with hypoxia-inducible factor-1α induction. These results suggested that ANXA1, which enhanced breast cancer invasion and metastasis under hypoxia, were significantly associated with the worst patient outcome. This is particularly noted in TNBC, the group of breast cancer with the worst outcome for which new therapeutic implications are required. PMID:25592491

  17. Stem cells as cellular vehicles for gene therapy against glioblastoma

    PubMed Central

    Wang, Wei; Liu, Fanlong; Xiang, Bingyu; Xiang, Charlie; Mou, Xiaozhou

    2015-01-01

    Glioblastoma (GBM) is the most common and deadliest primary tumor in adults, with current treatments having limited specific and efficient delivery of therapeutic drugs to tumor sites or cells. Therefore, the development of alternative treatment options is urgently needed. Stem cells are considered as ideal cellular vehicles for gene therapy against glioblastoma. In this paper, we reviewed the recent studies investigating the use of different types of stem cells as cellular vehicles and the gene of interests against the glioblastoma, as well as the future directions of the application of cellular vehicles mediated therapy for glioblastoma. PMID:26770303

  18. Cellular and Molecular Factors in Flexor Tendon Repair and Adhesions: A Histological and Gene Expression Analysis

    PubMed Central

    Juneja, Subhash C.; Schwarz, Edward M.; OKeefe, Regis J.; Awad, Hani A.

    2013-01-01

    Flexor tendon healing is mediated by cell proliferation, migration, and ECM synthesis that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor tendon adhesion formation has been linked to TGF-?. To elucidate the cellular and molecular events in this pathology, we implanted live FDL grafts from the reporter mouse Rosa26LacZ/+ in WT recipients, and used histological ?-galactosidase (?-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and RT-PCR to measure gene expression of TGF-? and its receptors, extracellular matrix (ECM) proteins, and MMPs and their regulators. Over the course of healing, graft cellularity and ?-gal activity progressively increased, and ?-gal-positive cells migrated out of the Rosa26LacZ/+ graft. In addition, there was evidence of influx of host cells (?-gal-negative) into the gliding space and the graft, suggesting that both graft and host cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1 expression was highest in the early phases of healing and gradually decreased thereafter, whereas Tgfb3 increased and remained upregulated later. The expression of TGF-? receptors was also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which inflammatory MMPs were highest early and matrix MMPs increased over time. These findings offer important insights into the complex cellular and molecular factors during flexor tendon healing. PMID:23586515

  19. Cocaine up-regulates Fra-2 and sigma-1 receptor gene and protein expression in brain regions involved in addiction and reward.

    PubMed

    Liu, Yun; Chen, Guang-Di; Lerner, Megan R; Brackett, Daniel J; Matsumoto, Rae R

    2005-08-01

    Sigma receptors have recently been implicated in the actions of cocaine, and antagonists of these receptors prevent many acute and subchronic cocaine effects. A previous study revealed that the immediate early gene fra-2 is up-regulated after cocaine administration, and this effect is prevented by the sigma-1 receptor antagonist BD1063 [1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine]. In the present study, the effects of cocaine and BD1063 on the expression of six fos and jun genes were evaluated in mouse brains using cDNA microarrays. Several of these genes were altered by cocaine, but only the alteration in fra-2 was prevented by BD1063. The time courses of fra-2 and sigma-1 receptor gene and protein expression in different brain regions were also determined. Cocaine up-regulated fra-2, which was followed by a later up-regulation of sigma-1 receptors. The cocaine-induced up-regulation of fra-2 and sigma-1 receptor genes and proteins were detected in whole brain, striatum, and cortex, but not in cerebellum. All of these cocaine-induced effects were prevented by BD1063. The interaction between cocaine, fra-2, and sigma-1 receptors involves brain regions that are established components of the neural circuit for reward, suggesting that they may contribute to the enduring changes that underlie the cellular basis of drug abuse. PMID:15879001

  20. Mechanisms of Hypoxic Up-Regulation of Versican Gene Expression in Macrophages

    PubMed Central

    Sotoodehnejadnematalahi, Fattah; Staples, Karl J.; Chrysanthou, Elvina; Pearson, Helen; Ziegler-Heitbrock, Loems; Burke, Bernard

    2015-01-01

    Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a range of hypoxia-inducible genes. The matrix proteoglycan versican has been identified as one such gene, but the mechanisms responsible for hypoxic induction are not fully characterised. Here we investigate the up-regulation of versican by hypoxia in primary human monocyte-derived macrophages (HMDM), and, intriguingly, show that versican mRNA is up-regulated much more highly (>600 fold) by long term hypoxia (5 days) than by 1 day of hypoxia (48 fold). We report that versican mRNA decay rates are not affected by hypoxia, demonstrating that hypoxic induction of versican mRNA is mediated by increased transcription. Deletion analysis of the promoter identified two regions required for high level promoter activity of luciferase reporter constructs in human macrophages. The hypoxia-inducible transcription factor HIF-1 has previously been implicated as a key potential regulator of versican expression in hypoxia, however our data suggest that HIF-1 up-regulation is unlikely to be principally responsible for the high levels of induction observed in HMDM. Treatment of HMDM with two distinct specific inhibitors of Phosphoinositide 3-kinase (PI3K), LY290042 and wortmannin, significantly reduced induction of versican mRNA by hypoxia and provides evidence of a role for PI3K in hypoxic up-regulation of versican expression. PMID:26057378

  1. Ascorbate up-regulates MLH1 (Mut L homologue-1) and p73: implications for the cellular response to DNA damage.

    PubMed

    Catani, M Valeria; Costanzo, Antonio; Savini, Isabella; Levrero, Massimo; de Laurenzi, Vincenzo; Wang, Jean Y J; Melino, Gerry; Avigliano, Luciana

    2002-06-01

    We have found previously that ascorbic acid (vitamin C), as well as acting as a radical scavenger, may modulate the expression of several genes [i.e. fra-1, glutathione S-transferase Pi (GSTpi) and Mut L homologue-1 (MLH1)] in human keratinocytes. In the present paper, we demonstrate that MLH1, as well as its downstream target p73, can be positively modulated by this antioxidant vitamin, indeed, up-regulation of the two mRNAs was observed after just 2 h, and increased further up to 16 h of treatment. Modulation of MLH1 and p73 gene expression improves cellular susceptibility to apoptosis triggered by the DNA-damaging agent cisplatin. Indeed, in ascorbate-supplemented cells, increased cisplatin-induced apoptosis was seen, involving activation of the MLH1/c-Abl/p73 signalling cascade. Our results were further confirmed by studies performed on genetically defined mutants, i.e. mouse embryo fibroblasts derived from knock-out animals for c-Abl or p53, as well as human colon carcinoma cell lines deficient in MLH1. The increased sensitivity to cisplatin observed in ascorbate-loaded cells appeared to be dependent exclusively on MLH1 and c-Abl expression, and independent of p53. These data suggest a potential mechanism accounting for the anti-carcinogenic and anti-cancer activities of vitamin C. PMID:12023887

  2. Ascorbate up-regulates MLH1 (Mut L homologue-1) and p73: implications for the cellular response to DNA damage.

    PubMed Central

    Catani, M Valeria; Costanzo, Antonio; Savini, Isabella; Levrero, Massimo; de Laurenzi, Vincenzo; Wang, Jean Y J; Melino, Gerry; Avigliano, Luciana

    2002-01-01

    We have found previously that ascorbic acid (vitamin C), as well as acting as a radical scavenger, may modulate the expression of several genes [i.e. fra-1, glutathione S-transferase Pi (GSTpi) and Mut L homologue-1 (MLH1)] in human keratinocytes. In the present paper, we demonstrate that MLH1, as well as its downstream target p73, can be positively modulated by this antioxidant vitamin, indeed, up-regulation of the two mRNAs was observed after just 2 h, and increased further up to 16 h of treatment. Modulation of MLH1 and p73 gene expression improves cellular susceptibility to apoptosis triggered by the DNA-damaging agent cisplatin. Indeed, in ascorbate-supplemented cells, increased cisplatin-induced apoptosis was seen, involving activation of the MLH1/c-Abl/p73 signalling cascade. Our results were further confirmed by studies performed on genetically defined mutants, i.e. mouse embryo fibroblasts derived from knock-out animals for c-Abl or p53, as well as human colon carcinoma cell lines deficient in MLH1. The increased sensitivity to cisplatin observed in ascorbate-loaded cells appeared to be dependent exclusively on MLH1 and c-Abl expression, and independent of p53. These data suggest a potential mechanism accounting for the anti-carcinogenic and anti-cancer activities of vitamin C. PMID:12023887

  3. Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 (CUG2)

    SciTech Connect

    Lee, Soojin . E-mail: leesoojin@cnu.ac.kr; Gang, Jingu; Jeon, Sun Bok; Jung, Jinyoung; Song, Si Young; Koh, Sang Seok . E-mail: sskoh@kribb.re.kr

    2007-08-31

    We examined genome-wide differences in gene expression between tumor biopsies and normal tissues in order to identify differentially regulated genes in tumors. Cancer-upregulated gene 2 (CUG2) was identified as an expressed sequence tag (EST) that exhibits significant differential expression in multiple human cancer types. CUG2 showed weak sequence homology with the down-regulator of transcription 1 (DR1) gene, a human transcription repressor. We found that EGFP-CUG2 fusion proteins were predominantly localized in the nucleus, suggesting their putative role in gene regulation. In addition, CUG2-overexpressing mouse fibroblast cells exhibited distinct cancer-specific phenotypes in vitro and developed into tumors in nude mice. Taken together, these findings suggest that CUG2 is a novel tumor-associated gene that is commonly activated in various human cancers and exhibits high transforming activities; it possibly belongs to a transcription regulator family that is involved in tumor biogenesis.

  4. The glutathione peroxidase homologous gene from Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet oxygen.

    PubMed

    Leisinger, U; Rfenacht, K; Fischer, B; Pesaro, M; Spengler, A; Zehnder, A J; Eggen, R I

    2001-07-01

    The glutathione peroxidase homologous gene (Gpxh gene) in Chlamydomonas reinhardtii is up-regulated under oxidative stress conditions. The Gpxh gene showed a remarkably strong and fast induction by the singlet oxygen-generating photosensitizers neutral red, methylene blue and rose Bengal. The Gpxh mRNA levels strongly increased, albeit much more slowly, upon exposure to the organic hydroperoxides tert-butyl hydroperoxide (t-BOOH) and cumene hydroperoxide. In contrast, the Gpxh mRNA levels were only weakly induced by exposure to the superoxide-generating compound paraquat and by hydrogen peroxide. A comparison of the Gpxh mRNA levels with those of the heat shock protein HSP70A and the iron superoxide dismutase gene showed qualitative and quantitative differences for the three genes under oxidative stress conditions tested. The Gpxh gene is specifically induced by singlet-oxygen photosensitizers and the relative induction by other compounds is much weaker for Gpxh than for the other genes investigated. Using Gpxh promoter fusions with the arylsulfatase reporter gene, we have shown that the Gpxh was transcriptionally up-regulated by singlet-oxygen photosensitizers. It is also shown that the Gpxh promoter contains a region between 104 and 179 bp upstream of the transcription start that is responsible for the mRNA up-regulation upon exposure to 1O2 but not t-BOOH. Within this region a regulatory sequence homologous to the mammalian cAMP response element (CRE) and activator protein 1 (AP-1) binding site was identified within a 16 bp palindrome. PMID:11485197

  5. [Separation of the up-regulated genes under nitrogen starvation from Phaeodactylum tricornutum by suppression subtractive hybridization technology].

    PubMed

    Tang, Jian-Xin; Chen, Zhuo; Hu, Han-Hua

    2009-08-01

    Sixteen cDNA fragments of Phaeodactylum tricornutum were isolated and identified to be up-regulated under nitrogen starvation by suppression subtractive hybridization (SSH) technology in order to elucidate the molecular mechanism of the diatom nitrogen utilization. Seven of them have high similarity with the functional genes related to the utilization of nitrogen. Northern blotting analysis indicated that 5 genes, nitrate transporter gene (nrt), nitrite reductase gene (nir), ammonium transporter gene (amt), ATP-binding cassette transporter gene (abc), and purine permease gene (pup), were significantly up-regulated under nitrogen starvation. PMID:19689950

  6. Astrocytes Upregulate Survival Genes in Tumor Cells and Induce Protection from Chemotherapy1

    PubMed Central

    Kim, Sun-Jin; Kim, Jang-Seong; Park, Eun Sung; Lee, Ju-Seog; Lin, Qingtang; Langley, Robert R; Maya, Marva; He, Junqin; Kim, Seung-Wook; Weihua, Zhang; Balasubramanian, Krishnakumar; Fan, Dominic; Mills, Gordon B; Hung, Mien-Chie; Fidler, Isaiah J

    2011-01-01

    In the United States, more than 40% of cancer patients develop brain metastasis. The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with conventional radiotherapy and chemotherapy. The growth and survival of metastasis depend on the interaction of tumor cells with host factors in the organ microenvironment. Brain metastases are surrounded and infiltrated by activated astrocytes and are highly resistant to chemotherapy. We report here that coculture of human breast cancer cells or lung cancer cells with murine astrocytes (but not murine fibroblasts) led to the up-regulation of survival genes, including GSTA5, BCL2L1, and TWIST1, in the tumor cells. The degree of up-regulation directly correlated with increased resistance to all tested chemotherapeutic agents. We further show that the up-regulation of the survival genes and consequent resistance are dependent on the direct contact between the astrocytes and tumor cells through gap junctions and are therefore transient. Knocking down these genes with specific small interfering RNA rendered the tumor cells sensitive to chemotherapeutic agents. These data clearly demonstrate that host cells in the microenvironment influence the biologic behavior of tumor cells and reinforce the contention that the organ microenvironment must be taken into consideration during the design of therapy. PMID:21390191

  7. Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

    SciTech Connect

    Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir; Noritake, Hidenao; Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 ; Kimura, Wataru; Wu, Yi-Xin; Kobayashi, Yoshimasa; Uezato, Tadayoshi; Miura, Naoyuki

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

  8. Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration

    PubMed Central

    Zaytseva, Yekaterina Y.; Harris, Jennifer W.; Mitov, Mihail I.; Kim, Ji Tae; Butterfield, D. Allan; Lee, Eun Y.; Weiss, Heidi L.; Gao, Tianyan; Evers, B. Mark

    2015-01-01

    Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC. PMID:25970773

  9. Increase in gene-transcript levels as indicators of up-regulation of the unfolded protein response in spontaneous canine tumors

    PubMed Central

    Elliot, Kirsten; MacDonald-Dickinson, Valerie; Linn, Kathleen; Simko, Elemir; Misra, Vikram

    2014-01-01

    The unfolded protein response (UPR), a conserved cellular response to stressors such as hypoxia and nutrient deprivation, is associated with angiogenesis and metastasis in tumor cells. This article discusses a pilot study conducted to determine whether components of the UPR could be identified in spontaneous canine tumors and whether they were up-regulated within tumor tissue compared with adjacent normal tissue. Tissue samples of various spontaneous canine neoplasms were taken from 13 dogs shortly after surgical excision or euthanasia; control samples were taken from adjacent normal tissue. RNA purification and real-time quantitative reverse-transcription polymerase chain reaction were done to measure the expression of 4 genes associated with the UPR (HERP, CHOP, GRP78, and XBP1s). The results indicated that UPR gene expression can be identified in spontaneous canine tumors and that the UPR is up-regulated, as indicated by significantly increased expression of CHOP and GRP78 within the tumor. PMID:24982546

  10. Screening of upregulated genes induced by high density in the vetch aphid Megoura crassicauda.

    PubMed

    Ishikawa, Asano; Ishikawa, Yuki; Okada, Yasukazu; Miyazaki, Satoshi; Miyakawa, Hitoshi; Koshikawa, Shigeyuki; Brisson, Jennifer A; Miura, Toru

    2012-03-01

    Aphids exhibit several polyphenisms in which discontinuous, alternative phenotypes are produced depending on environmental conditions. One representative example is the wing polyphenism, where winged and wingless females are produced through parthenogenesis. Previous work has shown that, in some aphid species, the density condition sensed by the mother aphid determines the developmental fate of embryos in her ovary, with high densities leading to winged progeny and low densities to wingless progeny. However, little is known about the molecular and physiological mechanisms underlying the wing polyphenism. To identify genes involved in the wing-morph determination in the vetch aphid, Megoura crassicauda, we compared maternal and embryonic transcripts between high- and low-density conditions using differential display, followed by quantitative real-time PCR (qRT-PCR). Under the high-density condition, two genes (Uba1 and Naca) were found to be upregulated in maternal tissues without ovaries, while one gene (ClpP) was upregulated in ovaries containing embryos. Uba1 and Naca encode factors that function in protein modification or transcriptional/translational regulation, respectively. In addition to differential display, candidate gene approaches focusing on morphogenetic and endocrine genes, i.e., wg, dpp, ap, hh, InR, IRS, Foxo, EcR, and USP, were also carried out. We found that wg was upregulated in maternal tissues under the high-density condition. The identified genes from both approaches are candidates for further study of their involvement in the transduction of density signals in mother aphids and/or the initial process of wing differentiation in embryos. PMID:22514053

  11. The Longitudinal Transcriptomic Response of the Substantia Nigra to Intrastriatal 6-Hydroxydopamine Reveals Significant Upregulation of Regeneration-Associated Genes

    PubMed Central

    Cole-Strauss, Allyson; Grabinski, Tessa; Mattingly, Zachary R.; Winn, Mary E.; Steece-Collier, Kathy; Sortwell, Caryl E.; Manfredsson, Fredric P.; Lipton, Jack W.

    2015-01-01

    We hypothesized that the study of gene expression at 1, 2, 4, 6 and 16 weeks in the substantia nigra (SN) after intrastriatal 6-OHDA in the Sprague-Dawley rat (rattus norvegicus) would identify cellular responses during the degenerative process that could be axoprotective. Specifically, we hypothesized that genes expressed within the SN that followed a profile of being highly upregulated early after the lesion (during active axonal degeneration) and then progressively declined to baseline over 16 weeks as DA neurons died are indicative of potential protective responses to the striatal 6-OHDA insult. Utilizing a ?-means cluster analysis strategy, we demonstrated that one such cluster followed this hypothesized expression pattern over time, and that this cluster contained several interrelated transcripts that are classified as regeneration-associated genes (RAGs) including Atf3, Sprr1a, Ecel1, Gadd45a, Gpnmb, Sox11, Mmp19, Srgap1, Rab15,Lifr, Trib3, Tgfb1, and Sema3c. All exemplar transcripts tested from this cluster (Sprr1a, Ecel1, Gadd45a, Atf3 and Sox11) were validated by qPCR and a smaller subset (Sprr1a, Gadd45a and Sox11) were shown to be exclusively localized to SN DA neurons using a dual label approach with RNAScope in situ hybridization and immunohistochemistry. Upregulation of RAGs is typically associated with the response to axonal injury in the peripheral nerves and was not previously reported as part of the axodegenerative process for DA neurons of the SN. Interestingly, as part of this cluster, other transcripts were identified based on their expression pattern but without a RAG provenance in the literature. These "RAG-like" transcripts need further characterization to determine if they possess similar functions to or interact with known RAG transcripts. Ultimately, it is hoped that some of the newly identified axodegeneration-reactive transcripts could be exploited as axoprotective therapies in PD and other neurodegenerative diseases. PMID:25992874

  12. 75 FR 65640 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-26

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... and Gene Therapies Advisory Committee. General Function of the Committee: To provide advice and... Tumor Vaccines and Biotechnology Branch, Office of Cellular, Tissue and Gene Therapies, Center...

  13. Identification of upregulated immune-related genes in Vibrio harveyi challenged Penaeus monodon postlarvae.

    PubMed

    Nayak, S; Singh, S K; Ramaiah, N; Sreepada, R A

    2010-09-01

    A subtracted cDNA library was constructed and analyzed to elucidate the response of Penaeus monodon postlarvae challenged with Vibrio harveyi. As many as 960 randomly selected cDNA fragments generated through suppression subtractive hybridization were single pass sequenced. Forty five genes and 20 hypothetical proteins were identified, a few being first reports from shrimps. The most abundant immune relevant genes were ferritin, hemocyanin, and TCTP (translationally controlled tumor protein) indicating their upregulation as also confirmed through qPCR. Post-infection qPCR analyses confirmed 2.04, 2.09, 3.28, 5.49, 6.47, and 11.63 fold rise respectively in ferritin, penaeidin, MnSOD, lysozyme, TCTP, and hemocyanin genes. These genes may be involved in the regulation of the host defense against V. harveyi. PMID:20580834

  14. 81 FR 3431 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2016-01-21

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... Branch and the Cellular and Tissue Therapy Branch of the Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapy, Center for Biologics Evaluation and Research, FDA. FDA...

  15. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    SciTech Connect

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-05-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  16. Urban air pollution produces up-regulation of myocardial inflammatory genes and dark chocolate provides cardioprotection.

    PubMed

    Villarreal-Calderon, Rodolfo; Reed, William; Palacios-Moreno, Juan; Keefe, Sheyla; Herritt, Lou; Brooks, Diane; Torres-Jardón, Ricardo; Calderón-Garcidueñas, Lilian

    2012-05-01

    Air pollution is a serious environmental problem. Elderly subjects show increased cardiac morbidity and mortality associated with air pollution exposure. Mexico City (MC) residents are chronically exposed to high concentrations of fine particulate matter (PM(2.5)) and PM-associated lipopolysaccharides (PM-LPS). To test the hypothesis that chronic exposure to urban pollution produces myocardial inflammation, female Balb-c mice age 4 weeks were exposed for 16 months to two distinctly different polluted areas within MC: southwest (SW) and northwest (NW). SW mice were given either no treatment or chocolate 2g/9.5 mg polyphenols/3 times per week. Results were compared to mice kept in clean air. Key inflammatory mediator genes: cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and the LPS receptor CD14 (cluster of differentiation antigen 14) were measured by real-time polymerase chain reaction. Also explored were target NFκB (nuclear factor κB), oxidative stress and antioxidant defense genes. TNF-α, IL-6, and COX-2 were significantly increased in both NW and SWMC mice (p=0.0001). CD14 was up-regulated in SW mice in keeping with the high exposures to particulate matter associated endotoxin. Chocolate administration resulted in a significant down-regulation of TNF-α (p<0.0001), IL-6 (p=0.01), and IL-1β (p=0.02). The up-regulation of antioxidant enzymes and the down-regulation of potent oxidases, toll-like receptors, and pro-apoptotic signaling genes completed the protective profile. Exposure to air pollution produces up-regulation of inflammatory myocardial genes and endotoxin plays a key role in the inflammatory response. Regular consumption of dark chocolate may reduce myocardial inflammation and have cardioprotective properties in the setting of air pollution exposures. PMID:20932730

  17. Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma

    PubMed Central

    2012-01-01

    Background The Breast Cancer Resistance Protein (BCRP/ABCG2) is one member of ABC transporters proteins super family responsible of drug resistance. Since data on ABCG2 expression in liver malignances are scanty, here we report the expression of ABCG2 in adult human hepatocellular carcinoma (HCC) in both in vivo and in vitro models with different degree of malignancy. Methods In cell lines derived from human hepatocellular carcinoma, ABCG2 gene expression was assessed by reverse transcription quantitative real time PCR and function by Hoechst 33342 efflux assay; protein content was assessed by SDS-PAGE Western blot. Results ABCG2 expression was found to be highest in the most undifferentiated cell lines, and this was related with a higher functional activity. ABCG2 expression was sensitive to antineoplastic drugs since exposure to 5 ?M doxorubicin for 24 hours resulted in significant up-regulations of ABCG2 in all cell lines, particularly in those lines with low basal ABCG2 expression (p<0.01). The gene expression was also investigated in 51 adult liver tissues with HCC and related cirrhosis; normal liver tissue was used as control. ABCG2 gene expression was higher in HCC than both cirrhotic paired tissue and normal tissue. This up-regulation was greater (p<0.05) in pathological poorly differentiated grade G3/G4 than in well-differentiated G1/G2 HCC. Conclusions Our results suggest a correlation of ABCG2 gene expression and differentiation stage both in human and HCC derived cell lines. The rapid up-regulation of ABCG2 to exposure to doxorubicin emphasizes the importance of this transporter in accounting for drug resistance in liver tumors. PMID:23153066

  18. Rapid systemic up-regulation of genes after heat-wounding and electrical stimulation

    NASA Technical Reports Server (NTRS)

    Davies, E.; Vian, A.; Vian, C.; Stankovic, B.

    1997-01-01

    When one leaf of a tomato plant is electrically-stimulated or heat-wounded, proteinase inhibitor genes are rapidly up-regulated in distant leaves. The identity of the systemic wound signal(s) is not yet known, but major candidates include hormones transmitted via the phloem or the xylem, the electrically-stimulated self-propagating electrical signal in the phloem (the action potential, AP), or the heat-wound-induced surge in hydraulic pressure in the xylem evoking a local change in membrane potential in adjacent living cells (the variation potential, VP). In order to discriminate between these signals we have adopted two approaches. The first approach involves applying stimuli that evoke known signals and determining whether these signals have similar effects on the "model" transcripts for proteinase inhibitors (pin) and calmodulin (cal). Here we show that a heat wound almost invariably evokes a VP, while an electrical stimulation occasionally evokes an AP, and both of these signals induce accumulation of transcripts encoding proteinase inhibitors. The second approach involves identifying the array of genes turned on by heat-wounding. To this end, we have constructed a subtractive library for heat-wounded tissue, isolated over 800 putatively up-regulated clones, and shown that all but two of the fifty that we have analyzed by Northern hybridization are, indeed, up-regulated. Here we show the early kinetics of up-regulation of three of these transcripts in the terminal (4th) leaf in response to heat-wounding the 3rd leaf, about 5 cm away. Even though these transcripts show somewhat different time courses of induction, with one peaking at 30 min, another at 15 min, and another at 5 min after flaming of a distant leaf, they all exhibit a similar pattern, i.e., a transient period of transcript accumulation preceding a period of transcript decrease, followed by a second period of transcript accumulation.

  19. PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival

    PubMed Central

    Panfil, Amanda R.; Al-Saleem, Jacob; Howard, Cory M.; Mates, Jessica M.; Kwiek, Jesse J.; Baiocchi, Robert A.; Green, Patrick L.

    2015-01-01

    Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL). This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5) on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i) in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment. PMID:26729154

  20. Interleukin-15 enhances cellular proliferation and upregulates CNS homing molecules in pre-B acute lymphoblastic leukemia.

    PubMed

    Williams, Mark T S; Yousafzai, Yasar; Cox, Charlotte; Blair, Allison; Carmody, Ruaidhrí; Sai, Shuji; Chapman, Karen E; McAndrew, Rachel; Thomas, Angela; Spence, Alison; Gibson, Brenda; Graham, Gerard J; Halsey, Christina

    2014-05-15

    Genome-wide association studies have consistently implicated the interleukin-15 (IL-15) gene in acute lymphoblastic leukemia (ALL) biology, including associations with disease susceptibility, and increased risk of central nervous system (CNS) involvement. However, whether pre-B ALL blasts directly respond to IL-15 is unknown. Here, we show that most pre-B ALL primary samples and cell lines express IL-15 and components of its receptor and that primary pre-B ALL cells show increased growth in culture in response to IL-15. Investigation of mechanisms of action using IL-15-responsive SD-1 cells shows this growth advantage is maximal under low-serum conditions, mimicking those found in cerebrospinal fluid. IL-15 also upregulates PSGL-1 and CXCR3, molecules associated with CNS trafficking. Investigation of downstream signaling pathways indicates that IL-15 induces signal transducer and activator of transcription 5 (STAT5), extracellular signal-regulated kinase (ERK) 1/2, and to a lesser extent phosphatidylinositol 3-kinase (PI3K) and nuclear factor κB (NF-κB) phosphorylation. The IL-15-mediated growth advantage is abolished by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), PI3K, and NF-κB inhibitors but preserved in the presence of STAT5 inhibition. Together, these observations provide a mechanistic link between increased levels of IL-15 expression and leukemogenesis, high-risk disease, and CNS relapse and suggest potential therapeutic targets. PMID:24700781

  1. Ciona intestinalis interleukin 17-like genes expression is upregulated by LPS challenge.

    PubMed

    Vizzini, Aiti; Di Falco, Felicia; Parrinello, Daniela; Sanfratello, Maria Antonietta; Mazzarella, Claudia; Parrinello, Nicol; Cammarata, Matteo

    2015-01-01

    In humans, IL-17 is a proinflammatory cytokine that plays a key role in the clearance of extracellular bacteria promoting cell infiltration and production of several cytokines and chemokines. Here, we report on three Ciona intestinalis IL-17 homologues (CiIL17-1, CiIL17-2, CiIL17-3). The gene organization, phylogenetic tree and modeling supported the close relationship with the mammalian IL-17A and IL-17F suggesting that the C. intestinalis IL-17 genes share a common ancestor in the chordate lineages. Real time PCR analysis showed a prompt expression induced by LPS inoculation suggesting that they are involved in the first phase of inflammatory response. In situ hybridization assays disclosed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels. PMID:25305501

  2. Identification of genes upregulated by pinewood nematode inoculation in Japanese red pine.

    PubMed

    Shin, Hanna; Lee, Hyoshin; Woo, Kwan-Soo; Noh, Eun-Woon; Koo, Yeong-Bon; Lee, Kyung-Joon

    2009-03-01

    Pine wilt disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, has destroyed huge areas of pine forest in East Asia, including Japan, China and Korea. No protection against PWN has been developed, and the responses of pine trees at the molecular level are unrecorded. We isolated and analyzed upregulated or newly induced genes from PWN-inoculated Japanese red pine (Pinus densiflora Sieb. et Zucc.) by using an annealing control primer system and suppression subtractive hybridization. Significant changes occurred in the transcript abundance of genes with functions related to defense, secondary metabolism and transcription, as the disease progressed. Other gene transcripts encoding pathogenesis-related proteins, pinosylvin synthases and metallothioneins were also more abundant in PWN-inoculated trees than in non-inoculated trees. Our report provides fundamental information on the molecular mechanisms controlling the biochemical and physiological responses of Japanese red pine trees to PWN invasion. PMID:19203959

  3. Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis.

    PubMed

    Lamore, Sarah D; Wondrak, Georg T

    2011-10-01

    Zinc ion homeostasis plays an important role in human cutaneous biology where it is involved in epidermal differentiation and barrier function, inflammatory and antimicrobial regulation, and wound healing. Zinc-based compounds designed for topical delivery therefore represent an important class of cutaneous therapeutics. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in over-the-counter topical antimicrobials, and has also been examined as an investigational therapeutic targeting psoriasis and UVB-induced epidermal hyperplasia. Recently, we have demonstrated that cultured primary human skin keratinocytes display an exquisite sensitivity to nanomolar ZnPT concentrations causing induction of heat shock response gene expression and poly(ADP-ribose) polymerase (PARP)-dependent cell death (Cell Stress Chaperones 15:309-322, 2010). Here we demonstrate that ZnPT causes rapid accumulation of intracellular zinc in primary keratinocytes as observed by quantitative fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS), and that PARP activation, energy crisis, and genomic impairment are all antagonized by zinc chelation. In epidermal reconstructs (EpiDerm) exposed to topical ZnPT (0.1-2% in Vanicream), ICP-MS demonstrated rapid zinc accumulation, and expression array analysis demonstrated upregulation of stress response genes encoding metallothionein-2A (MT2A), heat shock proteins (HSPA6, HSPA1A, HSPB5, HSPA1L, DNAJA1, HSPH1, HSPD1, HSPE1), antioxidants (SOD2, GSTM3, HMOX1), and the cell cycle inhibitor p21 (CDKN1A). IHC analysis of ZnPT-treated EpiDerm confirmed upregulation of Hsp70 and TUNEL-positivity. Taken together our data demonstrate that ZnPT impairs zinc ion homeostasis and upregulates stress response gene expression in primary keratinocytes and reconstructed human epidermis, activities that may underlie therapeutic and toxicological effects of this topical drug. PMID:21424779

  4. Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis

    PubMed Central

    Lamore, Sarah D.

    2014-01-01

    Zinc ion homeostasis plays an important role in human cutaneous biology where it is involved in epidermal differentiation and barrier function, inflammatory and antimicrobial regulation, and wound healing. Zinc-based compounds designed for topical delivery therefore represent an important class of cutaneous therapeutics. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in over-the-counter topical antimicrobials, and has also been examined as an investigational therapeutic targeting psoriasis and UVB-induced epidermal hyperplasia. Recently, we have demonstrated that cultured primary human skin keratinocytes display an exquisite sensitivity to nanomolar ZnPT concentrations causing induction of heat shock response gene expression and poly(ADP-ribose) polymerase (PARP)-dependent cell death (Cell Stress Chaperones 15:309322, 2010). Here we demonstrate that ZnPT causes rapid accumulation of intracellular zinc in primary keratinocytes as observed by quantitative fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS), and that PARP activation, energy crisis, and genomic impairment are all antagonized by zinc chelation. In epidermal reconstructs (EpiDerm) exposed to topical ZnPT (0.12% in Vanicream), ICP-MS demonstrated rapid zinc accumulation, and expression array analysis demonstrated upregulation of stress response genes encoding metallothionein-2A (MT2A), heat shock proteins (HSPA6, HSPA1A, HSPB5, HSPA1L, DNAJA1, HSPH1, HSPD1, HSPE1), antioxidants (SOD2, GSTM3, HMOX1), and the cell cycle inhibitor p21 (CDKN1A). IHC analysis of ZnPT-treated EpiDerm confirmed upregulation of Hsp70 and TUNEL-positivity. Taken together our data demonstrate that ZnPT impairs zinc ion homeostasis and upregulates stress response gene expression in primary keratinocytes and reconstructed human epidermis, activities that may underlie therapeutic and toxicological effects of this topical drug. PMID:21424779

  5. Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera.

    PubMed

    Mao, Wenfu; Schuler, Mary A; Berenbaum, May R

    2013-05-28

    As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by ∼60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses. PMID:23630255

  6. Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera

    PubMed Central

    Mao, Wenfu; Schuler, Mary A.; Berenbaum, May R.

    2013-01-01

    As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by ∼60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses. PMID:23630255

  7. Tissue-specific Forkhead protein FOXA2 up-regulates SOX14 gene expression.

    PubMed

    Popovic, Jelena; Klajn, Andrijana; Petrovic, Isidora; Stevanovic, Milena

    2010-01-01

    The expression of Sox14 gene in spinal cord explants was found to be regulated by Sonic hedgehog (SHH) in a dose-dependent manner, indicating that this signaling molecule might act as a regulator of Sox14-expressing interneuron differentiation. In the present study we identified the positive control element and provided the first evidence that FOXA2 is involved in up-regulation of SOX14 expression in HepG2 and U87MG cell lines. By functional analysis we demonstrated that mutation in FOXA2 binding site reduced the SOX14 reporter construct activity, and that FOXA2 over-expression increased endogenous SOX14 protein expression. Further, we have shown that human SOX14 expression is GLI1 dependent in U87MG cells and SHH-N dependent in U87MG and HepG2 cell lines. By applying siRNA silencing of FOXA2, we have demonstrated that upregulation of endogenous SOX14 gene expression by SHH is, at least in part, mediated by FOXA2. However, our data revealed that a positive regulatory region, containing functional FOXA2 site analyzed in this study, is not involved in mediation of SHH dependent SOX14 activation. Data presented here provide the initial insight into molecular mechanism underlying tissue and developmentally specific regulation of the SOX14 gene expression. PMID:20074681

  8. Cloning and analysizing the up-regulated expression of transthyretin-related gene (LR1) in rat liver regeneration following short interval successive partial hepatectomy

    PubMed Central

    Xu, Cun-Shuan; Li, Yu-Chang; Lin, Jun-Tang; Zhang, Hui-Yong; Zhang, Yun-Han

    2003-01-01

    AIM: Cloning and analysizing the up-regulated expression of transthyretin-related gene following short interval successive partial hepatectomy (SISPH) to elucidate the mechanism of differentiation, division, dedifferentiation and redifferentiation in rat liver regeneration (LR). METHODS: Lobus external sinister and lobus centralis sinister, lobus centralis, lobus dexter, lobus candatus were removed one by one from rat liver at four different time points 4, 36, 36 and 36 hr (total time: 4 hr, 40 hr, 76 hr, 112 hr) respectively. Suppression subtractive hybridization (SSH) was carried out by using normal rat liver tissue as driver and the tissue following short interval successive partial hepatectomy (SISPH) as tester to construct a highly efficient forward-subtractive cDNA library. After screening, an interested EST fragment was selected by SSH and primers were designed according to the sequence of the EST to clone the full-length cDNA fragment using RACE (rapid amplification of cDNA end). Homologous detection was performed between the full-lenth cDNA and Genbank. RESULTS: Forward suppression subtractive hybridization (FSSH) library between 0 h and 112 h following SISPH was constructed and an up-regulated full-length cDNA (named LR1), which was related with the transthyretin gene, was cloned by rapid amplification of cDNA end. It was suggested that the gene is involved in the cellular dedifferentiation in LR following SISPH. CONCLUSION: Some genes were up-regulated in 112 h following SISPH in rat. LR1 is one of these up-regulated expression genes which may play an important role in rat LR. PMID:12508371

  9. Bacterial Cellular Engineering by Genome Editing and Gene Silencing

    PubMed Central

    Nakashima, Nobutaka; Miyazaki, Kentaro

    2014-01-01

    Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption), knock-in (insertion), and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence) target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering. PMID:24552876

  10. [HIV-1 infection up-regulating expression of interferon-stimulated gene 15 in cell lines].

    PubMed

    Wu, Huan-mei; Sun, Jun; Meng, Zhe-feng; Zhang, Xiao-yan; Xu, Jian-qing

    2013-09-01

    To investigate whether HIV-1 infection affects expression of interferon-stimulated gene 15 (ISG15) and determine the antiviral effect of ISG15 in vitro, ISG15 expression at transcription and protein level and supernatant p24 of HIV-1 was detected in various HIV-1 infected or transfected cell lines, respec tively. HIV-1 molecular clone pNL4-3 was used to transfect 293T, TZM-bl and HeLa cells while HIV-1 pseudo-typed virus was used to infect Jurkat, MT-1 and THP-1 cells. After twenty-four hours post infection or transfection, cells were harvested for extraction of total RNAs and subsequently used in real time PCR for quantification of ISG15 transcriptional expression. After forty-eight hours post infection or transfection, cells were harvested for extraction of total proteins to detect ISG15 protein expression. A significant up-regulation of ISG15 at transcription level was observed in HIV-1 infected or transfected cell lines, particulaly in THP-1 and TZM-bl cells. Up-regulation of ISG15 protein was observed only in TZM-bl cell. Cotransfection of ISG15 and HIV-1 indicated that ISG15 inhibited production of HIV-1 progeny virus in a dose and time depend manner in 293T cell but not TZM-bl cell. These results revealed upregulating ISG15 expression in transcriptional level and potential antagonistic mechanism against ISG15 by HIV-1 infection, simultanelusly. PMID:24386835

  11. Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability.

    PubMed

    Liu, Gaowen; Yong, Mei Yun Jacy; Yurieva, Marina; Srinivasan, Kandhadayar Gopalan; Liu, Jaron; Lim, John Soon Yew; Poidinger, Michael; Wright, Graham Daniel; Zolezzi, Francesca; Choi, Hyungwon; Pavelka, Norman; Rancati, Giulia

    2015-12-01

    Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ?1,000 individual "essential" genes and found that ?9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance. PMID:26627736

  12. All-trans retinoic acid induces cellular senescence by up-regulating levels of p16 and p21 via promoter hypomethylation.

    PubMed

    Lim, Joo Song; Park, Sun-Hye; Jang, Kyung Lib

    2011-09-01

    All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor ?? whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy. PMID:21843507

  13. Insecticide-Mediated Up-Regulation of Cytochrome P450 Genes in the Red Flour Beetle (Tribolium castaneum)

    PubMed Central

    Liang, Xiao; Xiao, Da; He, Yanping; Yao, Jianxiu; Zhu, Guonian; Zhu, Kun Yan

    2015-01-01

    Some cytochrome P450 (CYP) genes are known for their rapid up-regulation in response to insecticide exposures in insects. To date, however, limited information is available with respect to the relationships among the insecticide type, insecticide concentration, exposure duration and the up-regulated CYP genes. In this study, we examined the transcriptional response of eight selected CYP genes, including CYP4G7, CYP4Q4, CYP4BR3, CYP12H1, CYP6BK11, CYP9D4, CYP9Z5 and CYP345A1, to each of four insecticides in the red flour beetle, Tribolium castaneum. Reverse transcription quantitative PCR (RT-qPCR) revealed that CYP4G7 and CYP345A1 can be significantly up-regulated by cypermethrin (1.97- and 2.06-fold, respectively), permethrin (2.00- and 2.03-fold) and lambda-cyhalothrin (1.73- and 1.81-fold), whereas CYP4BR3 and CYP345A1 can be significantly up-regulated by imidacloprid (1.99- and 1.83-fold) when 20-day larvae were exposed to each of these insecticides at the concentration of LC20 for 24 h. Our studies also showed that similar levels of up-regulation can be achieved for CYP4G7, CYP4BR3 and CYP345A1 by cypermethrin, permethrin, lambda-cyhalothrin or imidacloprid with approximately one fourth of LC20 in 6 h. Our study demonstrated that up-regulation of these CYP genes was rapid and only required low concentrations of insecticides, and the up-regulation not only depended on the CYP genes but also the type of insecticides. Our results along with those from previous studies also indicated that there were no specific patterns for predicting the up-regulation of specific CYP gene families based on the insecticide classification. PMID:25607733

  14. Insecticide-mediated up-regulation of cytochrome P450 genes in the red flour beetle (Tribolium castaneum).

    PubMed

    Liang, Xiao; Xiao, Da; He, Yanping; Yao, Jianxiu; Zhu, Guonian; Zhu, Kun Yan

    2015-01-01

    Some cytochrome P450 (CYP) genes are known for their rapid up-regulation in response to insecticide exposures in insects. To date, however, limited information is available with respect to the relationships among the insecticide type, insecticide concentration, exposure duration and the up-regulated CYP genes. In this study, we examined the transcriptional response of eight selected CYP genes, including CYP4G7, CYP4Q4, CYP4BR3, CYP12H1, CYP6BK11, CYP9D4, CYP9Z5 and CYP345A1, to each of four insecticides in the red flour beetle, Tribolium castaneum. Reverse transcription quantitative PCR (RT-qPCR) revealed that CYP4G7 and CYP345A1 can be significantly up-regulated by cypermethrin (1.97- and 2.06-fold, respectively), permethrin (2.00- and 2.03-fold) and lambda-cyhalothrin (1.73- and 1.81-fold), whereas CYP4BR3 and CYP345A1 can be significantly up-regulated by imidacloprid (1.99- and 1.83-fold) when 20-day larvae were exposed to each of these insecticides at the concentration of LC20 for 24 h. Our studies also showed that similar levels of up-regulation can be achieved for CYP4G7, CYP4BR3 and CYP345A1 by cypermethrin, permethrin, lambda-cyhalothrin or imidacloprid with approximately one fourth of LC20 in 6 h. Our study demonstrated that up-regulation of these CYP genes was rapid and only required low concentrations of insecticides, and the up-regulation not only depended on the CYP genes but also the type of insecticides. Our results along with those from previous studies also indicated that there were no specific patterns for predicting the up-regulation of specific CYP gene families based on the insecticide classification. PMID:25607733

  15. Korean Red Ginseng Up-regulates C21-Steroid Hormone Metabolism via Cyp11a1 Gene in Senescent Rat Testes.

    PubMed

    Kim, In-Hye; Kim, Si-Kwan; Kim, Eun-Hye; Kim, Sung-Won; Sohn, Sang-Hyun; Lee, Soo Cheol; Choi, Sangdun; Pyo, Suhkneung; Rhee, Dong-Kwon

    2011-09-01

    Ginseng (Panax ginseng Meyer) has been shown to have anti-aging effects in animal and clinical studies. However, the molecular mechanisms by which ginseng exerts these effects remain unknown. Here, the anti-aging effect of Korean red ginseng (KRG) in rat testes was examined by system biology analysis. KRG water extract prepared in feed pellets was administered orally into 12 month old rats for 4 months, and gene expression in testes was determined by microarray analysis. Microarray analysis identified 33 genes that significantly changed. Compared to the 2 month old young rats, 13 genes (Rps9, Cyp11a1, RT1-A2, LOC365778, Sv2b, RGD1565959, RGD1304748, etc.) were up-regulated and 20 genes (RT1-Db1, Cldn5, Svs5, Degs1, Vdac3, Hbb, LOC684355, Svs5, Tmem97, Orai1, Insl3, LOC497959, etc.) were down-regulated by KRG in the older rats. Ingenuity Pathway Analysis of untreated aged rats versus aged rats treated with KRG showed that the affected most was Cyp11a1, responsible for C21-steroid hormone metabolism, and the top molecular and cellular functions are organ morphology and reproductive system development and function. When genes in young rat were compared with those in the aged rat, sperm capacitation related genes were down-regulated in the old rat. However, when genes in the old rat were compared with those in the old rat treated with KRG, KRG treatment up-regulated C21-steroid hormone metabolism. Taken together, Cyp11a1 expression is decreased in the aged rat, however, it is up-regulated by KRG suggesting that KRG seems enhance testes function via Cyp11a1. PMID:23717070

  16. 70 FR 21799 - Cellular, Tissue and Gene Therapies Advisory Committee (Formerly Biological Response Modifiers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2005-04-27

    ... From the Federal Register Online via the Government Printing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee (Formerly.... Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee (formerly Biological...

  17. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    SciTech Connect

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian; Hou, Lichao; Chai, Yubo; Song, Qinghe; Chen, Sumin; Luo, Wenjing; Chen, Jingyuan

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  18. Upregulation of mitochondrial gene expression in PBMC from convalescent SARS patients.

    PubMed

    Shao, Hongwei; Lan, Dongming; Duan, Zhaohui; Liu, Zehuan; Min, Jun; Zhang, Lichun; Huang, Jian; Su, Jing; Chen, Shangwu; Xu, Anlong

    2006-11-01

    The observations that Lymphopenia is common in severe acute respiratory syndrome (SARS) patients and that peripheral blood mononuclear cell (PBMC) could be infected by SARS-CoV indicate that PBMC could be useful in identifying the gene expression profile in convalescent patients and tracing the host response to SARS-CoV infection. In this study, the altered genes expressions in the PBMC of convalescent SARS patients were investigated with suppression subtractive hybridization (SSH). We found that genes encoded by mitochondrial DNA (mtDNA) were obviously upregulated, while mitochondria were now found to be closely connected with antiviral immunity. The identification of a viral gene, M, in SSH cDNA library shows the long-term existence of SARS-CoV in vivo. In addition, some oxidative stress sensitive genes, heat shock proteins, transcription factors, and cytokines showed remarkable elevation. Thin-section electron microscope shows increased lysosome-like granule and mitochondria in PBMC of patients. These results provide important intracellular clue for tracing host response to SARS-CoV infection and suggest a role of mitochondria in that process. PMID:17024565

  19. Genetic determinants and cellular constraints in noisy gene expression

    PubMed Central

    Sanchez, Alvaro; Golding, Ido

    2014-01-01

    In individual cells, transcription is a random process obeying single-molecule kinetics. Often, it occurs in a bursty, intermittent manner. The frequency and size of these bursts affect the magnitude of temporal fluctuations in mRNA and protein content within a cell, creating variation or noise in gene expression. It is still unclear to what degree transcriptional kinetics are specific to each gene and determined by its promoter sequence. Alternative scenarios have been proposed, where the kinetics of transcription are governed by cellular constraints and follow universal rules across the genome. Evidence from genome-wide noise studies and from systematic perturbations of promoter sequences suggest that both scenariosnamely gene-specific versus genome-wide regulation of transcription kinetics may be present to different degrees in bacteria, yeast and animal cells. PMID:24311680

  20. Cellular senescence and tumor suppressor gene p16

    PubMed Central

    Rayess, Hani; Wang, Marilene B.; Srivatsan, Eri S.

    2011-01-01

    Cellular senescence is an irreversible arrest of cell growth. Biochemical and morphological changes occur during cellular senescence, including the formation of a unique cellular morphology such as flattened cytoplasm. Function of mitochondria, endoplasmic reticulum and lysosomes are affected resulting in the inhibition of lysosomal and proteosomal pathways. Cellular senescence can be triggered by a number of factors including, aging, DNA damage, oncogene activation and oxidative stress. While the molecular mechanism of senescence involves p16 and p53 tumor suppressor genes and telomere shortening, this review is focused on the mechanism of p16 control. The p16 mediated senescence acts through the retinoblastoma (Rb) pathway inhibiting the action of the cyclin dependant kinases leading to G1 cell cycle arrest. Rb is maintained in a hypophosphorylated state resulting in the inhibition of transcription factor E2F1. Regulation of p16 expression is complex and involves epigenetic control and multiple transcription factors. PRC1 (Pombe repressor complex 1) and PRC2 (Pombe repressor complex 2) proteins and histone deacetylases play an important role in the promoter hypermethylation for suppressing p16 expression. While transcription factors YY1 and Id1 suppress p16 expression, transcription factors CTCF, Sp1, and Ets family members activate p16 transcription. Senescence occurs with the inactivation of suppressor elements leading to the enhanced expression of p16. PMID:22025288

  1. 70 FR 51076 - Research Review Subcommittee of the Cellular, Tissue and Gene Therapies Advisory Committee...

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    .... Name of Subcommittee: Research Review Subcommittee of the Cellular, Tissue and Gene Therapies Advisory... to presentations about the research program at the Office of Cellular, Tissue and Gene Therapies... recommendations to the Cellular Tissue and Gene Therapies Advisory Committee at a future open meeting of the...

  2. 72 FR 71922 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

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    2007-12-19

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee... programs of the Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and...

  3. 72 FR 9766 - Cellular, Tissue, and Gene Therapies Advisory Committee; Notice of Meeting

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    2007-03-05

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue, and Gene Therapies Advisory Committee... hear overviews of research programs in the Division of Cellular and Gene Therapies, Center for... Division of Cellular and Gene Therapies. Persons attending FDA's advisory committee meetings are...

  4. 70 FR 7949 - Cellular, Tissue and Gene Therapies Advisory Committee (formerly the Biological Response...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2005-02-16

    ... SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee (formerly the... of meeting of the Cellular, Tissue and Gene Therapies Advisory Committee (formerly the Biological... announced that a meeting of the Cellular, Tissue and Gene Therapies Advisory Committee (formerly...

  5. 74 FR 19226 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2009-04-28

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee...: (1) Receive an update on Guidance documents from the Office of Cellular, Tissue and Gene...

  6. 71 FR 1432 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2006-01-09

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... update on the recent review of the research program of the Office of Cellular, Tissue and Gene Therapies... Subcommittee of the Cellular, Tissue and Gene Therapies Advisory Committee related to a review of the...

  7. 78 FR 44133 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-23

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee... on guidance documents issued from the Office of Cellular, Tissue and Gene Therapies, Center...

  8. Suppression subtraction hybridization and northern analysis reveal upregulation of heat shock, trkB, and sodium calcium exchanger genes following global cerebral ischemia in the rat.

    PubMed

    Majda, B T; Meloni, B P; Rixon, N; Knuckey, N W

    2001-09-30

    Driver (sham-operated) and tester (ischemic) hippocampal cDNAs were subtracted, and the resulting ischemia-induced upregulated gene expression was verified by northern analysis. cDNAs isolated corresponded to (1) genes known to be upregulated following ischemia, (hsc70, hsp90, hsp105 and trkB) and (2) a gene not previously implicated with cerebral ischemia, sodium calcium exchanger (ncx). Furthermore, upregulation of these genes was demonstrated following preconditioning transient global ischemia. PMID:11589994

  9. Depletion of the xynB2 gene upregulates ?-xylosidase expression in C. crescentus.

    PubMed

    Corra, Juliana Moo; Mingori, Moara Rodrigues; Gandra, Rinaldo Ferreira; Loth, Eduardo Alexandre; Seixas, Flvio Augusto Vicente; Simo, Rita de Cssia Garcia

    2014-01-01

    Caulobacter crescentus is able to express several enzymes involved in the utilization of lignocellulosic biomasses. Five genes, xynB1-5, that encode ?-xylosidases are present in the genome of this bacterium. In this study, the xynB2 gene, which encodes ?-xylosidase II (CCNA_02442), was cloned under the control of the PxylX promoter to generate the O-xynB2 strain, which overexpresses the enzyme in the presence of xylose. In addition, a null mutant strain, ?-xynB2, was created by two homologous recombination events where the chromosomal xynB2 gene was replaced by a copy that was disrupted by the spectinomycin-resistant cassette. We demonstrated that C. crescentus cells lacking ?-xylosidase II upregulates the xynB genes inducing ?-xylosidase activity. Transcriptional analysis revealed that xynB1 (RT-PCR analysis) and xynB2 (lacZ transcription fusion) gene expression was induced in the ?-xynB2 cells, and high ?-xylosidase activity was observed in the presence of different agro-industrial residues in the null mutant strain, a characteristic that can be explored and applied in biotechnological processes. In contrast, overexpression of the xynB2 gene caused downregulation of the expression and activity of the ?-xylosidase. For example, the ?-xylosidase activity that was obtained in the presence of sugarcane bagasse was 7-fold and 16-fold higher than the activity measured in the C. crescentus parental and O-xynB2 cells, respectively. Our results suggest that ?-xylosidase II may have a role in controlling the expression of the xynB1 and xynB2 genes in C. crescentus. PMID:24142353

  10. Dysfunctional chloroplasts up-regulate the expression of mitochondrial genes in Arabidopsis seedlings.

    PubMed

    Liao, Jo-Chien; Hsieh, Wei-Yu; Tseng, Ching-Chih; Hsieh, Ming-Hsiun

    2016-02-01

    Chloroplasts and mitochondria play important roles in maintaining metabolic and energy homeostasis in the plant cell. The interactions between these two organelles, especially photosynthesis and respiration, have been intensively studied. Still, little is known about the regulation of mitochondrial gene expression by chloroplasts and vice versa. The gene expression machineries in chloroplasts and mitochondria rely heavily on the nuclear genome. Thus, the interactions between nucleus and these organelles, including anterograde and retrograde regulation, have been actively investigated in the last two decades. Norflurazon (NF) and lincomycin (Lin) are two commonly used inhibitors to study chloroplast-to-nucleus retrograde signaling in plants. We used NF and Lin to block the development and functions of chloroplasts and examined their effects on mitochondrial gene expression, RNA editing and splicing. The editing of most mitochondrial transcripts was not affected, but the editing extents of nad4-107, nad6-103, and ccmFc-1172 decreased slightly in NF- and Lin-treated seedlings. While the splicing of mitochondrial transcripts was not significantly affected, steady-state mRNA levels of several mitochondrial genes increased significantly in NF- and Lin-treated seedlings. Moreover, Lin seemed to have more profound effects than NF on the expression of mitochondrial genes, indicating that signals derived from these two inhibitors might be distinct. NF and Lin also significantly induced the expression of nuclear genes encoding subunits of mitochondrial electron transport chain complexes. Thus, dysfunctional chloroplasts may coordinately up-regulate the expression of nuclear and mitochondrial genes encoding subunits of respiratory complexes. PMID:26008795

  11. Multiple β-defensin genes are upregulated by the vitamin D pathway in cattle.

    PubMed

    Merriman, Kathryn E; Kweh, Mercedes F; Powell, Jessica L; Lippolis, John D; Nelson, Corwin D

    2015-11-01

    Experimental models of bacterial and viral infections in cattle have suggested vitamin D has a role in innate immunity of cattle. The intracrine vitamin D pathway of bovine macrophages, however, has only been shown to activate a nitric oxide-mediated defense mechanism, as opposed to cathelicidin and β-defensin antimicrobial peptides in human macrophages. In this study we have investigated the actions of 1,25-dihydroxyvitamin D3 (1,25D) on a cluster of eleven bovine β-defensin genes on the basis of RNAseq data indicating they were targets of 1,25D in cattle. Treatment of bovine monocyte cultures with 1,25D (10 nM, 18 h) in the absence and presence of LPS stimulation increased the expression of bovine β-defensin 3 (BNBD3), BNBD4, BNBD6, BNBD7, and BNBD10 genes 5 to 10-fold compared to control (P<0.05). Treatment of lipopolysaccharide (LPS)-stimulated monocytes with 0-100 ng/mL 25-hydroxyvitamin D3 also increased BNBD3, BNBD4, BNBD7, and BNBD10 in a dose-dependent manner. Treatment of monocytes with the protein translation inhibitor, cycloheximide, however, blocked upregulation of the β-defensins in response to 1,25D suggesting the β-defensins in cattle are not direct targets of the vitamin D receptor. Furthermore, preliminary investigation of vitamin D's contribution to β-defensin expression in vivo revealed that intramammary 1,25D treatment of lactating cows increased BNBD7 expression in mammary macrophages. In conclusion, our data demonstrate that multiple β-defensin genes are upregulated by 1,25D in cattle, providing further indication that vitamin D contributes to bovine innate immunity. PMID:26255277

  12. Interleukin-6 upregulates paraoxonase 1 gene expression via an AKT/NF-κB-dependent pathway

    SciTech Connect

    Cheng, Chi-Chih; Hsueh, Chi-Mei; Chen, Chiu-Yuan; Chen, Tzu-Hsiu; Hsu, Shih-Lan; Department of Applied Chemistry, National Chi Nan University, Puli, Nantou, Taiwan

    2013-07-19

    Highlights: •IL-6 could induce PON1 gene expression. •IL-6 increased NF-κB protein expression and NF-κB-p50 and -p65 subunits nuclear translocation. •IL-6-induced PON1 up-regulation was through an AKT/NF-κB pathway. -- Abstract: The aim of this study is to investigate the relationship between paraoxonase 1 (PON1) and atherosclerosis-related inflammation. In this study, human hepatoma HepG2 cell line was used as a hepatocyte model to examine the effects of the pro-inflammatory cytokines on PON1 expression. The results showed that IL-6, but not TNF-α and IL-1β, significantly increased both the function and protein level of PON1; data from real-time RT-PCR analysis revealed that the IL-6-induced PON1 expression occurred at the transcriptional level. Increase of IκB kinase activity and IκB phosphorylation, and reduction of IκB protein level were also observed in IL-6-treated HepG2 cells compared with untreated culture. This event was accompanied by increase of NF-κB-p50 and -p65 nuclear translocation. Moreover, treatment with IL-6 augmented the DNA binding activity of NF-κB. Furthermore, pharmacological inhibition of NF-κB activation by PDTC and BAY 11-7082, markedly suppressed the IL-6-mediated PON1 expression. In addition, IL-6 increased the levels of phosphorylated protein kinase B (PKB, AKT). An AKT inhibitor LY294002 effectively suppressed IKK/IκB/NF-κB signaling and PON1 gene expression induced by IL-6. Our findings demonstrate that IL-6 upregulates PON1 gene expression through an AKT/NF-κB signaling axis in human hepatocyte-derived HepG2 cell line.

  13. Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1–HSF2 heterocomplexes

    PubMed Central

    Loison, Fabien; Debure, Laure; Nizard, Philippe; le Goff, Pascale; Michel, Denis; le Dréan, Yves

    2005-01-01

    Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between −218 and −106 bp, which contains a particular heat-shock element, named CLE for ‘clusterin element’. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1–HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity. PMID:16336210

  14. Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes.

    PubMed

    Loison, Fabien; Debure, Laure; Nizard, Philippe; le Goff, Pascale; Michel, Denis; le Dréan, Yves

    2006-04-01

    Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between -218 and -106 bp, which contains a particular heat-shock element, named CLE for 'clusterin element'. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1-HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity. PMID:16336210

  15. Nitrite reductase gene upregulated during conidiation is involved in macroconidium formation in Fusarium oxysporum.

    PubMed

    Iida, Y; Kurata, T; Harimoto, Y; Tsuge, T

    2008-10-01

    Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. We previously found that the transcript level of the nitrite reductase gene of F. oxysporum, named FoNIIA, was markedly upregulated during conidiation compared with during vegetative growth. FoNIIA was also found to be positively regulated by Ren1 that is a transcription regulator controlling development of microconidia and macroconidia. In this study, we analyzed the function of FoNIIA in conidiation of F. oxysporum. Conidiation cultures showed markedly higher level of accumulation of FoNiiA protein as well as FoNIIA mRNA than vegetative growth cultures. FoNIIA protein was significantly decreased in cultures of the REN1 disruption mutant compared with that of the wild type. These results confirmed that FoNIIA expression is upregulated during conidiation and is positively regulated by REN1. The FoNIIA disruption mutants produced microconidia, macroconidia, and chlamydospores, which were morphologically indistinguishable from those of the wild type. The mutants, however, produced significantly fewer macroconidia than the wild type, although the wild type and mutant strains produced similar numbers of microconidia and chlamydospores. These results demonstrate that nitrite reductase is involved in quantitative control of macroconidium formation as well as nitrate utilization in F. oxysporum. PMID:18943456

  16. A BTB domain-containing gene is upregulated by immune challenge.

    PubMed

    Wang, Gang; Liu, Peng-Cheng; Wang, Jin-Xing; Zhao, Xiao-Fan

    2011-06-01

    20-Hydroxyecdysone (20E) is an important hormone that regulates the development of insects. Although previous evidence revealed that 20E promotes innate immunity in insects, the mechanism involved is still unclear. In this study, the HaBBP gene from Helicoverpa armigera is cloned, which contains BTB (broad-complex, tramtrack, and bric-a-brac), a BACK (BTB and carboxyl-terminus kelch repeats), and PHR (PAM, highwire, and RPM) domains. RT-PCR analysis of HaBBP and western blot analysis of HaBBP show that the mRNA and protein level are higher in the fat body and hemocytes during the molting and metamorphic stages compared with the feeding stage. HaBBP was upregulated by 20E in hemocytes. Knockdown of the 20E receptor EcR-B1 and the heterodimeric partner ultraspiracle protein USP1 in an epidermal cell line (HaEpi) blocked the transcription of HaBBP. HaBBP is distributed in granulocytes and plasmatocytes. Immune stimulation by Escherichia coli caused the upregulation of HaBBP in both hemocytes and fat body. Thus, HaBBP is regulated by the 20E signaling pathway, and is likely involved in the insect innate immunity. PMID:21374716

  17. Bioefficacy of Graviola leaf extracts in scavenging free radicals and upregulating antioxidant genes.

    PubMed

    Son, Yu-Ra; Choi, Eun-Hye; Kim, Goon-Tae; Park, Tae-Sik; Shim, Soon-Mi

    2016-02-17

    The aims of this study were to determine bioactive components of Graviola leaf extracts and to examine the radical scavenging capacity, gene expression and transcription factors of antioxidant enzymes. Rutin, kaempferol-rutinoside, and vitamin U were identified from the steaming and 50% EtOH extracts of Graviola leaves. Graviola leaf extracts effectively scavenged peroxy and nitrogen radicals. 50% EtOH of Graviola leaves provided a 1-2.9 times higher trolox equivalent than the steaming extract. It also had a higher VCEAC. Graviola leaf extracts reduced the generation of reactive oxygen species (ROS) induced by H2O2 in a dose-dependent manner. The 50% EtOH extract of Graviola leaves upregulated SOD1 and Nrf2, but catalase and HMOX1 were not altered by the 50% EtOH extract of Graviola leaves. PMID:26674326

  18. Upregulation of human heme oxygenase gene expression by Ets-family proteins.

    PubMed

    Deramaudt, B M; Remy, P; Abraham, N G

    1999-03-01

    Overexpression of human heme oxygenase-1 has been shown to have the potential to promote EC proliferation and angiogenesis. Since Ets-family proteins have been shown to play an important role in angiogenesis, we investigated the presence of ETS binding sites (EBS), GGAA/T, and ETS protein contributing to human HO-1 gene expression. Several chloramphenicol acetyltransferase constructs were examined in order to analyze the effect of ETS family proteins on the transduction of HO-1 in Xenopus oocytes and in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by FLI-1ERGETS-1 protein(s). Chloramphenicol acetyltransferase (CAT) assays demonstrated that the promoter region (-1500 to +19) contains positive and negative control elements and that all three members of the ETS protein family were responsible for the up-regulation of HHO-1. Electrophoretic mobility shift assays (EMSA), performed with nuclear extracts from endothelial cells overexpressing HHO-1 gene, and specific HHO-1 oligonucleotides probes containing putative EBS resulted in a specific and marked bandshift. Synergistic binding was observed in EMSA between AP-1 on the one hand, FLI-1, ERG, and ETS-1 protein on the other. Moreover, 5'-deletion analysis demonstrated the existence of a negative control element of HHO-1 expression located between positions -1500 and -120 on the HHO-1 promoter. The presence of regulatory sequences for transcription factors such as ETS-1, FLI-1, or ERG, whose activity is associated with cell proliferation, endothelial cell differentiation, and matrix metalloproteinase transduction, may be an indication of the important role that HO-1 may play in coronary collateral circulation, tumor growth, angiogenesis, and hemoglobin-induced endothelial cell injuries. PMID:10022513

  19. Haplodeficiency of Klotho Gene Causes Arterial Stiffening via Upregulation of Scleraxis Expression and Induction of Autophagy.

    PubMed

    Chen, Kai; Zhou, Xiaoli; Sun, Zhongjie

    2015-11-01

    The prevalence of arterial stiffness increases with age, whereas the level of the aging-suppressor protein klotho decreases with age. The objective of this study is to assess whether haplodeficiency of klotho gene causes arterial stiffness and to investigate the underlying mechanism. Pulse wave velocity, a direct measure of arterial stiffness, was increased significantly in klotho heterozygous (klotho(+/-)) mice versus their age-matched wild-type (WT) littermates, suggesting that haplodeficiency of klotho causes arterial stiffening. Notably, plasma aldosterone levels were elevated significantly in klotho(+/-) mice. Treatment with eplerenone (6 mg/kg per day IP), an aldosterone receptor blocker, abolished klotho deficiency-induced arterial stiffening in klotho(+/-) mice. Klotho deficiency was associated with increased collagen and decreased elastin contents in the media of aortas. In addition, arterial matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-?1 expression and myofibroblast differentiation were increased in klotho(+/-) mice. These klotho deficiency-related changes can be blocked by eplerenone. Protein expression of scleraxis, a transcription factor for collagen synthesis, and LC3-II/LC3-I, an index of autophagy, were upregulated in aortas of klotho(+/-) mice, which can be abolished by eplerenone. In cultured mouse aortic smooth muscle cells, aldosterone increased collagen-1 expression that can be completely eliminated by small interfering RNA knockdown of scleraxis. Interestingly, aldosterone decreased elastin levels in smooth muscle cells, which can be abolished by small interfering RNA knockdown of Beclin-1, an autophagy-related gene. In conclusion, this study demonstrated for the first time that klotho deficiency-induced arterial stiffening may involve aldosterone-mediated upregulation of scleraxis and induction of autophagy, which led to increased collagen-1 expression and decreased elastin levels, respectively. PMID:26324504

  20. MANGANESE UPREGULATES CELLULAR PRION PROTEINS AND INHIBITS THE RATE OF PROTEINASE-K DEPENDENT LIMITED PROTEOLYSIS IN NEURONAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The key event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent cations such as copper to th...

  1. Increased expression of peripheral blood leukocyte genes implicate CD14+ tissue macrophages in cellular intestine allograft rejection.

    PubMed

    Ashokkumar, Chethan; Ningappa, Mylarappa; Ranganathan, Sarangarajan; Higgs, Brandon W; Sun, Qing; Schmitt, Lori; Snyder, Sara; Dobberstein, Jennifer; Branca, Maria; Jaffe, Ronald; Zeevi, Adriana; Squires, Robert; Alissa, Feras; Shneider, Benjamin; Soltys, Kyle; Bond, Geoffrey; Abu-Elmagd, Kareem; Humar, Abhinav; Mazariegos, George; Hakonarson, Hakon; Sindhi, Rakesh

    2011-10-01

    Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events. PMID:21854741

  2. Histone acetylation associated up-regulation of the cell wall related genes is involved in salt stress induced maize root swelling

    PubMed Central

    2014-01-01

    Background Salt stress usually causes crop growth inhibition and yield decrease. Epigenetic regulation is involved in plant responses to environmental stimuli. The epigenetic regulation of the cell wall related genes associated with the salt-induced cellular response is still little known. This study aimed to analyze cell morphological alterations in maize roots as a consequence of excess salinity in relation to the transcriptional and epigenetic regulation of the cell wall related protein genes. Results In this study, maize seedling roots got shorter and displayed swelling after exposure to 200 mM NaCl for 48 h and 96 h. Cytological observation showed that the growth inhibition of maize roots was due to the reduction in meristematic zone cell division activity and elongation zone cell production. The enlargement of the stele tissue and cortex cells contributed to root swelling in the elongation zone. The cell wall is thought to be the major control point for cell enlargement. Cell wall related proteins include xyloglucan endotransglucosylase (XET), expansins (EXP), and the plasma membrane proton pump (MHA). RT-PCR results displayed an up-regulation of cell wall related ZmEXPA1, ZmEXPA3, ZmEXPA5, ZmEXPB1, ZmEXPB2 and ZmXET1 genes and the down-regulation of cell wall related ZmEXPB4 and ZmMHA genes as the duration of exposure was increased. Histone acetylation is regulated by HATs, which are often correlated with gene activation. The expression of histone acetyltransferase genes ZmHATB and ZmGCN5 was increased after 200 mM NaCl treatment, accompanied by an increase in the global acetylation levels of histones H3K9 and H4K5. ChIP experiment showed that the up-regulation of the ZmEXPB2 and ZmXET1 genes was associated with the elevated H3K9 acetylation levels on the promoter regions and coding regions of these two genes. Conclusions These data suggested that the up-regulation of some cell wall related genes mediated cell enlargement to possibly mitigate the salinity-induced ionic toxicity, and different genes had specific function in response to salt stress. Histone modification as a mediator may contribute to rapid regulation of cell wall related gene expression, which reduces the damage of excess salinity to plants. PMID:24758373

  3. Cellular unfolded protein response against viruses used in gene therapy.

    PubMed

    Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R

    2014-01-01

    Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually "gutted" and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

  4. Cellular unfolded protein response against viruses used in gene therapy

    PubMed Central

    Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R.

    2014-01-01

    Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually gutted and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

  5. Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90)

    NASA Technical Reports Server (NTRS)

    Kano, Mihoko; Kitano, Takako; Ikemoto, Madoka; Hirasaka, Katsuya; Asanoma, Yuki; Ogawa, Takayuki; Takeda, Shinichi; Nonaka, Ikuya; Adams, Gregory R.; Baldwin, Kenneth M.; Oarada, Motoko; Kishi, Kyoichi; Nikawa, Takeshi

    2003-01-01

    We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle.

  6. Mammalian Mss51 is a skeletal muscle-specific gene modulating cellular metabolism

    PubMed Central

    Moyer, Adam L.; Wagner, Kathryn R.

    2015-01-01

    Background The transforming growth factor β (TGF-β) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-β1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator. Objective The objective of this work was to characterize mammalian Mss51. Methods Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism. Results Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-β family signaling. In human muscle, Mss51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, β-oxidation, glycolysis, and oxidative phosphorylation. Conclusions Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-β1 signaling. Unlike myostatin, TGF-β1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation. PMID:26634192

  7. Mouse Hepatitis Virus Infection Upregulates Genes Involved in Innate Immune Responses

    PubMed Central

    Chatterjee, Dhriti; Addya, Sankar; Khan, Reas S.; Kenyon, Lawrence C.; Choe, Alexander; Cohrs, Randall J.; Shindler, Kenneth S.; Sarma, Jayasri Das

    2014-01-01

    Neurotropic recombinant strain of Mouse Hepatitis Virus, RSA59, induces meningo-encephalitis, myelitis and demyelination following intracranial inoculation. RSA59 induced neuropathology is partially caused by activation of CNS resident microglia, as demonstrated by changes in cellular morphology and increased expression of a microglia/macrophage specific calcium ion binding factor, Iba1. Affymetrix Microarray analysis for mRNA expression data reveals expression of inflammatory mediators that are known to be released by activated microglia. Microglia-specific cell surface molecules, including CD11b, CD74, CD52 and CD68, are significantly upregulated in contrast to CD4, CD8 and CD19. Protein analysis of spinal cord extracts taken from mice 6 days post-inoculation, the time of peak inflammation, reveals robust expression of IFN-γ, IL-12 and mKC. Data suggest that activated microglia and inflammatory mediators contribute to a local CNS microenvironment that regulates viral replication and IFN-γ production during the acute phase of infection, which in turn can cause phagolysosome maturation and phagocytosis of the myelin sheath, leading to demyelination. PMID:25360880

  8. De-repressing LncRNA-Targeted Genes to Upregulate Gene Expression: Focus on Small Molecule Therapeutics

    PubMed Central

    Fatemi, Roya Pedram; Velmeshev, Dmitry; Faghihi, Mohammad Ali

    2014-01-01

    Non-protein coding RNAs (ncRNAs) make up the overwhelming majority of transcripts in the genome and have recently gained attention for their complex regulatory role in cells, including the regulation of protein-coding genes. Furthermore, ncRNAs play an important role in normal development and their expression levels are dysregulated in several diseases. Recently, several long noncoding RNAs (lncRNAs) have been shown to alter the epigenetic status of genomic loci and suppress the expression of target genes. This review will present examples of such a mechanism and focus on the potential to target lncRNAs for achieving therapeutic gene upregulation by de-repressing genes that are epigenetically silenced in various diseases. Finally, the potential to target lncRNAs, through their interactions with epigenetic enzymes, using various tools, such as small molecules, viral vectors and antisense oligonucleotides, will be discussed. We suggest that small molecule modulators of a novel class of drug targets, lncRNA-protein interactions, have great potential to treat some cancers, cardiovascular disease, and neurological disorders. PMID:25405465

  9. 72 FR 33764 - Cellular, Tissue, and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2007-06-19

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue, and Gene Therapies Advisory Committee... Therapies, Office of Cellular, Tissue, and Gene Therapies, Center for Biologics Evaluation and Research, FDA... portion of the meeting will be closed to the public. Name of Committee: Cellular, Tissue, and...

  10. 70 FR 3934 - Cellular, Tissue and Gene Therapies Advisory Committee (formerly the Biological Response...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2005-01-27

    ... SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee (formerly the... public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee (formerly the... cellular therapies for repair and regeneration of joint surfaces. The Committee will also receive...

  11. Thyroid hormone calorigenesis and mitochondrial redox signaling: upregulation of gene expression.

    PubMed

    Videla, Luis A; Fernández, Virginia; Tapia, Gladys; Varela, Patricia

    2007-01-01

    Thyroid hormone (TH, T3) is required for the normal function of most tissues, with major effects on O2 consumption and metabolic rate. These are due to transcriptional activation of respiratory genes through the interaction of T3-liganded TH receptors with TH response elements or the activation of intermediate factors, with the consequent higher rates of mitochondrial oxidative phosphorylation and reactive O2 species (ROS) generation and antioxidant depletion. The genomic effects of TH are accompanied by redox upregulation of the liver expression of cytokines (tumor necrosis factor-alpha [TNF-alpha]), enzymes (manganese superoxide dismutase), and anti-apoptotic proteins (Bcl-2), via a cascade initiated by TNF-alpha produced by Kupffer cells and involving inhibitor of kappa-B phosphorylation and nuclear factor-kappa-B activation. Thus, TH calorigenesis triggers non-genomic effects leading to an expression pattern that may represent an adaptive mechanism to re-establish redox homeostasis and promote cell survival under conditions of ROS toxicity secondary to TH-induced oxidative stress. Mechanisms of expression of respiratory and redox-sensitive genes may be functionally integrated, which could be of importance to understand the complexities of TH action and the outcome of thyroid gland dysfunction. PMID:17127375

  12. An excitable gene regulatory circuit induces transient cellular differentiation

    NASA Astrophysics Data System (ADS)

    Süel, Gürol M.; Garcia-Ojalvo, Jordi; Liberman, Louisa M.; Elowitz, Michael B.

    2006-03-01

    Certain types of cellular differentiation are probabilistic and transient. In such systems individual cells can switch to an alternative state and, after some time, switch back again. In Bacillus subtilis, competence is an example of such a transiently differentiated state associated with the capability for DNA uptake from the environment. Individual genes and proteins underlying differentiation into the competent state have been identified, but it has been unclear how these genes interact dynamically in individual cells to control both spontaneous entry into competence and return to vegetative growth. Here we show that this behaviour can be understood in terms of excitability in the underlying genetic circuit. Using quantitative fluorescence time-lapse microscopy, we directly observed the activities of multiple circuit components simultaneously in individual cells, and analysed the resulting data in terms of a mathematical model. We find that an excitable core module containing positive and negative feedback loops can explain both entry into, and exit from, the competent state. We further tested this model by analysing initiation in sister cells, and by re-engineering the gene circuit to specifically block exit. Excitable dynamics driven by noise naturally generate stochastic and transient responses, thereby providing an ideal mechanism for competence regulation.

  13. Cerulenin upregulates heat shock protein-70 gene expression in chicken muscle.

    PubMed

    Dridi, Sami; Decuypere, Eddy; Buyse, Johan

    2013-10-01

    Lines of evidence suggested that systems involved in the regulation of the stress responses and energy homeostasis are highly integrated. Because cerulenin, the natural antibiotic product of the fungus Cephalosporium ceruleans and a broad-spectrum fatty acid synthesis (FAS) inhibitor, has been shown to affect food intake and energy balance, and because the biomarker of stress Hsp-70 gene was found to interact directly with fatty acids, we hypothesized that cerulenin may regulate Hsp-70 gene expression. Therefore, the present study was undertaken to examine this issue. Cerulenin administration significantly (P < 0.05) decreased food intake and induced Hsp-70 mRNA levels in muscle, but not in liver or hypothalamus of 2-wk-old broiler chickens. These changes were accompanied by an unpregulation of muscle uncoupling protein and carnitine palmitoyltransferase 1 mRNA levels. This result indicated that the regulation of Hsp-70 gene expression in normal chickens, as estimated by oxidative stress indices [TBA reacting substances, ferric reducing/antioxidant power, and ceruloplasmin oxidase activity] levels, is tissue-specific. In attempt to discriminate between the effect of cerulenin and cerulenin-reduced food intake on Hsp-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses. Food deprivation for 16 h did not affect Hsp-70 gene expression in all tissues examined, indicating that the effect of cerulenin is independent of the inhibition of food intake. To ascertain whether the effect of cerulenin is direct or indirect, we carried out in vitro studies. Cerulenin treatment did not affect Hsp-70 gene expression in Leghorn male hepatoma and quail myoblast cell lines, suggesting that the observed effect in vivo may be mediated through the central nervous system. PMID:24046423

  14. Sampling-Dependent Up-regulation of Gene Expression in Sequential Samples of Human Airway Epithelial Cells

    PubMed Central

    Heguy, Adriana; Harvey, Ben-Gary; O’Connor, Timothy P; Hackett, Neil R; Crystal, Ronald G

    2003-01-01

    As part of a study of in vivo gene expression levels in the human airway epithelium in response to chronic cigarette smoking, we have identified a number of genes whose expression levels are altered in a time-dependent fashion resulting from the procedure used to sample epithelial cells. Fiberoptic bronchoscopy and airway epithelium brushing were used to obtain independent samples from a single individual, 1st from the right lung, followed by sampling of the left lung. We observed that a specific subset of early response genes encoding proteins involved in transcription, signal transduction, cell cycle/growth, and apoptosis were significantly up-regulated in the left lung samples (the 2nd region to be sampled) compared with the right lung samples (the 1st region to be sampled). This response was due to the temporal nature of the sampling procedure and not to inherent gene expression differences between airway epithelium of the right and left lungs. When the order of sampling was reversed, with the left airway epithelium sampled 1st, the same subset of genes were up-regulated in the samples obtained from the right airway epithelium. The time-dependent up-regulation of these genes was likely in response to the stress of the procedure and/or the anesthesia used. Sampling-dependent uncertainty of gene expression is likely a general phenomenon relevant to the procedures used for obtaining biological samples, particularly in humans where the sampling procedures are dependent on ensuring comfort and safety. PMID:15208741

  15. Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

    PubMed Central

    Finka, Andrija; Mattoo, Rayees U. H.

    2010-01-01

    Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0216-8) contains supplementary material, which is available to authorized users. PMID:20694844

  16. The Effect of Gravity Fields on Cellular Gene Expression

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, Millie

    1999-01-01

    Early theoretical analysis predicted that microgravity effects on the isolated cell would be minuscule at the subcellular level; however, these speculations have not proven true in the real world. Astronauts experience a significant bone and muscle loss in as little as 2 weeks of spaceflight and changes are seen at the cellular level soon after exposure to microgravity. Changes in biological systems may be primarily due to the lack of gravity and the resulting loss of mechanical stress on tissues and cells. Recent ground and flight studies examining the effects of gravity or mechanical stress on cells demonstrate marked changes in gene expression when relatively small changes in mechanical forces or gravity fields were made. Several immediate early genes (IEG) like c-fos and c-myc are induced by mechanical stimulation within minutes. In contrast, several investigators report that the absence of mechanical forces during space flight result in decreased sera response element (SRE) activity and attenuation of expression of IEGs such as c-fos, c-jun and cox-2 mRNAs. Clearly, these early changes in gene expression may have long term consequences on mechanically sensitive cells. In our early studies on STS-56, we reported four major changes in the osteoblast; 1) prostaglandin synthesis in flight, 2) changes in cellular morphology, 3) altered actin cytoskeleton and 4) reduced osteoblast growth after four days exposure to microgravity. Initially, it was believed that changes in fibronectin (FN) RNA, FN protein synthesis or subsequent FN matrix formation might account for the changes in cytoskeleton and/ or reduction of growth. However our recent studies on Biorack (STS-76, STS-81 and STS-84), using ground and in-flight 1-G controls, demonstrated that fibronectin synthesis and matrix formation were normal in microgravity. In addition, in our most recent Biorack paper, our laboratory has documented that relative protein synthesis and mRNA synthesis are not changed after 24 hours exposure to microgravity. We did, however, find significant changes in osteoblast gene expression of IEGs, c-fos and cox-2 in microgravity exposure as compared to ground and in-flight 1-G controls. Subsequent ground studies suggest that the molecular mechanism underlying these changes may involve prostaglandin c-AMP receptors (EPs) and/or subsequent alteration of intracellular signaling in the absence of gravity.

  17. Age-Associated Epigenetic Upregulation of the FKBP5 Gene Selectively Impairs Stress Resiliency

    PubMed Central

    Sabbagh, Jonathan J.; O'Leary, John C.; Blair, Laura J.; Klengel, Torsten; Nordhues, Bryce A.; Fontaine, Sarah N.; Binder, Elisabeth B.; Dickey, Chad A.

    2014-01-01

    Single nucleotide polymorphisms (SNPs) in the FK506 binding protein 5 (FKBP5) gene combine with traumatic events to increase risk for post-traumatic stress and major depressive disorders (PTSD and MDD). These SNPs increase FKBP51 protein expression through a mechanism involving demethylation of the gene and altered glucocorticoid signaling. Aged animals also display elevated FKBP51 levels, which contribute to impaired resiliency to depressive-like behaviors through impaired glucocorticoid signaling, a phenotype that is abrogated in FKBP5?/? mice. But the age of onset and progressive stability of these phenotypes remain unknown. Moreover, it is unclear how FKBP5 deletion affects other glucocorticoid-dependent processes or if age-associated increases in FKBP51 expression are mediated through a similar epigenetic process caused by SNPs in the FKBP5 gene. Here, we show that FKBP51-mediated impairment in stress resiliency and glucocorticoid signaling occurs by 10 months of age and this increased over their lifespan. Surprisingly, despite these progressive changes in glucocorticoid responsiveness, FKBP5?/? mice displayed normal longevity, glucose tolerance, blood composition and cytokine profiles across lifespan, phenotypes normally associated with glucocorticoid signaling. We also found that methylation of Fkbp5 decreased with age in mice, a process that likely explains the age-associated increases in FKBP51 levels. Thus, epigenetic upregulation of FKBP51 with age can selectively impair psychological stress-resiliency, but does not affect other glucocorticoid-mediated physiological processes. This makes FKBP51 a unique and attractive therapeutic target to treat PTSD and MDD. In addition, aged wild-type mice may be a useful model for investigating the mechanisms of FKBP5 SNPs associated with these disorders. PMID:25191701

  18. The effect of risedronate on osteogenic lineage is mediated by cyclooxygenase-2 gene upregulation

    PubMed Central

    2010-01-01

    Introduction The purpose of this study was to evaluate the effects of risedronate (Ris) in the modulation of bone formation in rats with glucocorticoid (GC)-induced osteoporosis by histomorphometric, immunohistochemical and gene expression analyses. Methods We analyzed structure, turnover and microarchitecture, cyclooxygenase 2 (COX-2) levels and osteocyte apoptosis in 40 female rats divided as follows: 1) vehicle of methylprednisolone (vGC) + vehicle of risedronate (vRis); 2) Ris 5 ?g/Kg + vGC; 3) methylprednisolone (GC) 7 mg/Kg + vRis; 4) GC 7 mg/Kg +Ris 5 ?g/Kg. In addition, we evaluated cell proliferation and expression of COX-2 and bone alkaline phosphatase (b-ALP) genes in bone marrow cells and MLO-y4 osteocytes treated with Ris alone or in co-treatment with the selective COX-2 inhibitor NS-398 or with dexametasone. Results Ris reduced apoptosis induced by GC of osteocytes (41% vs 86%, P < 0.0001) and increased COX-2 expression with respect to controls (Immuno-Hystochemical Score (IHS): 8.75 vs 1.00, P < 0.0001). These positive effects of Ris in bone formation were confirmed by in vitro data as the viability and expression of b-ALP gene in bone marrow cells resulted increased in a dose dependent manner. Conclusions These findings suggest a positive effect of Ris in bone formation and support the hypothesis that the up-regulation of COX-2 could be an additional mechanism of anabolic effect of Ris. PMID:20738860

  19. Osteopontin is up-regulated in chronic hepatitis C and is associated with cellular permissiveness for hepatitis C virus replication

    PubMed Central

    Choi, Steve S; Claridge, Lee C; Jhaveri, Ravi; Swiderska-Syn, Marzena; Clark, Paul; Suzuki, Ayako; Pereira, Thiago A; Mi, Zhiyong; Kuo, Paul C; Guy, Cynthia D; Pereira, Fausto EL; Diehl, Anna Mae; Patel, Keyur; Syn, Wing-Kin

    2014-01-01

    Background Osteopontin (OPN) is a Hedgehog (Hh)-regulated cytokine that is up-regulated during chronic liver injury, and directly promotes fibrosis. We reported that Hh-signaling enhances viral permissiveness and replication in HCV-infected cells. Hence, we hypothesized that OPN directly promotes HCV replication, and that targeting OPN could be beneficial in HCV. Methods We compared expression of OPN mRNA and protein in HCV (JFH1)-infected Huh7 and Huh7.5 cells, and evaluated if modulating OPN levels using exogenous OPN ligands (upregulate OPN) or OPN-specific RNA-aptamers (neutralize OPN), leads to changes in HCV expression. Sera and livers from patients with chronic HCV were analyzed to determine if OPN levels were associated with disease severity or response to therapy. Results Compared with Huh7, Huh7.5 support higher levels of HCV replication (15-fold), and expressed significantly more OPN mRNA (30-fold) and protein. Treating Huh7 with OPN ligands led to dose-related increase in HCV (15-fold) and OPN (8-fold) mRNA. Conversely, treating Huh7.5 with OPN-specific RNA-aptamers inhibited HCV RNA and protein by >50% and repressed OPN mRNA to basal levels. Liver OPN expression was significantly higher (3-fold) in patients with advanced fibrosis. Serum OPN positively correlated with fibrosis-stage (p=0.009), but negatively correlated with end-of-treatment (ET) biochemical-response (BCR), ET virological-response (VR), sustained (S)BCR, and SVR (p=0.007). The OPN-Fibrosis Score (serum OPN and presence of fibrosis ?F2) may be a predictor of SVR. Conclusions OPN is upregulated in the liver and serum of patients with chronic HCV, and supports increased viral replication. OPN neutralization may be a novel therapeutic strategy in chronic HCV. PMID:24438228

  20. 79 FR 50660 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2014-08-25

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee... entitled ``Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and...

  1. 73 FR 59635 - Draft Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2008-10-09

    ... Gene Therapy Products; Availability AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY... ``Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products'' dated October 2008. The draft guidance document provides manufacturers of cellular and gene therapy (CGT) products with...

  2. 78 FR 79699 - Cellular, Tissue, and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-31

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue, and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue, and Gene Therapies Advisory Committee..., Tissue, and Gene Therapies, Center for Biologics Evaluation and Research (CBER), FDA. On February...

  3. 75 FR 66381 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-28

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee... Lentiviral Vector Based Gene Therapy Products. FDA intends to make background material available to...

  4. 76 FR 22405 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-21

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee... gene therapy products for the treatment of retinal disorders. Topics to be considered include...

  5. Pea lectin receptor-like kinase functions in salinity adaptation without yield penalty, by alleviating osmotic and ionic stresses and upregulating stress-responsive genes.

    PubMed

    Vaid, Neha; Pandey, Prashant; Srivastava, Vineet Kumar; Tuteja, Narendra

    2015-05-01

    Lectin receptor-like kinases (LecRLKs) are members of RLK family composed of lectin-like extracellular recognition domain, transmembrane domain and cytoplasmic kinase domain. LecRLKs are plasma membrane proteins believed to be involved in signal transduction. However, most of the members of the protein family even in plants have not been functionally well characterized. Herein, we show that Pisum sativum LecRLK (PsLecRLK) localized in plasma membrane systems and/or other regions of the cell and its transcript upregulated under salinity stress. Overexpression of PsLecRLK in transgenic tobacco plants confers salinity stress tolerance by alleviating both the ionic as well the osmotic component of salinity stress. The transgenic plants show better tissue compartmentalization of Na(+) and higher ROS scavenging activity which probably results in lower membrane damage, improved growth and yield maintenance even under salinity stress. Also, expression of several genes involved in cellular homeostasis is perturbed by PsLecRLK overexpression. Alleviation of osmotic and ionic components of salinity stress along with reduced oxidative damage and upregulation of stress-responsive genes in transgenic plants under salinity stress conditions could be possible mechanism facilitating enhanced stress tolerance. This study presents PsLecRLK as a promising candidate for crop improvement and also opens up new avenue to investigate its signalling pathway. PMID:25863480

  6. 77 FR 71194 - Draft Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-29

    ...). The product areas covered by this guidance are cellular therapy, gene therapy, therapeutic vaccination..., therapeutic vaccination, and xenotransplantation. The guidance is intended to clarify current...

  7. Hfr-2, a wheat cytolytic toxin-like gene, is up-regulated by virulent Hessian fly larval feedingdouble dagger.

    PubMed

    Puthoff, David P; Sardesai, Nagesh; Subramanyam, Subhashree; Nemacheck, Jill A; Williams, Christie E

    2005-07-01

    SUMMARY Both yield and grain-quality are dramatically decreased when susceptible wheat (Triticum aestivum) plants are infested by Hessian fly (Mayetiola destructor) larvae. Examination of the changes in wheat gene expression during infestation by virulent Hessian fly larvae has identified the up-regulation of a gene, Hessian fly responsive-2 (Hfr-2), which contains regions similar to genes encoding seed-specific agglutinin proteins from Amaranthus. Hfr-2, however, did not accumulate in developing seeds, as do other wheat seed storage proteins. Additionally, a separate region of the HFR-2 predicted amino acid sequence is similar to haemolytic proteins, from both mushroom and bacteria, that are able to form pores in cell membranes of mammalian red blood cells. The involvement of Hfr-2 in interactions with insects was supported by experiments demonstrating its up-regulation by both fall armyworm (Spodoptera frugiperda) and bird cherry-oat aphid (Rhopalosiphum padi) infestations but not by virus infection. Examination of wheat defence response pathways showed Hfr-2 up-regulation following methyl jasmonate treatment and only slight up-regulation in response to salicylic acid, abscisic acid and wounding treatments. Like related proteins, HFR-2 may normally function in defence against certain insects or pathogens. However, we propose that as virulent Hessian fly larvae manipulate the physiology of the susceptible host, the HFR-2 protein inserts in plant cell membranes at the feeding sites and by forming pores provides water, ions and other small nutritive molecules to the developing larvae. PMID:20565667

  8. Carbon nanoparticle induced cytotoxicity in human mesenchymal stem cells through upregulation of TNF3, NFKBIA and BCL2L1 genes.

    PubMed

    Periasamy, Vaiyapuri S; Athinarayanan, Jegan; Alfawaz, Mohammed A; Alshatwi, Ali A

    2016-02-01

    Carbon based nanomaterials, including carbon nanotubes, graphene, nanodiamond and carbon nanoparticles, have emerged as potential candidates for a wide variety of applications because of their unusual electrical, mechanical, thermal and optical properties. However, our understanding of how increased usage of carbon based nanomaterials could lead to harmful effects in humans and other biological systems is inadequate. Our present investigation is focused on the cellular toxicity of carbon nanoparticles (CNPs) on human mesenchymal stem cells (hMSCs). Following exposure to CNPs, cell viability, nuclear morphological changes, apoptosis and cell cycle progression were monitored. Furthermore, the expression of genes involved in both cell death (e.g., P53, TNF3, CDKN1A, TNFRSF1A, TNFSF10, NFKBIA, BCL2L1) and cell cycle regulation (e.g., PCNA, EGR1, E2F1, CCNG1, CCND1, CCNC, CYCD3) were assessed using qPCR. Our results indicated that CNPs reduce cell viability and cause chromatin condensation and DNA fragmentation. Cell cycle analysis indicated that CNPs affect the cell cycle progression. However, the gene expression measurements confirmed that CNPs significantly upregulated the P53, TNF3, CDKNIA, and NFKBIA genes and downregulated the EGR1 gene in hMSCs. Our findings suggest that CNPs reduce cell viability by disrupting the expression of cell death genes in human mesenchymal stem cell (hMSC). The results of this investigation revealed that CNPs exhibited moderate toxicity on hMSCs. PMID:26364217

  9. Prolonged proteasome inhibition cyclically upregulates Oct3/4 and Nanog gene expression, but reduces induced pluripotent stem cell colony formation.

    PubMed

    Floyd, Z Elizabeth; Floyd, Elizabeth Z; Staszkiewicz, Jaroslaw; Power, Rachel A; Kilroy, Gail; Kirk-Ballard, Heather; Barnes, Christian W; Strickler, Karen L; Rim, Jong S; Harkins, Lettie L; Gao, Ru; Kim, Jeong; Eilertsen, Kenneth J

    2015-04-01

    There is ample evidence that the ubiquitin-proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. PMID:25826722

  10. MDM-2 Oncoprotein Overexpression, p53 Gene Mutation, and VEGF Up-Regulation in Angiosarcomas

    PubMed Central

    Zietz, Christian; Rssle, Matthias; Haas, Christian; Sendelhofert, Andrea; Hirschmann, Astrid; Strzl, Michael; Lhrs, Udo

    1998-01-01

    The endothelium is one of the largest cellular compartments of the human body and has a high proliferative potential. However, angiosarcomas are among the rarest malignancies. Despite this interesting contradiction, data on growth and angiogenesis control mechanisms of angiosarcomas are scarce. In this study of 19 angiosarcomas and 10 benign vascular control lesions we investigated the sequence and expression of the p53 tumor suppressor gene and the expression of the mdm-2 proto-oncogene, which is a negative regulator of p53 activity and of the vascular endothelial growth factor (VEGF), whose expression, among other factors, is regulated by the p53/MDM-2 pathway. Ten sarcomas (53%) exibited clear nuclear p53 protein accumulation. Two of these cases revealed mutations in the sequence-specific DNA binding domain of the p53 gene. Thirteen angiosarcomas (68%) showed an increased amount of MDM-2 protein. Elevated expression of p53 and MDM-2 protein correlated with increased VEGF expression, which was found in nearly 80% of the angiosarcoma cases. Negative or clearly lower immunostaining was obtained in cases from the benign control collective. Only one case of a juvenile hemangioma reached the cutoff value of p53 positivity coincidentally with high VEGF expression. Our data suggest that the p53/MDM-2 pathway is impaired in about two-thirds (14/19) of the angiosarcomas. This may be a key event in the pathogenesis of human angiosarcomas. The increased VEGF expression observed supports this hypothesis. PMID:9811333

  11. 77 FR 73472 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-10

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... and Gene Therapies Advisory Committee. General Function of the Committee: To provide advice...

  12. Maackiain is a novel antiallergic compound that suppresses transcriptional upregulation of the histamine H1 receptor and interleukin-4 genes

    PubMed Central

    Mizuguchi, Hiroyuki; Nariai, Yuki; Kato, Shuhei; Nakano, Tomohiro; Kanayama, Tomoyo; Kashiwada, Yoshiki; Nemoto, Hisao; Kawazoe, Kazuyoshi; Takaishi, Yoshihisa; Kitamura, Yoshiaki; Takeda, Noriaki; Fukui, Hiroyuki

    2015-01-01

    Kujin contains antiallergic compounds that inhibit upregulation of histamine H1 receptor (H1R) and interleukin (IL)-4 gene expression. However, the underlying mechanism remains unknown. We sought to identify a Kujin-derived antiallergic compound and investigate its mechanism of action. The H1R and IL-4 mRNA levels were determined by real-time quantitative RT-PCR. To investigate the effects of maackiain invivo, toluene-2,4-diisocyanate (TDI)-sensitized rats were used as a nasal hypersensitivity animal model. We identified (?)-maackiain as the responsible component. Synthetic maackiain showed stereoselectivity for the suppression of IL-4 gene expression but not for H1R gene expression, suggesting distinct target proteins for transcriptional signaling. (?)-Maackiain inhibited of PKC? translocation to the Golgi and phosphorylation of Tyr311 on PKC?, which led to the suppression of H1R gene transcription. However, (?)-maackiain did not show any antioxidant activity or inhibition of PKC? enzymatic activity per se. Pretreatment with maackiain alleviated nasal symptoms and suppressed TDI-induced upregulations of H1R and IL-4 gene expressions in TDI-sensitized rats. These data suggest that (?)-maackiain is a novel antiallergic compound that alleviates nasal symptoms in TDI-sensitized allergy model rats through the inhibition of H1R and IL-4 gene expression. The molecular mechanism underlying its suppressive effect for H1R gene expression is mediated by the inhibition of PKC? activation. PMID:26516579

  13. Lymphoid Gene Upregulation on Circulating Progenitors Participates in Their T-Lineage Commitment.

    PubMed

    Zepponi, Vanessa; Michaels Lopez, Victoria; Martinez-Cingolani, Carolina; Boudil, Amine; Pasqualetto, Valrie; Skhiri, Lamia; Gautreau, Laetitia; Legrand, Agns; Megret, Jerome; Zavala, Flora; Ezine, Sophie

    2015-07-01

    Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9(+) progenitors are more abundant in the blood than CCR7(+) progenitors. Second, although Flt3(-) CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25(+)-expressing cells and downregulated c-Kit and IL-7R? intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations' respective abilities to generate splenic T cell precursors (Lin(-)Thy1.2(+)CD25(+)IL7R?(+)) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3(-) CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement. PMID:26026063

  14. 72 FR 73850 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2007-12-28

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... portion of the meeting will be closed to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee. General Function of the Committee: To provide advice and recommendations...

  15. 79 FR 44184 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2014-07-30

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... portion of the meeting will be closed to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee. General Function of the Committee: To provide advice and recommendations...

  16. 70 FR 38689 - Cellular, Tissue and Gene Therapies Advisory Committee (formerly Biological Response Modifiers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2005-07-05

    ... From the Federal Register Online via the Government Printing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee (formerly.... Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee. General Function of...

  17. 71 FR 64541 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2006-11-02

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... portion of the meeting will be closed to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee. General Function of the Committee: To provide advice and recommendations...

  18. 74 FR 46435 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2009-09-09

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee... ISOLAGEN THERAPY, BLA 125348, Isolagen Technologies, Inc., for moderate to severe nasolabial fold...

  19. 76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-16

    ..., 2008 (73 FR 59635), FDA announced the availability of the draft guidance of the same title. FDA... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Potency Tests for Cellular and Gene... Industry: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance...

  20. 77 FR 65693 - Cellular, Tissue and Gene Therapies Advisory Committee; Amendment of Notice

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-30

    ... Administration (FDA) is announcing an amendment to the notice of a meeting of the Cellular, Tissue and Gene Therapies Advisory Committee. This meeting was announced in the Federal Register of October 17, 2012 (77 FR... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory...

  1. 76 FR 64951 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-19

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory...

  2. 76 FR 49774 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-11

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... be open to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory...

  3. N-acetylcysteine inhibits the upregulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically

    PubMed Central

    Caro, Andres A.; Bell, Matthew; Ejiofor, Shannon; Zurcher, Grant; Petersen, Dennis R.; Ronis, Martin J. J.

    2014-01-01

    Background Chronic ethanol administration to experimental animals induces hepatic oxidative stress and upregulates mitochondrial biogenesis. The mechanisms by which chronic ethanol upregulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative stress is a factor that triggers mitochondrial biogenesis after chronic ethanol feeding. If our hypothesis is correct, co-administration of antioxidants should prevent upregulation of mitochondrial biogenesis genes. Methods Rats were fed an ethanol-containing diet intragastrically by total enteral nutrition for 150 days, in the absence or presence of the antioxidant N-acetylcysteine (NAC) at 1.7 g/kg/day; control rats were administered isocaloric diets where carbohydrates substituted for ethanol calories. Results Ethanol administration significantly increased hepatic oxidative stress, evidenced as decreased liver total glutathione and GSH/GSSG ratio. These effects were inhibited by co-administration of ethanol and NAC. Chronic ethanol increased the expression of mitochondrial biogenesis genes including peroxisome proliferator activated receptor gamma-coactivator-1 alpha and mitochondrial transcription factor A, and mitochondrial DNA; co-administration of ethanol and NAC prevented these effects. Chronic ethanol administration was associated with decreased mitochondrial mass, inactivation and depletion of mitochondrial complex I and complex IV, and increased hepatic mitochondrial oxidative damage, effects that were not prevented by NAC. Conclusions These results suggest that oxidative stress caused by chronic ethanol triggered the upregulation of mitochondrial biogenesis genes in rat liver, because an antioxidant such as NAC prevented both effects. Because NAC did not prevent liver mitochondrial oxidative damage, extra-mitochondrial effects of reactive oxygen species may regulate mitochondrial biogenesis. In spite of the induction of hepatic mitochondrial biogenesis genes by chronic ethanol, mitochondrial mass and function decreased probably in association with mitochondrial oxidative damage. These results also predict that the effectiveness of NAC as an antioxidant therapy for chronic alcoholism will be limited by its limited antioxidant effects in mitochondria, and its inhibitory effect on mitochondrial biogenesis. PMID:25581647

  4. Non-Thermal Plasma Treatment Diminishes Fungal Viability and Up-Regulates Resistance Genes in a Plant Host

    PubMed Central

    Panngom, Kamonporn; Lee, Sang Hark; Park, Dae Hoon; Sim, Geon Bo; Kim, Yong Hee; Uhm, Han Sup; Park, Gyungsoon; Choi, Eun Ha

    2014-01-01

    Reactive oxygen and nitrogen species can have either harmful or beneficial effects on biological systems depending on the dose administered and the species of organism exposed, suggesting that application of reactive species can possibly produce contradictory effects in disease control, pathogen inactivation and activation of host resistance. A novel technology known as atmospheric-pressure non-thermal plasma represents a means of generating various reactive species that adversely affect pathogens (inactivation) while simultaneously up-regulating host defense genes. The anti-microbial efficacy of this technology was tested on the plant fungal pathogen Fusarium oxysporum f.sp. lycopersici and its susceptible host plant species Solanum lycopercicum. Germination of fungal spores suspended in saline was decreased over time after exposed to argon (Ar) plasma for 10 min. Although the majority of treated spores exhibited necrotic death, apoptosis was also observed along with the up-regulation of apoptosis related genes. Increases in the levels of peroxynitrite and nitrite in saline following plasma treatment may have been responsible for the observed spore death. In addition, increased transcription of pathogenesis related (PR) genes was observed in the roots of the susceptible tomato cultivar (S. lycopercicum) after exposure to the same Ar plasma dose used in fungal inactivation. These data suggest that atmospheric-pressure non-thermal plasma can be efficiently used to control plant fungal diseases by inactivating fungal pathogens and up-regulating mechanisms of host resistance. PMID:24911947

  5. Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease

    PubMed Central

    Choi, Christopher J.; Anantharam, Vellareddy; Martin, Dustin P.; Nicholson, Eric M.; Richt, Jrgen A.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2010-01-01

    Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrPC) into an abnormal form of scrapie prion (PrPSc). The cellular mechanisms underlying the misfolding of PrPC are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrPC processing in in vitro models of prion disease. Exposure to manganese significantly increased PrPC levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrPC upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrPC degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrPC turnover rate was significantly altered with manganese treatment, indicating increased stability of PrPC with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrPC. Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratoryinfected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis. PMID:20176619

  6. Virus-Induced Transcriptional Changes in the Brain Include the Differential Expression of Genes Associated with Interferon, Apoptosis, Interleukin 17 Receptor A, and Glutamate Signaling as Well as Flavivirus-Specific Upregulation of tRNA Synthetases

    PubMed Central

    Clarke, Penny; Leser, J. Smith; Bowen, Richard A.; Tyler, Kenneth L.

    2014-01-01

    ABSTRACT Flaviviruses, particularly Japanese encephalitis virus (JEV) and West Nile virus (WNV), are important causes of virus-induced central nervous system (CNS) disease in humans. We used microarray analysis to identify cellular genes that are differentially regulated following infection of the brain with JEV (P3) or WNV (New York 99). Gene expression data for these flaviviruses were compared to those obtained following infection of the brain with reovirus (type 3 Dearing), an unrelated neurotropic virus. We found that a large number of genes were up-regulated by all three viruses (using the criteria of a change of >2-fold and a P value of <0.001), including genes associated with interferon signaling, the immune system, inflammation, and cell death/survival signaling. In addition, genes associated with glutamate signaling were down-regulated in infections with all three viruses (criteria, a >2-fold change and a P value of <0.001). These genes may serve as broad-spectrum therapeutic targets for virus-induced CNS disease. A distinct set of genes were up-regulated following flavivirus infection but not following infection with reovirus. These genes were associated with tRNA charging and may serve as therapeutic targets for flavivirus-induced CNS disease. PMID:24618253

  7. Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

    PubMed

    Kumar, Gokhlesh; Sarker, Subhodeep; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

    2015-06-01

    Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan. PMID:25786607

  8. Rapid acclimation of juvenile corals to CO2 -mediated acidification by upregulation of heat shock protein and Bcl-2 genes.

    PubMed

    Moya, A; Huisman, L; Fort, S; Gattuso, J-P; Hayward, D C; Ball, E E; Miller, D J

    2015-01-01

    Corals play a key role in ocean ecosystems and carbonate balance, but their molecular response to ocean acidification remains unclear. The only previous whole-transcriptome study (Moya etal. Molecular Ecology, 2012; 21, 2440) documented extensive disruption of gene expression, particularly of genes encoding skeletal organic matrix proteins, in juvenile corals (Acropora millepora) after short-term (3d) exposure to elevated pCO2 . In this study, whole-transcriptome analysis was used to compare the effects of such 'acute' (3d) exposure to elevated pCO2 with a longer ('prolonged'; 9d) period of exposure beginning immediately post-fertilization. Far fewer genes were differentially expressed under the 9-d treatment, and although the transcriptome data implied wholesale disruption of metabolism and calcification genes in the acute treatment experiment, expression of most genes was at control levels after prolonged treatment. There was little overlap between the genes responding to the acute and prolonged treatments, but heat shock proteins (HSPs) and heat shock factors (HSFs) were over-represented amongst the genes responding to both treatments. Amongst these was an HSP70 gene previously shown to be involved in acclimation to thermal stress in a field population of another acroporid coral. The most obvious feature of the molecular response in the 9-d treatment experiment was the upregulation of five distinct Bcl-2 family members, the majority predicted to be anti-apoptotic. This suggests that an important component of the longer term response to elevated CO2 is suppression of apoptosis. It therefore appears that juvenile A.millepora have the capacity to rapidly acclimate to elevated pCO2 , a process mediated by upregulation of specific HSPs and a suite of Bcl-2 family members. PMID:25444080

  9. Screening of pro-apoptotic genes upregulated in an experimental street rabies virus-infected neonatal mouse brain.

    PubMed

    Ubol, Sukathida; Kasisith, Jitra; Pitidhammabhorn, Dhanesh; Tepsumethanol, Veera

    2005-01-01

    Rabies virus (RABV) is able to induce apoptotic death of target cells. The molecular pathway of RABV-induced cell death is partially known. In the present study, cDNA array analysis was used as a tool to screen for pro-apoptotic genes that may be involved in RABV induction. RNA was extracted from the infected CNS and from mock-infected controls. When the mean gene expression was compared between the infected group and controls, 21 potential apoptotic genes were identified that exhibited more than 2.5-fold difference in their expression levels. These 21 genes can be grouped into two groups, those genes that participate in the commitment phase and those that play a role as executioners. Examples of genes in commitment phase were death receptors (Fas-L receptor, TNF-receptor), lysosomal proteases, calpain, caspase-1, signaling molecules (ERK, p38MAPK) and bcl-2 family members. Cytochrome c and caspase-3 were representatives of executioners. Based on types of genes activated during the commitment phase, two independent apoptotic mechanisms may be activated in response to the RV infection. The first is immune-mediated death which may operate through the receptor-ligand pathway activated by caspase-1 and the pro-inflammatory cytokine, IL-1beta. The other mechanism is a protease-mediated process which involves lysosomal proteases and calcium-dependent neutral proteases. These two stimulating pathways were followed by Bad, Bak, Bid activation and subsequently the upregulation of cytochrome c and caspase-3. In addition, mobilization of K+ ion and other accessory apoptotic genes such as annexins and clusterin were also upregulated. PMID:15905604

  10. Survivin enhances telomerase activity via up-regulation of specificity protein 1- and c-Myc-mediated human telomerase reverse transcriptase gene transcription

    SciTech Connect

    Endoh, Teruo; Tsuji, Naoki; Asanuma, Koichi; Yagihashi, Atsuhito; Watanabe, Naoki . E-mail: watanabn@sapmed.ac.jp

    2005-05-01

    Suppression of apoptosis is thought to contribute to carcinogenesis. Survivin, a member of the inhibitor-of-apoptosis family, blocks apoptotic signaling activated by various cellular stresses. Since elevated expression of survivin observed in human cancers of varied origin was associated with poor patient survival, survivin has attracted growing attention as a potential target for cancer treatment. Immortalization of cells also is required for carcinogenesis; telomere length maintenance by telomerase is required for cancer cells to proliferate indefinitely. Yet how cancer cells activate telomerase remains unclear. We therefore examined possible interrelationships between survivin expression and telomerase activity. Correlation between survivin and human telomerase reverse transcriptase (hTERT) expression was observed in colon cancer tissues, and overexpression of survivin enhanced telomerase activity by up-regulation of hTERT expression in LS180 human colon cancer cells. DNA-binding activities of specificity protein 1 (Sp1) and c-Myc to the hTERT core promoter were increased in survivin gene transfectant cells. Phosphorylation of Sp1 and c-Myc at serine and threonine residues was enhanced by survivin, while total amounts of these proteins were unchanged. Further, 'knockdown' of survivin by a small inhibitory RNA decreased Sp1 and c-Myc phosphorylation. Thus survivin participates not only in inhibition of apoptosis, but also in prolonging cellular lifespan.

  11. Enhanced Cellular Responses and Distinct Gene Profiles in Human Fetoplacental Artery Endothelial Cells under Chronic Low Oxygen1

    PubMed Central

    Jiang, Yi-Zhou; Wang, Kai; Li, Yan; Dai, Cai-Feng; Wang, Ping; Kendziorski, Christina; Chen, Dong-Bao; Zheng, Jing

    2013-01-01

    ABSTRACT Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O2) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O2) for ∼20–25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O2 decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O2. PMID:24152727

  12. Parallel declines in cognition, motivation, and locomotion in aging mice: association with immune gene upregulation in the medial prefrontal cortex

    PubMed Central

    Bordner, Kelly A.; Kitchen, Robert R.; Carlyle, Becky; George, Elizabeth D.; Mahajan, Milind C.; Mane, Shrikant M.; Taylor, Jane R.; Simen, Arthur A.

    2013-01-01

    Aging in humans is associated with parallel changes in cognition, motivation, and motoric performance. Based on the human aging literature, we hypothesized that this constellation of age-related changes is mediated by the medial prefrontal cortex and that it would be observed in aging mice. Toward this end, we performed detailed assessments of cognition, motivation, and motoric behavior in aging mice. We assessed behavioral and cognitive performance in C57Bl/6 mice aged 6, 18, and 24 months, and followed this with microarray analysis of tissue from the medial prefrontal cortex and analysis of serum cytokine levels. Multivariate modeling of these data suggested that the age-related changes in cognition, motivation, motor performance, and prefrontal immune gene expression were highly correlated. Peripheral cytokine levels were also correlated with these variables, but less strongly than measures of prefrontal immune gene upregulation. To determine whether the observed immune gene expression changes were due to prefrontal microglial cells, we isolated CD11b-positive cells from the prefrontal cortex and subject them to next-generation RNA sequencing. Many of the immune changes present in whole medial prefrontal cortex were enriched in this cell population. These data suggest that, as in humans, cognition, motivation, and motoric performance in the mouse change together with age and are strongly associated with CNS immune gene upregulation. PMID:21453768

  13. Acute cold- and chronic heat-exposure upregulate hepatic leptin and muscle uncoupling protein (UCP) gene expression in broiler chickens.

    PubMed

    Dridi, Sami; Temim, Soraya; Derouet, Michel; Tesseraud, Sophie; Taouis, Mohammed

    2008-08-01

    Emerging evidence showed that variations in environmental temperature affect both leptin and uncoupling protein (UCP) gene expression in mammals, whereas a little is known about such interactions in birds. Thus, we conducted the present study to investigate the influence of acute (2 hours) cold (4 degrees C) and chronic (10 days) heat (32 degrees C) exposure on hepatic leptin and muscle UCP gene expression in 5-wk-old broiler chickens. Both cold- and heat-exposure significantly (P < 0.05 to P < 0.001) upregulated hepatic leptin (by 35 and 46%, respectively) and muscle UCP mRNA levels (by 71 and 71%, respectively) compared to the thermoneutrality (22 degrees C). This result suggests that leptin and UCP may be involved in the thermoregulation response of chickens to extreme climate (cold and hot temperatures). The upregulation of hepatic leptin gene expression was accompanied by an increase in plasma leptin levels, indicating that leptin may be regulated at transcriptional level. The increase of leptin and UCP mRNA abundance, and leptinemia we report here were not related to plasma glucose or insulin levels. In conclusion, the exposure of broiler chickens to extreme ambient temperatures (cold and heat) increases hepatic leptin and muscle UCP gene expression. PMID:18473347

  14. 77 FR 63840 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-17

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee..., Tissue and Gene Therapies Advisory Committee. General Function of the Committee: To provide advice and... in open session to hear updates of research programs in the Gene Transfer and Immunogenicity...

  15. 76 FR 81513 - Cellular, Tissue, and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-28

    ... HUMAN SERVICES Food and Drug Administration Cellular, Tissue, and Gene Therapies Advisory Committee..., Tissue, and Gene Therapies Advisory Committee. General Function of the Committee: To provide advice and... Gene Therapies, Center for Biologics Evaluation and Research, FDA. FDA intends to make...

  16. Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression

    PubMed Central

    Vink, Elizabeth I.; Zheng, Yueting; Yeasmin, Rukhsana; Stamminger, Thomas; Krug, Laurie T.; Hearing, Patrick

    2015-01-01

    The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation. PMID:25984715

  17. Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression.

    PubMed

    Vink, Elizabeth I; Zheng, Yueting; Yeasmin, Rukhsana; Stamminger, Thomas; Krug, Laurie T; Hearing, Patrick

    2015-05-01

    The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation. PMID:25984715

  18. Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized acti...

  19. Heteroconium chaetospira Induces Resistance to Clubroot via Upregulation of Host Genes Involved in Jasmonic Acid, Ethylene, and Auxin Biosynthesis

    PubMed Central

    Lahlali, Rachid; McGregor, Linda; Song, Tao; Gossen, Bruce D.; Narisawa, Kazuhiko; Peng, Gary

    2014-01-01

    An endophytic fungus, Heteroconium chaetospira isolate BC2HB1 (Hc), suppressed clubroot (Plasmodiophora brassicae -Pb) on canola in growth-cabinet trials. Confocal microscopy demonstrated that Hc penetrated canola roots and colonized cortical tissues. Based on qPCR analysis, the amount of Hc DNA found in canola roots at 14 days after treatment was negatively correlated (r = 0.92, P<0.001) with the severity of clubroot at 5 weeks after treatment at a low (2×105 spores pot−1) but not high (2×105 spores pot−1) dose of pathogen inoculum. Transcript levels of nine B. napus (Bn) genes in roots treated with Hc plus Pb, Pb alone and a nontreated control were analyzed using qPCR supplemented with biochemical analysis for the activity of phenylalanine ammonia lyases (PAL). These genes encode enzymes involved in several biosynthetic pathways related potentially to plant defence. Hc plus Pb increased the activity of PAL but not that of the other two genes (BnCCR and BnOPCL) involved also in phenylpropanoid biosynthesis, relative to Pb inoculation alone. In contrast, expression of several genes involved in the jasmonic acid (BnOPR2), ethylene (BnACO), auxin (BnAAO1), and PR-2 protein (BnPR-2) biosynthesis were upregulated by 63, 48, 3, and 3 fold, respectively, by Hc plus Pb over Pb alone. This indicates that these genes may be involved in inducing resistance in canola by Hc against clubroot. The upregulation of BnAAO1 appears to be related to both pathogenesis of clubroot and induced defence mechanisms in canola roots. This is the first report on regulation of specific host genes involved in induced plant resistance by a non-mycorrhizal endophyte. PMID:24714177

  20. 76 FR 18768 - Cellular, Tissue, and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-05

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Cellular, Tissue, and Gene Therapies Advisory Committee... portion of the meeting will be closed to the public. Name of Committee: Cellular, Tissue, and...

  1. 78 FR 15726 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-12

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Cellular, Tissue and Gene Therapies Advisory Committee... portion of the meeting will be closed to the public. Name of Committee: Cellular, Tissue and...

  2. 78 FR 70307 - Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-25

    ... Federal Register of November 29, 2012 (77 FR 71194), FDA announced the availability of the draft guidance... therapy, therapeutic vaccination, xenotransplantation, and certain biologic-device combination products... product areas covered by this guidance include cellular therapy, gene therapy, therapeutic...

  3. Mechanisms of miRNA-Mediated Gene Regulation from Common Downregulation to mRNA-Specific Upregulation

    PubMed Central

    Kazemzadeh-Bavili, Mina

    2014-01-01

    Discovered in 1993, micoRNAs (miRNAs) are now recognized as one of the major regulatory gene families in eukaryotes. To date, 24521 microRNAs have been discovered and there are certainly more to come. It was primarily acknowledged that miRNAs result in gene expression repression at both the level of mRNA stability by conducting mRNA degradation and the level of translation (at initiation and after initiation) by inhibiting protein translation or degrading the polypeptides through binding complementarily to 3′UTR of the target mRNAs. Nevertheless, some studies revealed that miRNAs have the capability of activating gene expression directly or indirectly in respond to different cell types and conditions and in the presence of distinct cofactors. This reversibility in their posttranslational gene regulatory natures enables the bearing cells to rapidly response to different cell conditions and consequently block unnecessary energy wastage or maintain the cell state. This paper provides an overview of the current understandings of the miRNA characteristics including their genes and biogenesis, as well as their mediated downregulation. We also review up-to-date knowledge of miRNA-mediated gene upregulation through highlighting some notable examples and discuss the emerging concepts of their associations with other posttranscriptional gene regulation processes. PMID:25180174

  4. Lipopolysaccharide pretreatment inhibits LPS-induced human umbilical cord mesenchymal stem cell apoptosis via upregulating the expression of cellular FLICE-inhibitory protein

    PubMed Central

    HOU, YU SEN; LIU, LING YING; CHAI, JIA KE; YU, YONG HUI; DUAN, HONG JIE; HU, QUAN; YIN, HUI NAN; WANG, YI HE; ZHUANG, SHU BO; FAN, JUN; CHU, WAN LI; MA, LI

    2015-01-01

    Mesenchymal stem cell (MSC)-based regenerative therapy is currently regarded as a novel approach with which to repair damaged tissues. However, the efficiency of MSC transplantation is limited due to the low survival rate of engrafted MSCs. Lipopolysaccharide (LPS) production is increased in numerous diseases and serves an essential function in the regulation of apoptosis in a variety of cell types. Previous studies have indicated that low-dose LPS pretreatment contributes to cytoprotection. In the current study, LPS was demonstrated to induce apoptosis in human umbilical cord mesenchymal stem cells (hUCMSCs) via the activation of caspase, in a dose-dependent manner. Low-dose LPS pretreatment may protect hUCMSCs against apoptosis induced by high-dose LPS, by upregulating the expression of cellular FADD-like IL-1?-converting enzyme-inhibitory protein (c-FLIP). The results of the present study indicate that pretreatment with an appropriate concentration of LPS may alleviate high-dose LPS-induced apoptosis. PMID:25955291

  5. Lipopolysaccharide pretreatment inhibits LPS-induced human umbilical cord mesenchymal stem cell apoptosis via upregulating the expression of cellular FLICE-inhibitory protein.

    PubMed

    Hou, Yu Sen; Liu, Ling Ying; Chai, Jia Ke; Yu, Yong Hui; Duan, Hong Jie; Hu, Quan; Yin, Hui Nan; Wang, Yi He; Zhuang, Shu Bo; Fan, Jun; Chu, Wan Li; Ma, Li

    2015-08-01

    Mesenchymal stem cell (MSC)-based regenerative therapy is currently regarded as a novel approach with which to repair damaged tissues. However, the efficiency of MSC transplantation is limited due to the low survival rate of engrafted MSCs. Lipopolysaccharide (LPS) production is increased in numerous diseases and serves an essential function in the regulation of apoptosis in a variety of cell types. Previous studies have indicated that low-dose LPS pretreatment contributes to cytoprotection. In the current study, LPS was demonstrated to induce apoptosis in human umbilical cord mesenchymal stem cells (hUCMSCs) via the activation of caspase, in a dose-dependent manner. Low-dose LPS pretreatment may protect hUCMSCs against apoptosis induced by high-dose LPS, by upregulating the expression of cellular FADD-like IL-1?-converting enzyme-inhibitory protein (c-FLIP). The results of the present study indicate that pretreatment with an appropriate concentration of LPS may alleviate high-dose LPS-induced apoptosis. PMID:25955291

  6. Upregulation of mitogen-inducible gene 6 triggers antitumor effect and attenuates progesterone resistance in endometrial carcinoma cells.

    PubMed

    Xu, W; Zhu, S; Zhou, Y; Jin, Y; Dai, H; Wang, X

    2015-11-01

    Researches regarding mitogen-inducible gene 6 (Mig-6) have confirmed its role as a tumor suppressor and progesterone resistance factor in endometrium. In this study, after confirming the downregulation of Mig-6 protein in endometrial carcinoma (EC) tissues, the expression of Mig-6 was upregulated in Ishikawa cells by pCMV6-Mig-6 plasmid. We observed the increased apoptosis, decreased proliferation and invasion potential of Ishikawa cells after upregulation of Mig-6. The proapoptosis ability of P4 significantly enhanced by 39.36%, the antiproliferation ability increased by 37.90% and the anti-invasion ability increased by 48.89%, suggesting the antiprogesterone resistance potential of Mig-6 in endometrium. In addition, the results suggested that Mig-6 may induce Ishikawa cell apoptosis through the mitochondrial pathway, inhibit cell proliferation via the extracellular signal-regulated kinase pathway and the anti-invasion potential may associate with matrix metalloproteinase (MMP)-2 and MMP-9 downexpression. Therefore, upregulation of Mig-6 may add a new strategy to suppress endometrial tumorigenesis and attenuate the progesterone resistance during P4 treatment. PMID:26450625

  7. C. elegans Metabolic Gene Regulatory Networks Govern the Cellular Economy

    PubMed Central

    Watson, Emma; Walhout, Albertha J.M.

    2014-01-01

    Diet greatly impacts metabolism in health and disease. In response to the presence or absence of specific nutrients, metabolic gene regulatory networks sense the metabolic state of the cell and regulate metabolic flux accordingly, for instance by the transcriptional control of metabolic enzymes. Here we discuss recent insights regarding metazoan metabolic regulatory networks using the nematode Caenorhabditis elegans as a model, including the modular organization of metabolic gene regulatory networks, the prominent impact of diet on the transcriptome and metabolome, specialized roles of nuclear hormone receptors in responding to dietary conditions, regulation of metabolic genes and metabolic regulators by microRNAs, and feedback between metabolic genes and their regulators. PMID:24731597

  8. Gene expression profiles induced by E6 from non-European HPV18 variants reveals a differential activation on cellular processes driving to carcinogenesis.

    PubMed

    Fragoso-Ontiveros, Vernica; Mara Alvarez-Garca, Rosa; Contreras-Paredes, Adriana; Vaca-Paniagua, Felipe; Alonso Herrera, Luis; Lpez-Camarillo, Cesar; Jacobo-Herrera, Nadia; Lizano-Sobern, Marcela; Prez-Plasencia, Carlos

    2012-10-10

    Cervical cancer in developed countries remains as a major concern on public health policies due to incidence and mortality rates. Persistent infection with high risk human papillomavirus is a necessary etiological agent in the progression to invasive cervical carcinoma. A proposed hypothesis is the association between more aggressive HPV variants and the risk to develop cervical cancer. In order to have a global perspective in terms of cellular transcripts and molecular pathways affected by HPV18 E6 intratype variants; we conducted a genome wide analysis of gene expression. Our results show that E6 derived from non-European variants are able to up-regulate cellular transcripts associated to the hallmarks of cancer; such as cell cycle, migration, Wnt pathway and mTor signaling. Moreover, we were able to show that HPV18 E6 from African variant had a major effect on cellular processes such as cell cycle and migration as confirmed by functional studies. PMID:22743128

  9. Comparison of the Essential Cellular Functions of the Two murA Genes of Bacillus anthracis▿

    PubMed Central

    Kedar, G. C.; Brown-Driver, Vickie; Reyes, Daniel R.; Hilgers, Mark T.; Stidham, Mark A.; Shaw, Karen Joy; Finn, John; Haselbeck, Robert J.

    2008-01-01

    Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug. PMID:18378720

  10. The bfl-1 Gene Is Transcriptionally Upregulated by the Epstein-Barr Virus LMP1, and Its Expression Promotes the Survival of a Burkitt's Lymphoma Cell Line

    PubMed Central

    D'Souza, Brendan; Rowe, Martin; Walls, Dermot

    2000-01-01

    The recently identified bfl-1 gene (also known as A1 or GRS), a homologue of bcl-2, encodes an antiapoptotic protein that suppresses apoptosis induced by the p53 tumor suppressor protein and exhibits proliferative and potent cooperative transforming activities. We show that elevated levels of bfl-1 mRNA are a feature of Epstein-Barr virus (EBV)-immortalized B-cell lines and Burkitt's lymphoma cell lines expressing the full spectrum of EBV latent proteins. Using an EBV-negative Burkitt's lymphoma cell line in which the expression of EBV latent membrane protein 1 (LMP1) is inducibly regulated by tetracycline, we demonstrate that LMP1 expression coincides with a dramatic increase in the level of bfl-1 mRNA. Also in this system, an increase in the level of Bcl-2 protein was seen to occur earlier than that of bcl-2 mRNA, suggesting that both transcriptional and translational mechanisms are involved in the control of Bcl-2 expression by LMP-1. We show that elevated bfl-1 mRNA stability can contribute to this effect of LMP-1, thus providing evidence of a novel mechanism of gene regulation by this EBV protein. Upregulation of bfl-1 by LMP1 was not observed in the T-cell line Jurkat or the epithelial cell line C33A. Ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type I infection protects against apoptosis induced by growth factor deprivation, thereby providing a functional role for Bfl-1 in this cellular context and adding Bfl-1 to the list of antiapoptotic proteins whose expression is modulated by EBV. This is the first report of the regulation of bfl-1 expression by a viral protein, and this novel finding may thus represent an important link between the EBV oncoprotein LMP1 and its cellular growth-transforming properties. PMID:10864681

  11. Transcriptome changes in foxtail millet genotypes at high salinity: identification and characterization of a PHGPX gene specifically upregulated by NaCl in a salt-tolerant line.

    PubMed

    Sreenivasulu, Nese; Miranda, Manoela; Prakash, Harischandra Sripathy; Wobus, Ulrich; Weschke, Winfriede

    2004-04-01

    Using a macro array filter with 711 cDNA inserts representing 620 unigenes selected from a barley EST collection, we identified transcripts differentially expressed in salt (NaCl)-treated tolerant (cv. Prasad) and sensitive (cv. Lepakshi) seedlings of foxtail millet (Setaria italica L.). Transcripts of hydrogen peroxide scavenging enzymes such as phospholipid hydroperoxide glutathione peroxidase (PHGPX), ascorbate peroxidase (APX) and catalase 1 (CAT1) in addition to some genes of cellular metabolism were found to be especially up-regulated at high salinity in the tolerant line. To analyse this process at the protein level we examined protein expression patterns under various stress conditions. A 25 kD protein with a pI of 4.8 was found to be induced prominently under high salt concentrations (250 mmol/L). This salt-induced 25 kD protein has been purified and identified by peptide sequencing as PHGPX protein. The increase of the PHGPX protein level under salt stress in the tolerant line parallels the PHGPX mRNA results of array analysis but was more pronounced. We cloned and characterized the foxtail millet PHGPX cDNA, which shows 85% and 95% homology at the DNA and protein level, respectively, to one stress-induced member of the small barley PHGPX gene family encoding non-selenium glutathione peroxidases. As shown by Southern blot analysis, a small family of PHGPX genes exists in foxtail millet, too. The specific expression pattern of the PHGPX gene in salt-induced tolerant millet seedlings suggests that its product plays an important role in the defense reaction against salt-induced oxidative damage and that the characterized glutathione peroxidase is one of the components conferring resistance against salt to the tolerant foxtail millet cultivar. PMID:15128034

  12. Trojan horse at cellular level for tumor gene therapies.

    PubMed

    Collet, Guillaume; Grillon, Catherine; Nadim, Mahdi; Kieda, Claudine

    2013-08-10

    Among innovative strategies developed for cancer treatments, gene therapies stand of great interest despite their well-known limitations in targeting, delivery, toxicity or stability. The success of any given gene-therapy is highly dependent on the carrier efficiency. New approaches are often revisiting the mythic trojan horse concept to carry therapeutic nucleic acid, i.e. DNAs, RNAs or small interfering RNAs, to pathologic tumor site. Recent investigations are focusing on engineering carrying modalities to overtake the above limitations bringing new promise to cancer patients. This review describes recent advances and perspectives for gene therapies devoted to tumor treatment, taking advantage of available knowledge in biotechnology and medicine. PMID:23542073

  13. Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine.

    PubMed

    Takarada, Takeshi; Ogura, Masato; Nakamichi, Noritaka; Kakuda, Takami; Nakazato, Ryota; Kokubo, Hiroshi; Ikeno, Shinsuke; Nakamura, Saki; Kutsukake, Takaya; Hinoi, Eiichi; Yoneda, Yukio

    2016-02-01

    We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100?M in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells. PMID:25957749

  14. A Novel Gene Amplification Causes Upregulation of the PatAB ABC Transporter and Fluoroquinolone Resistance in Streptococcus pneumoniae

    PubMed Central

    Baylay, Alison J.; Ivens, Alasdair

    2015-01-01

    Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism. PMID:25779578

  15. GENES FOR TUMOR MARKERS ARE CLUSTERED WITH CELLULAR PROTO-ONCOGENES ON HUMAN CHROMOSOMES

    EPA Science Inventory

    The relative mapping positions of genes for polypeptides expressed abnormally in tumors (tumor markers) and cellular proto-oncogenes were analyzed and a remarkable degree of co-mapping of tumor marker genes with oncogenes in the human karyotype were found. It is proposed that abe...

  16. Anaplasma phagocytophilum up-regulates some anti-apoptotic genes in neutrophils and pro-inflammatory genes in mononuclear cells of sheep.

    PubMed

    Woldehiwet, Z; Yavari, C

    2014-05-01

    Anaplasma phagocytophilum, the causative agent of tick-borne fever (TBF) in sheep and cattle and human granulocytic anaplasmosis, has the unique ability to selectively infect and multiply within the hostile environment of the neutrophil. Previous studies have shown that sheep with TBF are more susceptible to other infections and that infected neutrophils have reduced phagocytic ability and delayed apoptosis. This suggests that survival of A. phagocytophilum in these short-lived cells involves the ability to subvert or resist their bacterial killing, but also to modify the host cells such that the host cells survive long after infection. The present study shows that infection of sheep by A. phagocytophilum is characterized by up-regulation of some anti-apoptotic genes (BCL2, BIRC3 and CFLAR) in neutrophils and up-regulation of genes encoding the pro-inflammatory cytokines interferon-γ, interleukin (IL)-1β and IL-6 in mononuclear cells during the period of bacteraemia. Infection with A. phagocytophilum was also characterized by significant up-regulation of CYBB, which is associated with the respiratory burst of neutrophils. PMID:24602324

  17. Blood cell gene expression associated with cellular stress defense is modulated by antioxidant-rich food in a randomised controlled clinical trial of male smokers

    PubMed Central

    2010-01-01

    Background Plant-based diets rich in fruit and vegetables can prevent development of several chronic age-related diseases. However, the mechanisms behind this protective effect are not elucidated. We have tested the hypothesis that intake of antioxidant-rich foods can affect groups of genes associated with cellular stress defence in human blood cells. Trial registration number: NCT00520819 http://clinicaltrials.gov. Methods In an 8-week dietary intervention study, 102 healthy male smokers were randomised to either a diet rich in various antioxidant-rich foods, a kiwifruit diet (three kiwifruits/d added to the regular diet) or a control group. Blood cell gene expression profiles were obtained from 10 randomly selected individuals of each group. Diet-induced changes on gene expression were compared to controls using a novel application of the gene set enrichment analysis (GSEA) on transcription profiles obtained using Affymetrix HG-U133-Plus 2.0 whole genome arrays. Results Changes were observed in the blood cell gene expression profiles in both intervention groups when compared to the control group. Groups of genes involved in regulation of cellular stress defence, such as DNA repair, apoptosis and hypoxia, were significantly upregulated (GSEA, FDR q-values < 5%) by both diets compared to the control group. Genes with common regulatory motifs for aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (AhR/ARNT) were upregulated by both interventions (FDR q-values < 5%). Plasma antioxidant biomarkers (polyphenols/carotenoids) increased in both groups. Conclusions The observed changes in the blood cell gene expression profiles suggest that the beneficial effects of a plant-based diet on human health may be mediated through optimization of defence processes. PMID:20846424

  18. Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell

    SciTech Connect

    Chiou, S.-H. . E-mail: shchiou@vghtpe.gov.tw; Chen, S.-J. . E-mail: sjchen@vghtpe.gov.tw; Peng, C-H.; Chang, Y.-L.; Ku, H.-H.; Hsu, W.-M.; Ho, Larry L.-T.; Lee, C.-H.

    2006-05-05

    Fluoxetine is a widely used antidepressant compound which inhibits the reuptake of serotonin in the central nervous system. Recent studies have shown that fluoxetine can promote neurogenesis and improve the survival rate of neurons. However, whether fluoxetine modulates the proliferation or neuroprotection effects of neural stem cells (NSCs) needs to be elucidated. In this study, we demonstrated that 20 {mu}M fluoxetine can increase the cell proliferation of NSCs derived from the hippocampus of adult rats by MTT test. The up-regulated expression of Bcl-2, Bcl-xL and the cellular FLICE-inhibitory protein (c-FLIP) in fluoxetine-treated NSCs was detected by real-time RT-PCR. Our results further showed that fluoxetine protects the lipopolysaccharide-induced apoptosis in NSCs, in part, by activating the expression of c-FLIP. Moreover, c-FLIP induction by fluoxetine requires the activation of the c-FLIP promoter region spanning nucleotides -414 to -133, including CREB and SP1 sites. This effect appeared to involve the phosphatidylinositol-3-kinase-dependent pathway. Furthermore, fluoxetine treatment significantly inhibited the induction of proinflammatory factor IL-1{beta}, IL-6, and TNF-{alpha} in the culture medium of LPS-treated NSCs (p < 0.01). The results of high performance liquid chromatography coupled to electrochemical detection further confirmed that fluoxentine increased the functional production of serotonin in NSCs. Together, these data demonstrate the specific activation of c-FLIP by fluoxetine and indicate the novel role of fluoxetine for neuroprotection in the treatment of depression.

  19. Hypoxia upregulates the gene expression of mitochondrial aconitase in prostate carcinoma cells.

    PubMed

    Tsui, Ke-Hung; Chung, Li-Chuan; Wang, Shyi-Wu; Feng, Tsui-Hsia; Chang, Phei-Lang; Juang, Horng-Heng

    2013-01-01

    Hypoxia induces metabolic alteration in cancer cells by stabilizing hypoxia-inducible factor 1α (HIF-1α (HIF1A)), which regulates the bioenergetic genes of glycolysis and lipid metabolic pathways. However, the target genes of hypoxia-induced metabolic alterations in the prostate remain uncertain. Mitochondrial aconitase (mACON) (ACONM) is an enzyme that is central to carbohydrate and energy metabolism and is responsible for the interconversion of citrate to isocitrate as part of the citric acid cycle in the human prostate. We evaluated the effects of the molecular mechanisms of hypoxia on mACON gene expression in PC-3 and LNCaP human prostate carcinoma cells. Immunoblotting assays revealed that hypoxia modulated mACON and lactate dehydrogenase A (LDHA) protein expression, while these effects were attenuated when HIF-1α was knocked down. Hypoxia induced fatty acid synthase (FASN) in PC-3 cells while hypoxia blocked FASN gene expression in LNCaP cells after 24-h incubation. Results of real-time RT-qPCR, immunoblotting, and transient gene expression assays revealed that hypoxia treatment or co-transfection with HIF-1α expression vector enhanced gene expression of mACON, implying that hypoxia modulated mACON at the transcriptional level. Hypoxia-induced mACON promoter activity is dependent on the DNA fragment located at -1013 to -842 upstream of the translation initiation site. l-mimosine, an iron chelator, stabilized HIF-1α but downregulated mACON gene expression, suggesting that iron chelation blocked the hypoxia-induced mACON gene expression. These results suggest that hypoxia dysregulates the expressions of LDHA, FASN, and mACON genes, and the hypoxia-induced mACON gene expression is via the HIF-1α-dependent and iron-dependent pathways in prostate carcinoma cells. PMID:23709747

  20. Beta-lactam antibiotics modulate T-cell functions and gene expression via covalent binding to cellular albumin

    PubMed Central

    Mor, Felix; Cohen, Irun R.

    2013-01-01

    Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell–mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in nonobese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin. PMID:23382225

  1. Mitochondrial respiratory deficiencies signal up-regulation of genes for heat shock proteins.

    PubMed

    Kuzmin, Evgeny V; Karpova, Olga V; Elthon, Thomas E; Newton, Kathleen J

    2004-05-14

    The consequences of mitochondrial dysfunction are not limited to the development of oxidative stress or initiation of apoptosis but can result in the establishment of stress tolerance. Using maize mitochondrial mutants, we show that permanent mitochondrial deficiencies trigger novel Ca(2+)-independent signaling pathways, leading to constitutive expression of genes for molecular chaperones, heat shock proteins (HSPs) of different classes. The signaling to activate hsp genes appears to originate from a reduced mitochondrial transmembrane potential. Upon depolarization of mitochondrial membranes in transient assays, gene induction for mitochondrial HSPs occurred more rapidly than that for cytosolic HSPs. We also demonstrate that in the nematode Caenorhabditis elegans transcription of hsp genes can be induced by RNA interference of nuclear respiratory genes. In both organisms, activation of hsp genes in response to mitochondrial impairment is distinct from their responses to heat shock and is not associated with oxidative stress. Thus, mitochondria-to-nucleus signaling to express a hsp gene network is apparently a widespread retrograde mechanism to facilitate cell defense and survival. PMID:15016808

  2. Stromal upregulation of lateral epithelial adhesions: Gene expression analysis of signalling pathways in prostate epithelium

    PubMed Central

    2011-01-01

    Background Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. Methods Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. Results TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). Conclusions In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity. PMID:21696611

  3. Reference Genes to Study Herbicide Stress Response in Lolium sp.: Up-Regulation of P450 Genes in Plants Resistant to Acetolactate-Synthase Inhibitors

    PubMed Central

    Duhoux, Arnaud; Délye, Christophe

    2013-01-01

    Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants. PMID:23696834

  4. Reference genes to study herbicide stress response in Lolium sp.: up-regulation of P450 genes in plants resistant to acetolactate-synthase inhibitors.

    PubMed

    Duhoux, Arnaud; Délye, Christophe

    2013-01-01

    Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants. PMID:23696834

  5. Quantifying cellular capacity identifies gene expression designs with reduced burden.

    PubMed

    Ceroni, Francesca; Algar, Rhys; Stan, Guy-Bart; Ellis, Tom

    2015-05-01

    Heterologous gene expression can be a significant burden for cells. Here we describe an in vivo monitor that tracks changes in the capacity of Escherichia coli in real time and can be used to assay the burden imposed by synthetic constructs and their parts. We identify construct designs with reduced burden that predictably outperformed less efficient designs, despite having equivalent output. PMID:25849635

  6. Up-regulation of a magnesium transporter gene OsMGT1 is required for conferring aluminum tolerance in rice.

    PubMed

    Chen, Zhi Chang; Yamaji, Naoki; Motoyama, Ritsuko; Nagamura, Yoshiaki; Ma, Jian Feng

    2012-08-01

    Magnesium (Mg)-mediated alleviation of aluminum (Al) toxicity has been observed in a number of plant species, but the mechanisms underlying the alleviation are still poorly understood. When a putative rice (Oryza sativa) Mg transporter gene, Oryza sativa MAGNESIUM TRANSPORTER1 (OsMGT1), was knocked out, the tolerance to Al, but not to cadmium and lanthanum, was decreased. However, this inhibition could be rescued by addition of 10 ?m Mg, but not by the same concentration of barium or strontium. OsMGT1 was expressed in both the roots and shoots in the absence of Al, but the expression only in the roots was rapidly up-regulated by Al. Furthermore, the expression did not respond to low pH and other metals including cadmium and lanthanum, and was regulated by an Al-responsive transcription factor, AL RESISTANCE TRANSCRIPTION FACTOR1. An investigation of subcellular localization showed that OsMGT1 was localized to the plasma membrane. A short-term (30 min) uptake experiment with stable isotope (25)Mg showed that knockout of OsMGT1 resulted in decreased Mg uptake, but that the uptake in the wild type was enhanced by Al. Mg concentration in the cell sap of the root tips was also increased in the wild-type rice, but not in the knockout lines in the presence of Al. A microarray analysis showed that transcripts of genes related to stress were more up- and down-regulated in the knockout lines. Taken together, our results indicate that OsMGT1 is a transporter for Mg uptake in the roots and that up-regulation of this gene is required for conferring Al tolerance in rice by increasing Mg concentration in the cell. PMID:22732245

  7. Cellular Phenotype-Dependent and -Independent Effects of Vitamin C on the Renewal and Gene Expression of Mouse Embryonic Fibroblasts

    PubMed Central

    Kuo, Shiu-Ming; Burl, Lana R.; Hu, Zihua

    2012-01-01

    Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10−5 M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2−/− MEF did not respond to vitamin C. SVCT2−/− MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2−/− MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was discussed. PMID:22427916

  8. The Expression of Porcine Prdx6 Gene Is Up-Regulated by C/EBPβ and CREB

    PubMed Central

    Wu, Xinyu; Ji, Panlong; Zhang, Liang; Bu, Guowei; Gu, Hao; Wang, Xiaojing; Xiong, Yuanzhu; Zuo, Bo

    2015-01-01

    Peroxiredoxin6 (Prdx6) is one of the peroxiredoxin (Prdxs) family members that play an important role in maintaining cell homeostasis. Our previous studies demonstrated that Prdx6 was significantly associated with pig meat quality, especially meat tenderness. However, the transcriptional regulation of porcine Prdx6 remains unclear. In this study, we determined the transcription start site (TSS) of porcine Prdx6 gene by 5′ rapid-amplification of cDNA ends (5′ RACE). Several regulatory elements including CCAAT/enhancer-binding proteinβ (C/EBPβ), Myogenic Differentiation (MyoD), cAMP response element binding protein (CREB), stimulating protein1 (Sp1) and heat shock factor (HSF) binding sites were found by computational analyses together with luciferase reporter system. Overexpression and RNA interference experiments showed that C/EBPβ or CREB could up-regulate the expression of porcine Prdx6 gene at both mRNA and protein level. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP) confirmed that C/EBPβ and CREB could interact with Prdx6 promoter. Immuoprecipitation results also showed that C/EBPβ could interact with Prdx6 in vivo. Taken together, our findings identified C/EBPβ and CREB as the important regulators of porcine Prdx6 gene expression, and offered clues for further investigation of Prdx6 gene function. PMID:26659441

  9. A GNAS mutation found in pancreatic intraductal papillary mucinous neoplasms induces drastic alterations of gene expression profiles with upregulation of mucin genes.

    PubMed

    Komatsu, Hirotake; Tanji, Etsuko; Sakata, Naoaki; Aoki, Takeshi; Motoi, Fuyuhiko; Naitoh, Takeshi; Katayose, Yu; Egawa, Shinichi; Unno, Michiaki; Furukawa, Toru

    2014-01-01

    GNAS, a gene encoding G protein stimulating ? subunit, is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which are indolent and slow-growing pancreatic tumors that secrete abundant mucin. The GNAS mutation is not observed in conventional ductal adenocarcinomas of the pancreas. To determine the functional significance of the GNAS mutation in pancreatic ductal lineage cells, we examined in vitro phenotypes of cells of pancreatic ductal lineage, HPDE, PK-8, PCI-35, and MIA PaCa-2, with exogenous expression of either wild-type or mutated (R201H) GNAS. We found that exogenous GNAS upregulated intracellular cyclic adenine monophosphate (cAMP), particularly in mutated GNAS transfectants, and upregulated expression of MUC2 and MUC5AC in HPDE and PK-8 cells. By contrast, exogenous GNAS inhibited expression of mucin genes in PCI-35 and MIA PaCa-2 cells, despite upregulation of cAMP. We examined global gene expression profiles of some of the cells transfected with exogenous mutated GNAS (PK-8, PCI-35, and MIA PaCa-2), and found that PK-8 cells exhibited drastic alterations of the gene expression profile, which contrasted with modest alterations in PCI-35 and MIA PaCa-2 cells. To identify a cause of these different effects of exogenous mutated GNAS on phenotypes of the cells, we examined effects of interactions of the signaling pathways of G protein-coupled receptor (GPCR), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) on expression of mucin genes. The MAPK and PI3K pathways significantly influenced the expression of mucin genes. Exogenous GNAS did not promote cell growth but suppressed it in some of the cells. In conclusion, mutated GNAS found in IPMNs may extensively alter gene expression profiles, including expression of mucin genes, through the interaction with MAPK and PI3K pathways in pancreatic ductal cells; these changes may determine the characteristic phenotype of IPMN. PK-8 cells expressing exogenous mutated GNAS may be an ideal in vitro model of IPMN. PMID:24498386

  10. A GNAS Mutation Found in Pancreatic Intraductal Papillary Mucinous Neoplasms Induces Drastic Alterations of Gene Expression Profiles with Upregulation of Mucin Genes

    PubMed Central

    Komatsu, Hirotake; Tanji, Etsuko; Sakata, Naoaki; Aoki, Takeshi; Motoi, Fuyuhiko; Naitoh, Takeshi; Katayose, Yu; Egawa, Shinichi; Unno, Michiaki; Furukawa, Toru

    2014-01-01

    GNAS, a gene encoding G protein stimulating ? subunit, is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which are indolent and slow-growing pancreatic tumors that secrete abundant mucin. The GNAS mutation is not observed in conventional ductal adenocarcinomas of the pancreas. To determine the functional significance of the GNAS mutation in pancreatic ductal lineage cells, we examined in vitro phenotypes of cells of pancreatic ductal lineage, HPDE, PK-8, PCI-35, and MIA PaCa-2, with exogenous expression of either wild-type or mutated (R201H) GNAS. We found that exogenous GNAS upregulated intracellular cyclic adenine monophosphate (cAMP), particularly in mutated GNAS transfectants, and upregulated expression of MUC2 and MUC5AC in HPDE and PK-8 cells. By contrast, exogenous GNAS inhibited expression of mucin genes in PCI-35 and MIA PaCa-2 cells, despite upregulation of cAMP. We examined global gene expression profiles of some of the cells transfected with exogenous mutated GNAS (PK-8, PCI-35, and MIA PaCa-2), and found that PK-8 cells exhibited drastic alterations of the gene expression profile, which contrasted with modest alterations in PCI-35 and MIA PaCa-2 cells. To identify a cause of these different effects of exogenous mutated GNAS on phenotypes of the cells, we examined effects of interactions of the signaling pathways of G protein-coupled receptor (GPCR), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) on expression of mucin genes. The MAPK and PI3K pathways significantly influenced the expression of mucin genes. Exogenous GNAS did not promote cell growth but suppressed it in some of the cells. In conclusion, mutated GNAS found in IPMNs may extensively alter gene expression profiles, including expression of mucin genes, through the interaction with MAPK and PI3K pathways in pancreatic ductal cells; these changes may determine the characteristic phenotype of IPMN. PK-8 cells expressing exogenous mutated GNAS may be an ideal in vitro model of IPMN. PMID:24498386

  11. Lactate Up-Regulates the Expression of Lactate Oxidation Complex-Related Genes in Left Ventricular Cardiac Tissue of Rats

    PubMed Central

    Gabriel-Costa, Daniele; da Cunha, Telma Fatima; Bechara, Luiz Roberto Grassmann; Fortunato, Rodrigo Soares; Bozi, Luiz Henrique Marchesi; Coelho, Marcele de Almeida; Barreto-Chaves, Maria Luiza; Brum, Patricia Chakur

    2015-01-01

    Background Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex. Methods/Principal Findings Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O2●-/H2O2) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O2●-/H2O2 levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O2●-/H2O2. Conclusions Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O2●-/H2O2 driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle. PMID:25996919

  12. CEP290 gene transfer rescues Leber Congenital Amaurosis cellular phenotype

    PubMed Central

    Burnight, E.R.; Wiley, L.A.; Drack, A.V.; Braun, T.A.; Anfinson, K.R.; Kaalberg, E.E.; Halder, J.A.; Affatigato, L.M.; Mullins, R.F.; Stone, E.M.; Tucker, B.A.

    2014-01-01

    Mutations in CEP290 are the most common cause of Leber congenital amaurosis (LCA), a severe inherited retinal degenerative disease for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene-replacement therapy, and cells derived from affected individuals give researchers the ability to study human disease and therapeutic gene correction in vitro. Here we report the development of lentiviral vectors carrying full-length CEP290 for the purpose of correcting the CEP290 disease-specific phenotype in human cells. A lentiviral vector containing CMV-driven human full-length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, rt-PCR analysis and western blotting revealed vector-derived expression. Because CEP290 is important in ciliogenesis, the ability of fibroblast cultures from CEP290-associated LCA patients to form cilia was investigated. In cultures derived from these patients, fewer cells formed cilia compared to unaffected controls. Cilia that were formed were shorter in patient derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect. The successful construction and viral transfer of full-length CEP290 brings us closer to the goal of providing gene- and cell- based therapies for patients affected with this common form of LCA. PMID:24807808

  13. VCAM-1 is a TGF-?1 inducible gene upregulated in idiopathic pulmonary fibrosis.

    PubMed

    Agassandian, Marianna; Tedrow, John R; Sembrat, John; Kass, Daniel J; Zhang, Yingze; Goncharova, Elena A; Kaminski, Naftali; Mallampalli, Rama K; Vuga, Louis J

    2015-12-01

    Idiopathic pulmonary fibrosis (IPF) is a chronic lethal interstitial lung disease of unknown etiology. We previously reported that high plasma levels of vascular cell adhesion molecule 1 (VCAM-1) predict mortality in IPF subjects. Here we investigated the cellular origin and potential role of VCAM-1 in regulating primary lung fibroblast behavior. VCAM-1 mRNA was significantly increased in lungs of subjects with IPF compared to lungs from control subjects (p=0.001), and it negatively correlated with two markers of lung function, forced vital capacity (FVC) and pulmonary diffusion capacity for carbon monoxide (DLCO). VCAM-1 protein levels were highly expressed in IPF subjects where it was detected in fibrotic foci and blood vessels of IPF lung. Treatment of human lung fibroblasts with TGF-?1 significantly increased steady-state VCAM1 mRNA and protein levels without affecting VCAM1 mRNA stability. Further, cellular depletion of VCAM-1 inhibited fibroblast cell proliferation and reduced G2/M and S phases of the cell cycle suggestive of cell cycle arrest. These effects on cell cycle progression triggered by VCAM1 depletion were associated with reductions in levels of phosphorylated extracellular regulated kinase 1/2 and cyclin D1. Thus, these observations suggest that VCAM-1 is a TGF-?1 responsive mediator that partakes in fibroblast proliferation in subjects with IPF. PMID:26386411

  14. Myocardial Gene Transfer: Routes and Devices for Regulation of Transgene Expression by Modulation of Cellular Permeability

    PubMed Central

    Katz, Michael G.; Bridges, Charles R.

    2013-01-01

    Abstract Heart diseases are major causes of morbidity and mortality in Western society. Gene therapy approaches are becoming promising therapeutic modalities to improve underlying molecular processes affecting failing cardiomyocytes. Numerous cardiac clinical gene therapy trials have yet to demonstrate strong positive results and advantages over current pharmacotherapy. The success of gene therapy depends largely on the creation of a reliable and efficient delivery method. The establishment of such a system is determined by its ability to overcome the existing biological barriers, including cellular uptake and intracellular trafficking as well as modulation of cellular permeability. In this article, we describe a variety of physical and mechanical methods, based on the transient disruption of the cell membrane, which are applied in nonviral gene transfer. In addition, we focus on the use of different physiological techniques and devices and pharmacological agents to enhance endothelial permeability. Development of these methods will undoubtedly help solve major problems facing gene therapy. PMID:23427834

  15. Identification of genes co-upregulated with Arc during BDNF-induced long-term potentiation in adult rat dentate gyrus in vivo.

    PubMed

    Wibrand, Karin; Messaoudi, Elhoucine; Hvik, Bjarte; Steenslid, Vibeke; Lvlie, Roger; Steen, Vidar M; Bramham, Clive R

    2006-03-01

    Brain-derived neurotrophic factor (BDNF) is a critical regulator of transcription-dependent adaptive neuronal responses, such as long-term potentiation (LTP). Brief infusion of BDNF into the dentate gyrus of adult anesthetized rats triggers stable LTP at medial perforant path-granule synapses that is transcription-dependent and requires induction of the immediate early gene Arc. Rather than acting alone, Arc is likely to be part of a larger BDNF-induced transcriptional program. Here, we used cDNA microarray expression profiling to search for genes co-upregulated with Arc 3 h after BDNF-LTP induction. Of nine cDNAs encoding for known genes and up-regulated more than four-fold, we selected five genes, Narp, neuritin, ADP-ribosylation factor-like protein-4 (ARL4L), TGF-beta-induced immediate early gene-1 (TIEG1) and CARP, for further validation. Real-time PCR confirmed robust up-regulation of these genes in an independent set of BDNF-LTP experiments, whereas infusion of the control protein cytochrome C had no effect. In situ hybridization histochemistry further revealed up-regulation of all five genes in somata of post-synaptic granule cells following both BDNF-LTP and high-frequency stimulation-induced LTP. While Arc synthesis is critical for local actin polymerization and stable LTP formation, several of the co-upregulated genes have known functions in excitatory synaptogenesis, axon guidance and glutamate receptor clustering. These results provide novel insight into gene expression responses underlying BDNF-induced synaptic consolidation in the adult brain in vivo. PMID:16553613

  16. Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression

    SciTech Connect

    Olszewski, Pawel K.; Fredriksson, Robert; Eriksson, Jenny D.; Mitra, Anaya; Radomska, Katarzyna J.; Gosnell, Blake A.; Solvang, Maria N.; Levine, Allen S.; Department of Food Science and Nutrition, Saint Paul, MN 55108 ; Schioeth, Helgi B.

    2011-05-13

    Highlights: {yields} The majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto. {yields} The level of colocalization is similar in the male and female brain. {yields} Fto overexpression in hypothalamic neurons increases oxytocin mRNA levels by 50%. {yields} Oxytocin does not affect Fto expression through negative feedback mechanisms. -- Abstract: Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance through a functional relationship with specific genes encoding feeding-related peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin.

  17. Hyperosmotic shock adaptation by cortisol involves upregulation of branchial osmotic stress transcription factor 1 gene expression in Mozambique Tilapia.

    PubMed

    McGuire, Alison; Aluru, Neelakanteswar; Takemura, Akihiro; Weil, Roxana; Wilson, Jonathan M; Vijayan, Mathilakath M

    2010-01-15

    The Mozambique tilapia (Oreochromis mossambicus) is a euryhaline species that does not survive direct seawater exposure. Cortisol is involved in re-establishing electrolyte homeostasis in seawater and is thought to play a role in allowing tilapia to cope with abrupt seawater exposure, but the mechanism(s) are far from clear. Recently, osmotic stress transcription factor 1 (OSTF1) was identified as a key signaling molecule involved in hyperosmotic stress adaptation in tilapia. Consequently, we tested the hypothesis that upregulation of OSTF1 expression by cortisol is a key response for hyperosmotic stress adaptation in tilapia. Fish were exposed to different salinities over a 24h period, while a major electrolyte disturbance and mortality was observed only with full-strength seawater exposure. Therefore, we administered cocoa butter implants of cortisol (50mg/kg) intraperitoneally to tilapia maintained in fresh water and after three days exposed these fish to full-strength seawater. There was 50% mortality in the control fish upon seawater exposure, but this was abolished by cortisol treatment. Abrupt seawater exposure did not affect plasma cortisol levels, while, as expected, exogenous administration of this steroid elevated plasma cortisol levels both in fresh water and seawater. Cortisol treatment significantly induced OSTF1 gene expression in fresh water tilapia, and also enhanced further the seawater-induced OSTF1 mRNA abundance. Plasma osmolality decreased, while gill Na(+)/K(+)-ATPase activity was suppressed in the cortisol group in seawater compared to the sham group. This corresponded with a significant reduction in gill ionocyte size and Na(+)/K(+)-ATPase activity and protein expression after seawater exposure. Cortisol did not modify liver metabolism, but significantly suppressed gill metabolic capacity in seawater. Overall, cortisol adapts tilapia to a hyperosmotic shock associated with abrupt seawater exposure. This involves upregulation of OSTF1 gene expression and a concomitant suppression of branchial metabolism in tilapia. PMID:19651127

  18. 80 FR 33271 - Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2015-06-11

    ... of Cellular and Gene Therapy Products; Guidance for Industry; Availability AGENCY: Food and Drug... and Gene Therapy Products; Guidance for Industry.'' The guidance document is to assist sponsors and investigators in designing early-phase clinical trials for cellular therapy (CT) and gene therapy (GT)...

  19. Dietary fish oil replacement with canola oil up-regulates glutathione peroxidase 1 gene expression in yellowtail kingfish (Seriola lalandi).

    PubMed

    Bowyer, Jenna N; Rout-Pitt, Nathan; Bain, Peter A; Stone, David A J; Schuller, Kathryn A

    2012-08-01

    The marine carnivore yellowtail kingfish (YTK, Seriola lalandi) was fed diets containing 5% residual fish oil (from the dietary fish meal) plus either 20% fish oil (FO), 20% canola oil (CO), 20% poultry oil (PO), 10% fish oil plus 10% canola oil (FO/CO) or 10% fish oil plus 10% poultry oil (FO/PO) and the effects on fish growth and hepatic expression of two glutathione peroxidase (GPx 1 and GPx 4) and two peroxiredoxin (Prx 1 and Prx 4) antioxidant genes were investigated. Partial (50%) replacement of the added dietary fish oil with poultry oil significantly improved fish growth whereas 100% replacement with canola oil significantly depressed fish growth. The fatty acid profiles of the fish fillets generally reflected those of the dietary oils except that there was apparent selective utilization of palmitic acid (16:0) and oleic acid (18:1n-9) and apparent selective retention of eicospentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The Prx 1 and 4 genes were expressed at 10- and 100-fold the level of the GPx 4 and 1 genes, respectively, and at one-tenth the level of the highly expressed β-actin reference gene. Dietary fish oil replacement with canola oil significantly up-regulated GPx 1 gene expression and there was a non-significant tendency towards down-regulation of Prx 1 and Prx 4. The results are discussed in terms of the effects of fish oil replacement on the peroxidation index of the diets and the resulting effects on the target antioxidant enzymes. PMID:22521527

  20. Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways.

    PubMed

    Ahmad, Rasheed; Ennaciri, Jamila; Cordeiro, Paulo; El Bassam, Souad; Menezes, Jos

    2007-03-16

    IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda. PMID:17239392

  1. Evidence for functional convergence in genes upregulated by herbivores ingesting plant secondary compounds

    PubMed Central

    2014-01-01

    Background Nearly 40 years ago, Freeland and Janzen predicted that liver biotransformation enzymes dictated diet selection by herbivores. Despite decades of research on model species and humans, little is known about the biotransformation mechanisms used by mammalian herbivores to metabolize plant secondary compounds (PSCs). We investigated the independent evolution of PSC biotransformation mechanisms by capitalizing on a dramatic diet change event—the dietary inclusion of creosote bush (Larrea tridentata)—that occurred in the recent evolutionary history of two species of woodrats (Neotoma lepida and N. bryanti). Results By comparing gene expression profiles of two populations of woodrats with evolutionary experience to creosote and one population naïve to creosote, we identified genes either induced by a diet containing creosote PSCs or constitutively higher in populations with evolutionary experience of creosote. Although only one detoxification gene (an aldo-keto reductase) was induced by both experienced populations, these populations converged upon functionally equivalent strategies to biotransform the PSCs of creosote bush by constitutively expressing aldehyde and alcohol dehydrogenases, Cytochromes P450s, methyltransferases, glutathione S-transferases and sulfotransferases. The response of the naïve woodrat population to creosote bush was indicative of extreme physiological stress. Conclusions The hepatic detoxification system of mammals is notoriously complex, with hundreds of known biotransformation enzymes. The comparison herein of woodrat taxa that differ in evolutionary and ecological experience with toxins in creosote bush reveals convergence in the overall strategies used by independent species after a historical shift in diet. In addition, remarkably few genes seemed to be important in this dietary shift. The research lays the requisite groundwork for future studies of specific biotransformation pathways used by woodrats to metabolize the toxins in creosote and the evolution of diet switching in woodrats. On a larger level, this work advances our understanding of the mechanisms used by mammalian herbivores to process toxic diets and illustrates the importance of the selective relationship of PSCs in shaping herbivore diversity. PMID:25123454

  2. Target Genes of Neuron-Restrictive Silencer Factor Are Abnormally Up-Regulated in Human Myotilinopathy

    PubMed Central

    Barrachina, Marta; Moreno, Jesús; Juvés, Salvador; Moreno, Dolores; Olivé, Montse; Ferrer, Isidre

    2007-01-01

    Myotilinopathy is a subgroup of myofibrillar myopathies caused by mutations in the myotilin gene in which there is aggregation of abnormal cytoskeletal proteins and ubiquitin. We report here on the accumulation of neuron-related proteins such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1), synaptosomal-associated protein 25, synaptophysin, and α-internexin in aberrant protein aggregates in myotilinopathy. We have determined that the neuron-restrictive silencer factor (NRSF)/RE1 silencing transcription factor (REST), a transcription factor expressed in non-neuronal tissues repressing the expression of several neuronal genes, is reduced in myotilinopathies. Moreover, NRSF transfection reduces UCHL1, synaptosomal-associated protein 25, synaptophysin, and α-internexin mRNA levels in DMS53 cells, whereas short interferring NRSF transfection increases UCHL1 and synaptophysin mRNA levels in U87-MG cells. Chromatin immunoprecipitation assays have shown that NRSF interacts with the UCHL1 promoter in U87-MG and HeLa cells. In silico analysis of the UCHL1 gene promoter sequence using the MatInspector software has predicted three potential neuron-restrictive silencer elements (NRSEs): NRSE1 located in the complementary DNA chain and NRSE2 and NRSE3 in intron 1, in the coding and complementary chains, respectively. Together, these findings show, for the first time, abnormal regulation of NRSF/REST as a mechanism associated with the aberrant expression of selected neuron-related proteins, which in turn accumulate in abnormal protein aggregates, in myotilinopathy. PMID:17823282

  3. Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women.

    PubMed

    Maningat, Patricia D; Sen, Partha; Rijnkels, Monique; Hadsell, Darryl L; Bray, Molly S; Haymond, Morey W

    2011-04-27

    Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7-10 days. PMID:21205870

  4. Neonatal cellular and gene therapies for mucopolysaccharidoses: the earlier the better?

    PubMed

    Tomatsu, Shunji; Azario, Isabella; Sawamoto, Kazuki; Pievani, Alice Silvia; Biondi, Andrea; Serafini, Marta

    2016-03-01

    Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs). The increasing interest in newborn screening procedures for LSDs underlines the need for alternative cellular and gene therapy approaches to be developed during the perinatal period, supporting the treatment of MPS patients before the onset of clinical signs and symptoms. The rationale for considering these early therapies results from the clinical experience in the treatment of MPSs and other genetic disorders. The normal or gene-corrected hematopoiesis transplanted in patients can produce the missing protein at levels sufficient to improve and/or halt the disease-related abnormalities. However, these current therapies are only partially successful, probably due to the limited efficacy of the protein provided through the hematopoiesis. An alternative explanation is that the time at which the cellular or gene therapy procedures are performed could be too late to prevent pre-existing or progressive organ damage. Considering these aspects, in the last several years, novel cellular and gene therapy approaches have been tested in different animal models at birth, a highly early stage, showing that precocious treatment is critical to prevent long-term pathological consequences. This review provides insights into the state-of-art accomplishments made with neonatal cellular and gene-based therapies and the major barriers that need to be overcome before they can be implemented in the medical community. PMID:26578156

  5. Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP**C) into an abnormal form of scrapie prion (PrP**Sc). The cellular mechanisms underlying the misfolding of PrP**C are not well understood. Since cellular prion proteins harbor divalent metal b...

  6. Involvement of IL-1 genes in the cellular responses to carbon nanotube exposure.

    PubMed

    Arnoldussen, Yke Jildouw; Skogstad, Asbjrn; Skaug, Vidar; Kasem, Mayes; Haugen, Aage; Benker, Nathalie; Weinbruch, Stephan; Apte, Ron N; Zienolddiny, Shanbeh

    2015-05-01

    The interleukin-1 (IL-1) family has been implicated in cellular responses to nanoparticles including carbon nanotubes (CNTs). IL-1? and ? are key proinflammatory cytokines important in inflammatory and oxidative stress responses. The aim of this study was to characterize the role of IL-1 in cellular responses of CNTs in cells from IL-1?/? wild type (IL1-WT) mice and cells with reduced inflammatory potential from IL-1?/? deficient (IL1-KO) mice. Two multi-walled CNTs, CNT-1 containing long and thick fibers and CNT-2 containing short and thin fibers, were compared to UICC crocidolite asbestos fibers. Upon CNT exposure toxicity and apoptosis were affected differently in IL1-WT and IL1-KO cells. Upregulation of TNF? and IL-1? mRNA expression in IL1-WT cells was dependent on the type of CNT. On the contrary precursor IL-1? protein was downregulated after 24h. The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) was activated in IL1-KO cells and regulated by CNTs, whereas no significant changes of extracellular regulated kinase (ERK) were observed when comparing IL1-WT and IL1-KO cells. In summary, the results presented here indicate that IL-1 contributes to the cellular and molecular effects of CNT exposure and that the type of CNT has an important effect on the cellular response. PMID:25748835

  7. Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair

    PubMed Central

    Aparicio, Oscar; Carnero, Elena; Abad, Xabier; Razquin, Nerea; Guruceaga, Elizabeth; Segura, Victor; Fortes, Puri

    2010-01-01

    Adenovirus virus-associated (VA) RNAs are processed to functional viral miRNAs or mivaRNAs. mivaRNAs are important for virus production, suggesting that they may target cellular or viral genes that affect the virus cell cycle. To look for cellular targets of mivaRNAs, we first identified genes downregulated in the presence of VA RNAs by microarray analysis. These genes were then screened for mivaRNA target sites using several bioinformatic tools. The combination of microarray analysis and bioinformatics allowed us to select the splicing and translation regulator TIA-1 as a putative mivaRNA target. We show that TIA-1 is downregulated at mRNA and protein levels in infected cells expressing functional mivaRNAs and in transfected cells that express mivaRNAI-138, one of the most abundant adenoviral miRNAs. Also, reporter assays show that TIA-1 is downregulated directly by mivaRNAI-138. To determine whether mivaRNAs could target other cellular genes we analyzed 50 additional putative targets. Thirty of them were downregulated in infected or transfected cells expressing mivaRNAs. Some of these genes are important for cell growth, transcription, RNA metabolism and DNA repair. We believe that a mivaRNA-mediated fine tune of the expression of some of these genes could be important in adenovirus cell cycle. PMID:19933264

  8. cDNA microarray analysis reveals that antioxidant and immune genes are upregulated during involution of the bovine mammary gland.

    PubMed

    Singh, K; Davis, S R; Dobson, J M; Molenaar, A J; Wheeler, T T; Prosser, C G; Farr, V C; Oden, K; Swanson, K M; Phyn, C V C; Hyndman, D L; Wilson, T; Henderson, H V; Stelwagen, K

    2008-06-01

    We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N(1)-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution. PMID:18487646

  9. Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator

    PubMed Central

    Gondcaille, Catherine; Depreter, Marianne; Fourcade, Stphane; Lecca, Maria Rita; Leclercq, Sabrina; Martin, Pascal G.P.; Pineau, Thierry; Cadepond, Franoise; ElEtr, Martine; Bertrand, Nathalie; Beley, Alain; Duclos, Sandrine; De Craemer, Dirk; Roels, Frank; Savary, Stphane; Bugaut, Maurice

    2005-01-01

    X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPAR?-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPAR? independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition. PMID:15809314

  10. Mycoplasma gallisepticum Lipid Associated Membrane Proteins Up-regulate Inflammatory Genes in Chicken Tracheal Epithelial Cells via TLR-2 Ligation through an NF-κB Dependent Pathway

    PubMed Central

    Majumder, Sanjukta; Zappulla, Frank; Silbart, Lawrence K.

    2014-01-01

    Mycoplasma gallisepticum-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (Rlow) or a non-virulent (Rhigh) strain were incubated with primary TECs or chicken tracheae ex vivo. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. Ex-vivo exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1β, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, Rlow exposed epithelial cells both in vitro and ex vivo showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1β and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2–3 fold. Conversely, an NF-κB inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either Rlow or Rhigh exposure. Taken together we conclude that LAMPs isolated from both Rhigh and Rlow induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-κB dependent pathway. PMID:25401327

  11. Mycoplasma gallisepticum lipid associated membrane proteins up-regulate inflammatory genes in chicken tracheal epithelial cells via TLR-2 ligation through an NF-?B dependent pathway.

    PubMed

    Majumder, Sanjukta; Zappulla, Frank; Silbart, Lawrence K

    2014-01-01

    Mycoplasma gallisepticum-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (R(low)) or a non-virulent (R(high)) strain were incubated with primary TECs or chicken tracheae ex vivo. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. Ex-vivo exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1?, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, R(low) exposed epithelial cells both in vitro and ex vivo showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1? and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2-3 fold. Conversely, an NF-?B inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either R(low) or R(high) exposure. Taken together we conclude that LAMPs isolated from both R(high) and R(low) induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-?B dependent pathway. PMID:25401327

  12. Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation.

    PubMed Central

    Majano, P L; Garca-Monzn, C; Lpez-Cabrera, M; Lara-Pezzi, E; Fernndez-Ruiz, E; Garca-Iglesias, C; Borque, M J; Moreno-Otero, R

    1998-01-01

    Increased nitric oxide (NO) production may contribute to the pathological changes featuring in some inflammatory diseases, but the role of NO in chronic viral hepatitis is still unknown. We compared the inducible NO synthase (NOS2) expression in the liver of patients with chronic viral hepatitis with that of both nonviral liver disease and histologically normal liver. NOS2 expression was assessed by immunohistochemical and in situ hybridization studies of liver biopsy sections. An intense hepatocellular NOS2 reactivity was detected in chronic viral hepatitis, whereas it was weakly or not observed in nonviral liver disease or normal liver, respectively. In addition, we determined whether the hepatitis B virus (HBV) might regulate the synthesis of this enzyme. NOS2 mRNA and protein levels as well as enzyme activity were assessed in cytokine-stimulated HBV-transfected and untransfected hepatoma cells. Transfection with either HBV genome or HBV X gene resulted in induction of NOS2 mRNA expression, and the maximal induction of this transcript and NO production was observed in cytokine-stimulated HBV-transfected cells. These results indicate that hepatotropic viral infections are able to upregulate the NOS2 gene expression in human hepatocytes, suggesting that NO may mediate important pathogenic events in the course of chronic viral hepatitis. PMID:9525976

  13. The high-mobility group A1 gene up-regulates cyclooxygenase 2 expression in uterine tumorigenesis.

    PubMed

    Tesfaye, Abeba; Di Cello, Francescopaolo; Hillion, Joelle; Ronnett, Brigitte M; Elbahloul, Ossama; Ashfaq, Raheela; Dhara, Surajit; Prochownik, Edward; Tworkoski, Kathryn; Reeves, Raymond; Roden, Richard; Ellenson, Lora Hedrick; Huso, David L; Resar, Linda M S

    2007-05-01

    Uterine cancer is the most common cancer of the female genital tract and is the fourth most frequent cause of cancer death in women in the U.S. Despite the high prevalence of uterine cancers, the molecular events that lead to neoplastic transformation in the uterus are poorly understood. Moreover, there are limited mouse models to study these malignancies. We generated transgenic mice with high-mobility group A1 gene (HMGA1a) expression targeted to uterine tissue and all female mice developed tumors by 9 months of age. Histopathologically, the tumors resemble human uterine adenosarcoma and are transplantable. To determine whether these findings are relevant to human disease, we evaluated primary human uterine neoplasms and found that HMGA1a mRNA and protein levels are increased in most high-grade neoplasms but not in normal uterine tissue, benign tumors, or most low-grade neoplasms. We also found that HMGA1a up-regulates cyclooxygenase 2 (COX-2) expression in transgenic tumors. Moreover, both HMGA1a and COX-2 expression are up-regulated in high-grade human leiomyosarcomas. Using chromatin immunoprecipitation, HMGA1a binds directly to the COX-2 promoter in human uterine cancer cells in vivo and activates its expression in transfection experiments. We also show that blocking either HMGA1a or COX-2 in high-grade human uterine cancer cells blocks anchorage-independent cell growth in methylcellulose. These findings show that HMGA1a functions as an oncogene when overexpressed in the uterus and contributes to the pathogenesis of human uterine cancer by activating COX-2 expression. Although a larger study is needed to confirm these results, HMGA1a may be a useful marker for aggressive human uterine cancers. PMID:17483309

  14. Acquisition of docetaxel resistance in breast cancer cells reveals upregulation of ABCB1 expression as a key mediator of resistance accompanied by discrete upregulation of other specific genes and pathways.

    PubMed

    Hansen, Stine Ninel; Westergaard, David; Thomsen, Mathilde Borg Houlberg; Vistesen, Mette; Do, Khoa Nguyen; Fogh, Louise; Belling, Kirstine C; Wang, Jun; Yang, Huanming; Gupta, Ramneek; Ditzel, Henrik J; Moreira, Jos; Brnner, Nils; Stenvang, Jan; Schrohl, Anne-Sofie

    2015-06-01

    The microtubule-targeting taxanes are important in breast cancer therapy, but no predictive biomarkers have yet been identified with sufficient scientific evidence to allow clinical routine use. The purposes of the present study were to develop a cell-culture-based discovery platform for docetaxel resistance and thereby identify key molecular mechanisms and predictive molecular characteristics to docetaxel resistance. Two docetaxel-resistant cell lines, MCF7RES and MDARES, were generated from their respective parental cell lines MCF-7 and MDA-MB-231 by stepwise selection in docetaxel dose increments over 15 months. The cell lines were characterized regarding sensitivity to docetaxel and other chemotherapeutics and subjected to transcriptome-wide mRNA microarray profiling. MCF7RES and MDARES exhibited a biphasic growth inhibition pattern at increasing docetaxel concentrations. Gene expression analysis singled out ABCB1, which encodes permeability glycoprotein (Pgp), as the top upregulated gene in both MCF7RES and MDARES. Functional validation revealed Pgp as a key resistance mediator at low docetaxel concentrations (first-phase response), whereas additional resistance mechanisms appeared to be prominent at higher docetaxel concentrations (second-phase response). Additional resistance mechanisms were indicated by gene expression profiling, including genes in the interferon-inducible protein family in MCF7RES and cancer testis antigen family in MDARES. Also, upregulated expression of various ABC transporters, ECM-associated proteins, and lysosomal proteins was identified in both resistant cell lines. Finally, MCF7RES and MDARES presented with cross-resistance to epirubicin, but only MDARES showed cross-resistance to oxaliplatin. In conclusion, Pgp was identified as a key mediator of resistance to low docetaxel concentrations with other resistance mechanisms prominent at higher docetaxel concentrations. Supporting Pgp upregulation as one major mechanism of taxane resistance and cell-line-specific alterations as another, both MCF7RES and MDARES were cross-resistant to epirubicin (Pgp substrate), but only MDARES was cross-resistant to oxaliplatin (non-Pgp substrate). PMID:25596703

  15. Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model

    PubMed Central

    Garcia-Manteiga, Jose M.; Bonfiglio, Silvia; Malosio, Maria Luisa; Lazarevic, Dejan; Stupka, Elia; Cittaro, Davide; Meldolesi, Jacopo

    2015-01-01

    Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation. PMID:26413508

  16. Computational evaluation of cellular metabolic costs successfully predicts genes whose expression is deleterious

    PubMed Central

    Wagner, Allon; Zarecki, Raphy; Reshef, Leah; Gochev, Camelia; Sorek, Rotem; Gophna, Uri; Ruppin, Eytan

    2013-01-01

    Gene suppression and overexpression are both fundamental tools in linking genotype to phenotype in model organisms. Computational methods have proven invaluable in studying and predicting the deleterious effects of gene deletions, and yet parallel computational methods for overexpression are still lacking. Here, we present Expression-Dependent Gene Effects (EDGE), an in silico method that can predict the deleterious effects resulting from overexpression of either native or foreign metabolic genes. We first test and validate EDGE’s predictive power in bacteria through a combination of small-scale growth experiments that we performed and analysis of extant large-scale datasets. Second, a broad cross-species analysis, ranging from microorganisms to multiple plant and human tissues, shows that genes that EDGE predicts to be deleterious when overexpressed are indeed typically down-regulated. This reflects a universal selection force keeping the expression of potentially deleterious genes in check. Third, EDGE-based analysis shows that cancer genetic reprogramming specifically suppresses genes whose overexpression impedes proliferation. The magnitude of this suppression is large enough to enable an almost perfect distinction between normal and cancerous tissues based solely on EDGE results. We expect EDGE to advance our understanding of human pathologies associated with up-regulation of particular transcripts and to facilitate the utilization of gene overexpression in metabolic engineering. PMID:24198337

  17. Buyang Huanwu decoction up-regulates Notch1 gene expression in injured spinal cord

    PubMed Central

    Guo, Zhan-peng; Huang, Mi-na; Liu, An-qi; Yuan, Ya-jiang; Zhao, Jian-bo; Mei, Xi-fan

    2015-01-01

    Expression of genes in the Notch signaling pathway is altered in the injured spinal cord, which indicates that Notch participates in repair after spinal cord injury. Buyang Huanwu decoction, a traditional Chinese herbal preparation, can promote the growth of nerve cells and nerve fibers; however, it is unclear whether Buyang Huanwu decoction affects the Notch signaling pathway in injured spinal cord. In this study, a rat model was established by injuring the T10 spinal cord. At 2 days after injury, rats were intragastrically administered 2 mL of 0.8 g/mL Buyang Huanwu decoction daily until sacrifice. Real-time reverse transcription polymerase chain reaction analysis demonstrated that at 7, 14 and 28 days after injury, the expression of Notch1 was increased in the Buyang Huanwu decoction group compared with controls. These findings confirm that Buyang Huanwu decoction can promote the expression of Notch1 in rats with incomplete spinal cord injury, and may indicate a mechanism to promote the repair of spinal cord injury. PMID:26487863

  18. Gene markers of cellular aging in human multipotent stromal cells in culture

    PubMed Central

    2014-01-01

    Introduction Human multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined. Methods Commercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology. Results The phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7. Conclusions Our results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs before clinical use. PMID:24780490

  19. Thyroid Hormone Upregulates Hypothalamic kiss2 Gene in the Male Nile Tilapia, Oreochromis niloticus

    PubMed Central

    Ogawa, Satoshi; Ng, Kai We; Xue, Xiaoyu; Ramadasan, Priveena Nair; Sivalingam, Mageswary; Li, Shuisheng; Levavi-Sivan, Berta; Lin, Haoran; Liu, Xiaochun; Parhar, Ishwar S.

    2013-01-01

    Kisspeptin has recently been recognized as a critical regulator of reproductive function in vertebrates. During the sexual development, kisspeptin neurons receive sex steroids feedback to trigger gonadotropin-releasing hormone (GnRH) neurons. In teleosts, a positive correlation has been found between the thyroid status and the reproductive status. However, the role of thyroid hormone in the regulation of kisspeptin system remains unknown. We cloned and characterized a gene encoding kisspeptin (kiss2) in a cichlid fish, the Nile tilapia (Oreochromis niloticus). Expression of kiss2 mRNA in the brain was analyzed by in situ hybridization. The effect of thyroid hormone (triiodothyronine, T3) and hypothyroidism with methimazole (MMI) on kiss2 and the three GnRH types (gnrh1, gnrh2, and gnrh3) mRNA expression was analyzed by real-time PCR. Expression of thyroid hormone receptor mRNAs were analyzed in laser-captured kisspeptin and GnRH neurons by RT-PCR. The kiss2 mRNA expressing cells were seen in the nucleus of the lateral recess in the hypothalamus. Intraperitoneal administration of T3 (5 μg/g body weight) to sexually mature male tilapia significantly increased kiss2 and gnrh1 mRNA levels at 24 h post injection (P < 0.001), while the treatment with an anti-thyroid, MMI (100 ppm for 6 days) significantly reduced kiss2 and gnrh1 mRNA levels (P < 0.05). gnrh2, gnrh3, and thyrotropin-releasing hormone mRNA levels were insensitive to the thyroid hormone manipulations. Furthermore, RT-PCR showed expression of thyroid hormone receptor mRNAs in laser-captured GnRH neurons but not in kiss2 neurons. This study shows that GnRH1 may be directly regulated through thyroid hormone, while the regulation of Kiss2 by T3 is more likely to be indirect. PMID:24324459

  20. 80 FR 7872 - Joint Meeting of the Cellular, Tissue and Gene Therapies Advisory Committee and the Oncologic...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2015-02-12

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Joint Meeting of the Cellular, Tissue and Gene Therapies...: Cellular, Tissue and Gene Therapies Advisory Committee and the Oncologic Drug Advisory Committee....

  1. 80 FR 58261 - Joint Meeting of the Cellular, Tissue, and Gene Therapies Advisory Committee and the Oncologic...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2015-09-28

    ... From the Federal Register Online via the Government Publishing Office ] DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Joint Meeting of the Cellular, Tissue, and Gene Therapies...: Cellular, Tissue, and Gene Therapies Advisory Committee and the Oncologic Drugs Advisory Committee....

  2. Expression of costimulatory molecules: B7 and ICAM up-regulation after treatment with a suicide gene.

    PubMed

    Ramesh, R; Munshi, A; Abboud, C N; Marrogi, A J; Freeman, S M

    1996-01-01

    The herpes simplex virus thymidine kinase gene (HSV-TK) in combination with ganciclovir (GCV), is currently being used in gene therapy-based clinical trials for cancer treatment. Its therapeutic effect is based on a "bystander effect" whereby HSV-TK gene-modified tumor cells are toxic to nearby unmodified tumor cells when exposed to the antiviral drug GCV. We have recently hypothesized that the in vivo mechanism of this bystander effect is due to alterations in the tumor microenvironment in response to release of cytokines and an infiltration of leukocytes after treatment with HSV-TK gene-modified tumor cells and GCV, which results in tumor regression. Expression of B7, a recently identified costimulatory molecule that is important for T-cell stimulation, has been shown to be modulated by stimulatory cytokines interferon-gamma, tumor necrosis factor-alpha, and inhibited by interleukin-10. In the present study, we investigated whether the cytokines released after HSV-TK and GCV treatment could include the expression of the costimulatory molecules B7-1 and B7-2 and the adhesion molecule (ICAM)-1 in the tumor. Furthermore, we investigated whether this altered environment affected the antitumor properties of host lymphocytes. An in vitro model was developed to establish the effects of HSV-TK gene-modified tumor cells and GCV on tumor infiltrating cells. The murine macrophage cell line (IC21) was exposed to either supernatants or cell lysates collected from a mixture of HSV-TK-transduced (KBALB-STK) and non-transduced (KBALB) murine fibrosarcoma tumor cells previously exposed to GCV (experimental). Immunohistochemical analysis showed a significant expression (P < .0001) of B7-1 and B7-2 post exposure of IC21 cells to either supernatant or lysate. In contrast, the level of expression in IC21 cells exposed to the control lysate or supernatant remained unchanged for B7-1 and B7-2. In vivo analysis for B7-1 and B7-2 expression by immunohistochemistry in tumor tissues from experimental mice receiving HSV-TK gene-modified tumor cells and GCV treatment showed a significant expression of B7.1 (35%, P < .0001) and B7.2 (38.2%, P < .0001) on tumor-infiltrating mononuclear cells. In contrast, tumor-bearing control animals showed low levels of B7-2 expression (5.8%), whereas B7-1 was undetectable, as confirmed by reverse-transcriptase polymerase chain reaction. In addition, a significant up-regulation of ICAM expression (50%) on tumor tissues was observed in the experimental group (P = .0317) as compared with the control group (25%). Furthermore, T cells isolated from experimental mice showed a significant in vitro proliferative response (p = .0202) when exposed to syngeneic tumor cells as compared with the control group. These data demonstrated that the use of HSV-TK gene-modified tumor cells and GCV as a suicide gene in the treatment of an intraperitoneal tumor resulted in the expression of the B7 costimulatory molecules and ICAM-1 adhesion molecule and enhanced proliferative response of host T cells. These findings help to understand the mechanism of tumor cell killing in vivo using HSV-TK gene-modified tumor cells. PMID:8988840

  3. Virus-induced transcriptional changes in the brain include the differential expression of genes associated with interferon, apoptosis, interleukin 17 receptor A, and glutamate signaling as well as flavivirus-specific upregulation of tRNA synthetases.

    PubMed

    Clarke, Penny; Leser, J Smith; Bowen, Richard A; Tyler, Kenneth L

    2014-01-01

    Flaviviruses, particularly Japanese encephalitis virus (JEV) and West Nile virus (WNV), are important causes of virus-induced central nervous system (CNS) disease in humans. We used microarray analysis to identify cellular genes that are differentially regulated following infection of the brain with JEV (P3) or WNV (New York 99). Gene expression data for these flaviviruses were compared to those obtained following infection of the brain with reovirus (type 3 Dearing), an unrelated neurotropic virus. We found that a large number of genes were up-regulated by all three viruses (using the criteria of a change of >2-fold and a P value of <0.001), including genes associated with interferon signaling, the immune system, inflammation, and cell death/survival signaling. In addition, genes associated with glutamate signaling were down-regulated in infections with all three viruses (criteria, a >2-fold change and a P value of <0.001). These genes may serve as broad-spectrum therapeutic targets for virus-induced CNS disease. A distinct set of genes were up-regulated following flavivirus infection but not following infection with reovirus. These genes were associated with tRNA charging and may serve as therapeutic targets for flavivirus-induced CNS disease. IMPORTANCE Viral infections of the central nervous system (CNS) are an important cause of morbidity and mortality. Treatment options for virus-induced CNS disease are limited, and for many clinically important neurotropic viruses, no specific therapy of proven benefit is currently available. We performed microarray analysis to identify genes that are differentially regulated in the brain following virus infection in order to identify pathways that might provide novel therapeutic targets for virus-induced CNS disease. Although several studies have described gene expression changes following virus infection of the brain, this report is the first to directly compare large-scale gene expression data from different viruses. We identified genes that are differentially regulated in infection of the brain with viruses from different families and those which appear to be specific to flavivirus infections. PMID:24618253

  4. Control of late cornified envelope genes relevant to psoriasis risk: upregulation by 1,25-dihydroxyvitamin D3 and plant-derived delphinidin.

    PubMed

    Hoss, Elika; Austin, Heather R; Batie, Shane F; Jurutka, Peter W; Haussler, Mark R; Whitfield, G Kerr

    2013-12-01

    Psoriasis is a chronic inflammatory skin disease featuring abnormal keratinocyte proliferation and differentiation. A genetic risk factor for psoriasis (PSORS4) is a deletion of LCE3B and LCE3C genes encoding structural proteins in terminally differentiated keratinocytes. Because analogs of 1,25-dihydroxyvitamin D3 (1,25D) are used in psoriasis treatment, we hypothesized that 1,25D acts via the vitamin D receptor (VDR) to upregulate expression of LCE 3A/3D/3E genes, potentially mitigating the absence of LCE3B/LCE3C gene products. Results in a human keratinocyte line, HaCaT, suggested that 1,25D, low affinity VDR ligands docosahexaenoic acid and curcumin, along with a novel candidate ligand, delphinidin, induce LCE transcripts as monitored by qPCR. Further experiments in primary human keratinocytes preincubated with 1.2mM calcium indicated that 1,25D and 10?M delphinidin upregulate all five LCE3 genes (LCE3A-E). Competition binding assays employing radiolabeled 1,25D revealed that delphinidin binds VDR weakly (IC50?1mM). However, 20?M delphinidin was capable of upregulating a luciferase reporter gene in a VDRE-dependent manner in a transfected keratinocyte cell line (KERTr). These results are consistent with a scenario in which delphinidin is metabolized to an active compound that then stimulates LCE3 transcription in a VDR/VDRE-dependent manner. We propose that upregulation of LCE genes may be part of the therapeutic effect of 1,25D to ameliorate psoriasis by providing sufficient LCE proteins, especially in individuals missing the LCE3B and 3C genes. Results with delphinidin further suggest that this compound or its metabolite(s) might offer an alternative to 1,25D in psoriasis therapy. PMID:23839497

  5. An Arabidopsis thaliana pectin acetylesterase gene is upregulated in nematode feeding sites induced by root-knot and cyst nematodes.

    PubMed

    Vercauteren, Isabel; de Almeida Engler, Janice; De Groodt, Ruth; Gheysen, Godelieve

    2002-04-01

    By using differential display, gene expression was investigated in Arabidopsis thaliana roots shortly after nematode infection, and a putative pectin acetylesterase (PAE) homolog (DiDi 9C-12) was found to be upregulated. PAEs catalyze the deacetylation of pectin, a major compound of primary cell walls. mRNA in situ hybridization experiments showed that the expression of DiDi 9C-12 was enhanced very early after infection in initiating giant-cells and in cells surrounding the nematodes. Later on, the level of DiDi 9C-12 mRNA was lower in giant-cells and transcripts were mainly found in parenchyma, endodermis, and pericycle cells of the root gall. Twenty days after infection, DiDi 9C-12 transcripts could no longer be detected. DiDi 9C-12 transcripts were also found in young syncytia and in the cells surrounding the expanding syncytium. Our results suggest that plant parasitic nematodes can modulate the rapid growth of the feeding cells and the expansion of the root gall by triggering the expression of DiDi 9C-12. PAEs, which probably act together with a range of other pectin-degrading enzymes, could be involved in softening and loosening the primary cell wall in nematode-infected plant roots. PMID:12026180

  6. Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases

    PubMed Central

    Wang, Haiyan; Zheng, Yanshan; He, Shaoheng

    2006-01-01

    Background Hypersecretion of cytokines and serine proteinases has been observed in asthma. Since protease-activated receptors (PARs) are receptors of several serine proteinases and airway epithelial cells are a major source of cytokines, the influence of serine proteinases and PARs on interleukin (IL)-8 secretion and gene expression in cultured A549 cells was examined. Results A549 cells express all four PARs at both protein and mRNA levels as assessed by flow cytometry, immunofluorescence microscopy and reverse transcription polymerase chain reaction (PCR). Thrombin, tryptase, elastase and trypsin induce a up to 8, 4.3, 4.4 and 5.1 fold increase in IL-8 release from A549 cells, respectively following 16 h incubation period. The thrombin, elastase and trypsin induced secretion of IL-8 can be abolished by their specific inhibitors. Agonist peptides of PAR-1, PAR-2 and PAR-4 stimulate up to 15.6, 6.6 and 3.5 fold increase in IL-8 secretion, respectively. Real time PCR shows that IL-8 mRNA is up-regulated by the serine proteinases tested and by agonist peptides of PAR-1 and PAR-2. Conclusion The proteinases, possibly through activation of PARs can stimulate IL-8 release from A549 cells, suggesting that they are likely to contribute to IL-8 related airway inflammatory disorders in man. PMID:16696869

  7. Nephroblastoma overexpressed gene (NOV) enhances RCC cell motility through upregulation of ICAM-1 and COX-2 expression via Akt pathway

    PubMed Central

    Liu, Shuai; Han, Liping; Wang, Xiaoqing; Liu, Zheng; Ding, Sentai; Lu, Jiaju; Bi, Dongbin; Mei, Yikun; Niu, Zhihong

    2015-01-01

    Renal cell carcinoma (RCC) carries a high risk of malignancy and metastasis. The inducible isoform of prostaglandin synthase, cyclooxygenase (COX)-2, and ICAM-1 may be involved in tumor metastasis. CCN3, also called nephroblastoma overexpressed gene (NOV), has been found to regulate the proliferation and differentiation of cancer cells. The effects of NOV on RCC cell migration and expression of COX-2 and ICAM-1 have not described yet in detail. But here, NOV was found to promote the migration and expression of COX-2 and ICAM-1 in human RCC cells. Akt inhibitor was found to interfere with this NOV-induced migration and up-regulation of COX-2 and ICAM-1 in RCC cells. NOV stimulation was here found to promote the phosphorylation of Akt. RCC tissue chips were subjected to IHC staining, which showed COX-2 expression in RCC tissues to be a significantly closely correlated with NOV expression, with significance determined using Pearson correlation testing (P < 0.05). The results of the current work indicate that NOV activates COX-2 and ICAM-1 through Akt, promoting the migration of RCC cells. PMID:25973014

  8. A cellular enhancer of retrovirus gene expression in embryonal carcinoma cells.

    PubMed Central

    Taketo, M; Tanaka, M

    1987-01-01

    Murine embryonal carcinoma (EC) cells are refractory to infection by retroviruses because retroviral long terminal repeat (LTR) enhancers have little activity in EC cells. A previous report described the isolation of clonal EC cell lines that express the integrated neomycin-resistance gene (neo) linked to the Moloney murine leukemia virus LTR. The expression of the neo gene was explained by a cis-acting mechanism [Taketo, M., Gilboa, E. & Sherman, M. I. (1985) Proc. Natl. Acad. Sci. USA 82, 2422-2426]. From one such EC cell line, we isolated the flanking cellular sequence 5' to the proviral genome, ligated it to various test constructs, and transfected into the parental EC cells. The cellular sequence increased expression of the LTR-linked neo gene significantly, in a manner independent of its orientation and position. The neo mRNA was initiated at the bona fide promoter of the LTR. By deletion analyses, we defined a region of DNA essential for the enhancer activity and determined its sequence. This region contains distinctly characteristic stretches as well as some similarity to various viral and cellular enhancers. Thus the LTR-linked neo gene is expressed because the provirus is integrated in the vicinity of this enhancer that is active in undifferentiated EC cells. Images PMID:3473480

  9. N-acetylcysteine inhibits the up-regulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Chronic ethanol (EtOH) administration to experimental animals induces hepatic oxidative stress and up-regulates mitochondrial biogenesis. The mechanisms by which chronic EtOH up-regulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative ...

  10. A rare sugar, d-allose, confers resistance to rice bacterial blight with upregulation of defense-related genes in Oryza sativa.

    PubMed

    Kano, Akihito; Gomi, Kenji; Yamasaki-Kokudo, Yumiko; Satoh, Masaru; Fukumoto, Takeshi; Ohtani, Kouhei; Tajima, Shigeyuki; Izumori, Ken; Tanaka, Keiji; Ishida, Yutaka; Tada, Yasuomi; Nishizawa, Yoko; Akimitsu, Kazuya

    2010-01-01

    We investigated responses of rice plant to three rare sugars, d-altrose, d-sorbose, and d-allose, due to establishment of mass production methods for these rare sugars. Root growth and shoot growth were significantly inhibited by d-allose but not by the other rare sugars. A large-scale gene expression analysis using a rice microarray revealed that d-allose treatment causes a high upregulation of many defense-related, pathogenesis-related (PR) protein genes in rice. The PR protein genes were not upregulated by other rare sugars. Furthermore, d-allose treatment of rice plants conferred limited resistance of the rice against the pathogen Xanthomonas oryzae pv. oryzae but the other tested sugars did not. These results indicate that d-allose has a growth inhibitory effect but might prove to be a candidate elicitor for reducing disease development in rice. PMID:19968553

  11. Building quantitative, three dimensional atlases of gene expression and morphology at cellular resolution

    PubMed Central

    Knowles, David W.; Biggin, Mark D.

    2013-01-01

    Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy based approaches to establish permanent, quantitative datasets—atlases—that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization and quantitative analysis. PMID:24123936

  12. Gene expression profiling for mechanistic understanding of cellular aggregation in mammalian cell perfusion cultures.

    PubMed

    Liu, Meile; Goudar, Chetan T

    2013-02-01

    Aggregation of baby hamster kidney (BHK) cells cultivated in perfusion mode for manufacturing recombinant proteins was characterized. The potential impact of cultivation time on cell aggregation for an aggregating culture (cell line A) was studied by comparing expression profiles of 84 genes in the extracellular adhesion molecules (ECM) pathway by qRT-PCR from 9 and 25 day shake flask samples and 80 and 94 day bioreactor samples. Significant up-regulation of THBS2 (4.4- to 6.9-fold) was seen in both the 25 day shake flask and 80 and 94 day bioreactor samples compared to the 9 day shake flask while NCAM1 was down-regulated 5.1- to 8.9-fold in the 80 and 94 day bioreactor samples. Subsequent comparisons were made between cell line A and a non-aggregating culture (cell line B). A 65 day perfusion bioreactor sample from cell line B served as the control for 80 and 94 day samples from four different perfusion bioreactors for cell line A. Of the 84 genes in the ECM pathway, four (COL1A1, COL4A1, THBS2, and VCAN) were consistently up-regulated in cell line A while two (NCAM1 and THBS1) were consistently down-regulated. The magnitudes of differential gene expression were much higher when cell lines were compared (4.1- to 44.6-fold) than when early and late cell line B samples were compared (4.4- to 6.9-fold) indicating greater variability between aggregating and non-aggregating cell lines. Based on the differential gene expression results, two mechanistic models were proposed for aggregation of BHK cells in perfusion cultures. PMID:23007466

  13. Quality controls in cellular immunotherapies: rapid assessment of clinical grade dendritic cells by gene expression profiling.

    PubMed

    Castiello, Luciano; Sabatino, Marianna; Zhao, Yingdong; Tumaini, Barbara; Ren, Jiaqiang; Ping, Jin; Wang, Ena; Wood, Lauren V; Marincola, Francesco M; Puri, Raj K; Stroncek, David F

    2013-02-01

    Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers. PMID:23147403

  14. Gene-specific DNA repair of pyrimidine dimers does not decline during cellular aging in vitro.

    PubMed

    Christiansen, M; Stevnsner, T; Bohr, V A; Clark, B F; Rattan, S I

    2000-04-10

    A large number of studies have demonstrated that various kinds of DNA damage accumulate during aging and one of the causes for this could be a decrease in DNA repair capacity. However, the level of total genomic repair has not been strongly correlated with aging. DNA repair of certain kinds of damage is known to be closely connected to the transcription process; thus, we chose to investigate the level of gene-specific repair of UV-induced damage using in vitro aging of human diploid skin fibroblasts and trabecular osteoblasts as model systems for aging. We find that the total genomic repair is not significantly affected during cellular aging of cultures of both human skin fibroblasts and trabecular osteoblasts. Gene-specific repair was analyzed during cellular aging in the dihydrofolate reductase housekeeping gene, the p53 tumor suppressor gene, and the inactive region X(754). There was no clear difference in the capacity of young and old cells to repair UV-induced pyrimidine dimers in any of the analyzed genes. Thus, in vitro senescent cells can sustain the ability to repair externally induced damage. PMID:10739678

  15. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

    SciTech Connect

    Verma, Saguna; Ziegler, Katja; Ananthula, Praveen; Co, Juliene K.G.; Frisque, Richard J.; Yanagihara, Richard; Nerurkar, Vivek R. . E-mail: nerurkar@pbrc.hawaii.edu

    2006-02-20

    Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

  16. Dietary Fucoxanthin Increases Metabolic Rate and Upregulated mRNA Expressions of the PGC-1alpha Network, Mitochondrial Biogenesis and Fusion Genes in White Adipose Tissues of Mice

    PubMed Central

    Wu, Meng-Ting; Chou, Hong-Nong; Huang, Ching-jang

    2014-01-01

    The mechanism for how fucoxanthin (FX) suppressed adipose accumulation is unclear. We aim to investigate the effects of FX on metabolic rate and expressions of genes related to thermogenesis, mitochondria biogenesis and homeostasis. Using a 2 2 factorial design, four groups of mice were respectively fed a high sucrose (50% sucrose) or a high-fat diet (23% butter + 7% soybean oil) supplemented with or without 0.2% FX. FX significantly increased oxygen consumption and carbon dioxide production and reduced white adipose tissue (WAT) mass. The mRNA expressions of peroxisome proliferator-activated receptor (PPAR) ? coactivator-1? (PGC-1?), cell death-inducing DFFA-like effecter a (CIDEA), PPAR?, PPAR?, estrogen-related receptor ? (ERR?), ?3-adrenergic receptor (?3-AR) and deiodinase 2 (Dio2) were significantly upregulated in inguinal WAT (iWAT) and epididymal WAT (eWAT) by FX. Mitochondrial biogenic genes, nuclear respiratory factor 1 (NRF1) and NRF2, were increased in eWAT by FX. Noticeably, FX upregulated genes of mitochondrial fusion, mitofusin 1 (Mfn1), Mfn2 and optic atrophy 1 (OPA1), but not mitochondrial fission, Fission 1, in both iWAT and eWAT. In conclusion, dietary FX enhanced the metabolic rate and lowered adipose mass irrespective of the diet. These were associated with upregulated genes of the PGC-1? network and mitochondrial fusion in eWAT and iWAT. PMID:24534841

  17. Dietary fucoxanthin increases metabolic rate and upregulated mRNA expressions of the PGC-1alpha network, mitochondrial biogenesis and fusion genes in white adipose tissues of mice.

    PubMed

    Wu, Meng-Ting; Chou, Hong-Nong; Huang, Ching-jang

    2014-02-01

    The mechanism for how fucoxanthin (FX) suppressed adipose accumulation is unclear. We aim to investigate the effects of FX on metabolic rate and expressions of genes related to thermogenesis, mitochondria biogenesis and homeostasis. Using a 2 2 factorial design, four groups of mice were respectively fed a high sucrose (50% sucrose) or a high-fat diet (23% butter + 7% soybean oil) supplemented with or without 0.2% FX. FX significantly increased oxygen consumption and carbon dioxide production and reduced white adipose tissue (WAT) mass. The mRNA expressions of peroxisome proliferator-activated receptor (PPAR) ? coactivator-1? (PGC-1?), cell death-inducing DFFA-like effecter a (CIDEA), PPAR?, PPAR?, estrogen-related receptor ? (ERR?), ?3-adrenergic receptor (?3-AR) and deiodinase 2 (Dio2) were significantly upregulated in inguinal WAT (iWAT) and epididymal WAT (eWAT) by FX. Mitochondrial biogenic genes, nuclear respiratory factor 1 (NRF1) and NRF2, were increased in eWAT by FX. Noticeably, FX upregulated genes of mitochondrial fusion, mitofusin 1 (Mfn1), Mfn2 and optic atrophy 1 (OPA1), but not mitochondrial fission, Fission 1, in both iWAT and eWAT. In conclusion, dietary FX enhanced the metabolic rate and lowered adipose mass irrespective of the diet. These were associated with upregulated genes of the PGC-1? network and mitochondrial fusion in eWAT and iWAT. PMID:24534841

  18. Human p38{delta} MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

    SciTech Connect

    Ozawa, Shigeyuki; Department of Biochemistry and Molecular Biology; Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580 ; Ito, Shin; Kato, Yasumasa; Department of Biochemistry and Molecular Biology ; Kubota, Eiro; Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580 ; Hata, Ryu-Ichiro; Department of Biochemistry and Molecular Biology

    2010-06-11

    The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38{alpha}, {beta}, {gamma} and {delta}. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38{alpha} and {beta}, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38{gamma} and/or {delta} was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38{delta} attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38{delta} with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38{delta} isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38{alpha} and/or {beta} isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

  19. Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program

    PubMed Central

    Thiru, Prathapan; Kern, David M.; McKinley, Kara L.; Monda, Julie K.; Rago, Florencia; Su, Kuan-Chung; Tsinman, Tonia; Yarar, Defne; Bell, George W.; Cheeseman, Iain M.

    2014-01-01

    The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNAmicrotubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis. PMID:24829384

  20. Epigenetic regulations in the IFN? signalling pathway: IFN?-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes

    PubMed Central

    Vlkov, Veronika; t?pnek, Ivan; Hrukov, Veronika; enigl, Filip; Mayerov, Veronika; rmek, Martin; mov, Jana; Bieblov, Jana; Indrov, Marie; Hejhal, Tom; Drian, Nicolas; Klatzmann, David; Six, Adrien; Reini, Milan

    2014-01-01

    Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFN?. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFN? treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFN?-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFN? or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFN? acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes. PMID:25071011

  1. Epigenetic regulations in the IFN? signalling pathway: IFN?-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes.

    PubMed

    Vlkov, Veronika; t?pnek, Ivan; Hrukov, Veronika; enigl, Filip; Mayerov, Veronika; rmek, Martin; mov, Jana; Bieblov, Jana; Indrov, Marie; Hejhal, Tom; Drian, Nicolas; Klatzmann, David; Six, Adrien; Reini, Milan

    2014-08-30

    Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFN?. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFN? treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFN?-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFN? or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFN? acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes. PMID:25071011

  2. Sunflower-seed oil, rapidly-degradable starch, and adiposity up-regulate leptin gene expression in lactating goats.

    PubMed

    Bonnet, M; Delavaud, C; Bernard, L; Rouel, J; Chilliard, Y

    2009-08-01

    We conducted experiments to evaluate the effects of lipid supplementation and the nature of starchy concentrate on the regulation of leptin synthesis in lactating goats. Multiparous goats in mid- to late lactation received diets based on different forages and containing plant oil or seeds rich in either 18:1c9, 18:2n-6 or 18:3n-3 corresponding to 3%-7% dry matter (DM) as lipid supplements, or diets based on concentrate as either rapidly or slowly degradable starch. The isoenergetic replacement of a part of the concentrate by either oleic sunflower-seed oil, formaldehyde-treated linseeds, or linseed oil did not modify leptinemia and the leptin mRNA concentration in adipose tissues, suggesting a lack of effect of 18:1c9, 18:3n-3, or their biohydrogenation products. Conversely, leptinemia and the leptin mRNA abundance were increased (by 20% and 140%, respectively, P<0.05) in goats fed sunflower-seed oil under a grassland hay-based diet but not a maize silage-based diet, at similar energy intakes and adiposity. Thus, 18:2n-6 per se may up-regulate leptin gene expression, but the effect could be blunted by other fatty acids formed during the ruminal digestion of sunflower-seed oil when combined with maize silage. Consumption of rapidly but not slowly degradable starch increased (by 17%, P<0.05) leptinemia. Moreover, during lactation, plasma leptin was positively correlated (P<0.05) to adiposity parameters and negatively correlated to fiber intake. The results suggest that leptinemia responds poorly to nutritional factors in lactating goats, thus highlighting the physiological need to sustain hypoleptinemia during lactation. PMID:19446425

  3. Common Genetic Variation In Cellular Transport Genes and Epithelial Ovarian Cancer (EOC) Risk

    PubMed Central

    Chornokur, Ganna; Lin, Hui-Yi; Tyrer, Jonathan P.; Lawrenson, Kate; Dennis, Joe; Amankwah, Ernest K.; Qu, Xiaotao; Tsai, Ya-Yu; Jim, Heather S. L.; Chen, Zhihua; Chen, Ann Y.; Permuth-Wey, Jennifer; Aben, Katja KH.; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V.; Bean, Yukie T.; Beckmann, Matthias W.; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A.; Brooks-Wilson, Angela; Bunker, Clareann H.; Butzow, Ralf; Campbell, Ian G.; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S.; Cramer, Daniel W.; Cunningham, Julie M.; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Dicks, Ed; Doherty, Jennifer A.; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F.; Eccles, Diana M.; Edwards, Robert P.; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goodman, Marc T.; Gronwald, Jacek; Harrington, Patricia; Harter, Philipp; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A. T.; Hillemanns, Peter; Hogdall, Claus K.; Hogdall, Estrid; Hosono, Satoyo; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y.; Kelemen, Linda E.; Kellar, Mellissa; Kiemeney, Lambertus A.; Krakstad, Camilla; Kjaer, Susanne K.; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F. A. G.; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; McNeish, Iain; Menon, Usha; Milne, Roger L.; Modugno, Francesmary; Moysich, Kirsten B.; Ness, Roberta B.; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Paul, James; Pearce, Celeste L.; Pejovic, Tanja; Pelttari, Liisa M.; Pike, Malcolm C.; Poole, Elizabeth M.; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Schernhammer, Eva; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B.; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C.; Spiewankiewicz, Beata; Sucheston, Lara; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J.; Thomsen, Lotte; Tangen, Ingvild L.; Tworoger, Shelley S.; van Altena, Anne M.; Vierkant, Robert A.; Vergote, Ignace; Walsh, Christine S.; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Wu, Anna H.; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Hasmad, Hanis N.; Berchuck, Andrew; Iversen, Edwin S.; Schildkraut, Joellen M.; Ramus, Susan J.; Goode, Ellen L.; Monteiro, Alvaro N. A.; Gayther, Simon A.; Narod, Steven A.; Pharoah, Paul D. P.; Sellers, Thomas A.; Phelan, Catherine M.

    2015-01-01

    Background Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. As DNA damage and uncontrolled proliferation are hallmarks of cancer, including epithelial ovarian cancer (EOC), we hypothesized that inherited variation in the cellular transport genes contributes to EOC risk. Methods In total, DNA samples were obtained from 14,525 case subjects with invasive EOC and from 23,447 controls from 43 sites in the Ovarian Cancer Association Consortium (OCAC). Two hundred seventy nine SNPs, representing 131 genes, were genotyped using an Illumina Infinium iSelect BeadChip as part of the Collaborative Oncological Gene-environment Study (COGS). SNP analyses were conducted using unconditional logistic regression under a log-additive model, and the FDR q<0.2 was applied to adjust for multiple comparisons. Results The most significant evidence of an association for all invasive cancers combined and for the serous subtype was observed for SNP rs17216603 in the iron transporter gene HEPH (invasive: OR = 0.85, P = 0.00026; serous: OR = 0.81, P = 0.00020); this SNP was also associated with the borderline/low malignant potential (LMP) tumors (P = 0.021). Other genes significantly associated with EOC histological subtypes (p<0.05) included the UGT1A (endometrioid), SLC25A45 (mucinous), SLC39A11 (low malignant potential), and SERPINA7 (clear cell carcinoma). In addition, 1785 SNPs in six genes (HEPH, MGST1, SERPINA, SLC25A45, SLC39A11 and UGT1A) were imputed from the 1000 Genomes Project and examined for association with INV EOC in white-European subjects. The most significant imputed SNP was rs117729793 in SLC39A11 (per allele, OR = 2.55, 95% CI = 1.5-4.35, p = 5.66x10-4). Conclusion These results, generated on a large cohort of women, revealed associations between inherited cellular transport gene variants and risk of EOC histologic subtypes. PMID:26091520

  4. Induction of Cellular Immune Response by DNA Vaccine Coexpressing E. acervulina 3-1E Gene and Mature CHIl-15 Gene

    PubMed Central

    Ma, Dexing; Ma, Chunli; Gao, Mingyang; Li, Guangxing; Niu, Ze; Huang, Xiaodan

    2012-01-01

    We previously reported that the chimeric DNA vaccine pcDNA-3-1E-linker-mChIL-15, fused through linking Eimeria acervulina 3-1E encoding gene and mature chicken IL-15 (mChIL-15) gene with four flexible amino acid SPGS, could significantly offer protection against homologous challenge. In the present study, the induction of cellular immune response induced by the chimeric DNA vaccine pcDNA-3-1E-linker-mChIL-15 was investigated. Spleen lymphocyte subpopulations were characterized by flow cytometric analysis. The spleen lymphocyte proliferation assays were measured by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT) method. The mRNA profiles of ChIL-2 and ChIFN-γ in spleen were characterized by means of real-time PCR. Chickens immunized with pcDNA-3-1E-linker-mChIL-15 exhibited significant upregulated level of ChIL-2 and ChIFN-γ transcripts in spleen following two immunizations compared with chickens in other groups (P < 0.01). In comparison with pcDNA3.1-immunized and control groups, lymphocyte proliferation, percentage of CD8α+ cell, and levels of ChIL-2 and ChIFN-γ transcripts in the group immunized with pcDNA-3-1E-linker-mChIL-15 were significantly increased on day 6 following challenge (P < 0.05, P < 0.01, and P < 0.01, resp.). Our data suggested that the fusion antigen 3-1E-linker-mChIL-15 could be a potential candidate for E. acervulina vaccine development. PMID:22754694

  5. MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

    SciTech Connect

    Wang, Jie; Yan, Cheng-Hui; Li, Yang; Xu, Kai; Tian, Xiao-Xiang; Peng, Cheng-Fei; Tao, Jie; Sun, Ming-Yu; Han, Ya-Ling

    2013-05-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC phenotypic modulation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases. - Highlights: ► MiR-31 modulates CREG expression by binding directly to the human CREG mRNA 3′-UTR. ► MiR-31 mediates the human VSMC phenotypic modulation by regulating the expression of human CREG. ► Serum miR-31 may act as an important biomarker in diseases involving in stent restenosis after PCI.

  6. Patients with Dilated Cardiomyopathy and Sustained Monomorphic Ventricular Tachycardia Show Up-Regulation of KCNN3 and KCNJ2 Genes and CACNG8-Linked Left Ventricular Dysfunction

    PubMed Central

    Rosell-Llet, Esther; Gil-Cayuela, Carolina; Lago, Francisca; Gonzlez-Juanatey, Jose-Ramn; Cinca, Juan; Jorge, Esther; Martnez-Dolz, Luis; Portols, Manuel; Rivera, Miguel

    2015-01-01

    Aims Disruptions in cardiac ion channels have shown to influence the impaired cardiac contraction in heart failure. We sought to determine the altered gene expression profile of this category in dilated cardiomyopathy (DCM) patients and relate the altered gene expression with the clinical signs present in our patients, such as ventricular dysfunction and sustained monomorphic ventricular tachycardia (SMVT). Methods and Results Left ventricular (LV) tissue samples were used in RNA-sequencing technique to elucidate the transcriptomic changes of 13 DCM patients compared to controls (n = 10). We analyzed the differential gene expression of cardiac ion channels, and we found a total of 34 altered genes. We found that the calcium channel CACNG8 mRNA and protein levels were down-regulated and highly and inversely related with LV ejection fraction (LVEF) (r = 0.78, P<0.01). Furthermore, the potassium channels KCNN3 and KCNJ2 mRNA and protein levels were up-regulated and showed also a significant and inverse correlation with LVEF (r = 0.61, P<0.05; r = 0.60, P<0.05) in patients with SMVT. Conclusion A broad set of deregulated genes have been identified by RNA-sequencing technique. The relationship of CACNG8, KCNN3 and KCNJ2 with LVEF, and the up-regulation of KCNN3 and KCNJ2 in all patients with SMVT, irrespective of CACNG8 expression, suggest a significant role for these three ion flux related genes in the LV dysfunction present in this cardiomyopathy and an important relationship between KCNN3 and KCNJ2 up-regulation and the presence of SMVT. PMID:26710323

  7. L-Mimosine blocks cell proliferation via upregulation of B-cell translocation gene 2 and N-myc downstream regulated gene 1 in prostate carcinoma cells.

    PubMed

    Chung, Li-Chuan; Tsui, Ke-Hung; Feng, Tsui-Hsia; Lee, Shiow-Ling; Chang, Phei-Lang; Juang, Horng-Heng

    2012-02-15

    L-Mimosine, an iron chelator and a prolyl 4-hydroxylase inhibitor, blocks many cancer cells at the late G1 phase. B-cell translocation gene 2 (Btg2) regulates the G1/S transition phases of the cell cycle. N-myc downstream regulated gene 1 (Ndrg1) is a differentiation-inducing gene upregulated by hypoxia. We evaluated the molecular mechanisms of L-mimosine on cell cycle modulation in PC-3 and LNCaP prostate carcinoma cells. The effect of L-mimosine on cell proliferation of prostate carcinoma cells was determined by the [3H]thymidine incorporation and flow cytometry assays. L-Mimosine arrested the cell cycle at the G1 phase in PC-3 cells and at the S phase in LNCaP cells, thus attenuating cell proliferation. Immunoblot assays indicated that hypoxia and L-mimosine stabilized hypoxia-inducible factor-1α (HIF-1α) and induced Btg2 and Ndrg1 protein expression, but downregulated protein levels of cyclin A in both PC-3 and LNCaP cells. L-Mimosine treatment decreased cyclin D1 protein in PC-3 cells, but not in LNCaP cells. Dimethyloxalylglycine, a pan-prolyl hydroxylase inhibitor, also induced Btg2 and Ndrg1 protein expression in LNCaP cells. The transient gene expression assay revealed that L-mimosine treatment or cotransfection with HIF-1α expression vector enhanced the promoter activities of Btg2 and Ndrg1 genes. Knockdown of HIF-1α attenuated the increasing protein levels of both Btg2 and Ndrg1 by hypoxia or L-mimosine in LNCaP cells. Our results indicated that hypoxia and L-mimosine modulated Btg2 and Ndrg1 at the transcriptional level, which is dependent on HIF-1α. L-Mimosine enhanced expression of Btg2 and Ndrg1, which attenuated cell proliferation of the PC-3 and LNCaP prostate carcinoma cells. PMID:22116304

  8. 125I seed irradiation induces up-regulation of the genes associated with apoptosis and cell cycle arrest and inhibits growth of gastric cancer xenografts

    PubMed Central

    2012-01-01

    Background Iodine 125 (125I) seed irradiation can be used as an important supplementary treatment for unresectable advanced gastric cancer. Here, we aim to comprehensively elucidate the biological effects induced by 125I seed irradiation in human gastric cancer xenograft model by using global expression and DNA methylation analyses. Methods The 48 mice bearing NCI-N87 gastric cancer xenografts were randomly separated into 2 groups: sham seeds (O mCi) were implanted into the control group (n?=?24); 125?l seeds (0.9?mCi) were implanted into the treatment group (n?=?24). The mitotic index and apoptotic index were evaluated by quantitative morphometric analysis of the expression of proliferating cell nuclear antigen (PCNA) and in situ terminal transferase-mediated fluorescein deoxy- UTP nick end labeling (TUNEL), respectively. Global gene expression changes induced by 125I seed irradiation were analyzed by using Nimblegen Human gene expression array. DNA methylation profile in the tumors from control group was investigated with methylated DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays. The changes in the methylation status of selected genes were further investigated by using MeDIP-PCR. Results 125I seed irradiation suppresses the growth of gastric cancer xenografts in nude mice. PCNA staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis contribute to the 125I-induced tumor suppression in nude mouse model. Gene expression profiles revealed that the expression levels of several hundred genes, many of which are associated with apoptosis or cell cycle arrest, including BMF, MAPK8, BNIP3, RFWD3, CDKN2B and WNT9A, were upregulated following 125I seed irradiation. Furthermore, the up-regulation of some of these genes, such as BNIP3 and WNT9A, was found to be associated with irradiation-induced DNA demethylation. Conclusions This study revealed that 125I seed irradiation could significantly induce the up-regulation of apoptosis- and cell cycle-related genes in human gastric cancer xenografts. And some of the up-regulation might be attributed to 125I-irradiation induced demethylation in gene promoter regions. Collectively, these findings provided evidence for the efficacy of this modality for the treatment of gastric cancer. PMID:22827957

  9. Identification of immediate early gene X-1 as a cellular target gene of hcmv-mir-UL148D.

    PubMed

    Wang, Yue-Ping; Qi, Ying; Huang, Yu-Jing; Qi, Man-Long; Ma, Yan-Ping; He, Rong; Ji, Yao-Hua; Sun, Zheng-Rong; Ruan, Qiang

    2013-04-01

    Human cytomegalovirus (HCMV) is a herpesvirus that causes congenital diseases and opportunistic infections in immunocompromised individuals. Its functional proteins and microRNAs (miRNAs) facilitate efficient viral propagation by altering host cell behavior. The identification of functional target genes of miRNAs is an important step in the study of HCMV pathogenesis. HCMV encodes at least 14 miRNAs, including hcmv-mir-UL148D, which resides in the HCMV UL/b' region. hcmv-mir-UL148D is the only miRNA encoded by the HCMV UL/b' region; however, its targets and functional effects have not yet been eludidated. In this study, hybrid-PCR screening was used to identify target genes and dual luciferase reporter assay was used to evaluate the binding effect of hcmv-miR-UL148D to the 3' untranslated region (3'UTR) of IEX-1. In addition, western blot analysis was used to detect the expression kinetics of IEX-1 protein and apoptosis assay was used to identify the effects of hcmv-miR-UL148D on cell apoptosis. The hybrid-PCR results showed that only one binding site in the 3'UTR of the cellular gene, human immediate early gene X-1 (IEX-1), was completely complementary to an 11 nucleotide (nt) sequence in the 5' terminus of hcmv-mir-UL148D, including the entire seed region. The binding site was demonstrated to be functional by dual luciferase reporter assay with a 47% repression of the relative luciferase activity. In an in vitro system of human embryonic kidney 293 (HEK293) cells, the ectopically expressed hcmv-mir-UL148D exhibited a downregulatory effect on IEX-1 expression, and decreased the cell apoptosis induced by transfected IEX-1. Our data demonstrate that hcmv-mir-UL148D targets the cellular gene, IEX-1, downregulating its expression and thus results in anti-apoptotic effects. PMID:23403649

  10. GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

    PubMed

    Reshi, Latif; Wu, Horng-Cherng; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-04-01

    Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these results suggested that aquatic GSIV ST kinase could induce apoptosis via upregulation of p53 and Bax expression, resulting in mitochondrial disruption, which activated a downstream caspases-mediated cell death pathway. PMID:26833308

  11. Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes.

    PubMed

    Dusanic, Daliborka; Bencina, Dusan; Oven, Irena; Cizelj, Ivanka; Bencina, Mojca; Narat, Mojca

    2012-01-01

    The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans. PMID:22280251

  12. Modified poly(lactic-co-glycolic acid) nanoparticles for enhanced cellular uptake and gene editing in the lung.

    PubMed

    Fields, Rachel J; Quijano, Elias; McNeer, Nicole Ali; Caputo, Christina; Bahal, Raman; Anandalingam, Kavi; Egan, Marie E; Glazer, Peter M; Saltzman, W Mark

    2015-02-18

    Surface-modified poly(lactic-co-glycolic acid) (PLGA)/poly(?-aminoester)(PBAE)nanoparticles (NPs) have shown great promise in gene delivery. In this work, the pulmonary cellular uptake of these NPs is evaluated and surface-modified PLGA/PBAE NPs are shown to achieve higher cellular association and gene editing than traditional NPs composed of PLGA or PLGA/PBAE blends alone. PMID:25156908

  13. Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

    PubMed Central

    Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

    2015-01-01

    Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression. PMID:26583057

  14. Seasonal expressed sequence tags of rainbow smelt (Osmerus mordax) revealed by subtractive hybridization and the identification of two genes up-regulated during winter.

    PubMed

    Richards, Robert C; Achenbach, John C; Short, Connie E; Kimball, Jennifer; Reith, Michael E; Driedzic, William R; Ewart, K Vanya

    2008-11-15

    The rainbow smelt (Osmerus mordax) is freeze-resistant and maintains swimming and feeding activity during winter. In order to identify genes differentially expressed in smelt liver response to winter water temperatures, a large-scale analysis of gene expression using suppression subtractive hybridization was carried out using samples obtained in fall and winter. Forward and reverse subtractions were performed, subtraction-enriched products were cloned, and clones were sequenced from both of the resulting libraries. When 27 of these genes were screened by semi-quantitative RT-PCR to identify candidates for differential expression based generally on 2-fold changes in expression, one encoding FK506-binding protein 5 was classified as up-regulated in response to seasonal change, another encoding the mitochondrial solute carrier 25 member 25 (ATP-Mg/Pi carrier) was similarly classified with seasonal change and low temperature shift, and the one encoding the 78 kDa glucose-regulated protein was provisionally classified as down-regulated with low temperature shift. Analysis of fall (warm) and winter (cold) seasonal samples by quantitative PCR (qPCR) revealed significant up-regulation of genes encoding FK506-binding protein 51 and the mitochondrial solute carrier, whereas the gene encoding the glucose-regulated protein showed no significant change in expression. The mitochondrial solute carrier and FK506-binding protein results may relate to changes in cortisol action, as both are regulated by cortisol in other species. PMID:18761395

  15. A gene deletion that up-regulates viral gene expression yields an attenuated RSV vaccine with improved antibody responses in children.

    PubMed

    Karron, Ruth A; Luongo, Cindy; Thumar, Bhagvanji; Loehr, Karen M; Englund, Janet A; Collins, Peter L; Buchholz, Ursula J

    2015-11-01

    Respiratory syncytial virus (RSV) is the leading viral cause of severe pediatric respiratory illness, and a safe and effective vaccine for use in infancy and early childhood is needed. We previously showed that deletion of the coding sequence for the viral M2-2 protein (ΔM2-2) down-regulated viral RNA replication and up-regulated gene transcription and antigen synthesis, raising the possibility of development of an attenuated vaccine with enhanced immunogenicity. RSV MEDI ΔM2-2 was therefore evaluated as a live intranasal vaccine in adults, RSV-seropositive children, and RSV-seronegative children. When results in RSV-seronegative children were compared to those achieved with the previous leading live attenuated RSV candidate vaccine, vaccine virus shedding was significantly more restricted, yet the postvaccination RSV-neutralizing serum antibody achieved [geometric mean titer (GMT) = 1:97] was significantly greater. Surveillance during the subsequent RSV season showed that several seronegative RSV MEDI ΔM2-2 recipients had substantial antibody rises without reported illness, suggesting that the vaccine was protective yet primed for anamnestic responses to RSV. Rational design appears to have yielded a candidate RSV vaccine that is intrinsically superior at eliciting protective antibody in RSV-naïve children and highlights an approach for the development of live attenuated RSV vaccines. PMID:26537255

  16. Generally detected genes in comparative transcriptomics in bivalves: toward the identification of molecular markers of cellular stress response.

    PubMed

    Miao, Jingjing; Chi, Luping; Pan, Luqing; Song, Ying

    2015-01-01

    The specificity and representativeness of protein-coding genes identified by transcriptomics as biomarkers for environmental toxicological stress is crucial. We extracted the differential gene expression profile data from 49 published comparative transcriptomic studies of bivalves from January 2004 till November 2014 performed in 15 different bivalve species. Among the studies, 77 protein-coding genes were frequently detected when we use threefold of the average detection frequency as cut-off. Cellular organization and communication, protein and energy metabolism, stress response are the main functional classes of these proteins. We consider if these protein-coding genes represent common cellular stress responses of bivalves. PMID:25601151

  17. Modified pectin-based carrier for gene delivery: Cellular barriers in gene delivery course

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of biodegradable and biocompatible polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches rich in galactose residues which serve as potential liga...

  18. Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

    PubMed Central

    Mendoza-Rodriguez, Mnica; Arreola, Hugo; Valdivia, Alejandra; Peralta, Ral; Serna, Humberto; Villegas, Vanessa; Romero, Pablo; Alvarado-Hernndez, Beatriz; Paniagua, Lucero; Marrero-Rodrguez, Daniel; Meraz, Marco A; Salcedo, Mauricio

    2013-01-01

    Aims: Cervical Cancer (CC) is one of the most important health problems in women. It frequently presents genetic changes at chromosome region 3q21. This region contains the Cellular Retinol Binding Protein 1 gene (CRBP1) which has been implicated as an important element in the development of other types of cancer. The main goal of the present work was to determine the molecular alterations of CRBP1 and its relationship to CC. Methods: To determine the molecular alterations of CRBP1 gene in CC; twenty-six CC and twenty-six healthy cervix samples were evaluated for: 1) Copy number gain by real-time PCR analysis, 2) expression levels by an immunohistochemistry assay on tissue microarray, and 3) the methylation status of the CRBP1 promoter region. Results: The increase in CRBP1 copy number was observed in 10 out of the 26 CC samples analyzed, while healthy cervices samples showed no changes in the copy number. In addition, there was a lack of expression of the CRBP1 gene in an important number of the CC samples (17/26), and the CRBP1 gene promoter was methylated in 15/26 of the CC samples. Interestingly, there was a significant association between the lack of expression of the CRBP1 gene and its methylation status. Conclusions: The data indicates that, both activating and inactivating changes in the CRBP1 gene could be significant events in the development and progression of CC, and the lack of expression of the CRBP1 protein could be related with to the development of CC. We believe that there is enough evidence to consider to CRBP1 gene as a tumor suppressor gene for CC. PMID:24040446

  19. REST-Governed Gene Expression Profiling in a Neuronal Cell Model Reveals Novel Direct and Indirect Processes of Repression and Up-Regulation

    PubMed Central

    Garcia-Manteiga, Jose M.; Bonfiglio, Silvia; Folladori, Lucrezia; Malosio, Maria L.; Lazarevic, Dejan; Stupka, Elia; Cittaro, Davide; Meldolesi, Jacopo

    2015-01-01

    The role of REST changes in neurons, including the rapid decrease of its level during differentiation and its fluctuations during many mature functions and diseases, is well established. However, identification of many thousand possible REST-target genes, mostly based on indirect criteria, and demonstration of their operative dependence on the repressor have been established for only a relatively small fraction. In the present study, starting from our recently published work, we have expanded the identification of REST-dependent genes, investigated in two clones of the PC12 line, a recognized neuronal cell model, spontaneously expressing different levels of REST: very low as in neurons and much higher as in most non-neural cells. The molecular, structural and functional differences of the two PC12 clones were shown to depend largely on their different REST level and the ensuing variable expression of some dependent genes. Comprehensive RNA-Seq analyses of the 13,700 genes expressed, validated by parallel RT-PCR and western analyses of mRNAs and encoded proteins, identified in the high-REST clone two groups of almost 900 repressed and up-regulated genes. Repression is often due to direct binding of REST to target genes; up-regulation to indirect mechanism(s) mostly mediated by REST repression of repressive transcription factors. Most, but not all, genes governing neurosecretion, excitability, and receptor channel signaling were repressed in the high REST clone. The genes governing expression of non-channel receptors (G protein-coupled and others), although variably affected, were often up-regulated together with the genes of intracellular kinases, small G proteins, cytoskeleton, cell adhesion, and extracellular matrix proteins. Expression of REST-dependent genes governing functions other than those mentioned so far were also identified. The results obtained by the parallel investigation of the two PC12 clones revealed the complexity of the REST molecular and functional role, deciphering new aspects of its participation in neuronal functions. The new findings could be relevant for further investigation and interpretation of physiological processes typical of neurons. Moreover, they could be employed as tools in the study of neuronal diseases recently shown to depend on REST for their development. PMID:26617488

  20. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    NASA Astrophysics Data System (ADS)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-03-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly (P < 0.05) higher during the thermal stress. Pearson correlation coefficient analysis revealed that the expression of ATPase ?1, ATPase B2, and ATPase B3 is highly correlated (P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  1. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    NASA Astrophysics Data System (ADS)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P < 0.05) higher during the thermal stress. Pearson correlation coefficient analysis revealed that the expression of ATPase Β1, ATPase B2, and ATPase B3 is highly correlated ( P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  2. Lentiviral gene therapy using cellular promoters cures type 1 Gaucher disease in mice.

    PubMed

    Dahl, Maria; Doyle, Alexander; Olsson, Karin; Månsson, Jan-Eric; Marques, André R A; Mirzaian, Mina; Aerts, Johannes M; Ehinger, Mats; Rothe, Michael; Modlich, Ute; Schambach, Axel; Karlsson, Stefan

    2015-05-01

    Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease. PMID:25655314

  3. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls.

    PubMed

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T V; Alyethodi, Rafeeque R; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly (P?genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle. PMID:25875448

  4. Impact of Cytokine and Cytokine Receptor Gene Polymorphisms on Cellular Immunity after Smallpox Vaccination

    PubMed Central

    Ovsyannikova, Inna G.; Haralambieva, Iana H.; Kennedy, Richard B.; Pankratz, V. Shane; Vierkant, Robert A.; Jacobson, Robert M.; Poland, Gregory A.

    2012-01-01

    We explored associations between SNPs in cytokine/cytokine receptor genes and cellular immunity in subjects following primary smallpox vaccination. We also analyzed the genotype-phenotype associations discovered in the Caucasian subjects among a cohort of African-Americans. In Caucasians we found 277 associations (p<0.05) between gene SNPs and inter-individual variations in IFN-?, IL-12p40, IL-1?, IL-2, and TNF-? secretion levels. A collection of SNPs in the IL1RN, IL2RB, IL4R, IL6, IL10RB, IL12A, and IL12RB2 genes had consistent associations among both Caucasians and African-Americans. A regulatory SNP (rs452204) in the IL1RN gene was significantly associated with higher levels of IL-2 secretion in an allele dose-dependent manner in both race groups (p=0.05 for Caucasians and p=0.002 for African-Americans). IL12RB2 polymorphism rs3790567 was associated with a dose-related decrease in IL-1? secretion (p=0.009 for Caucasians and p=0.01 for African-Americans). Our results demonstrate that variations in smallpox vaccine-induced cytokine responses are modulated by genetic polymorphisms in cytokine and cytokine receptor genes. PMID:23009887

  5. The dormancy-breaking stimuli "chilling, hypoxia and cyanamide exposure" up-regulate the expression of ?-amylase genes in grapevine buds.

    PubMed

    Rubio, Sebastin; Donoso, Amanda; Prez, Francisco J

    2014-03-15

    It has been suggested that respiratory stress is involved in the mechanism underlying the dormancy-breaking effect of hydrogen cyanamide (H2CN2) and sodium azide in grapevine buds; indeed, reductions in oxygen levels (hypoxia) and inhibitors of respiration promote bud-break in grapevines. In this study, we showed that, hypoxia increased starch hydrolysis soluble sugar consumption and up-regulated the expression of ?-amylase genes (Vv?-AMYs) in grapevine buds, suggesting that these biochemical changes induced by hypoxia, may play a relevant role in the release of buds from endodormancy (ED). Three of the four Vv?-AMY genes that are expressed in grapevine buds were up-regulated by hypoxia and a correlation between changes in sugar content and level of Vv?-AMY gene expression during the hypoxia treatment was found, suggesting that soluble sugars mediate the effect of hypoxia on Vv?-AMY gene expression. Exogenous applications of soluble sugars and sugar analogs confirmed this finding and revealed that osmotic stress induces the expression of Vv?-AMY1 and Vv?-AMY3 and that soluble sugars induces Vv?-AMY2 and Vv?-AMY4 gene expression. Interestingly, the plant hormone gibberellic acid (GA3) induced the expression of Vv?-AMY3 and Vv?-AMY4 genes, while dormancy breaking stimuli, chilling and cyanamide exposure, mainly induced the expression of Vv?-AMY1 and Vv?-AMY2 genes, suggesting that these two ?-amylase genes might be involved in the release of grapevine buds from the ED. PMID:24594388

  6. Implication of p53-dependent cellular senescence related gene, TARSH in tumor suppression

    SciTech Connect

    Wakoh, Takeshi; Uekawa, Natsuko; Terauchi, Kunihiko; Sugimoto, Masataka; Ishigami, Akihito; Shimada, Jun-ichi; Maruyama, Mitsuo

    2009-03-20

    A novel target of NESH-SH3 (TARSH) was identified as a cellular senescence related gene in mouse embryonic fibroblasts (MEFs) replicative senescence, the expression of which has been suppressed in primary clinical lung cancer specimens. However, the molecular mechanism underlying the regulation of TARSH involved in pulmonary tumorigenesis remains unclear. Here we demonstrate that the reduction of TARSH gene expression by short hairpin RNA (shRNA) system robustly inhibited the MEFs proliferation with increase in senescence-associated {beta}-galactosidase (SA-{beta}-gal) activity. Using p53{sup -/-} MEFs, we further suggest that this growth arrest by loss of TARSH is evoked by p53-dependent p21{sup Cip1} accumulation. Moreover, we also reveal that TARSH reduction induces multicentrosome in MEFs, which is linked in chromosome instability and tumor development. These results suggest that TARSH plays an important role in proliferation of replicative senescence and may serve as a trigger of tumor development.

  7. Identification of genes up-regulated by retinoic-acid-induced differentiation of the human neuronal precursor cell line NTERA-2 cl.D1.

    PubMed

    Leypoldt, F; Lewerenz, J; Methner, A

    2001-02-01

    The human teratocarcinoma cell line NTERA-2 cl.D1 (NT2 cells) can be induced with retinoic acid and cell aggregation to yield postmitotic neurones. This seems to model the in vivo situation, as high concentrations of retinoic acid, retinoic acid binding proteins, and receptors have been detected in the embryonic CNS and the developing spinal cord suggesting a role for retinoic acid in neurogenesis. Suppression subtractive hybridization was used to detect genes up-regulated by this paradigm of neuronal differentiation. Microfibril-associated glycoprotein 2 was found to be drastically up-regulated and has not been implicated in neuronal differentiation before. Suppression subtractive hybridization also identified DYRK4, a homologue of the Drosophila gene minibrain. Minibrain mutations result in specific defects in the development of the fly central nervous system. In adult rats, DYRK4 is only expressed in testis, but our results suggest an additional role for DYRK4 in neuronal differentiation. We have shown that suppression subtractive hybridization in conjunction with an efficient screening procedure is a valuable tool to produce a repertoire of differentially expressed genes and propose a new physiological role for several identified genes and expressed sequence tags. PMID:11158252

  8. A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

    PubMed Central

    2014-01-01

    Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cus positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilsons disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

  9. Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures

    PubMed Central

    Zeng, Hongkui; Shen, Elaine H.; Hohmann, John G.; Oh, Wook Seung; Bernard, Amy; Royall, Joshua J.; Glattfelder, Katie J.; Sunkin, Susan M.; Morris, John A.; Guillozet-Bongaarts, Angela L.; Smith, Kimberly A.; Ebbert, Amanda J.; Swanson, Beryl; Kuan, Leonard; Page, Damon T.; Overly, Caroline C.; Lein, Ed S.; Hawrylycz, Michael J.; Hof, Patrick R.; Hyde, Thomas M.; Kleinman, Joel E.; Jones, Allan R.

    2012-01-01

    Summary Although there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of ~1,000 genes important for neural functions, by in situ hybridization with cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual genes expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain. PMID:22500809

  10. p53 Protein-mediated Up-regulation of MAP Kinase Phosphatase 3 (MKP-3) Contributes to the Establishment of the Cellular Senescent Phenotype through Dephosphorylation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2)*

    PubMed Central

    Zhang, Hui; Chi, Yuan; Gao, Kun; Zhang, Xiling; Yao, Jian

    2015-01-01

    Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced ?-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogen-activated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-?. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence. PMID:25414256

  11. Mitigating effects of L-selenomethionine on low-dose iron ion radiation-induced changes in gene expression associated with cellular stress.

    PubMed

    Nuth, Manunya; Kennedy, Ann R

    2013-07-01

    Ionizing radiation associated with highly energetic and charged heavy (HZE) particles poses a danger to astronauts during space travel. The aim of the present study was to evaluate the patterns of gene expression associated with cellular exposure to low-dose iron ion irradiation, in the presence and absence of L-selenomethionine (SeM). Human thyroid epithelial cells (HTori-3) were exposed to low-dose iron ion (1 GeV/n) irradiation at 10 or 20 cGy with or without SeM pretreatment. The cells were harvested 6 and 16 h post-irradiation and analyzed by the Affymetrix U133Av2 gene chip arrays. Genes exhibiting a 1.5-fold expression cut-off and 5% false discovery rate (FDR) were considered statistically significant and subsequently analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) for pathway analysis. Representative genes were further validated by real-time RT-PCR. Even at low doses of radiation from iron ions, global genome profiling of the irradiated cells revealed the upregulation of genes associated with the activation of stress-related signaling pathways (ubiquitin-mediated proteolysis, p53 signaling, cell cycle and apoptosis), which occurred in a dose-dependent manner. A 24-h pretreatment with SeM was shown to reduce the radiation effects by mitigating stress-related signaling pathways and downregulating certain genes associated with cell adhesion. The mechanism by which SeM prevents radiation-induced transformation in vitro may involve the suppression of the expression of genes associated with stress-related signaling and certain cell adhesion events. PMID:23946774

  12. Gene Expression of Glucose Transporter 1 (GLUT1), Hexokinase 1 and Hexokinase 2 in Gastroenteropancreatic Neuroendocrine Tumors: Correlation with F-18-fluorodeoxyglucose Positron Emission Tomography and Cellular Proliferation

    PubMed Central

    Binderup, Tina; Knigge, Ulrich Peter; Federspiel, Birgitte; Sommer, Peter; Hasselby, Jane Preuss; Loft, Annika; Kjaer, Andreas

    2013-01-01

    Neoplastic tissue exhibits high glucose utilization and over-expression of glucose transporters (GLUTs) and hexokinases (HKs), which can be imaged by 18F-Fluorodeoxyglucose-positron emission tomography (FDG-PET). The aim of the present study was to investigate the expression of glycolysis-associated genes and to compare this with FDG-PET imaging as well as with the cellular proliferation index in two cancer entities with different malignant potential. Using real-time PCR, gene expression of GLUT1, HK1 and HK2 were studied in 34 neuroendocrine tumors (NETs) in comparison with 14 colorectal adenocarcinomas (CRAs). The Ki67 proliferation index and, when available, FDG-PET imaging was compared with gene expression. Overexpression of GLUT1 gene expression was less frequent in NETs (38%) compared to CRAs (86%), P = 0.004. HK1 was overexpressed in 41% and 71% of NETs and CRAs, respectively (P = 0.111) and HK2 was overexpressed in 50% and 64% of NETs and CRAs, respectively (P = 0.53). There was a significant correlation between the Ki67 proliferation index and GLUT1 gene expression for the NETs (R = 0.34, P = 0.047), but no correlation with the hexokinases. FDG-PET identified foci in significantly fewer NETs (36%) than CRAs (86%), (P = 0.04). The gene expression results, with less frequent GLUT1 and HK1 upregulation in NETs, confirmed the lower metabolic activity of NETs compared to the more aggressive CRAs. In accordance with this, fewer NETs were FDG-PET positive compared to CRA tumors and FDG uptake correlated with GLUT1 gene expression.

  13. A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes

    PubMed Central

    Suzuki, Oscar T.; Frick, Amber; Parks, Bethany B.; Trask, O. Joseph; Butz, Natasha; Steffy, Brian; Chan, Emmanuel; Scoville, David K.; Healy, Eric; Benton, Cristina; McQuaid, Patricia E.; Thomas, Russell S.; Wiltshire, Tim

    2014-01-01

    New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways. PMID:25221565

  14. Identification, expression pattern, cellular location and potential role of the caveolin-1 gene from Artemia sinica.

    PubMed

    Li, Xuejie; Yao, Feng; Zhang, Wei; Cheng, Cheng; Chu, Bing; Liu, Yan; Mei, Yanli; Wu, Yang; Zou, Xiangyang; Hou, Lin

    2014-05-01

    Caveolins are integral membrane proteins that serve as scaffolds to recruit numerous signaling molecules. Caveolins play an important role in membrane trafficking, signal transduction, substrate transport and endocytosis in differentiated cells. In this study, a caveolin-1 gene from Artemia sinica (As-cav-1) was successfully cloned for the first time. The full-length cDNA of As-cav-1 comprises 974 bp, with a 675 bp open reading frame (ORF) that encodes a polypeptide of 224 amino acids with a caveolin scaffolding domain (CSD) and two transmembrane domains. Multiple sequence alignment revealed that the putative As-CAV-1 protein sequence was relatively conserved across species, especially in the CSD domain. Real-time PCR revealed high levels of the As-cav-1 transcript at 0h of embryo development. Furthermore, As-cav-1 transcripts were highly upregulated under high salinity (200) and low temperature stresses (15C). To further characterize As-cav-1, recombinant pET30a-cav-1 protein was expressed using a prokaryotic expression system. The recombinant protein comprised 290 amino acids with a theoretical molecular weight of 32kDa, and a predicted isoelectric point of 5.6. Western blotting of the expression levels of As-CAV-1 during different embryo development stages revealed that As-CAV-1 levels decreased gradually during development stages from 0 h to 40 h, and increased at 3d. Furthermore, western blotting showed that As-CAV-1 was upregulated to its highest expression level by low temperature stress (15C) and high salinity. Confocal laser microscopy analysis, using antibodies generated against the recombinant As-CAV-1 protein, showed that As-CAV-1 was mostly located in the cell membrane. Our results suggested that As-cav-1 plays a vital role in protecting embryos from high salt damage and low temperature stress, especially during post-diapause embryonic development. PMID:24583171

  15. Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos.

    PubMed

    Edman, R M; Linger, R J; Belikoff, E J; Li, F; Sze, S-H; Tarone, A M; Scott, M J

    2015-02-01

    The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making 'male-only' strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests. PMID:25225046

  16. Gene expression patterns in Euglena gracilis: insights into the cellular response to environmental stress.

    PubMed

    Dos Santos Ferreira, Vernica; Rocchetta, Iara; Conforti, Visitacin; Bench, Shellie; Feldman, Robert; Levin, Mariano J

    2007-03-15

    To better understand Euglena gracilis gene expression under different stress conditions (Chromium, Streptomycin or darkness), we undertook a survey of the E. gracilis transcriptome by cDNA sequencing and microarray analysis. First, we constructed a non-normalized cDNA library from the E. gracilis UTEX strain and sequenced a total of 1000 cDNAs. Six hundred and ten of these ESTs were similar to either Plantae or Protistae genes (e-valuegenes in E. gracilis. Finally, we identified 23 unknown ESTs (U-ESTs) following the expression profiles of these putative stress-related genes suggesting that they could be related to the cellular mechanism of stress response. PMID:17197134

  17. BRCA1 Haploinsufficiency Leads to Altered Expression of Genes Involved in Cellular Proliferation and Development

    PubMed Central

    Feilotter, Harriet E.; Michel, Claire; Uy, Paolo; Bathurst, Lauren; Davey, Scott

    2014-01-01

    The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes. PMID:24950059

  18. BRCA1 haploinsufficiency leads to altered expression of genes involved in cellular proliferation and development.

    PubMed

    Feilotter, Harriet E; Michel, Claire; Uy, Paolo; Bathurst, Lauren; Davey, Scott

    2014-01-01

    The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes. PMID:24950059

  19. Cellular defense system gene expression profiling of human whole blood: opportunities to predict health benefits in response to diet.

    PubMed

    Drew, Janice E

    2012-07-01

    Diet is a critical factor in the maintenance of human cellular defense systems, immunity, inflammation, redox regulation, metabolism, and DNA repair that ensure optimal health and reduce disease risk. Assessment of dietary modulation of cellular defense systems in humans has been limited due to difficulties in accessing target tissues. Notably, peripheral blood gene expression profiles associated with nonhematologic disease are detectable. Coupled with recent innovations in gene expression technologies, gene expression profiling of human blood to determine predictive markers associated with health status and dietary modulation is now a feasible prospect for nutrition scientists. This review focuses on cellular defense system gene expression profiling of human whole blood and the opportunities this presents, using recent technological advances, to predict health status and benefits conferred by diet. PMID:22797985

  20. Cellular Defense System Gene Expression Profiling of Human Whole Blood: Opportunities to Predict Health Benefits in Response to Diet12

    PubMed Central

    Drew, Janice E.

    2012-01-01

    Diet is a critical factor in the maintenance of human cellular defense systems, immunity, inflammation, redox regulation, metabolism, and DNA repair that ensure optimal health and reduce disease risk. Assessment of dietary modulation of cellular defense systems in humans has been limited due to difficulties in accessing target tissues. Notably, peripheral blood gene expression profiles associated with nonhematologic disease are detectable. Coupled with recent innovations in gene expression technologies, gene expression profiling of human blood to determine predictive markers associated with health status and dietary modulation is now a feasible prospect for nutrition scientists. This review focuses on cellular defense system gene expression profiling of human whole blood and the opportunities this presents, using recent technological advances, to predict health status and benefits conferred by diet. PMID:22797985

  1. IL-17 downregulates filaggrin and affects keratinocyte expression of genes associated with cellular adhesion.

    PubMed

    Gutowska-Owsiak, Danuta; Schaupp, Anna L; Salimi, Maryam; Selvakumar, Tharini A; McPherson, Tess; Taylor, Stephen; Ogg, Graham S

    2012-02-01

    Atopic eczema and psoriasis are common skin diseases. While it is well established that the pathogenesis of these diseases varies, both are characterized by impairment in epidermal barrier function and abnormal IL-17 expression in the skin and peripheral blood. Recent findings indicated that filaggrin is essential during barrier formation and its insufficiency underlies the pathogenesis of atopic eczema. Filaggrin downregulation has also been reported in psoriasis. It is clear that Th1/Th2 bias influences expression of the protein, but an analysis of the effects of interleukin-17 (IL-17) on the expression of the protein and profilaggrin-processing enzymes has not yet been reported. In addition, the effect of the cytokine on components of functional epidermal barrier, tight junctions and adhesion/desmosomal proteins, has not been elucidated. Keratinocytes were exposed to interleukin-17A, and microarray analysis was performed. Filaggrin protein level was assessed by western blot. We have observed a significant decrease in profilaggrin mRNA level in interleukin-17A-exposed cultures (P = 0.008). Expression of processing enzymes was also altered, indicating an indirect effect of the cytokine on filaggrin production/degradation. Moreover, expression of many genes involved in cellular adhesion was also decreased. A significant downregulation of filaggrin at the protein level was detected by western blot in immortal and primary keratinocytes. Gene ontology analysis indicated changes in keratinization, epidermal differentiation and formation of the cornified envelope. We conclude that IL-17A downregulates the expression of filaggrin and genes important for cellular adhesion which could affect epidermal barrier formation. This effect potentially contributes to barrier dysfunction and could become a possible therapeutic target. PMID:22229441

  2. Resveratrol Reverses Cadmium Chloride-induced Testicular Damage and Subfertility by Downregulating p53 and Bax and Upregulating Gonadotropins and Bcl-2 gene Expression

    PubMed Central

    ELEAWA, Samy M; ALKHATEEB, Mahmoud A; ALHASHEM, Fahaid H; BIN-JALIAH, Ismaeel; SAKR, Hussein F; ELREFAEY, Hesham M; ELKARIB, Abbas O; ALESSA, Riyad M; HAIDARA, Mohammad A; SHATOOR, Abdullah S.; KHALIL, Mohammad A

    2014-01-01

    This study was performed to investigate the protective and therapeutic effects of resveratrol (RES) against CdCl2-induced toxicity in rat testes. Seven experimental groups of adult male rats were formulated as follows: A) controls+NS, B) control+vehicle (saline solution of hydroxypropyl cyclodextrin), C) RES treated, D) CdCl2+NS, E) CdCl2+vehicle, F) RES followed by CdCl2 and M) CdCl2 followed by RES. At the end of the protocol, serum levels of FSH, LH and testosterone were measured in all groups, and testicular levels of TBARS and superoxide dismutase (SOD) activity were measured. Epididymal semen analysis was performed, and testicular expression of Bcl-2, p53 and Bax was assessed by RT-PCR. Also, histopathological changes of the testes were examined microscopically. Administration of RES before or after cadmium chloride in rats improved semen parameters including count, motility, daily sperm production and morphology, increased serum concentrations of gonadotropins and testosterone, decreased testicular lipid peroxidation and increased SOD activity. RES not only attenuated cadmium chloride-induced testicular histopathology but was also able to protect against the onset of cadmium chloride testicular toxicity. Cadmium chloride downregulated the anti-apoptotic gene Bcl2 and upregulated the expression of pro-apoptotic genes p53 and Bax. Resveratrol protected against and partially reversed cadmium chloride testicular toxicity via upregulation of Bcl2 and downregulation of p53 and Bax gene expression. The antioxidant activity of RES protects against cadmium chloride testicular toxicity and partially reverses its effect via upregulation of BCl2 and downregulation of p53 and Bax expression. PMID:24492640

  3. Growth differentiation factor-15: a p53- and demethylation-upregulating gene represses cell proliferation, invasion, and tumorigenesis in bladder carcinoma cells

    PubMed Central

    Tsui, Ke-Hung; Hsu, Shu-Yuan; Chung, Li-Chuan; Lin, Yu-Hsiang; Feng, Tsui-Hsia; Lee, Tzu-Yi; Chang, Phei-Lang; Juang, Horng-Heng

    2015-01-01

    Growth differentiation factor-15 (GDF15), a member of the TGF-β superfamily, affects tumor biology of certain cancers, but remains poorly understood in bladder cancer cells. This study determined the expression, regulation, function, and potential downstream target genes of GDF15 in bladder carcinoma cells. The transitional papilloma carcionoma cells (RT4) expressed higher levels of GDF15 as compared with the bladder carcinoma cells (HT1376 and T24). Treatments of recombinant human GDF15 (rhGDF15) reduced the proliferations of HT1376 and T24 cells. Expression of GDF15 was upregulated via DNA demethylation and p53. The cell proliferation, invasion, and tumorigenesis were reduced in ectopic overexpression of GDF15, while enhanced in GDF15 knockdown. The expressions of mammary serine protease inhibitor (MASPIN) and N-myc downstream-regulated family genes (NDRG1, NDRG2, and NDRG3) were upregulated by GDF15 overexpressions and rhGDF15 treatments in bladder carcinoma cells. GDF15 knockdown induced epithelial-mesenchymal transition (EMT) and F-actin polarization in HT1376 cells. Our results suggest that enhanced expressions of MASPIN and N-myc downstream-regulated family genes and the modulation of EMT may account for the inhibitory functions of GDF15 in the cell proliferation, invasion, and tumorigenesis of bladder carcinoma cells. The GDF15 should be considered as a tumor suppressor in human bladder carcinoma cells. PMID:26249737

  4. Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells

    PubMed Central

    Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

    2015-01-01

    Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer. PMID:25692008

  5. Dissociation of Oct-1 from the Nuclear Peripheral Structure Induces the Cellular Aging-associated Collagenase Gene Expression

    PubMed Central

    Imai, Shin-ichiro; Nishibayashi, Seiji; Takao, Koji; Tomifuji, Masayuki; Fujino, Tadahiro; Hasegawa, Mayumi; Takano, Toshiya

    1997-01-01

    The cellular aging-associated transcriptional repressor that we previously named as Orpheus was identical to Oct-1, a member of the POU domain family. Oct-1 represses the collagenase gene, one of the cellular aging-associated genes, by interacting with an AT-rich cis-element in the upstream of the gene in preimmortalized cells at earlier population-doubling levels and in immortalized cells. In these stages of cells, considerable fractions of the Oct-1 protein were prominently localized in the nuclear periphery and colocalized with lamin B. During the cellular aging process, however, this subspecies of Oct-1 disappeared from the nuclear periphery. The cells lacking the nuclear peripheral Oct-1 protein exhibited strong collagenase expression and carried typical senescent morphologies. Concomitantly, the binding activity and the amount of nuclear Oct-1 protein were reduced in the aging process and resumed after immortalization. However, the whole cellular amounts of Oct-1 protein were not significantly changed during either process. Thus, the cellular aging-associated genes including the collagenase gene seemed to be derepressed by the dissociation of Oct-1 protein from the nuclear peripheral structure. Oct-1 may form a transcriptional repressive apparatus by anchoring nuclear matrix attachment regions onto the nuclear lamina in the nuclear periphery. PMID:9398664

  6. Coordinate up-regulation of low-density lipoprotein receptor and cyclo-oxygenase-2 gene expression in human colorectal cells and in colorectal adenocarcinoma biopsies

    NASA Technical Reports Server (NTRS)

    Lum, D. F.; McQuaid, K. R.; Gilbertson, V. L.; Hughes-Fulford, M.

    1999-01-01

    Many colorectal cancers have high levels of cyclo-oxygenase 2 (COX-2), an enzyme that metabolizes the essential fatty acids into prostaglandins. Since the low-density lipoprotein receptor (LDLr) is involved in the uptake of essential fatty acids, we studied the effect of LDL on growth and gene regulation in colorectal cancer cells. DiFi cells grown in lipoprotein-deficient sera (LPDS) grew more slowly than cells with LDL. LDLr antibody caused significant inhibition of tumor cell growth but did not affect controls. In addition, LDL uptake did not change in the presence of excess LDL, suggesting that ldlr mRNA lacks normal feedback regulation in some colorectal cancers. Analysis of the ldlr mRNA showed that excess LDL in the medium did not cause down-regulation of the message even after 24 hr. The second portion of the study examined the mRNA expression of ldlr and its co-regulation with cox-2 in normal and tumor specimens from patients with colorectal adenocarcinomas. The ratio of tumor:paired normal mucosa of mRNA expression of ldlr and of cox-2 was measured in specimens taken during colonoscopy. ldlr and cox-2 transcripts were apparent in 11 of 11 carcinomas. There was significant coordinate up-regulation both of ldlr and of cox-2 in 6 of 11 (55%) tumors compared with normal colonic mucosa. There was no up-regulation of cox-2 without concomitant up-regulation of ldlr. These data suggest that the LDLr is abnormally regulated in some colorectal tumors and may play a role in the up-regulation of cox-2. Copyright 1999 Wiley-Liss, Inc.

  7. Identification of genes upregulated by the transcription factor Bcr1 that are involved in impermeability, impenetrability, and drug resistance of Candida albicans a/α biofilms.

    PubMed

    Srikantha, Thyagarajan; Daniels, Karla J; Pujol, Claude; Kim, Elena; Soll, David R

    2013-06-01

    Candida albicans forms two types of biofilm, depending upon the configuration of the mating type locus. Although architecturally similar, a/α biofilms are impermeable, impenetrable, and drug resistant, whereas a/a and α/α biofilms lack these traits. The difference appears to be the result of an alternative matrix. Overexpression in a/a cells of BCR1, a master regulator of the a/α matrix, conferred impermeability, impenetrability, and drug resistance to a/a biofilms. Deletion of BCR1 in a/α cells resulted in the loss of these a/α-specific biofilm traits. Using BCR1 overexpression in a/a cells, we screened 107 genes of interest and identified 8 that were upregulated by Bcr1. When each was overexpressed in a/a biofilms, the three a/α traits were partially conferred, and when each was deleted in a/α cells, the traits were partially lost. Five of the eight genes have been implicated in iron homeostasis, and six encode proteins that are either in the wall or plasma membrane or secreted. All six possess sites for O-linked and N-linked glycosylation that, like glycosylphosphatidylinositol (GPI) anchors, can cross-link to the wall and matrix, suggesting that they may exert a structural role in conferring impermeability, impenetrability, and drug resistance, in addition to their physiological functions. The fact that in a screen of 107 genes, all 8 of the Bcr1-upregulated genes identified play a role in impermeability, impenetrability, and drug resistance suggests that the formation of the a/α matrix is highly complex and involves a larger number of genes than the initial ones identified here. PMID:23563485

  8. Transcriptional up-regulation of antioxidant genes by PPAR{delta} inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells

    SciTech Connect

    Kim, Hyo Jung; Ham, Sun Ah; Paek, Kyung Shin; Hwang, Jung Seok; Jung, Si Young; Kim, Min Young; Jin, Hanna; Kang, Eun Sil; Woo, Im Sun; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Han, Chang Woo; Seo, Han Geuk

    2011-03-25

    Research highlights: {yields} Activation of PPAR{delta} by GW501516 significantly inhibited Ang II-induced premature senescence in hVSMCs. {yields} Agonist-activated PPAR{delta} suppressed generation of Ang II-triggered ROS with a concomitant reduction in DNA damage. {yields} GW501516 up-regulated expression of antioxidant genes, such as GPx1, Trx1, Mn-SOD and HO-1. {yields} Knock-down of these antioxidant genes abolished the effects of GW501516 on ROS production and premature senescence. -- Abstract: This study evaluated peroxisome proliferator-activated receptor (PPAR) {delta} as a potential target for therapeutic intervention in Ang II-induced senescence in human vascular smooth muscle cells (hVSMCs). Activation of PPAR{delta} by GW501516, a specific agonist of PPAR{delta}, significantly inhibited the Ang II-induced premature senescence of hVSMCs. Agonist-activated PPAR{delta} suppressed the generation of Ang II-triggered reactive oxygen species (ROS) with a concomitant reduction in DNA damage. Notably, GW501516 up-regulated the expression of antioxidant genes, such as glutathione peroxidase 1, thioredoxin 1, manganese superoxide dismutase and heme oxygenase 1. siRNA-mediated down-regulation of these antioxidant genes almost completely abolished the effects of GW501516 on ROS production and premature senescence in hVSMCs treated with Ang II. Taken together, the enhanced transcription of antioxidant genes is responsible for the PPAR{delta}-mediated inhibition of premature senescence through sequestration of ROS in hVSMCs treated with Ang II.

  9. Expression of Senescence-Associated microRNAs and Target Genes in Cellular Aging and Modulation by Tocotrienol-Rich Fraction

    PubMed Central

    2014-01-01

    Emerging evidences highlight the implication of microRNAs as a posttranscriptional regulator in aging. Several senescence-associated microRNAs (SA-miRNAs) are found to be differentially expressed during cellular senescence. However, the role of dietary compounds on SA-miRNAs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on SA-miRNAs (miR-20a, miR-24, miR-34a, miR-106a, and miR-449a) and established target genes of miR-34a (CCND1, CDK4, and SIRT1) during replicative senescence of human diploid fibroblasts (HDFs). Primary cultures of HDFs at young and senescent were incubated with TRF at 0.5?mg/mL. Taqman microRNA assay showed significant upregulation of miR-24 and miR-34a and downregulation of miR-20a and miR-449a in senescent HDFs (P < 0.05). TRF reduced miR-34a expression in senescent HDFs and increased miR-20a expression in young HDFs and increased miR-449a expression in both young and senescent HDFs. Our results also demonstrated that ectopic expression of miR-34a reduced the expression of CDK4 significantly (P < 0.05). TRF inhibited miR-34a expression thus relieved its inhibition on CDK4 gene expression. No significant change was observed on the expression of CCND1, SIRT1, and miR-34a upstream transcriptional regulator, TP53. In conclusion tocotrienol-rich fraction prevented cellular senescence of human diploid fibroblasts via modulation of SA-miRNAs and target genes expression. PMID:25132913

  10. Cellular adhesion gene SELP is associated with rheumatoid arthritis and displays differential allelic expression.

    PubMed

    Burkhardt, Jana; Blume, Mechthild; Petit-Teixeira, Elisabeth; Hugo Teixeira, Vitor; Steiner, Anke; Quente, Elfi; Wolfram, Grit; Scholz, Markus; Pierlot, Cline; Migliorini, Paola; Bombardieri, Stefano; Balsa, Alejandro; Westhovens, Ren; Barrera, Pilar; Radstake, Timothy R D J; Alves, Helena; Bardin, Thomas; Prum, Bernard; Emmrich, Frank; Cornelis, Franois; Ahnert, Peter; Kirsten, Holger

    2014-01-01

    In rheumatoid arthritis (RA), a key event is infiltration of inflammatory immune cells into the synovial lining, possibly aggravated by dysregulation of cellular adhesion molecules. Therefore, single nucleotide polymorphisms of 14 genes involved in cellular adhesion processes (CAST, ITGA4, ITGB1, ITGB2, PECAM1, PTEN, PTPN11, PTPRC, PXN, SELE, SELP, SRC, TYK2, and VCAM1) were analyzed for association with RA. Association analysis was performed consecutively in three European RA family sample groups (Nfamilies?=?407). Additionally, we investigated differential allelic expression, a possible functional consequence of genetic variants. SELP (selectin P, CD62P) SNP-allele rs6136-T was associated with risk for RA in two RA family sample groups as well as in global analysis of all three groups (ptotal?=?0.003). This allele was also expressed preferentially (p<10-6) with a two- fold average increase in regulated samples. Differential expression is supported by data from Genevar MuTHER (p1?=?0.004; p2?=?0.0177). Evidence for influence of rs6136 on transcription factor binding was also found in silico and in public datasets reporting in vitro data. In summary, we found SELP rs6136-T to be associated with RA and with increased expression of SELP mRNA. SELP is located on the surface of endothelial cells and crucial for recruitment, adhesion, and migration of inflammatory cells into the joint. Genetically determined increased SELP expression levels might thus be a novel additional risk factor for RA. PMID:25147926

  11. Cellular Adhesion Gene SELP Is Associated with Rheumatoid Arthritis and Displays Differential Allelic Expression

    PubMed Central

    Petit-Teixeira, Elisabeth; Hugo Teixeira, Vitor; Steiner, Anke; Quente, Elfi; Wolfram, Grit; Scholz, Markus; Pierlot, Cline; Migliorini, Paola; Bombardieri, Stefano; Balsa, Alejandro; Westhovens, Ren; Barrera, Pilar; Radstake, Timothy R. D. J.; Alves, Helena; Bardin, Thomas; Prum, Bernard; Emmrich, Frank; Cornelis, Franois

    2014-01-01

    In rheumatoid arthritis (RA), a key event is infiltration of inflammatory immune cells into the synovial lining, possibly aggravated by dysregulation of cellular adhesion molecules. Therefore, single nucleotide polymorphisms of 14 genes involved in cellular adhesion processes (CAST, ITGA4, ITGB1, ITGB2, PECAM1, PTEN, PTPN11, PTPRC, PXN, SELE, SELP, SRC, TYK2, and VCAM1) were analyzed for association with RA. Association analysis was performed consecutively in three European RA family sample groups (Nfamilies?=?407). Additionally, we investigated differential allelic expression, a possible functional consequence of genetic variants. SELP (selectin P, CD62P) SNP-allele rs6136-T was associated with risk for RA in two RA family sample groups as well as in global analysis of all three groups (ptotal?=?0.003). This allele was also expressed preferentially (p<10?6) with a two- fold average increase in regulated samples. Differential expression is supported by data from Genevar MuTHER (p1?=?0.004; p2?=?0.0177). Evidence for influence of rs6136 on transcription factor binding was also found in silico and in public datasets reporting in vitro data. In summary, we found SELP rs6136-T to be associated with RA and with increased expression of SELP mRNA. SELP is located on the surface of endothelial cells and crucial for recruitment, adhesion, and migration of inflammatory cells into the joint. Genetically determined increased SELP expression levels might thus be a novel additional risk factor for RA. PMID:25147926

  12. Tumor cells as cellular vehicles to deliver gene therapies to metastatic tumors.

    PubMed

    García-Castro, Javier; Martínez-Palacio, Jesús; Lillo, Rosa; García-Sánchez, Félix; Alemany, Ramón; Madero, Luis; Bueren, Juan A; Ramírez, Manuel

    2005-04-01

    A long-pursued goal in cancer treatment is to deliver a therapy specifically to metastases. As a result of the disseminated nature of the metastatic disease, carrying the therapeutic agent to the sites of tumor growth represents a major step for success. We hypothesized that tumor cells injected intravenously (i.v.) into an animal with metastases would respond to many of the factors driving the metastatic process, and would target metastases. Using a model of spontaneous metastases, we report here that i.v. injected tumor cells localized on metastatic lesions. Based on this fact, we used genetically transduced tumor cells for tumor targeting of anticancer agents such as a suicide gene or an oncolytic virus, with evident antitumoral effect and negligible systemic toxicity. Therefore, autologous tumor cells may be used as cellular vehicles for systemic delivery of anticancer therapies to metastatic tumors. PMID:15650763

  13. Perturbations at the ribosomal genes loci are at the centre of cellular dysfunction and human disease

    PubMed Central

    2014-01-01

    Ribosomal RNA (rRNA) gene (rDNA) transcription by RNA Polymerase I (Pol I) drives cell growth and underlies nucleolar structure and function, indirectly coordinating many fundamental cellular processes. The importance of keeping rDNA transcription under tight control is reflected by the fact that deranged Pol I transcription is a feature of cancer and other human disorders. In this review, we discuss multiple aspects of rDNA function including the relationship between Pol I transcription and proliferative capacity, the role of Pol I transcription in mediating nucleolar structure and integrity, and rDNA/nucleolar interactions with the genome and their influence on heterochromatin and global genome stability. Furthermore, we discuss how perturbations in the structure of the rDNA loci might contribute to human disease, in some cases independent of effects on ribosome biogenesis. PMID:25949792

  14. Genes Related to Ion-Transport and Energy Production Are Upregulated in Response to CO2-Driven pH Decrease in Corals: New Insights from Transcriptome Analysis

    PubMed Central

    Vidal-Dupiol, Jeremie; Zoccola, Didier; Tambutté, Eric; Grunau, Christoph; Cosseau, Céline; Smith, Kristina M.; Freitag, Michael; Dheilly, Nolwenn M.; Allemand, Denis; Tambutté, Sylvie

    2013-01-01

    Since the preindustrial era, the average surface ocean pH has declined by 0.1 pH units and is predicted to decline by an additional 0.3 units by the year 2100. Although subtle, this decreasing pH has profound effects on the seawater saturation state of carbonate minerals and is thus predicted to impact on calcifying organisms. Among these are the scleractinian corals, which are the main builders of tropical coral reefs. Several recent studies have evaluated the physiological impact of low pH, particularly in relation to coral growth and calcification. However, very few studies have focused on the impact of low pH at the global molecular level. In this context we investigated global transcriptomic modifications in a scleractinian coral (Pocillopora damicornis) exposed to pH 7.4 compared to pH 8.1during a 3-week period. The RNAseq approach shows that 16% of our transcriptome was affected by the treatment with 6% of upregulations and 10% of downregulations. A more detailed analysis suggests that the downregulations are less coordinated than the upregulations and allowed the identification of several biological functions of interest. In order to better understand the links between these functions and the pH, transcript abundance of 48 candidate genes was quantified by q-RT-PCR (corals exposed at pH 7.2 and 7.8 for 3 weeks). The combined results of these two approaches suggest that pH≥7.4 induces an upregulation of genes coding for proteins involved in calcium and carbonate transport, conversion of CO2 into HCO3− and organic matrix that may sustain calcification. Concomitantly, genes coding for heterotrophic and autotrophic related proteins are upregulated. This can reflect that low pH may increase the coral energy requirements, leading to an increase of energetic metabolism with the mobilization of energy reserves. In addition, the uncoordinated downregulations measured can reflect a general trade-off mechanism that may enable energy reallocation. PMID:23544045

  15. Epstein–Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression

    PubMed Central

    Ramasubramanyan, Sharada; Osborn, Kay; Al-Mohammad, Rajaei; Naranjo Perez-Fernandez, Ijiel B.; Zuo, Jianmin; Balan, Nicolae; Godfrey, Anja; Patel, Harshil; Peters, Gordon; Rowe, Martin; Jenner, Richard G.; Sinclair, Alison J.

    2015-01-01

    Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements. PMID:25779048

  16. Activation of TrkA by nerve growth factor upregulates expression of the cholinergic gene locus but attenuates the response to ciliary neurotrophic growth factor.

    PubMed Central

    Berse, B; Lopez-Coviella, I; Blusztajn, J K

    1999-01-01

    Nerve growth factor (NGF) stimulates the expression of the cholinergic gene locus, which encodes choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), the proteins necessary for the synthesis and storage of the neurotransmitter acetylcholine (ACh). To determine whether this action of NGF is mediated by the p140TrkA NGF receptor (a member of the Trk tyrosine kinase family) we used a murine basal forebrain cholinergic cell line, SN56, stably transfected with rat trkA cDNA. Treatment of these transfectants with NGF activated mitogen-activated protein kinase and increased cytosolic free calcium concentrations, confirming the reconstitution of TrkA-mediated signalling pathways. The expression of ChAT and VAChT mRNA, as well as ACh content, were coordinately up-regulated by NGF in SN56-trkA transfectants. None of these responses occurred in the parental SN56 cells, which do not express endogenous TrkA, indicating that these actions of NGF required TrkA. We previously reported that ciliary neurotrophic factor (CNTF) upregulates the expression of ChAT and VAChT, as well as ACh production, in SN56 cells. The combined treatment of SN56-trkA cells with CNTF and NGF revealed a complex interaction of these factors in the regulation of cholinergic gene locus expression. At low concentrations of CNTF (<1 ng/ml), the upregulation of ACh synthesis evoked by these factors was additive. However, at higher concentrations of CNTF (>1 ng/ml), NGF attenuated the stimulatory effect of CNTF on ChAT and VAChT mRNA and ACh content. This attenuation was not due to interference with early steps of CNTF receptor signalling, as pre-treatment of SN56-trkA cells with NGF did not affect the nuclear translocation of the transcription factor, Stat3, evoked by CNTF. PMID:10455015

  17. Upregulation of Mouse Genes in HSV-1 Latent TG after Butyrate Treatment Implicates the Multiple Roles of the LAT-ICP0 Locus

    PubMed Central

    Clement, Christian; Bhattacharjee, Partha S.; Kumar, Manish; Foster, Timothy P.; Thompson, Hilary W.

    2011-01-01

    Purpose. To determine host response by gene expression in HSV-1 latent trigeminal ganglia (TG) after sodium butyrate (NaBu) treatment. Methods. Corneas of 6-week-old female BALB/c mice were scarified and inoculated with HSV-1 17Syn+ (high phenotypic reactivator) or its mutant 17ΔPst(LAT−) (low phenotypic reactivator) at 104 plaque-forming units/eye. NaBu-induced viral reactivation was by intraperitoneal (IP) administration at postinfection (PI) day 28, followed by euthanasia after 1 hour. NaBu-treated, uninfected mice served as the control. The resultant labeled cRNA from TG isolated total RNA was hybridized to gene microarray chips containing 14,000 mouse genes. Quantitative real-time PCR was performed to confirm gene expression. Results. Differential induction of gene expression between 17Syn+ and its mutant 17ΔPst(LAT−) was designated as NaBu-induced gene expression and yielded significant upregulation of 2- to 16-fold of 0.4% (56/14,000) host genes probed, comprising mainly nucleosome assembly and binding, central nervous system structural activity, hormonal activity, and signaling activity. Approximately 0.2% (24/14,000) of the host genes, mainly of the same functional categories were downregulated 3- to 11-fold. Immune activity was minor in comparison to our reports on gene expression during latency and heat stress induction. Euchromatin analysis revealed that the LAT-ICP0 locus is amenable to the effects of NaBu. Histone activity was detected by early transcription of histone cluster 2 H2be (Hist2h2be). Conclusions. NaBu-induced reactivation of HSV-1 is twofold: drug action involving significant moderation of specific host epigenetic changes and failure to elicit or suppress immune activity at the early time point of 1 hour. PMID:20881297

  18. The effects of local bFGF release and uniaxial strain on cellular adaptation and gene expression in a 3D environment: implications for ligament tissue engineering.

    PubMed

    Petrigliano, Frank A; English, Christopher S; Barba, David; Esmende, Sean; Wu, Benjamin M; McAllister, David R

    2007-11-01

    The objectives of this investigation were (1) to characterize the growth factor release profile of a basic fibroblast growth factor (bFGF)-coated three-dimensional (3D) polymer scaffold under static and cyclically strained conditions, and (2) to delineate the individual and collective contributions of locally released bFGF and mechanical strain on cellular morphology and gene expression in this 3D system. Scaffolds were treated with I(125)-bFGF and subjected to mechanical strain or maintained in a static environment and the media sampled for factor release over a period of 6 days. Over the first 10 hours, a burst release of 25% of the incorporated growth factor into the surrounding media was noted. At 24 hours, approximately 40% of the bFGF was released into the media, after which steady state was achieved and minimal subsequent release was noted. Mechanical stimulation had no effect on growth factor release from the scaffold in this system. To test the concerted effects of bFGF and mechanical stimulation on bone marrow stromal cells (BMSCs), scaffolds were loaded with 0, 100, or 500 ng of bFGF, seeded with cells, and subjected to mechanical strain or maintained in a static environment. Scaffolds were harvested at 1, 7, and 21 days for RT-PCR and histomorphometry. All scaffolds subjected to growth factor and/or mechanical stimulation demonstrated cellular adherence and spreading at 21 days. Conversely, in the absence of both bFGF and mechanical stimulation, cells demonstrated minimal cytoplasmic spread. Moreover, at 21 days, cells subjected to both mechanical stimulation and bFGF (500 ng) demonstrated the highest upregulation of stress-resistive (collagen I, III) and stress-responsive proteins (tenascin-C). The effect of growth factor may be dose sensitive, however, as unstrained scaffolds treated with 100 ng of bFGF demonstrated upregulation of gene expression comparable to strained scaffolds treated with lower doses of bFGF (0 or 100 ng). In conclusion, results from this study suggest that the stimulatory effects of bFGF are dose sensitive and appear to be influenced by the addition of mechanical strain. The concurrent application of biochemical and mechanical stimuli may be important in promoting the adaptation of BMSCs and driving the transcription of genes essential for synthesis of a functional ligament replacement tissue. PMID:17727336

  19. Striated muscle activator of Rho signalling (STARS) is a PGC-1?/oestrogen-related receptor-? target gene and is upregulated in human skeletal muscle after endurance exercise.

    PubMed

    Wallace, Marita A; Hock, M Benjamin; Hazen, Bethany C; Kralli, Anastasia; Snow, Rod J; Russell, Aaron P

    2011-04-15

    The striated muscle activator of Rho signalling (STARS) is an actin-binding protein specifically expressed in cardiac, skeletal and smooth muscle. STARS has been suggested to provide an important link between the transduction of external stress signals to intracellular signalling pathways controlling genes involved in the maintenance of muscle function. The aims of this study were firstly, to establish if STARS, as well as members of its downstream signalling pathway, are upregulated following acute endurance cycling exercise; and secondly, to determine if STARS is a transcriptional target of peroxisome proliferator-activated receptor gamma co-activator 1-? (PGC-1?) and oestrogen-related receptor-? (ERR?). When measured 3 h post-exercise, STARS mRNA and protein levels as well as MRTF-A and serum response factor (SRF) nuclear protein content, were significantly increased by 140, 40, 40 and 40%, respectively. Known SRF target genes, carnitine palmitoyltransferase-1? (CPT-1?) and jun B proto-oncogene (JUNB), as well as the exercise-responsive genes PGC-1? mRNA and ERR? were increased by 2.3-, 1.8-, 4.5- and 2.7-fold, 3 h post-exercise. Infection of C2C12 myotubes with an adenovirus-expressing human PGC-1? resulted in a 3-fold increase in Stars mRNA, a response that was abolished following the suppression of endogenous ERR?. Over-expression of PGC-1? also increased Cpt-1?, Cox4 and Vegf mRNA by 6.2-, 2.0- and 2.0-fold, respectively. Suppression of endogenous STARS reduced basal Cpt-1? levels by 8.2-fold and inhibited the PGC-1?-induced increase in Cpt-1? mRNA. Our results show for the first time that the STARS signalling pathway is upregulated in response to acute endurance exercise. Additionally, we show in C2C12 myotubes that the STARS gene is a PGC-1?/ERR? transcriptional target. Furthermore, our results suggest a novel role of STARS in the co-ordination of PGC-1?-induced upregulation of the fat oxidative gene, CPT-1?. PMID:21486805

  20. Quaternized starch-based carrier for siRNA delivery: from cellular uptake to gene silencing.

    PubMed

    Amar-Lewis, Eliz; Azagury, Aharon; Chintakunta, Ramesh; Goldbart, Riki; Traitel, Tamar; Prestwood, Jackson; Landesman-Milo, Dalit; Peer, Dan; Kost, Joseph

    2014-07-10

    RNAi therapeutics is a powerful tool for treating diseases by sequence-specific targeting of genes using siRNA. Since its discovery, the need for a safe and efficient delivery system for siRNA has increased. Here, we have developed and characterized a delivery platform for siRNA based on the natural polysaccharide starch in an attempt to address unresolved delivery challenges of RNAi. Modified potato starch (Q-starch) was successfully obtained by substitution with quaternary reagent, providing Q-starch with cationic properties. The results indicate that Q-starch was able to bind siRNA by self-assembly formation of complexes. For efficient and potent gene silencing we monitored the physical characteristics of the formed nanoparticles at increasing N/P molar ratios. The minimum ratio for complete entrapment of siRNA was 2. The resulting complexes, which were characterized by a small diameter (~30 nm) and positive surface charge, were able to protect siRNA from enzymatic degradation. Q-starch/siRNA complexes efficiently induced P-glycoprotein (P-gp) gene silencing in the human ovarian adenocarcinoma cell line, NCI-ADR/Res (NAR), over expressing the targeted gene and presenting low toxicity. Additionally, Q-starch-based complexes showed high cellular uptake during a 24-hour study, which also suggested that intracellular siRNA delivery barriers governed the kinetics of siRNA transfection. In this study, we have devised a promising siRNA delivery vector based on a starch derivative for efficient and safe RNAi application. PMID:24794893

  1. Cot, a novel kinase of histone H3, induces cellular transformation through up-regulation of c-fos transcriptional activity

    PubMed Central

    Choi, Hong Seok; Kang, Bong Seok; Shim, Jung-Hyun; Cho, Yong-Yeon; Choi, Bu Young; Bode, Ann M.; Dong, Zigang

    2010-01-01

    Post-translational modification of histones is critical for gene expression, mitosis, cell growth, apoptosis, and cancer development. Thus, finding protein kinases that are responsible for the phosphorylation of histones at critical sites is considered an important step in understanding the process of histone modification. The serine/threonine kinase Cot is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family. We show here that Cot can phosphorylate histone H3 at Ser-10 in vivo and in vitro, and that the phosphorylation of histone H3 at Ser-10 is required for Cot-induced cell transformation. We found that activated Cot is recruited to the c-fos promoter resulting in increased activator protein-1 (AP-1) transactivation. The formation of the Cot-c-fos promoter complex was also apparent when histone H3 was phosphorylated at Ser-10. Furthermore, the use of dominant negative mutants of histone H3 revealed that Cot was required for phosphorylation of histone H3 at Ser-10 to induce neoplastic cell transformation. These results revealed an important function of Cot as a newly discovered histone H3 kinase. Moreover, the transforming ability of Cot results from the coordinated activation of histone H3, which ultimately converges on the regulation of the transcriptional activity of the c-fos promoter, followed by AP-1 transactivation activity. PMID:17724252

  2. Upregulation of B-cell translocation gene 2 by epigallocatechin-3-gallate via p38 and ERK signaling blocks cell proliferation in human oral squamous cell carcinoma cells.

    PubMed

    Lee, Jehn-Chuan; Chung, Li-Chuan; Chen, Yu-Jen; Feng, Tsui-Hsia; Chen, Wen-Tsung; Juang, Horng-Heng

    2015-05-01

    Oral squamous cell carcinoma (OSCC) is a well-known malignancy that accounts for the majority of oral cancers. B-cell translocation gene 2 (BTG2) is an important regulator of cell cycle dynamics in cancer cells. However, the role of BTG2 in OSCC cells and the influences of epigallocatechin-3-gallate (EGCG) on BTG2 gene expressions have not been well evaluated. The objectives of this study were to examine the effect of EGCG-induced BTG2 expression and the potential signal pathways involved. The (3)H-thymidine incorporation and Western-blot assays revealed cell proliferation was attenuated by EGCG via upregulation of BTG2 expression causing cell cycle G1 phase arrest in OSCC cells. BTG2 overexpression decreased tumor cell growth, while BTG2 knockdown illuminated the opposite effect in xenograft animal studies. Overexpressed BTG2 arrested the cell cycle at the G1 phase and downregulated protein expressions of cyclin A, cyclin D, and cyclin E. Western-blot assays indicated that EGCG induced phosphorylation of p38, JNK, and ERK. However, pretreatments with selective mitogen-activated protein kinase (MAPK) inhibitors, SB203580 (p38 inhibitor) and PD0325901 (ERK1/2 inhibitor), significantly suppressed the activation of EGCG on BTG2 expression. Our results indicate that EGCG attenuates cell proliferation of OSCC cells by upregulating BTG2 expression via p38 and ERK pathways. PMID:25721086

  3. DaTrypsin, a novel clip-domain serine proteinase gene up-regulated during winter and summer diapauses of the onion maggot, Delia antiqua.

    PubMed

    Chen, Bin; Kayukawa, Takumi; Jiang, Haobo; Monteiro, Antnia; Hoshizaki, Sugihiko; Ishikawa, Yukio

    2005-02-28

    Diapause prepares insects and other arthropods to survive in harsh environments. To explore the molecular basis of winter (WD) and summer diapauses (SD), we screened for diapause-specific genes in the onion maggot, Delia antiqua, that diapauses as a pupa in both summer and winter. A diapause-induced transcript, DaTrypsin, was identified through differential display, and examined by Northern blot, quantitative real-time PCR and sequence analyses. The full-length cDNA, 1379 bp long, encodes 384 a.a. with a molecular mass of 43,005 Da. The protein contains a 20-a.a. secretion peptide, followed by an amino-terminal clip domain and a carboxyl-terminal serine proteinase domain. With Ser, His and Asp as catalytic residues and Asp, Gly and Ser as specificity determinants, DaTrypsin is anticipated to be a trypsin-like enzyme. DaTrypsin transcription is up-regulated in both SD and WD pupae with higher mRNA levels during WD than SD. Heat shock further elevated gene transcription in both SD and WD pupae, whereas cold shock reduced DaTrypsin expression in SD pupae and had no significant effect on WD pupae. In SD pupae, DaTrypsin transcripts gradually build up during diapause, and after temperature shocks, whereas in WD pupae DaTrypsin mRNA levels are high at the beginning of diapause and immediately after a temperature shock and then gradually decrease with time. DaTrypsin represents the first serine proteinase gene expressed during diapause as well as the first gene up-regulated in both SD and WD. It may participate in the host's immune defense and/or maintain the developmental status in the diapausing pupae. PMID:15715962

  4. The MAT1-2-1 mating-type gene upregulates photo-inducible carotenoid biosynthesis in Fusarium verticillioides.

    PubMed

    Adm, Attila L; Garca-Martnez, Jorge; Szucs, Endre P; Avalos, Javier; Hornok, Lszl

    2011-05-01

    Filamentous ascomycetes, including mitotic holomorphs, have constitutively transcribed MAT (mating type) genes. These genes encode transcription factors considered to be the major regulators of sexual communication. The proven targets of the MAT transcription factors are pheromone precursor and pheromone receptor genes. However, recent studies demonstrated that MAT proteins may also affect other genes not involved directly in the mating process. When grown in the light, Fusarium verticillioides produces the acidic xanthophyll neurosporoxanthin and lower amounts of nonpolar precursor carotenes, such as phytoene, torulene, ?-carotene, and ?-carotene. Depending on the illumination conditions, a drastic decrease or the absence of light-inducible carotenoid accumulation was detected in three independent ?FvMAT1-2-1 knockout mutants of F. verticillioides as compared with the parental wild-type strain. Transcript levels of the carB, carRA, and carT genes, encoding key enzymes of the carotenoid biosynthetic pathway, were also significantly reduced in the mutants. The downregulation of these genes in the ?FvMAT1-2-1 mutant indicates that MAT genes play a role in the control of carotenogenesis in Fusarium. The finding that mating-type genes regulate important processes unrelated to sex helps to understand the presence of functional MAT genes in asexually reproducing fungus populations. PMID:21314709

  5. Tolerance in banana to Fusarium wilt is associated with early up-regulation of cell wall-strengthening genes in the roots.

    PubMed

    VAN DEN Berg, Nolani; Berger, Dave K; Hein, Ingo; Birch, Paul R J; Wingfield, Michael J; Viljoen, Altus

    2007-05-01

    SUMMARY Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of bananas. In the tropics and subtropics, Cavendish banana varieties are highly susceptible to Foc race 4 (VCG 0120). Cavendish selection GCTCV-218 was shown to have significantly lower disease severity and incidence compared with susceptible cultivar Williams in replicated greenhouse and field trials. Suppression subtractive hybridization (SSH) was previously carried out to identify genes induced in roots of GCTCV-218, but not in Williams, after infection with Foc'subtropical' race 4. Seventy-nine SSH clones were sequenced and revealed 13 non-redundant gene fragments, several of which showed homology to defence-associated genes, including cell wall-strengthening genes. Quantitative RT-PCR was used to confirm up-regulation and differential expression of a number of genes throughout a time-course, following Foc infection in the tolerant GCTCV-218 when compared with susceptible cv. Williams. Tolerance of GCTCV-218 was linked to significantly increased induction of cell wall-associated phenolic compounds. PMID:20507503

  6. Mechanistic links between cellular trade-offs, gene expression, and growth

    PubMed Central

    Oyarzún, Diego A.; Danos, Vincent; Swain, Peter S.

    2015-01-01

    Intracellular processes rarely work in isolation but continually interact with the rest of the cell. In microbes, for example, we now know that gene expression across the whole genome typically changes with growth rate. The mechanisms driving such global regulation, however, are not well understood. Here we consider three trade-offs that, because of limitations in levels of cellular energy, free ribosomes, and proteins, are faced by all living cells and we construct a mechanistic model that comprises these trade-offs. Our model couples gene expression with growth rate and growth rate with a growing population of cells. We show that the model recovers Monod’s law for the growth of microbes and two other empirical relationships connecting growth rate to the mass fraction of ribosomes. Further, we can explain growth-related effects in dosage compensation by paralogs and predict host–circuit interactions in synthetic biology. Simulating competitions between strains, we find that the regulation of metabolic pathways may have evolved not to match expression of enzymes to levels of extracellular substrates in changing environments but rather to balance a trade-off between exploiting one type of nutrient over another. Although coarse-grained, the trade-offs that the model embodies are fundamental, and, as such, our modeling framework has potentially wide application, including in both biotechnology and medicine. PMID:25695966

  7. Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting

    PubMed Central

    Zheng, Nan; Yin, Lichen; Song, Ziyuan; Ma, Liang; Tang, Haoyu; Gabrielson, Nathan P.; Lu, Hua; Cheng, Jianjun

    2014-01-01

    The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors: vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors. PMID:24211080

  8. Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting.

    PubMed

    Zheng, Nan; Yin, Lichen; Song, Ziyuan; Ma, Liang; Tang, Haoyu; Gabrielson, Nathan P; Lu, Hua; Cheng, Jianjun

    2014-01-01

    The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors. Vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors. PMID:24211080

  9. Transcriptome Profiling of Botrytis cinerea Conidial Germination Reveals Upregulation of Infection-Related Genes during the Prepenetration Stage

    PubMed Central

    Leroch, Michaela; Kleber, Astrid; Silva, Evelyn; Coenen, Tina; Koppenhfer, Dieter; Shmaryahu, Amir; Valenzuela, Pablo D. T.

    2013-01-01

    Botrytis cinerea causes gray mold on a great number of host plants. Infection is initiated by airborne conidia that invade the host tissue, often by penetration of intact epidermal cells. To mimic the surface properties of natural plant surfaces, conidia were incubated on apple wax-coated surfaces, resulting in rapid germination and appressorium formation. Global changes in gene expression were analyzed by microarray hybridization between conidia incubated for 0 h (dormant), 1 h (pregermination), 2.5 h (postgermination), 4 h (appressoria), and 15 h (early mycelium). Considerable changes were observed, in particular between 0 h and 1 h. Genes induced during germination were enriched in those genes encoding secreted proteins, including lytic enzymes. Comparison of wild-type and a nonpathogenic MAP kinase mutant (bmp1) revealed marked differences in germination-related gene expression, in particular related to secretory proteins. Using promoter-GFP reporter strains, we detected a strictly germination-specific expression pattern of a putative chitin deacetylase gene (cda1). In contrast, a cutinase gene (cutB) was found to be expressed only in the presence of plant lipids, in a developmentally less stringent pattern. We also identified a coregulated gene cluster possibly involved in secondary metabolite synthesis which was found to be controlled by a transcription factor also encoded in this cluster. Our data demonstrate that early conidial development in B. cinerea is accompanied by rapid shifts in gene expression that prepare the fungus for germ tube outgrowth and host cell invasion. PMID:23417562

  10. 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.

  11. Alteration of cellular mediated cytotoxicity, T cell receptor zeta (TcR zeta) and apoptosis related gene expression in nasopharyngeal carcinoma (NPC) patients: possible clinical relevance.

    PubMed

    Laytragoon-Lewin, N; Porwit-MacDonald, A; Mellstedt, H; Lewin, F

    2000-01-01

    We have investigated apoptosis related gene expression in tumour cells, phenotype and function of blood mononuclear cells at diagnosis in relation to clinical response in three patients with nasopharyngeal carcinoma (NPC). We have focused our study on the Epstein Barr virus latent membrane protein-1 (LMP-1) and Bcl-2 expression in the tumour cells, the essential signal-transducing zeta molecule of T cell receptor (TcR zeta) and cellular mediated cytolysis of the blood mononuclear cells. The carcinoma cells of the patients were Bcl-2 negative. They were heterogeneous with regard to the expression of LMP-1 and the number of proliferating or apoptotic cells. Decrease in the expression of mature T cells (CD3, CD4, and CD8), TcR zeta and cellular mediated cytotoxicity was detected in blood mononuclear cells of the patients. IL-2 up-regulated these phenotypes and the cytolytic capacity of the blood mononuclear cells. The patient with LMP-1 negative carcinoma cells, down-regulated TcR zeta expression and impaired IL-2 mediated cytolysis, had the worst clinical outcome. Another patient with low apoptotic, highly proliferating and LMP-1 positive carcinoma cells had recurrent disease only in the irradiated area. Interestingly, NPC with high apoptotic and few LMP-1 expressing cells was detected in the patient with a normal level of TcR zeta expression and cytolytic functions in blood mononuclear cells at the time of diagnosis. After combination treatment with chemotherapy followed by radiotherapy, this patient is still alive with complete remission and disease-free at 36 months. Suppression of the immunological functions may occur in NPC patients. Our study suggests that the immunological functions and apoptosis related gene expression in the carcinoma cells may be used as prognostic factors and help in the decision of therapy of patients with nasopharyngeal cancer. PMID:10810402

  12. Lack of nAChR Activity Depresses Cochlear Maturation and Up-Regulates GABA System Components: Temporal Profiling of Gene Expression in α9 Null Mice

    PubMed Central

    Turcan, Sevin; Slonim, Donna K.; Vetter, Douglas E.

    2010-01-01

    Background It has previously been shown that deletion of chrna9, the gene encoding the α9 nicotinic acetylcholine receptor (nAChR) subunit, results in abnormal synaptic terminal structure. Additionally, all nAChR-mediated cochlear activity is lost, as characterized by a failure of the descending efferent system to suppress cochlear responses to sound. In an effort to characterize the molecular mechanisms underlying the structural and functional consequences following loss of α9 subunit expression, we performed whole-transcriptome gene expression analyses on cochleae of wild type and α9 knockout (α9−/−) mice during postnatal days spanning critical periods of synapse formation and maturation. Principal Findings Data revealed that loss of α9 receptor subunit expression leads to an up-regulation of genes involved in synaptic transmission and ion channel activity. Unexpectedly, loss of α9 receptor subunit expression also resulted in an increased expression of genes encoding GABA receptor subunits and the GABA synthetic enzyme, glutamic acid decarboxylase. These data suggest the existence of a previously unrecognized association between the nicotinic cholinergic and GABAergic systems in the cochlea. Computational analyses have highlighted differential expression of several gene sets upon loss of nicotinic cholinergic activity in the cochlea. Time-series analysis of whole transcriptome patterns, represented as self-organizing maps, revealed a disparate pattern of gene expression between α9−/− and wild type cochleae at the onset of hearing (P13), with knockout samples resembling immature postnatal ages. Conclusions We have taken a systems biology approach to provide insight into molecular programs influenced by the loss of nicotinic receptor-based cholinergic activity in the cochlea and to identify candidate genes that may be involved in nicotinic cholinergic synapse formation, stabilization or function within the inner ear. Additionally, our data indicate a change in the GABAergic system upon loss of α9 nicotinic receptor subunit within the cochlea. PMID:20140217

  13. Upregulation of human angiotensinogen (AGT) gene transcription by interferon-gamma: involvement of the STAT1-binding motif in the AGT promoter.

    PubMed

    Jain, Sudhir; Shah, Mehul; Li, Yanna; Vinukonda, Govindaiah; Sehgal, Pravin B; Kumar, Ashok

    2006-07-01

    Mechanisms to maintain blood pressure in the face of infection are critical to survival. The angiotensinogen (AGT) gene locus is an important component of this response. Thus the AGT gene, expressed predominantly by liver cells, is known to be a positive acute phase reactant. We have previously demonstrated activation of the AGT promoter in hepatocytes through the IL6/STAT3 signaling mechanism. We have now investigated whether IFN-gamma, a cytokine also induced in response to diverse infections, can regulate AGT gene expression, and have elucidated the molecular mechanism involved. IFN gamma treatment up-regulated AGT mRNA level and promoter activity in Hep3B hepatocytes. Sequential deletion of the promoter from the 5' side suggested the major IFN gamma responsive DNA element to be between -303 and -103. This region contained a candidate STAT1-binding site between -271 and -279. EMSA and chromatin immuno-precipitation (ChIP) assays confirmed that IFN-gamma treatment induced the binding of STAT1 to this element. Reporter constructs containing this AGT promoter derived element in a multimerized context but not a mutant version were responsive to IFN gamma. Moreover mutating this STAT1 element in the context of the wild-type AGT holo promoter reduced responsiveness to IFN gamma. In contrast to the clear synergism between dexamethasone and IL 6 in the upregulation of the AGT promoter (through interaction between GR and STAT3), the combination of IFN gamma with IL 6 or with dexamethasone did not further increase AGT promoter activity suggesting that the IFN gamma/STAT1 pathway represents a separate signaling mechanism. These data highlight the redundancy in cytokine-mediated host response pathways aimed at the maintenance of blood pressure during infection. PMID:16949687

  14. Up-Regulation of a Magnesium Transporter Gene OsMGT1 Is Required for Conferring Aluminum Tolerance in Rice1[W][OA

    PubMed Central

    Chen, Zhi Chang; Yamaji, Naoki; Motoyama, Ritsuko; Nagamura, Yoshiaki; Ma, Jian Feng

    2012-01-01

    Magnesium (Mg)-mediated alleviation of aluminum (Al) toxicity has been observed in a number of plant species, but the mechanisms underlying the alleviation are still poorly understood. When a putative rice (Oryza sativa) Mg transporter gene, Oryza sativa MAGNESIUM TRANSPORTER1 (OsMGT1), was knocked out, the tolerance to Al, but not to cadmium and lanthanum, was decreased. However, this inhibition could be rescued by addition of 10 μm Mg, but not by the same concentration of barium or strontium. OsMGT1 was expressed in both the roots and shoots in the absence of Al, but the expression only in the roots was rapidly up-regulated by Al. Furthermore, the expression did not respond to low pH and other metals including cadmium and lanthanum, and was regulated by an Al-responsive transcription factor, AL RESISTANCE TRANSCRIPTION FACTOR1. An investigation of subcellular localization showed that OsMGT1 was localized to the plasma membrane. A short-term (30 min) uptake experiment with stable isotope 25Mg showed that knockout of OsMGT1 resulted in decreased Mg uptake, but that the uptake in the wild type was enhanced by Al. Mg concentration in the cell sap of the root tips was also increased in the wild-type rice, but not in the knockout lines in the presence of Al. A microarray analysis showed that transcripts of genes related to stress were more up- and down-regulated in the knockout lines. Taken together, our results indicate that OsMGT1 is a transporter for Mg uptake in the roots and that up-regulation of this gene is required for conferring Al tolerance in rice by increasing Mg concentration in the cell. PMID:22732245

  15. Correlation of glucocorticoid-mediated E4BP4 upregulation with altered expression of pro- and anti-apoptotic genes in CEM human lymphoblastic leukemia cells.

    PubMed

    Beach, Jessica A; Nary, Laura J; Hovanessian, Rebeka; Medh, Rheem D

    2014-08-29

    In Caenorhabditiselegans, motorneuron apoptosis is regulated via a ces-2-ces-1-egl-1 pathway. We tested whether human CEM lymphoblastic leukemia cells undergo apoptosis via an analogous pathway. We have previously shown that E4BP4, a ces-2 ortholog, mediates glucocorticoid (GC)-dependent upregulation of BIM, an egl-1 ortholog, in GC-sensitive CEM C7-14 cells and in CEM C1-15mE#3 cells, which are sensitized to GCs by ectopic expression of E4BP4. In the present study, we demonstrate that the human ces-1 orthologs, SLUG and SNAIL, are not significantly repressed in correlation with E4BP4 expression. Expression of E4BP4 homologs, the PAR family genes, especially HLF, encoding a known anti-apoptotic factor, was inverse to that of E4BP4 and BIM. Expression of pro- and anti-apoptotic genes in CEM cells was analyzed via an apoptosis PCR Array. We identified BIRC3 and BIM as genes whose expression paralleled that of E4BP4, while FASLG, TRAF4, BCL2A1, BCL2L1, BCL2L2 and CD40LG as genes whose expression was opposite to that of E4BP4. PMID:25101525

  16. Association of Bone Loss with the Upregulation of Survival-Related Genes and Concomitant Downregulation of Mammalian Target of Rapamycin and Osteoblast Differentiation-Related Genes in the Peripheral Blood of Late Postmenopausal Osteoporotic Women

    PubMed Central

    Tchetina, Elena V.; Maslova, Karina A.; Krylov, Mikhail Y.; Myakotkin, Valery A.

    2015-01-01

    We aimed to identify bone related markers in the peripheral blood of osteoporotic (OP) patients that pointed toward molecular mechanisms underlying late postmenopausal bone loss. Whole blood from 22 late postmenopausal OP patients and 26 healthy subjects was examined. Bone mineral density (BMD) was measured by DXA. Protein levels of p70-S6K, p21, MMP-9, TGF?1, and caspase-3 were quantified by ELISA. Gene expression was measured using real-time RT-PCR. OP registered by low BMD indices in late postmenopausal patients was associated with a significant upregulation of autophagy protein ULK1, cyclin-dependent kinase inhibitor p21, and metalloproteinase MMP-9 gene expression in the blood compared to the healthy controls and in a significant downregulation of mTOR (mammalian target of rapamycin), RUNX2, and ALPL gene expression, while expression of cathepsin K, caspase-3, transforming growth factor (TGF) ?1, interleukin- (IL-) 1?, and tumor necrosis factor ? (TNF?) was not significantly affected. We also observed a positive correlation between TGF?1 and RUNX2 expression and BMD at femoral sites in these patients. Therefore, bone loss in late postmenopausal OP patients is associated with a significant upregulation of survival-related genes (ULK1 and p21) and MMP-9, as well as the downregulation of mTOR and osteoblast differentiation-related genes (RUNX2 and ALPL) in the peripheral blood compared to the healthy controls. PMID:25759764

  17. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway

    PubMed Central

    Awortwe, Charles; Manda, Vamshi K.; Avonto, Cristina; Khan, Shabana I.; Khan, Ikhlas A.; Walker, Larry A.; Bouic, Patrick J.; Rosenkranz, Bernd

    2015-01-01

    This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. Thus E. purpurea preparations cause herb–drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  18. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway.

    PubMed

    Awortwe, Charles; Manda, Vamshi K; Avonto, Cristina; Khan, Shabana I; Khan, Ikhlas A; Walker, Larry A; Bouic, Patrick J; Rosenkranz, Bernd

    2015-03-01

    1.This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. 2.Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. 3. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. 4.Thus E. purpurea preparations cause herb-drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  19. Genome wide identification of Fruitless targets suggests a role in upregulating genes important for neural circuit formation

    PubMed Central

    Vernes, Sonja C.

    2014-01-01

    The fruitless gene (fru) encodes a set of transcription factors (Fru) that display sexually dimorphic gene expression in the brain of the fruit-fly; Drosophila melanogaster. Behavioural studies have demonstrated that fru is essential for courtship behaviour in the male fly and is thought to act by directing the development of sex-specific neural circuitry that encodes this innate behavioural response. This study reports the identification of direct regulatory targets of the sexually dimorphic isoforms of the Fru protein using an in vitro model system. Genome wide binding sites were identified for each of the isoforms using Chromatin Immunoprecipitation coupled to deep sequencing (ChIP-Seq). Putative target genes were found to be involved in processes such as neurotransmission, ion-channel signalling and neuron development. All isoforms showed a significant bias towards genes located on the X-chromosome, which may reflect a specific role for Fru in regulating x-linked genes. Taken together with expression analysis carried out in Fru positive neurons specifically isolated from the male fly brain, it appears that the Fru protein acts as a transcriptional activator. Understanding the regulatory cascades induced by Fru will help to shed light on the molecular mechanisms that are important for specification of neural circuitry underlying complex behaviour. PMID:24642956

  20. Comparative gene expression analysis of blood and brain provides concurrent validation of SELENBP1 up-regulation in schizophrenia.

    PubMed

    Glatt, Stephen J; Everall, Ian P; Kremen, William S; Corbeil, Jacques; Sásik, Roman; Khanlou, Negar; Han, Mark; Liew, Choong-Chin; Tsuang, Ming T

    2005-10-25

    Microarray techniques hold great promise for identifying risk factors for schizophrenia (SZ) but have not yet generated widely reproducible results due to methodological differences between studies and the high risk of type I inferential errors. Here we established a protocol for conservative analysis and interpretation of gene expression data from the dorsolateral prefrontal cortex of SZ patients using statistical and bioinformatic methods that limit false positives. We also compared brain gene expression profiles with those from peripheral blood cells of a separate sample of SZ patients to identify disease-associated genes that generalize across tissues and populations and further substantiate the use of gene expression profiling of blood for detecting valid SZ biomarkers. Implementing this systematic approach, we: (i) discovered 177 putative SZ risk genes in brain, 28 of which map to linked chromosomal loci; (ii) delineated six biological processes and 12 molecular functions that may be particularly disrupted in the illness; (iii) identified 123 putative SZ biomarkers in blood, 6 of which (BTG1, GSK3A, HLA-DRB1, HNRPA3, SELENBP1, and SFRS1) had corresponding differential expression in brain; (iv) verified the differential expression of the strongest candidate SZ biomarker (SELENBP1) in blood; and (v) demonstrated neuronal and glial expression of SELENBP1 protein in brain. The continued application of this approach in other brain regions and populations should facilitate the discovery of highly reliable and reproducible candidate risk genes and biomarkers for SZ. The identification of valid peripheral biomarkers for SZ may ultimately facilitate early identification, intervention, and prevention efforts as well. PMID:16223876

  1. A Sexual Shift Induced by Silencing of a Single Insulin-Like Gene in Crayfish: Ovarian Upregulation and Testicular Degeneration

    PubMed Central

    Rosen, Ohad; Manor, Rivka; Weil, Simy; Gafni, Ohad; Linial, Assaf; Aflalo, Eliahu D.; Ventura, Tomer; Sagi, Amir

    2010-01-01

    In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins. PMID:21151555

  2. Regulation of Viral and Cellular Gene Expression by Kaposi's Sarcoma-Associated Herpesvirus Polyadenylated Nuclear RNA

    PubMed Central

    Rossetto, Cyprian C.; Tarrant-Elorza, Margaret; Verma, Subhash; Purushothaman, Pravinkumar

    2013-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphoma. In cell culture, KSHV results in a latent infection, and lytic reactivation is usually induced with the expression of K-Rta or by treatment with phorbol 12-myristate 13-acetate (TPA) and/or n-butyrate. Lytic infection is marked by the activation of the entire viral genomic transcription cascade and the production of infectious virus. KSHV-infected cells express a highly abundant, long, noncoding transcript referred to as polyadenylated nuclear RNA (PAN RNA). PAN RNA interacts with specific demethylases and physically binds to the KSHV genome to mediate activation of viral gene expression. A recombinant BACmid lacking the PAN RNA locus fails to express K-Rta and does not produce virus. We now show that the lack of PAN RNA expression results in the failure of the initiation of the entire KSHV transcription program. In addition to previous findings of an interaction with demethylases, we show that PAN RNA binds to protein components of Polycomb repression complex 2 (PRC2). RNA-Seq analysis using cell lines that express PAN RNA shows that transcription involving the expression of proteins involved in cell cycle, immune response, and inflammation is dysregulated. Expression of PAN RNA in various cell types results in an enhanced growth phenotype, higher cell densities, and increased survival compared to control cells. Also, PAN RNA expression mediates a decrease in the production of inflammatory cytokines. These data support a role for PAN RNA as a major global regulator of viral and cellular gene expression. PMID:23468496

  3. Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling

    PubMed Central

    Morgan, Charles W.; Diaz, Juan E.; Zeitlin, Samantha G.; Gray, Daniel C.; Wells, James A.

    2015-01-01

    Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies. PMID:26106156

  4. Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling.

    PubMed

    Morgan, Charles W; Diaz, Juan E; Zeitlin, Samantha G; Gray, Daniel C; Wells, James A

    2015-07-01

    Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies. PMID:26106156

  5. Spatial association of apoptosis-related gene expression and cellular death in clinical neuroblastoma.

    PubMed Central

    Hoehner, J. C.; Gestblom, C.; Olsen, L.; Påhlman, S.

    1997-01-01

    Several unique features of neuroblastoma (NB), including the capacity for spontaneous regression and maturation to benign pathology, suggest that genes that regulate cellular proliferation, survival and differentiation may be involved in directing clinical tumour aggressiveness. The in situ expression of Bcl-2, Rb, p21, p53 and Bax proteins, as well as the proliferation marker proliferating cell nuclear antigen (PCNA) were examined immunocytochemically in a selection of 38 stage- and outcome-identified NB tumours. Apoptotic cells were identified morphologically and by a DNA fragmentation labelling technique (TUNEL). Although the tumour cell density of Bcl-2, p53, Bax, PCNA and TUNEL positivity correlated with patient survival, a spatially organized expression pattern was further recognized in stroma-poor differentiating tumours. Immature tumour cells adjacent to thin fibrovascular stroma are proliferating, as evidenced by PCNA positivity, and often express Bcl-2. At increasing distance from this fibrovascular stroma, intermediately differentiated tumour cells express Rb, while with more advanced differentiation, proliferation ceases and Bcl-2 immunoreactivity is lost. The most differentiated tumour cells, which often express p53, and occasionally p21 and Bax, lie adjacent to TUNEL-positive, morphologically apoptotic cells. This spatial organization in favourable outcome NB tumours suggests that physiological regulation of differentiation and apoptosis may be involved in tumour regression. Images Figure 2 PMID:9099968

  6. Cellular distribution and gene expression profile during flexor tendon graft repair: A novel tissue engineering approach*

    PubMed Central

    2013-01-01

    To understand scar and adhesion formation during postsurgical period of intrasynovial tendon graft healing, a murine model of flexor digitorum longus tendon graft repair was developed, by utilizing flexor digitorum longus tendon allograft from donor Rosa26/+ mouse, and the healing process at days 3, 7, 14, 21, 28, and 35 post surgery of host wild-type mouse was followed. Using X-gal staining, ?-galactosidase positive cells of allograft origin were detectable in tissue sections of grafted tendon post surgery. Graft healing was assessed for the cellular density, scar and adhesion formation, and their interaction with surrounding tissue. From histological analysis, it was evident that the healing of intrasynovial flexor digitorum longus tendon graft takes place in an interactive environment of donor graft, host tendon, and host surrounding tissue. A total of 32 genes, analyzed by RNA analysis, expressed during healing process. Particularly, Alk1, Postn, Tnc, Tppp3, and Mkx will be further investigated for therapeutical value in reducing scars and adhesions. PMID:23762501

  7. The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

    PubMed

    Czosnek, H H; Bienz, B; Givol, D; Zakut-Houri, R; Pravtcheva, D D; Ruddle, F H; Oren, M

    1984-08-01

    The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed. PMID:6387444

  8. Cloning and characterization of up-regulated HbSINA4 gene induced by drought stress in Tibetan hulless barley.

    PubMed

    Yuan, H J; Luo, X M; Nyima, T S; Wang, Y L; Xu, Q J; Zeng, X Q

    2015-01-01

    Hulless barley is an important crop cereal in Tibetan, China. Drought is a major abiotic stress in barley production. In this study, we cloned the drought-related HbSINA4 gene from the variety 'Himalaya 10' and analyzed its expression patterns under different drought and rehydration conditions. The cDNA of HbSINA4 was 1052 bp long, including an open reading frame of 771 bp that encoded a protein of 256 amino acids. The molecular weight of HbSINA4 protein was predicted to be 29.53 kDa and the theoretical pI was 8.32. Bioinformatic analysis showed that the HbSINA4 gene contained a protein kinase domain profile family signature motif, with high similarity to that of Oryza sativa and Brachypodium distachyon. Real-time polymerase chain reaction (PCR) assays revealed that gene expression declined rapidly with increasing drought stress; in contrast, its expression increased after rehydration treatment. Therefore, the HbSINA4 gene responds to the drought stress and plays an important role in barely drought resistance. Furthermore, our results provide information which may be useful in other temperate crop studies and in aiding resistance to drought. PMID:26634495

  9. Lactoferrin up-regulates intestinal gene expression of brain-derived neurotrophic factors BDNF, UCHL1 and alkaline phosphatase activity to alleviate early weaning diarrhea in postnatal piglets.

    PubMed

    Yang, Changwei; Zhu, Xi; Liu, Ni; Chen, Yue; Gan, Hexia; Troy, Frederic A; Wang, Bing

    2014-08-01

    The molecular mechanisms underlying how dietary lactoferrin (Lf) impacts gut development and maturation and protects against early weaning diarrhea are not well understood. In this study, we supplemented postnatal piglets with an Lf at a dose level of 155 and 285 mg/kg/day from 3 to 38 days following birth. Our findings show that the high dose of Lf up-regulated messenger RNA expression levels of genes encoding brain-derived neurotrophic factor (BDNF) and ubiquitin carboxy-terminal hydrolase L1 (ubiquitin thiolesterase (UCHL1) and, to a lesser extent, glial cell line-derived neurotrophic factor, in the duodenum (P<.05). Piglets in the high and low Lf group had 30% and 7% larger jejunal crypts compared with the control group (P<.05). Escherichia coli 16S rRNA copy number per gram of ascending colon contents was significantly reduced (P=.001), while the copy number of Bifidobacteria and Lactobacillus spp. was not affected. In addition, Lf increased intestinal alkaline phosphatase activity (P<.05) and delayed the onset of food transitional diarrhea, reducing its frequency and duration (P<.05). The incidence of diarrhea in the high and low Lf groups was decreased 54% and 15%, respectively, compared with the control group (P=.035). In summary, these findings provide new evidence that dietary Lf supplementation up-regulated gene expression of BDNF and UCHL1, decreased the colon microbiota of E. coli, improved gut maturation and reduced early weaning diarrhea in piglets. The molecular basis underlying these findings suggests that Lf may enhance gut development and immune function by providing new insight into the gut-brain-microbe axis that has not been previously reported. PMID:24824862

  10. Tumour necrosis factor superfamily member 15 (Tnfsf15) facilitates lymphangiogenesis via up-regulation of Vegfr3 gene expression in lymphatic endothelial cells.

    PubMed

    Qin, Ting-Ting; Xu, Guo-Ce; Qi, Jian-Wei; Yang, Gui-Li; Zhang, Kun; Liu, Hai-Lin; Xu, Li-Xia; Xiang, Rong; Xiao, Guozhi; Cao, Huiling; Wei, Yuquan; Zhang, Qiang-Zhe; Li, Lu-Yuan

    2015-11-01

    Lymphangiogenesis is essential in embryonic development but is rare in adults. It occurs, however, in many disease conditions including cancers. Vascular endothelial growth factor-C/D (VEGF-C/D) and VEGF receptor-3 (Vegfr3) play a critical role in the regulation of lymphangiogenesis. We investigated how the VEGF-C/Vegfr3 signalling system is regulated by tumour necrosis factor superfamily member 15 (Tnfsf15), an endothelium-derived cytokine. We report here that Tnfsf15, which is known to induce apoptosis in vascular endothelial cells, can promote lymphatic endothelial cell (LEC) growth and migration, stimulate lymphangiogenesis, and facilitate lymphatic circulation. Treatment of mouse LECs with Tnfsf15 results in up-regulation of Vegfr3 expression; this can be inhibited by gene silencing of death domain-containing receptor-3 (DR3; Tnfrsf25), a cell surface receptor for Tnfsf15, with siRNA, or by blocking Tnfsf15-DR3 interaction with a Tnfsf15 neutralizing antibody, 4-3H. Additionally, Tnfsf15/DR3 signalling pathways in LECs include activation of NF-κB. Tnfsf15-overexpressing transgenic mice exhibit a marked enhancement of lymph drainage; this is confirmed by treatment of wild-type mice with intraperitoneal injection of recombinant Tnfsf15. Moreover, systemic treatment of pregnant Tnfsf15 transgenic mice with 4-3H leads to inhibition of embryonic lymphangiogenesis. Our data indicate that Tnfsf15, a cytokine produced largely by endothelial cells, facilitates lymphangiogenesis by up-regulating Vegfr3 gene expression in LECs, contributing to the maintenance of the homeostasis of the circulatory system. This finding also suggests that Tnfsf15 may be of potential value as a therapeutic tool for the treatment of lymphoedema. PMID:26096340

  11. Cadmium up-regulates transcription of the steroidogenic acute regulatory protein (StAR) gene through phosphorylated CREB rather than SF-1 in K28 cells.

    PubMed

    Park, Soo-Yun; Gomes, Cynthia; Oh, Sung-Dug; Soh, Jaemog

    2015-04-01

    Cadmium is a widely used heavy metal in industry and affects the male reproductive system of animals, including humans, as a result of occupational and environmental exposures. However, the molecular mechanism underlying its effect on steroidogenesis in gonads remains unclear. In this study, we demonstrated that exposure of K28 mouse testicular Leydig tumor cells to cadmium led to a significant increase in the mRNA level, promoter activity and protein level of the steroidogenic acute regulatory protein (StAR), an essential factor for steroid biosynthesis. It has been well documented that StAR gene transcription is regulated by multiple transcription factors, including cAMP-responsive element binding protein (CREB) family members and SF-1. Cadmium treatment caused an increase in CREB phosphorylation but did not alter the CREB protein level in the nucleus. EMSA studies revealed that cadmium-induced phosphorylated CREB formed specific complexes with the proximal region of the StAR gene promoter. Furthermore, co-transfection with a CREB expression plasmid significantly increased cadmium-induced StAR promoter activity. However, the nuclear level and the affinity of SF-1 protein for the StAR proximal promoter were dramatically decreased upon exposure to cadmium. Taken together, these results suggest that cadmium up-regulates StAR gene expression through phosphorylated CREB rather than through SF-1 in mouse testicular Leydig cells. PMID:25786521

  12. Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways.

    PubMed

    Fuentes, Lida Q; Reyes, Carlos E; Sarmiento, Jos M; Villanueva, Carolina I; Figueroa, Carlos D; Navarro, Javier; Gonzlez, Carlos B

    2008-09-01

    Activation of V(1a) receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. We found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V(1a) receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and beta-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

  13. CD40 Is Essential in the Upregulation of TRAF Proteins and NF-KappaB-Dependent Proinflammatory Gene Expression after Arterial Injury

    PubMed Central

    Yu, Shiyong; Rivet, Joshua J.; Smyth, Susan S.; Nanda, Anil; Granger, D. Neil; Li, Guohong

    2011-01-01

    Despite extensive investigations, restenosis, which is characterized primarily by neointima formation, remains an unsolved clinical problem after vascular interventions. A recent study has shown that CD40 signaling through TNF receptor associated factor 6 (TRAF6) plays a key role in neointima formation after carotid artery injury; however, underlying mechanisms are not clearly elucidated. Because neointima formation may vary significantly depending on the type of injury, we first assessed the effect of CD40 deficiency on neointima formation in 2 injury models, carotid artery ligation and femoral artery denudation injury. Compared with wild-type mice, CD40 deficiency significantly reduced neointima formation and lumen stenosis in two different models. Further, we investigated the mechanism by which CD40 signaling affects neointima formation after arterial injury. In wild-type mice, the expression levels of CD40, several TRAF proteins, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6, as well as total NF-kB p65 and phospho-NF-kB p65, in the carotid artery were markedly upregulated within 37 days after carotid ligation. Deficiency of CD40 abolished the injury-induced upregulation of TRAFs including TRAF6 and NF-kB-p65 in the injured vessel wall. Further, CD40?/? mice showed a significant decrease in the recruitment of neutrophils (at 3, 7d) and macrophages (at 7, 21d) into injured artery; this effect was most likely attributed to inhibition of NF-kB activation and marked downregulation of NF-kB-related gene expression, including cytokines (TNF?, IL-1?, IL-6), chemokines (MCP-1), and adhesion molecules (ICAM-1, VCAM-1). Moreover, neutrophil recruitment in a model of thioglycollate-induced peritonitis is impaired in CD40-deficient mice. In vitro data revealed that CD40 deficiency blocked CD40L-induced NF-kB p65 nuclear translocation in leukocytes. Altogether, our data identified for the first time that CD40 is essential in the upregulation of TRAF6, NF-kB activation, and NF-kB-dependent proinflammatory genes in vivo. Our findings firmly established the role for CD40 in neointima formation in 2 distinct injury models. PMID:21876738

  14. The Neurospora crassa chr-1 gene is up-regulated by chromate and its encoded CHR-1 protein causes chromate sensitivity and chromium accumulation.

    PubMed

    Flores-Alvarez, Luis J; Corrales-Escobosa, Alma R; Corts-Penagos, Carlos; Martnez-Pacheco, Mauro; Wrobel-Zasada, Kazimierz; Wrobel-Kaczmarczyk, Katarzyna; Cervantes, Carlos; Gutirrez-Corona, Flix

    2012-12-01

    The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate resistance by exporting chromate ions from the cell's cytoplasm. The Neurospora crassa strain 74-A chr-1 gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the chr-1 gene was up-regulated by chromate exposure of N. crassa cultures. Introduction in N. crassa of sense and antisense fragments of the chr-1 gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in N crassa strains deleted in the genomic chr-1 gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from N. crassa chr-1 gene (Ncchr-1) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast Saccharomyces cerevisiae. Galactose-induced S. cerevisiae transformants expressing Ncchr-1 were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect S. cerevisiae chr-1 transformants from chromate toxicity. These data indicate that the N. crassa CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein. PMID:23085746

  15. Identification of SNPs in Cellular Retinol Binding Protein 1 and Cellular Retinol Binding Protein 3 Genes and Their Associations with Laying Performance Traits in Erlang Mountainous Chicken

    PubMed Central

    Wang, Yan; Xiao, Li-Hua; Zhao, Xiao-Ling; Liu, Yi-Ping; Zhu, Qing

    2014-01-01

    CRBP1 (cellular retinol binding protein 1) and CRBP3 (cellular retinol binding protein 3), are important components of the retinoid signaling pathway and take part in vitamin A absorption, transport and metabolism. Based on the role of vitamin A in chicken laying performance, we investigated the polymorphism of CRBP1 and CRBP3 genes in 349 chickens using single strand conformation polymorphism and DNA sequencing methods. Only one polymorphism was identified in the third intron of CRBP1, two polymorphisms were detected in CRBP3; they were located in the second intron and the third intron respectively. The association studies between these three SNPs and laying performance traits were performed in Erlang mountainous chicken. Notably, the SNP g.14604G>T of CRBP1 was shown to be significantly associated with body weight at first egg (BWFE), age at first egg (AFE), weight at first egg (WFE) and total number of eggs with 300 age (EN). The CRBP3 polymorphism g.934C>G was associated with AFE, and the g.1324A>G was associated with AFE and BWFE, but none of these polymorphisms were associated with egg quality traits. Haplotype combinations constructed on these two SNPs of CRBP3 gene were associated with BWFE and AFE. In particular, diplotype H2H2 had positive effect on AFE, BWFE, EN, and average egg-laying interval. We herein describe for the first time basic research on the polymorphism of chicken CRBP1 and CRBP3 genes that is predictive of genetic potential for laying performance in chicken. PMID:25083100

  16. A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets

    PubMed Central

    Gren, Susanne T.; Rasmussen, Thomas B.; Janciauskiene, Sabina; Hkansson, Katarina; Gerwien, Jens G.; Grip, Olof

    2015-01-01

    Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NF?B1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NF?B1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies. PMID:26650546

  17. Transactivation of the major capsid protein gene of herpes simplex virus type 1 requires a cellular transcription factor.

    PubMed

    Chen, S; Mills, L; Perry, P; Riddle, S; Wobig, R; Lown, R; Millette, R L

    1992-07-01

    The purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP). PMID:1318406

  18. Enhanced hypocholesterolemic effects of interesterified oils are mediated by upregulating LDL receptor and cholesterol 7-?- hydroxylase gene expression in rats.

    PubMed

    Reena, Malongil B; Gowda, Lalitha R; Lokesh, Belur R

    2011-01-01

    The concentration of LDL cholesterol in plasma is strongly influenced by the amount and type of lipid in the diet. Our studies have shown that positional changes in the fatty acids in blended oil introduced using lipase-catalyzed interesterification differentially modulate circulating LDL levels in rats compared with those observed in rats given a physical blend of oils. To investigate the molecular basis of these differences, transcriptional profiling of genes involved in cholesterol homeostasis was studied after feeding rats with a semipurified diet containing 10% fat from native oils; coconut oil (CNO), rice bran oil (RBO), or sesame oil (SESO); blended (B); CNO+RBO(B) or CNO+SESO(B) and interesterified oil (I); CNO+RBO(I) or CNO+SESO(I) for 60 d. Hepatic LDL receptor (LDL-R) expression significantly increased in rats fed interesterified oils by 100-200% compared with rats fed blended oils and by 400-500% compared with rats fed CNO. Positional alteration in fatty acids of oils used in the diet induced changes in LDL-R expression, which was accompanied by parallel changes in cholesterol-7?-hydroxylase (CYP7A1) and SREBP-2 genes. This suggested that not only the fatty acid type but also its position in the TG of dietary lipids play an important role in maintaining plasma cholesterol levels by suitably modulating gene expression for LDL-R in rat liver. PMID:21106933

  19. Identification of genes that regulate multiple cellular processes/responses in the context of lipotoxicity to hepatoma cells

    PubMed Central

    Srivastava, Shireesh; Li, Zheng; Yang, Xuerui; Yedwabnick, Matthew; Shaw, Stephen; Chan, Christina

    2007-01-01

    Background In order to devise efficient treatments for complex, multi-factorial diseases, it is important to identify the genes which regulate multiple cellular processes. Exposure to elevated levels of free fatty acids (FFAs) and tumor necrosis factor alpha (TNF-?) alters multiple cellular processes, causing lipotoxicity. Intracellular lipid accumulation has been shown to reduce the lipotoxicity of saturated FFA. We hypothesized that the genes which simultaneously regulate lipid accumulation as well as cytotoxicity may provide better targets to counter lipotoxicity of saturated FFA. Results As a model system to test this hypothesis, human hepatoblastoma cells (HepG2) were exposed to elevated physiological levels of FFAs and TNF-?. Triglyceride (TG) accumulation, toxicity and the genomic responses to the treatments were measured. Here, we present a framework to identify such genes in the context of lipotoxicity. The aim of the current study is to identify the genes that could be altered to treat or ameliorate the cellular responses affected by a complex disease rather than to identify the causal genes. Genes that regulate the TG accumulation, cytotoxicity or both were identified by a modified genetic algorithm partial least squares (GA/PLS) analysis. The analyses identified NADH dehydrogenase and mitogen activated protein kinases (MAPKs) as important regulators of both cytotoxicity and lipid accumulation in response to FFA and TNF-? exposure. In agreement with the predictions, inhibiting NADH dehydrogenase and c-Jun N-terminal kinase (JNK) reduced cytotoxicity significantly and increased intracellular TG accumulation. Inhibiting another MAPK pathway, the extracellular signal regulated kinase (ERK), on the other hand, improved the cytotoxicity without changing TG accumulation. Much greater reduction in the toxicity was observed upon inhibiting the NADH dehydrogenase and MAPK (which were identified by the dual-response analysis), than for the stearoyl-CoA desaturase (SCD) activation (which was identified for the TG-alone analysis). Conclusion These results demonstrate the applicability of GA/PLS in identifying the genes that regulate multiple cellular responses of interest and that genes regulating multiple cellular responses may be better candidates for countering complex diseases. PMID:17925029

  20. Androgens upregulate cyp19a1b (aromatase B) gene expression in the brain of zebrafish (Danio rerio) through estrogen receptors.

    PubMed

    Mouriec, Karen; Gueguen, Marie-Madeleine; Manuel, Christelle; Percevault, Frdric; Thieulant, Marie-Lise; Pakdel, Farzad; Kah, Olivier

    2009-05-01

    The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared with other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (AREs) on this promoter, in vivo and in vitro effects of androgens were studied. Using immunohistochemistry and quantitative PCR on zebrafish embryos, we found that cyp19a1b is upregulated by testosterone, an aromatizable androgen, and by 5alpha-dihydrotestosterone (DHT), a nonaromatizable androgen, suggesting a potential androgenic regulation of cyp19a1b through androgen receptors (ARs). To assess a putative direct regulation of the cyp19a1b gene by ARs, we transfected U251MG cells with zebrafish AR together with a luciferase reporter gene driven by 3000 bp of the proximal cyp19a1b promoter containing the ERE and potential AREs. Interestingly, although zebrafish AR activated luciferase reporter genes controlled by AREs, they failed to induce the cyp19a1b-luciferase construct. These data suggest that the androgenic regulation of cyp19a1b does not involve AR. We further showed that regulation of the cyp19a1b gene by testosterone is, in fact, due to aromatization, whereas the effect of DHT involves conversion into 5alpha-androstane-3beta,17beta-diol (betadiol), a metabolite of DHT with known estrogenic activity. The blockage of the androgen regulation of cyp19a1b expression using antiestrogens further confirmed the involvement of estrogen receptors in mediating these effects. PMID:19129512

  1. Cancer/Testis OIP5 and TAF7L Genes are Up-Regulated in Breast Cancer.

    PubMed

    Mobasheri, Maryam Beigom; Shirkoohi, Reza; Modarressi, Mohammad Hossein

    2015-01-01

    Breast cancer still remains as the most frequent cancer with second mortality rate in women worldwide. There are no validated biomarkers for detection of the disease in early stages with effective power in diagnosis and therapeutic approaches. Cancer/testis antigens are recently promising tumor antigens and suitable candidates for targeted therapies and generating cancer vaccines. We conducted the present study to analyze transcript changes of two cancer/testis antigens, OIP5 and TAF7L, in breast tumors and cell lines in comparison with normal breast tissues by quantitative real time RT-PCR for the first time. Significant over-expression of OIP5 was observed in breast tumors and three out of six cell lines including MDA-MB-468, T47D and SKBR3. Not significant expression of TAF7L was evident in breast tumors but significant increase was noted in three out of six cell lines including MDA-MB-231, BT474 and T47D. OIP5 has ssignificant role in chromatin organization and cell cycle control during cell cycle exit and normal chromosome segregation during mitosis and TAF7L is a component of the transcription factor ??D, which is involved in transcription initiation of most protein coding genes. TAF7Lis located at X chromosome and belongs to the CT-X gene family of cancer/testis antigens which contains about 50% of CT antigens, including those which have been used in cancer immunotherapy. PMID:26107214

  2. Pteromalus puparum venom impairs host cellular immune responses by decreasing expression of its scavenger receptor gene.

    PubMed

    Fang, Qi; Wang, Lei; Zhu, Yangkeng; Stanley, David W; Chen, Xuexin; Hu, Cui; Ye, Gongyin

    2011-11-01

    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. Although there is a rich literature on these systems, parasitoid immune-disabling mechanisms have not been fully elucidated. Here we report on a newly discovered immune-disabling mechanism in the Pieris rapae/Pteromalus puparum host/parasitoid system. Because venom injections and parasitization suppresses host phagocytosis, we turned attention to the P. rapae scavenger receptor (Pr-SR), posing the hypothesis that P. puparum venom suppresses expression of the host Pr-SR gene. To test our hypothesis, we cloned a full-length cDNA of the Pr-SR. Multiple sequences alignment showed the deduced amino acid sequence of Pr-SR is similar to scavenger receptors of other lepidopterans. Bacterial and bead injections induced Pr-SR mRNA and protein expression, which peaked at 4h post-bead injection. Venom injection inhibited Pr-SR expression. Pr-SR was specifically expressed in granulocytes compared to plasmatocytes. We localized the Pr-SR protein in cytoplasm and cellular membrane, with no evidence of secretion into host plasma. Double-strand RNA designed to Pr-SR mRNA silenced expression of Pr-SR and significantly impaired host phagocytosis and encapsulation reactions. Venom injections similarly silenced Pr-SR expression during the first 8h post-treatment, after which the silencing effects gradually abated. We infer from these findings that one mechanism of impairing P. rapae hemocytic immune reactions is by silencing expression of Pr-SR. PMID:21802512

  3. Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation

    PubMed Central

    Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

    2015-01-01

    The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process. PMID:26161530

  4. Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation.

    PubMed

    Hespeels, Boris; Li, Xiang; Flot, Jean-Franois; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

    2015-01-01

    The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process. PMID:26161530

  5. Hepatitis E genotype 4 virus from feces of monkeys infected experimentally can be cultured in PLC/PRF/5 cells and upregulate host interferon-inducible genes.

    PubMed

    Zhang, Feng; Qi, Ying; Harrison, Tim J; Luo, Baobin; Zhou, Yan; Li, Xiuhua; Song, Aijing; Huang, Weijin; Wang, Youchun

    2014-10-01

    The understanding of the interaction between hepatitis E virus (HEV) and its host cells has been impeded greatly by the absence of a cell culture system. In this study, an efficient cultivation method was developed in PLC/PRF/5 cells for HEV genotype 4 from the feces of monkeys infected experimentally. Compared to minimal essential medium (MEM), mixed Dulbecco's Modified Eagle's Medium (DMEM)/M199 improved the infection efficiency of HEV in PLC/PRF/5 cells. The incubation time and temperature were set at 6 hr and 40°C, respectively. Compared to a 100% ELISA positive ratio (EPR) of 1 × 10(6)  copies/ml HEV inoculated flasks, the ELISA positive ratio was 100%, 75%, 37.5%, and 100% for flasks inoculated with HEV incubated for 30 min under the conditions of pH 3.0, pH 11.0, 56°C and delipidation treatment, respectively. Gene expression profiles of HEV inoculated and control PLC/PRF/5 cells were assayed using a microarray. Four interferon-inducible genes, IFI27, IFI6, Mx1, and CMPK2, were up-regulated during HEV-infection. Furthermore, the replication of HEV was inhibited at 3-14 days after treatment with 500 IU/ml IFN-α2b. PMID:25042677

  6. Intermittent neonatal hypoxia elicits the upregulation of inflammatory-related genes in adult male rats through long-lasting programming effects.

    PubMed

    Gehrand, Ashley L; Kaldunski, Mary L; Bruder, Eric D; Jia, Shuang; Hessner, Martin J; Raff, Hershel

    2015-12-01

    The long-term effects of neonatal intermittent hypoxia (IH), an accepted model of apnea-induced hypoxia, are unclear. We have previously shown lasting "programming" effects on the HPA axis in adult rats exposed to neonatal IH. We hypothesized that neonatal rat exposure to IH will subsequently result in a heightened inflammatory state in the adult. Rat pups were exposed to normoxia (control) or six cycles of 5% IH or 10% IH over one hour daily from postnatal day 2-6. Plasma samples from blood obtained at 114days of age were analyzed by assessing the capacity to induce transcription in a healthy peripheral blood mononuclear cell (PBMC) population and read using a high-density microarray. The analysis of plasma from adult rats previously exposed to neonatal 5% IH versus 10% IH resulted in 2579 significantly regulated genes including increased expression of Cxcl1, Cxcl2, Ccl3, Il1a, and Il1b. We conclude that neonatal exposure to intermittent hypoxia elicits a long-lasting programming effect in the adult resulting in an upregulation of inflammatory-related genes. PMID:26660555

  7. NF-kappa B mediates up-regulation of CFTR gene expression in Calu-3 cells by interleukin-1beta.

    PubMed

    Brouillard, F; Bouthier, M; Leclerc, T; Clement, A; Baudouin-Legros, M; Edelman, A

    2001-03-23

    Inflammation of the airways is a major feature of the inherited disease cystic fibrosis. Previous studies have shown that the pro-inflammatory cytokines tumor necrosis factor alpha and interferon gamma reduce the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR) in HT-29 and T84 cells by acting post-transcriptionally. We have investigated the effect of the pro-inflammatory peptide interleukin 1beta (IL-1beta) on the expression of the CFTR in Calu-3 cells. IL-1beta increased the production of CFTR mRNA in a dose- and time-dependent manner. Its action was inhibited by inhibitors of the NF-kappaB pathway, including N-acetyl-l-cysteine, pyrrolidine dithiocarbamate, and a synthetic cell-permeable peptide containing the NF-kappaB nuclear localization signal sequence. Gel shift analysis showed that IL-1beta activated NF-kappaB in Calu-3 cells, and transfection experiments using p50 and RelA expressing vectors showed that exogenous transfected NF-kappaB subunits increased the concentration of CFTR mRNA. Gel shift analysis with antibody supershifting also showed that IL-1beta caused the binding of NF-kappaB to a kappaB-like response element at position -1103 to -1093 in the CFTR 5'-flanking region. Transfection experiments using -2150 to +52 CFTR reporter gene constructs showed that the activity of the CFTR promoter is enhanced by exogenous transfected NF-kappaB and IL-1beta and that this enhancement is due, at least in part, to the -1103 to -1093 kappaB site. We conclude that the intracellular signaling that leads to increased CFTR mRNA in response to IL-1beta in Calu-3 cells includes the binding of NF-kappaB to the -1103 kappaB element and a subsequent increase in CFTR promoter activity. PMID:11114294

  8. An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

    NASA Technical Reports Server (NTRS)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

  9. FLI1 expression is correlated with breast cancer cellular growth, migration, and invasion and altered gene expression.

    PubMed

    Scheiber, Melissa N; Watson, Patricia M; Rumboldt, Tihana; Stanley, Connor; Wilson, Robert C; Findlay, Victoria J; Anderson, Paul E; Watson, Dennis K

    2014-10-01

    ETS factors have been shown to be dysregulated in breast cancer. ETS factors control the expression of genes involved in many biological processes, such as cellular proliferation, differentiation, and apoptosis. FLI1 is an ETS protein aberrantly expressed in retrovirus-induced hematological tumors, but limited attention has been directed towards elucidating the role of FLI1 in epithelial-derived cancers. Using data mining, we show that loss of FLI1 expression is associated with shorter survival and more aggressive phenotypes of breast cancer. Gain and loss of function cellular studies indicate the inhibitory effect of FLI1 expression on cellular growth, migration, and invasion. Using Fli1 mutant mice and both a transgenic murine breast cancer model and an orthotopic injection of syngeneic tumor cells indicates that reduced Fli1 contributes to accelerated tumor growth. Global expression analysis and RNA-Seq data from an invasive human breast cancer cell line with over expression of either FLI1 and another ETS gene, PDEF, shows changes in several cellular pathways associated with cancer, such as the cytokine-cytokine receptor interaction and PI3K-Akt signaling pathways. This study demonstrates a novel role for FLI1 in epithelial cells. In addition, these results reveal that FLI1 down-regulation in breast cancer may promote tumor progression. PMID:25379017

  10. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

    NASA Astrophysics Data System (ADS)

    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02395a

  11. The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage

    PubMed Central

    Sappino, A-P; Buser, R; Seguin, Q; Fernet, M; Lesne, L; Gumy-Pause, F; Reith, W; Favaudon, V; Mandriota, S J

    2012-01-01

    The p53 tumor-suppressor protein has a key role in the induction of cellular senescence, an important barrier to cancer development. However, very little is known about the physiological mediators of cellular senescence induced by p53. CEACAM1 is an immunoglobulin superfamily member whose expression is frequently lost in human tumors and exhibits tumor-suppressor features in several experimental systems, including Ceacam1 knockout mice. There is currently little understanding of the pathways and mechanisms by which CEACAM1 exerts its tumor-suppressor function. Here we report that CEACAM1 is strongly upregulated during the cellular response to DNA double-strand breaks (DSBs) starting from the lowest doses of DSB inducers used, and that upregulation is mediated by the ataxia telangiectasia mutated (ATM)/p53 pathway. Stable silencing of CEACAM1 showed that CEACAM1 is required for p53-mediated cellular senescence, but not initial cell growth arrest, in response to DNA damage. These findings identify CEACAM1 as a key component of the ATM/p53-mediated cellular response to DNA damage, and as a tumor suppressor mediating cellular senescence downstream of p53. PMID:23552604

  12. Cysteine-Rich Secretory Protein-3 (CRISP3) Is Strongly Up-Regulated in Prostate Carcinomas with the TMPRSS2-ERG Fusion Gene

    PubMed Central

    Costa, Vera L.; Barros-Silva, João D.; Ramalho-Carvalho, João; Jerónimo, Carmen; Henrique, Rui; Lind, Guro E.; Skotheim, Rolf I.; Lothe, Ragnhild A.; Teixeira, Manuel R.

    2011-01-01

    A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16) and without (n = 8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (rs = 0.65, p<0.001) and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene. PMID:21814574

  13. Cysteine-rich secretory protein-3 (CRISP3) is strongly up-regulated in prostate carcinomas with the TMPRSS2-ERG fusion gene.

    PubMed

    Ribeiro, Franclim R; Paulo, Paula; Costa, Vera L; Barros-Silva, João D; Ramalho-Carvalho, João; Jerónimo, Carmen; Henrique, Rui; Lind, Guro E; Skotheim, Rolf I; Lothe, Ragnhild A; Teixeira, Manuel R

    2011-01-01

    A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16) and without (n = 8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (r(s) = 0.65, p<0.001) and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene. PMID:21814574

  14. Cellular Transcriptome Analysis Reveals Differential Expression of Pro- and Antiapoptosis Genes by Varicella-Zoster Virus-Infected Neurons and Fibroblasts

    PubMed Central

    Markus, Amos; Waldman Ben-Asher, Hiba; Kinchington, Paul R.

    2014-01-01

    Transcriptional changes following varicella-zoster virus (VZV) infection of cultured human neurons derived from embryonic stem cells were compared to those in VZV-infected human foreskin fibroblasts. Transcription of 340 neuronal genes significantly altered by VZV infection included 223 transcript changes unique to neurons. Strikingly, genes inhibiting apoptosis were upregulated in neurons, while proapoptotic gene transcription was increased in fibroblasts. These data are a basis for discovery of differences in virus-host interactions between these VZV targets. PMID:24741086

  15. Two c-Myc binding sites are crucial in upregulating the expression of human phospholipid scramblase 1 gene.

    PubMed

    Vinnakota, Janaki Manoja; Gummadi, Sathyanarayana N

    2016-01-15

    Human phospholipid scramblase 1 (hPLSCR1) is a type II endofacial membrane protein which mediates bi-directional transport of phospholipids across the plasma membrane. hPLSCR1, a multifunctional protein with variety of roles in apoptosis, tumor progression, cell signaling and anti-viral defense. The expression of such a multifunctional protein should be under tight regulation. Apart from a single report showing snail mediated down regulation of hPLSCR1, the molecular mechanisms regulating the expression of scramblases are not well elucidated. In this study we identified c-Myc as a transcriptional regulator of hPLSCR1. Transcription factor prediction tool ConSite predicted three binding sites for c-Myc. Reporter gene assays and western blot analysis revealed c-Myc mediated up regulation of hPLSCR1 expression. Deletion construct-790 lacking one c-Myc binding site showed a 27% decrease in promoter activity while deletion construct-469 lacking two c-Myc binding sites showed a 62% decrease in promoter activity. Site directed mutagenesis revealed the importance of c-Myc binding sites from-751 to-756 and-548 to-553 on the promoter of hPLSCR1in transcriptionally regulating the expression of hPLSCR1. The results were further confirmed by shRNA mediated knock down of endogenous c-Myc and invivo interactions by ChIP assay. PMID:26679604

  16. Induced thiacloprid insensitivity in honeybees (Apis mellifera L.) is associated with up-regulation of detoxification genes.

    PubMed

    Alptekin, S; Bass, C; Nicholls, C; Paine, M J I; Clark, S J; Field, L; Moores, G D

    2016-04-01

    Honey bees, Apis mellifera, are markedly less sensitive to neonicotinoid insecticides containing a cyanoimino pharmacophore than to those with a nitroimino group. Although previous work has suggested that this results from enhanced metabolism of the former by detoxification enzymes, the specific enzyme(s) involved remain to be characterized. In this work, a pretreatment of honey bees with a sublethal dose of thiacloprid resulted in induced insensitivity to the same compound immediately following thiacloprid feeding. A longer pretreatment time resulted in no, or increased, sensitivity. Transcriptome profiling, using microarrays, identified a number of genes encoding detoxification enzymes that were over-expressed significantly in insecticide-treated bees compared with untreated controls. These included five P450s, CYP6BE1, CYP305D1, CYP6AS5, CYP315A1, CYP301A1, and a carboxyl/cholinesterase (CCE) CCE8. Four of these P450s were functionally expressed in Escherichia coli and their ability to metabolize thiacloprid examined by liquid chromatography-mass spectrometry (LC-MS) analysis. PMID:26790026

  17. Tumour necrosis factor-alpha up-regulates decay-accelerating factor gene expression in human intestinal epithelial cells.

    PubMed Central

    Andoh, A; Fujiyama, Y; Sumiyoshi, K; Sakumoto, H; Okabe, H; Bamba, T

    1997-01-01

    The increased expression of decay-accelerating factor (DAF) has been detected in intestinal epithelial cells at the inflamed mucosa. In this study, we examined the effects of tumour necrosis factor (TNF)-alpha on DAF expression in three intestinal epithelial cell lines. DAF mRNA expression was evaluated by Northern blot analysis, and DAF protein expression was analysed by biotin labelling and immunoprecipitation. TNF-alpha induced a marked increase in DAF mRNA and protein expression in HT-29, T84 and Caco-2 cells. In HT-29 cells, the effects of TNF-a on DAF mRNA accumulation were observed in a dose-dependent manner; DAF mRNA accumulation reached a maximum at 3-6 hr, and then gradually decreased. These effects of TNF-alpha required de novo protein synthesis. Messenger RNA stability studies suggested that TNF-alpha partially regulated DAF gene expression by a posttranscriptional mechanism. Moreover, the combination of TNF-alpha and interleukin (IL)-4 induced an additive increase in DAF mRNA accumulation in HT-29 and T84 cells. In human intestinal epithelial cells, TNF-alpha acts as a potent inducer of DAF mRNA expression, indicating an important role for TNF-alpha in the regulation of DAF expression at the inflamed mucosa. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 PMID:9155641

  18. Prosaposin is an AR-target gene and its neurotrophic domain upregulates AR expression and activity in prostate stromal cells.

    PubMed

    Koochekpour, S; Lee, T-J; Sun, Y; Hu, S; Grabowski, G A; Liu, Z; Garay, J

    2008-08-15

    Recent studies have introduced prosaposin (PSAP) as a pleiotrophic growth factor for prostate cancer (PCa). We have previously reported that PSAP or one of its known active molecular derivatives, saposin C functions as an androgen-agonist and androgen-regulated gene (ARG) for androgen-sensitive (AS) PCa cell lines. Due to the potential significance of androgen receptor (AR)-expressing stroma in PCa, we evaluated a possible bi-directional paracrine regulatory interactions between DHT and PSAP in AR-positive prostate stromal (PrSt) cells. We report that saposin C in a ligand-independent manner increased AR expression, its nuclear content, and tyrosine phosphorylation. DHT treatment of PrSt cells increased PSAP expression. We also demonstrated both serum- and androgen-inducibility of a previously characterized hormone-responsive element (HRE) located in the proximal region of PSAP promoter. In addition, conditioned-media derived from PrSt cells and bone fibroblasts (i.e., MSF) differentially increased PSAP-promoter activity in androgen-independent (AI) PC-3 and AS LNCaP cells. Our data for the first time demonstrate that not only saposin C or PSAP regulates AR expression/activity, but also function as an ARG in PrSt. Ligand-independent activation of AR by PSAP or saposin C in PCa and stromal cells may contribute not only to prostate carcinogenesis at an early stage, but also in AI progression of the disease in an androgen-deprived tumor microenvironment. PMID:18481277

  19. Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

    PubMed

    Holland, Tai C; Killilea, David W; Shenvi, Swapna V; King, Janet C

    2015-12-01

    A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells. PMID:26420239

  20. 2009 Pandemic H1N1 Influenza Virus Causes Disease and Upregulation of Genes Related to Inflammatory and Immune Responses, Cell Death, and Lipid Metabolism in Pigs▿

    PubMed Central

    Ma, Wenjun; Belisle, Sarah E.; Mosier, Derek; Li, Xi; Stigger-Rosser, Evelyn; Liu, Qinfang; Qiao, Chuanling; Elder, Jake; Webby, Richard; Katze, Michael G.; Richt, Juergen A.

    2011-01-01

    There exists limited information about whether adaptation is needed for cross-species transmission of the 2009 pandemic H1N1 influenza virus (pH1N1). Here, we compare the pathogenesis of two pH1N1 viruses, one derived from a human patient (A/CA/04/09 [CA09]) and the other from swine (A/swine/Alberta/25/2009 [Alb09]), with that of the 1918-like classical swine influenza virus (A/swine/Iowa/1930 [IA30]) in the pig model. Both pH1N1 isolates induced clinical symptoms such as coughing, sneezing, decreased activity, fever, and labored breathing in challenged pigs, but IA30 virus did not cause any clinical symptoms except fever. Although both the pH1N1 viruses and the IA30 virus caused lung lesions, the pH1N1 viruses were shed from the nasal cavities of challenged pigs whereas the IA30 virus was not. Global gene expression analysis indicated that transcriptional responses of the viruses were distinct. pH1N1-infected pigs had an upregulation of genes related to inflammatory and immune responses at day 3 postinfection that was not seen in the IA30 infection, and expression levels of genes related to cell death and lipid metabolism at day 5 postinfection were markedly different from those of IA30 infection. These results indicate that both pH1N1 isolates are more virulent due in part to differences in the host transcriptional response during acute infection. Our study also indicates that pH1N1 does not need prior adaptation to infect pigs, has a high potential to be maintained in naïve swine populations, and might reassort with currently circulating swine influenza viruses. PMID:21900171

  1. Panax ginseng extract modulates oxidative stress, DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B 1.

    PubMed

    Hassan, Aziza M; Abdel-Aziem, Sekena H; El-Nekeety, Aziza A; Abdel-Wahhab, Mosaad A

    2015-10-01

    Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on DNA and gene expression in rat. Female Sprague-Dawley rats were divided into eight groups (ten rats/group) and treated for 12weeks including the control group, the group having received AFB1 (80g/kg bw), the group having received FB1 (100g/kg bw), the group having received AFB1 plus FB1 and the groups having received PGE (20mg/kg bw) alone or with AFB1 and/or FB1. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB1 and/or FB1 plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB1 and FB1 have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties. PMID:24748134

  2. 2009 pandemic H1N1 influenza virus causes disease and upregulation of genes related to inflammatory and immune responses, cell death, and lipid metabolism in pigs.

    PubMed

    Ma, Wenjun; Belisle, Sarah E; Mosier, Derek; Li, Xi; Stigger-Rosser, Evelyn; Liu, Qinfang; Qiao, Chuanling; Elder, Jake; Webby, Richard; Katze, Michael G; Richt, Juergen A

    2011-11-01

    There exists limited information about whether adaptation is needed for cross-species transmission of the 2009 pandemic H1N1 influenza virus (pH1N1). Here, we compare the pathogenesis of two pH1N1 viruses, one derived from a human patient (A/CA/04/09 [CA09]) and the other from swine (A/swine/Alberta/25/2009 [Alb09]), with that of the 1918-like classical swine influenza virus (A/swine/Iowa/1930 [IA30]) in the pig model. Both pH1N1 isolates induced clinical symptoms such as coughing, sneezing, decreased activity, fever, and labored breathing in challenged pigs, but IA30 virus did not cause any clinical symptoms except fever. Although both the pH1N1 viruses and the IA30 virus caused lung lesions, the pH1N1 viruses were shed from the nasal cavities of challenged pigs whereas the IA30 virus was not. Global gene expression analysis indicated that transcriptional responses of the viruses were distinct. pH1N1-infected pigs had an upregulation of genes related to inflammatory and immune responses at day 3 postinfection that was not seen in the IA30 infection, and expression levels of genes related to cell death and lipid metabolism at day 5 postinfection were markedly different from those of IA30 infection. These results indicate that both pH1N1 isolates are more virulent due in part to differences in the host transcriptional response during acute infection. Our study also indicates that pH1N1 does not need prior adaptation to infect pigs, has a high potential to be maintained in nave swine populations, and might reassort with currently circulating swine influenza viruses. PMID:21900171

  3. Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

    PubMed Central

    Jamsheer K, Muhammed; Laxmi, Ashverya

    2015-01-01

    Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response. PMID:26442059

  4. Inhibin beta E is upregulated by drug-induced endoplasmic reticulum stress as a transcriptional target gene of ATF4

    SciTech Connect

    Brüning, Ansgar Matsingou, Christina; Brem, German Johannes; Rahmeh, Martina; Mylonas, Ioannis

    2012-10-15

    Inhibins and activins are gonadal peptide hormones of the transforming growth factor-β super family with important functions in the reproductive system. By contrast, the recently identified inhibin βE subunit, primarily expressed in liver cells, appears to exert functions unrelated to the reproductive system. Previously shown downregulation of inhibin βE in hepatoma cells and anti-proliferative effects of ectopic inhibin βE overexpression indicated growth-regulatory effects of inhibin βE. We observed a selective re-expression of the inhibin βE subunit in HepG2 hepatoblastoma cells, MCF7 breast cancer cells, and HeLa cervical cancer cells under endoplasmic reticulum stress conditions induced by tunicamycin, thapsigargin, and nelfinavir. Analysis of XPB1 splicing and ATF4 activation revealed that inhibin βE re-expression was associated with induction of the endoplasmic reticulum stress reaction by these drugs. Transfection of an ATF4 expression plasmid specifically induced inhibin βE expression in HeLa cells and indicates inhibin βE as a hitherto unidentified target gene of ATF4, a key transcription factor of the endoplasmic reticulum stress response. Therefore, the inhibin βE subunit defines not only a new player but also a possible new marker for drug-induced endoplasmic reticulum stress. -- Highlights: ► Endoplasmic reticulum stress induces inhibin beta E expression. ► Inhibin beta E is regulated by the transcription factor ATF4. ► Inhibin beta E expression can be used as a marker for drug-induced ER stress.

  5. Profiling of BoNT/C3-reversible gene expression induced by lysophosphatidic acid: ephrinB1 gene up-regulation underlying neuropathic hyperalgesia and allodynia.

    PubMed

    Uchida, Hitoshi; Matsumoto, Misaki; Ueda, Hiroshi

    2009-01-01

    Lysophosphatidic acid (LPA) signaling, through LPA(1) receptor and its downstream RhoA, has been reported to initiate nerve injury-induced neuropathic pain. In the present study, we performed gene expression profiling of the dorsal root ganglion (DRG) to identify genes induced by intrathecal injection of LPA in a botulinum toxin C3 (BoNT/C3)-reversible manner. We selected and functionally characterized ephrinB1 from 82 identified genes as a potential gene involved in pain transmission, since ephrinB1 is implicated to modulate N-methyl-d-aspartate (NMDA) receptor functions in spinal pain transmission. The LPA-induced and BoNT/C3-reversible ephrinB1 gene expression was confirmed by quantitative real-time PCR. Furthermore, treatments with an antisense oligodeoxynucleotide for ephrinB1 largely abolished the LPA-induced thermal hyperalgesia and allodynia in response to mechanical or Abeta-fiber-mediated electrical stimuli on day 1 after the injection. In addition, intrathecal treatment with a soluble ligand, ephrinB1-Fc, caused similar neuropathic pain-like behaviors in a manner that was reversible by the NMDA receptor antagonist MK-801. These results suggest that ephrinB1 plays a crucial role in LPA-induced neuropathic pain. In addition, the present study may provide a new strategy to identify unique neuropathic pain-related genes. PMID:19111589

  6. Dnmt3a1 upregulates transcription of distinct genes and targets chromosomal gene clusters for epigenetic silencing in mouse embryonic stem cells.

    PubMed

    Kotini, Andriana G; Mpakali, Anastasia; Agalioti, Theodora

    2011-04-01

    Dnmt3a1 and Dnmt3a2 are two de novo DNA methyltransferases expressed in mouse embryonic stem cells (mESCs). They differ in that a 219-amino-acid (aa) amino (N)-terminal noncatalytic domain is present only in Dnmt3a1. Here, we examined the unique functions of Dnmt3a1 in mESCs by targeting the coding sequence of the Dnmt3a1 N-terminal domain tagged with enhanced green fluorescent protein (GFP) for insertion into the mouse Rosa26 locus. Using these targeted cells (GFP-3a1Nter), we showed that Dnmt3a1 was efficiently recruited to the silenced Oct3/4 and activated Vtn (vitronectin) gene promoters via its unique N-terminal domain. This recruitment affected the two genes in contrasting ways, compromising Oct3/4 gene promoter DNA methylation to prevent consolidation of the silent state while significantly reducing Vtn transcription. We used this negative effect of the Dnmt3a1 N-terminal domain to investigate the extent of transcriptional regulation by Dnmt3a1 in mESCs by using microarrays. A small group of all-trans retinoic acid (tRA)-inducible genes had lower transcript levels in GFP-3a1Nter cells than in wild-type mESCs. Intriguingly, this group included genes that are important for fetal nutrition, placenta development, and metabolic functions and is enriched for a distinct set of imprinted genes. We also identified a larger group of genes that showed higher transcript levels in the GFP-3a1Nter-expressing cells than in wild-type mESCs, including pluripotency factors and key regulators of primordial germ cell differentiation. Thus, Dnmt3a1 in mESCs functions primarily as a negative and to a lesser extent as a positive regulator of transcription. Our findings suggest that Dnmt3a1 positively affects transcription of specific genes at the promoter level and targets chromosomal domains to epigenetically silence gene clusters in mESCs. PMID:21262766

  7. Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

    PubMed Central

    2011-01-01

    Background During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. Results We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. Conclusions Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells. PMID:21663599

  8. Cellular Gene Expression upon Human Immunodeficiency Virus Type 1 Infection of CD4+-T-Cell Lines

    PubMed Central

    van 't Wout, Anglique B.; Lehrman, Ginger K.; Mikheeva, Svetlana A.; O'Keeffe, Gemma C.; Katze, Michael G.; Bumgarner, Roger E.; Geiss, Gary K.; Mullins, James I.

    2003-01-01

    The expression levels of ?4,600 cellular RNA transcripts were assessed in CD4+-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1BRU) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1BRU infection, consistent with the G2 arrest of HIV-1-infected cells induced by Vpr. These included genes involved in cell division and transcription, a family of DEAD-box proteins (RNA helicases), and all genes involved in translation and splicing. However, the overall level of cell activation and signaling was increased in infected cells, consistent with strong virus production. These included a subgroup of transcription factors, including EGR1 and JUN, suggesting they play a specific role in the HIV-1 life cycle. Some regulatory changes were cell line specific; however, the majority, including enzymes involved in cholesterol biosynthesis, of changes were regulated in most infected cell lines. Compendium analysis comparing gene expression profiles of our HIV-1 infection experiments to those of cells exposed to heat shock, interferon, or influenza A virus indicated that HIV-1 infection largely induced specific changes rather than simply activating stress response or cytokine response pathways. Thus, microarray analysis confirmed several known HIV-1 host cell interactions and permitted identification of specific cellular pathways not previously implicated in HIV-1 infection. Continuing analyses are expected to suggest strategies for impacting HIV-1 replication in vivo by targeting these pathways. PMID:12502855

  9. The hepatitis B virus X protein increases the cellular level of TATA-binding protein, which mediates transactivation of RNA polymerase III genes

    SciTech Connect

    Wang, Horng-Dar; Johnson, D.L.; Yuh, Chio-Hwa

    1995-12-01

    This report decribes the mechanism by which the hepatitis B virus X gene product induces RNA polymerase III genes. The RNA pol III transcription system serves as model for understanding the mechanism of X in the transactivation of cellular genes in both Drosophila and rat cell lines. 53 refs., 7 figs., 1 tab.

  10. A Prolyl-Hydroxylase Inhibitor, Ethyl-3,4-Dihydroxybenzoate, Induces Cell Autophagy and Apoptosis in Esophageal Squamous Cell Carcinoma Cells via Up-Regulation of BNIP3 and N-myc Downstream-Regulated Gene-1

    PubMed Central

    Han, Bo; Li, Wei; Sun, Yulin; Zhou, Lanping; Xu, Yang; Zhao, Xiaohang

    2014-01-01

    The protocatechuic acid ethyl ester ethyl-3,4-dihydroxybenzoate is an antioxidant found in the testa of peanut seeds. Previous studies have shown that ethyl-3,4-dihydroxybenzoate can effectively reduce breast cancer cell metastasis by inhibiting prolyl-hydroxylase. In this study, we investigated the cytotoxic effect of ethyl-3,4-dihydroxybenzoate on esophageal squamous cell carcinoma cells in vitro and identified key regulators of ethyl-3,4-dihydroxybenzoate-induced esophageal cancer cell death through transcription expression profiling. Using flow cytometry analysis, we found that ethyl-3,4-dihydroxybenzoate induced S phase accumulation, a loss in mitochondrial membrane permeabilization, and caspase-dependent apoptosis. Moreover, an expression profile analysis identified 46 up- and 9 down-regulated genes in esophageal cancer KYSE 170 cells treated with ethyl-3,4-dihydroxybenzoate. These differentially expressed genes are involved in several signaling pathways associated with cell cycle regulation and cellular metabolism. Consistent with the expression profile results, the transcriptional and protein expression levels of candidate genes NDRG1, BNIP3, AKR1C1, CCNG2 and VEGFA were found to be significantly increased in treated KYSE 170 cells by reverse-transcription PCR and western blot analysis. We also found that protein levels of hypoxia-inducible factor-1α, BNIP3, Beclin and NDRG1 were increased and that enriched expression of BNIP3 and Beclin caused autophagy mediated by microtubule-associated protein 1 light chain 3 in the treated cells. Autophagy and apoptosis were activated together in esophageal cancer cells after exposed to ethyl-3,4-dihydroxybenzoate. Furthermore, knock-down of NDRG1 expression by siRNA significantly attenuated apoptosis in the cancer cells, implying that NDRG1 may be required for ethyl-3,4-dihydroxybenzoate-induced apoptosis. Together, these results suggest that the cytotoxic effects of ethyl-3,4-dihydroxybenzoate were mediated by the up-regulation of NDRG1, BNIP3, Beclin and hypoxia-inducible factor-1α, initiating BNIP3 and Beclin mediated autophagy at an early stage and ultimately resulting in esophageal cancer cell apoptosis. PMID:25232961

  11. Expression patterns of GASA genes in Arabidopsis thaliana: the GASA4 gene is up-regulated by gibberellins in meristematic regions.

    PubMed

    Aubert, D; Chevillard, M; Dorne, A M; Arlaud, G; Herzog, M

    1998-04-01

    The GASA gene family previously identified in Arabidopsis belongs to a wide-spread class of genes found in mono- and dicotyledonous plants, all structurally related to the original GA-regulated GAST1 gene from tomato. They encode small peptides (97 to 112 residues) of unknown function sharing a 60 amino acid conserved C-terminal domain comprising twelve conserved cysteine residues which define a pattern not related to other known cysteine-rich motifs. Northern blot hybridization analysis revealed sequential expression of three genes during flowering, silique development and seed germination. GASA4 transcripts were detected in flower buds. GASA1 transcripts markedly accumulated in siliques, about five days after pollination, and correlated with the peak of GA biosynthesis at this stage of silique development. GASA3 transcripts accumulated at the end of the maturation stage of the silique, and transcripts were still present in dry seeds but degraded rapidly during imbibition. In addition, the GASA4 gene was again actively transcribed after germination and this expression was shown to be dependent on the presence of GAs in GA-deficient mutants. Immunoblot analysis confirmed the presence of the GASA4 gene product in flower buds, seedlings and roots. We focused on the GASA4 gene and characterized its expression. The upstream region (-890 to +128) was fused to the GUS reporter gene. GASA4/GUS expression was detected in transgenic Arabidopsis primarily in all meristematic regions, including vegetative, inflorescence and floral meristems, as well as primary and lateral root tips. In a GA-deficient background (ga1-3), GUS activity in the vegetative meristem was detected only in the presence of supplied GA. In root and flower meristems, basal GUS activity was slightly enhanced by exogenous GA. Interestingly, GA strongly inhibit GUS activity in expanding cotyledons and leaves in ga1-3 mutants supplied with exogenous GAs, as well as in the wild type. The GA-dependent meristem-specific expression pattern suggests that the GASA4 protein plays a role in dividing cells rather than in elongating cells. PMID:9520278

  12. Dietary wolfberry up-regulates carotenoid metabolic genes and enhances mitochondrial biogenesis in the retina of db/db diabetic mice

    PubMed Central

    Yu, Huifeng; Wark, Logan; Ji, Hua; Willard, Lloyd; Jaing, Yu; Han, Jing; He, Hui; Ortiz, Edlin; Zhang, Yunong; Medeiros, Denis M; Lin, Dingbo

    2013-01-01

    Scope Our aim was to investigate whether dietary wolfberry altered carotenoid metabolic gene expression and enhanced mitochondrial biogenesis in the retina of diabetic mice. Methods and Results Six-week-old male db/db and wild type mice were fed the control or wolfberry diets for 8 weeks. At study termination, liver and retinal tissues were collected for analysis by transmission electron microscopy, real-time PCR, immunoprecipitation, Western blot, and HPLC. Wolfberry elevated zeaxanthin and lutein levels in the liver and retinal tissues and stimulated expression of retinal scavenger receptor class B type I, glutathione S-transferase Pi 1, and β,β-carotene 9’,10’-oxygenase 2, and induced activation and nuclear enrichment of retinal AMP-activated protein kinase α2 (AMPKα2). Furthermore, wolfberry attenuated hypoxia and mitochondrial stress as demonstrated by declined expression of hypoxia-inducible factor-1α, vascular endothelial growth factor, and heat shock protein 60. Wolfberry enhanced retinal mitochondrial biogenesis in diabetic retinas as demonstrated by reversed mitochondrial dispersion in the retinal pigment epithelium, increased mitochondrial copy number, elevated citrate synthase activity, and up-regulated expression of peroxisome proliferator-activated receptor γ co-activator 1 α, nuclear respiratory factor 1, and mitochondrial transcription factor A. Conclusion Consumption of dietary wolfberry could be beneficial to retinoprotection through reversal of mitochondrial function in diabetic mice. PMID:23505020

  13. Increasing of temperature induces pathogenicity of Streptococcus agalactiae and the up-regulation of inflammatory related genes in infected Nile tilapia (Oreochromis niloticus).

    PubMed

    Kayansamruaj, Pattanapon; Pirarat, Nopadon; Hirono, Ikuo; Rodkhum, Channarong

    2014-08-01

    Temperature strongly affects the health of aquatic poikilotherms. In Nile tilapia (Oreochromis niloticus), elevated water temperatures increase the severity of streptococcosis. Here we investigated the effects of temperature on the vulnerability and inflammatory response of Nile tilapia to Streptococcus agalactiae (Group B streptococci; GBS). At 35 and 28 C, GBS took 4 and 7h, respectively to reach the log-phase and, when incubated with tilapia whole blood, experienced survival rates of 97% and 2%, respectively. The hemolysis activity of GBS grown at 35 C was five times higher than that of GBS grown at 28 C. GBS expressed cylE (?-hemolysin/cytolysin), cfb (CAMP factor) and PI-2b (pili-backbone) much more strongly at 35 C than at 28 C. Challenging Nile tilapia reared at 35 and 28 C with GBS resulted in accumulated mortalities of about 85% and 45%, respectively. At 35 C, infected tilapia exhibited tremendous inflammatory responses due to a dramatic up-regulation (30-40-fold) of inflammatory-related genes (cyclooxygenase-2, IL-1? and TNF-?) between 6 and 96 h-post infection. These results suggest that the increase of GBS pathogenicity to Nile tilapia induced by elevated temperature is associated with massive inflammatory responses, which may lead to acute mortality. PMID:24856132

  14. Apoptosis Induction of Human Bladder Cancer Cells by Sanguinarine through Reactive Oxygen Species-Mediated Up-Regulation of Early Growth Response Gene-1

    PubMed Central

    Han, Min Ho; Park, Cheol; Jin, Cheng-Yun; Kim, Gi-Young; Chang, Young-Chae; Moon, Sung-Kwon; Kim, Wun-Jae; Choi, Yung Hyun

    2013-01-01

    Although the effects of sanguinarine, a benzophenanthridine alkaloid, on the inhibition of some kinds of cancer cell growth have been established, the underlying mechanisms are not completely understood. This study investigated possible mechanisms by which sanguinarine exerts its anticancer action in cultured human bladder cancer cell lines (T24, EJ, and 5637). Sanguinarine treatment resulted in concentration-response growth inhibition of the bladder cancer cells by inducing apoptosis. Sanguinarine-induced apoptosis was correlated with the up-regulation of Bax, the down-regulation of Bid and XIAP, the activation of caspases (-3, -8, and -9), and the generation of increased reactive oxygen species (ROS). The ROS scavenger N-acetyl cysteine (NAC) completely reversed the sanguinarine-triggered apoptotic events. In addition, sanguinarine effectively increased the activation of the c-Jun N-terminal kinase (JNK) and the expression of the early growth response gene-1 (Egr-1), which was recovered by pretreatment with NAC. Furthermore, knockdown of Egr-1 expression by small interfering RNA attenuated sanguinarine-induced apoptosis, but not the JNK inhibitor, indicating that the interception of ROS generation blocked the sanguinarine-induced apoptotic effects via deregulation of the expression of Egr-1 proteins. Taken together, the data provide evidence that sanguinarine is a potent anticancer agent, which inhibits the growth of bladder cancer cells and induces their apoptosis through the generation of free radicals. PMID:23717422

  15. Green and Red Light Reduces the Disease Severity by Pseudomonas cichorii JBC1 in Tomato Plants via Upregulation of Defense-Related Gene Expression.

    PubMed

    Nagendran, Rajalingam; Lee, Yong Hoon

    2015-04-01

    Light influences many physiological processes in most organisms. To investigate the influence of light on plant and pathogen interaction, we challenged tomato seedlings with Pseudomonas cichorii JBC1 by flood inoculation and incubated the seedlings under different light conditions. Tomato seedlings exposed to green or red light showed a significant reduction in disease incidence compared with those grown under white light or dark conditions. To understand the underlying mechanisms, we investigated the effects of each light wavelength on P. cichorii JBC1 and tomato plants. Treatment with various light wavelengths at 120 mol m(-2) s(-1) revealed no significant difference in growth, swarming motility, or biofilm formation of the pathogen. In addition, when we vacuum-infiltrated P. cichorii JBC1 into tomato plants, green and red light also suppressed disease incidence which indicated that the reduced disease severity was not from direct influence of light on the pathogen. Significant upregulation of the defense-related genes, phenylalanine ammonia-lyase (PAL) and pathogenesis-related protein 1a (PR-1a) was observed in P. cichorii JBC1-infected tomato seedlings grown under green or red light compared with seedlings grown under white light or dark conditions. The results of this study indicate that light conditions can influence plant defense mechanisms. In particular, green and red light increase the resistance of tomato plants to infection by P. cichorii. PMID:25536016

  16. Exposure to Diflubenzuron Results in an Up-Regulation of a Chitin Synthase 1 Gene in Citrus Red Mite, Panonychus citri (Acari: Tetranychidae)

    PubMed Central

    Xia, Wen-Kai; Ding, Tian-Bo; Niu, Jin-Zhi; Liao, Chong-Yu; Zhong, Rui; Yang, Wen-Jia; Liu, Bin; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB. PMID:24590130

  17. Interferon-Stimulated Gene 15 Upregulation Precedes the Development of Blood-Brain Barrier Disruption and Cerebral Edema after Traumatic Brain Injury in Young Mice.

    PubMed

    Rossi, Janet L; Todd, Tracey; Daniels, Zachary; Bazan, Nicolas G; Belayev, Ludmila

    2015-07-15

    Recent studies show that myosin light chain kinase (MLCK) plays a pivotal role in development of cerebral edema, a known complication following traumatic brain injury (TBI) in children and a contributing factor to worsened neurologic recovery. Interferon-stimulated gene 15 (ISG15) is upregulated after cerebral ischemia and is neuroprotective. The significant role of ISG15 after TBI has not been studied. Postnatal Day (PND) 21 and PND24 mice were subjected to lateral closed-skull injury with impact depth of 2.0 or 2.25 mm. Behavior was examined at 7 d using two-object novel recognition and Wire Hang tests. Mice were sacrificed at 6 h, 12 h, 24 h, 48 h, 72 h, and 7 d. ISG15 and MLCK were analyzed by Western blot and immunohistochemistry, blood-brain barrier (BBB) disruption with Evans Blue (EB), and cerebral edema with wet/dry weights. EB extravasation and edema peaked at 72 h in both ages. PND21 mice had more severe neurological deficits, compared with PND24 mice. PND24 mice showed peak ISG15 expression at 6 h, and PND21 mice at 72 h. MLCK peaked in both age groups at 12 h and co-localized with ISG15 on immunohistochemistry and co-immunoprecipitation. These studies provide evidence, ISG15 is elevated following TBI in mice, preceding MLCK elevation, development of BBB disruption, and cerebral edema. PMID:25669448

  18. Knockdown of Litopenaeus vannamei HtrA2, an up-regulated gene in response to WSSV infection, leading to delayed shrimp mortality.

    PubMed

    Peepim, Termsri; Phiwsaiya, Kornsunee; Charoensapsri, Walaiporn; Khunrae, Pongsak; Senapin, Saengchan; Rattanarojpong, Triwit

    2016-02-10

    HtrA2 is an apoptosis-activating gene that enhances the apoptotic process by preventing the formation of the IAP-caspase complex, thereby freeing caspase to trigger the apoptosis pathway. In this study, we presented the full-length cDNA sequence of HtrA2 from Litopenaeus vannamei (LvHtrA2). The full-length LvHtrA2 was 1335bp, encoding 444 amino acids. This deduced amino acid sequence contained five conserved domains: a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a trimerization motif, a serine protease domain, and a PDZ domain normally found in the HtrA2 proteins of other organisms. A phylogenetic analysis revealed that LvHtrA2 clustered with the HtrA2 from other invertebrates and was closely related to Penaeus monodon HtrA2 (PmHtrA2). RT-PCR with RNA extracts from L. vannamei revealed that LvHtrA2 expression was found in several tissues, including the lymphoid organs, the haemocytes, the hepatopancreas, the gill, and the stomach, with different expression levels. When determining the role of LvHtrA2 in WSSV infection, it was found that LvHtrA2 transcription was early up-regulated in the WSSV-infected shrimp at 8h post-infection (p.i.) and expression still remained high at 48h p.i.. It also demonstrated that dsRNA specific to LvHtrA2 reduced the cumulative mortality in the WSSV-infected shrimp compared with the control group. Additionally, depletion of the LvHtrA2 transcripts reduced expression levels for caspase-3 (Cap-3) gene in shrimp. This result could suggest that LvHtrA2 may involved in apoptosis mediated mortality rather than providing immune protection during WSSV infection. PMID:26712477

  19. Leukemogenesis as a new approach to investigate the correlation between up regulated gene 4/upregulator of cell proliferation (URG4/URGCP) and signal transduction genes in leukemia.

    PubMed

    Dodurga, Yavuz; Oymak, Yeşim; Gündüz, Cumhur; Satıroglu-Tufan, N Lale; Vergin, Canan; Cetingül, Nazan; Biray Avci, Cığır; Topçuoğlu, Nejat

    2013-04-01

    The aim of the study is to the determine the profiles of cell cycle genes and a new candidate oncogene of URG4/URGCP which play role in leukemia, establishing the association between the early prognosis of cancer and the quantitation of genetic changes, and bringing a molecular approach to definite diagnosis. In this study, 36 newly diagnosed patients' with ALL-AML in the range of 0-18 years and six control group patients' bone marrow samples were included. Total RNA was isolated from samples and then complementary DNA synthesis was performed. The obtained cDNAs have been installed 96 well plates after prepared appropriate mixtures and assessed with LightCycler(®) 480 Real-Time PCR quantitatively. CHEK1, URG4/URGCP, CCNG1, CCNC, CDC16, KRAS, CDKN2D genes in the T-ALL group; CCND2, ATM, CDK8, CHEK1, TP53, CHEK2, CCNG2, CDK4, CDKN2A, E2F4, CCNC, KRAS genes in the precursor B-ALL group and CCND2, CDK6 genes in the AML group have shown significant increase in mRNA expression level. In the featured role of acute leukemia the regulating signaling pathways of leukemogenesis partially defined, although identification of new genetic markers in acute leukemia subgroups, will allow the development of early diagnostic and new treatment protocols. PMID:23266667

  20. Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample

    SciTech Connect

    Suzuki, M.T.; Rappe, M.S.; Haimberger, Z.W.

    1997-03-01

    Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods. However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases. To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample. The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library. An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the {gamma} subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%). In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the {gamma} subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%). Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases. 34 refs., 4 figs., 3 tabs.

  1. First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

    PubMed

    Colson-Proch, Cline; Morales, Anne; Hervant, Frdric; Konecny, Lara; Moulin, Colette; Douady, Christophe J

    2010-05-01

    Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms. PMID:19777376

  2. PATZ1 interacts with p53 and regulates expression of p53-target genes enhancing apoptosis or cell survival based on the cellular context

    PubMed Central

    Valentino, T; Palmieri, D; Vitiello, M; Pierantoni, G M; Fusco, A; Fedele, M

    2013-01-01

    PATZ1 is a transcriptional factor functioning either as an activator or a repressor of gene transcription depending upon the cellular context. It appears to have a dual oncogenic/anti-oncogenic activity. Indeed, it is overexpressed in colon carcinomas, and its silencing inhibits colon cancer cell proliferation or increases sensitivity to apoptotic stimuli of glioma cells, suggesting an oncogenic role. Conversely, the development of B-cell lymphomas, sarcomas, hepatocellular carcinomas and lung adenomas in Patz1-knockout (ko) mice supports its tumour suppressor function. PATZ1 role in mouse lymphomagenesis is mainly because of the involvement of PATZ1 in BCL6-negative autoregulation. However, this does not exclude that PATZ1 may be involved in tumorigenesis by other mechanisms. Here, we report that PATZ1 interacts with the tumour suppressor p53 and binds p53-dependent gene promoters, including those of BAX, CDKN1A and MDM2. Knockdown of PATZ1 in HEK293 cells reduces promoter activity of these genes and inhibits their expression, suggesting a role of PATZ in enhancing p53 transcriptional activity. Consistently, Patz1-ko mouse embryonic fibroblasts (MEFs) show decreased expression of Bax, Cdkn1a and Mdm2 compared with wild-type (wt) MEFs. Moreover, Patz1-ko MEFs show a decreased percentage of apoptotic cells, either spontaneous or induced by treatment with 5-fluorouracil (5FU), compared with wt controls, suggesting a pro-apoptotic role for PATZ1 in these cells. However, PATZ1 binds p53-target genes also independently from p53, exerting, in the absence of p53, an opposite function on their expression. Indeed, knockdown of PATZ1 in p53-null osteosarcoma cells upregulates BAX expression and decreases survival of 5FU-treated cells, then suggesting an anti-apoptotic role of PATZ1 in p53-null cancer cells. Therefore, these data support a PATZ1 tumour-suppressive function based on its ability to enhance p53-dependent transcription and apoptosis. Conversely, its opposite and anti-apoptotic role in p53-null cancer cells provides the perspective of PATZ1 silencing as a possible adjuvant in the treatment of p53-null cancer. PMID:24336083

  3. Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

    PubMed Central

    Muyan, Mesut; Gpr, Gizem; Ya?ar, Pelin; Ayaz, Gamze; User, S?rma Damla; Kazan, Hasan Hseyin; Huang, Yanfang

    2015-01-01

    Estrogen receptor ? (ER?), as a ligand-dependent transcription factor, mediates 17?-estradiol (E2) effects. ER? is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ER? dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ER?-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ER?. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ER? or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ER? or monotransactivator. Our results indicate that an activator or a repressor possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

  4. L-Arginine ameliorates cardiac left ventricular oxidative stress by upregulating eNOS and Nrf2 target genes in alloxan-induced hyperglycemic rats

    SciTech Connect

    Ramprasath, Tharmarajan; Hamenth Kumar, Palani; Syed Mohamed Puhari, Shanavas; Senthil Murugan, Ponniah; Vasudevan, Varadaraj; Selvam, Govindan Sadasivam

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer L-Arginine treatment reduced the metabolic disturbances in diabetic animals. Black-Right-Pointing-Pointer Antioxidant marker proteins were found high in myocardium by L-arginine treatment. Black-Right-Pointing-Pointer Elevated antioxidant status, mediates the reduced TBA-reactivity in left ventricle. Black-Right-Pointing-Pointer L-Arginine treatment enhanced the Nrf2 and eNOS signaling in left ventricle. Black-Right-Pointing-Pointer Improved cell survival signaling by arginine, offers a novel tactic for targeting. -- Abstract: Hyperglycemia is independently related with excessive morbidity and mortality in cardiovascular disorders. L-Arginine-nitric oxide (NO) pathway and the involvement of NO in modulating nuclear factor-E2-related factor-2 (Nrf2) signaling were well established. In the present study we investigated, whether L-arginine supplementation would improve the myocardial antioxidant defense under hyperglycemia through activation of Nrf2 signaling. Diabetes was induced by alloxan monohydrate (90 mg kg{sup -1} body weight) in rats. Both non-diabetic and diabetic group of rats were divided into three subgroups and they were administered either with L-arginine (2.25%) or L-NAME (0.01%) in drinking water for 12 days. Results showed that L-arginine treatment reduced the metabolic disturbances in diabetic rats. Antioxidant enzymes and glutathione levels were found to be increased in heart left ventricles, thereby reduction of lipid peroxidation by L-arginine treatment. Heart histopathological analysis further validates the reversal of typical diabetic characteristics consisting of alterations in myofibers and myofibrillary degeneration. qRT-PCR studies revealed that L-arginine treatment upregulated the transcription of Akt and downregulated NF-{kappa}B. Notably, transcription of eNOS and Nrf2 target genes was also upregulated, which were accompanied by enhanced expression of Nrf2 in left ventricular tissue from diabetic and control rats. Under these findings, we suggest that targeting of eNOS and Nrf2 signaling by L-arginine supplementation could be used as a potential treatment method to alleviate the late diabetic complications.

  5. Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase.

    PubMed Central

    Peleman, J; Boerjan, W; Engler, G; Seurinck, J; Botterman, J; Alliotte, T; Van Montagu, M; Inz, D

    1989-01-01

    S-Adenosylmethionine serves as a methyl group donor in numerous transmethylation reactions and plays a role in the biosynthesis of polyamines and ethylene. We have cloned and sequenced an S-adenosylmethionine synthetase gene (sam-1) of Arabidopsis thaliana. The deduced polypeptide sequence of the enzyme has extensive homology with the corresponding enzymes of Escherichia coli and yeast. Genomic hybridization indicates the presence of two adenosylmethionine synthetase genes per haploid Arabidopsis genome. RNA gel blot analysis shows that adenosylmethionine synthetase mRNA levels are high in stems and roots, correlating well with the higher enzyme activity in stems, compared with leaves. Histochemical analysis of transgenic Arabidopsis plants transformed with a chimeric beta-glucuronidase gene, under the control of 748-base pair 5' sequences of the sam-1 gene, demonstrates that the gene is expressed primarily in vascular tissues. In addition, high expression was observed in sclerenchyma and in the root cortex. A hypothesis for the strong cellular preference in the expression of the sam-1 gene is presented. PMID:2535470

  6. A Digital Framework to Build, Visualize and Analyze a Gene Expression Atlas with Cellular Resolution in Zebrafish Early Embryogenesis

    PubMed Central

    Castro-Gonzlez, Carlos; Luengo-Oroz, Miguel A.; Duloquin, Louise; Savy, Thierry; Rizzi, Barbara; Desnoulez, Sophie; Doursat, Ren; Kergosien, Yannick L.; Ledesma-Carbayo, Mara J.; Bourgine, Paul

    2014-01-01

    A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages. PMID:24945246

  7. Adipose depots differ in cellularity, adipokines produced, gene expression, and cell systems.

    PubMed

    Dodson, Michael V; Du, Min; Wang, Songbo; Bergen, Werner G; Fernyhough-Culver, Melinda; Basu, Urmila; Poulos, Sylvia P; Hausman, Gary J

    2014-01-01

    The race to manage the health concerns related to excess fat deposition has spawned a proliferation of clinical and basic research efforts to understand variables including dietary uptake, metabolism, and lipid deposition by adipocytes. A full appreciation of these variables must also include a depot-specific understanding of content and location in order to elucidate mechanisms governing cellular development and regulation of fat deposition. Because adipose tissue depots contain various cell types, differences in the cellularity among and within adipose depots are presently being documented to ascertain functional differences. This has led to the possibility of there being, within any one adipose depot, cellular distinctions that essentially result in adipose depots within depots. The papers comprising this issue will underscore numerous differences in cellularity (development, histogenesis, growth, metabolic function, regulation) of different adipose depots. Such information is useful in deciphering adipose depot involvement both in normal physiology and in pathology. Obesity, diabetes, metabolic syndrome, carcass composition of meat animals, performance of elite athletes, physiology/pathophysiology of aging, and numerous other diseases might be altered with a greater understanding of adipose depots and the cells that comprise them-including stem cells-during initial development and subsequent periods of normal/abnormal growth into senescence. Once thought to be dormant and innocuous, the adipocyte is emerging as a dynamic and influential cell and research will continue to identify complex physiologic regulation of processes involved in adipose depot physiology. PMID:26317047

  8. Adipose depots differ in cellularity, adipokines produced, gene expression, and cell systems

    PubMed Central

    Dodson, Michael V; Du, Min; Wang, Songbo; Bergen, Werner G; Fernyhough-Culver, Melinda; Basu, Urmila; Poulos, Sylvia P; Hausman, Gary J

    2014-01-01

    The race to manage the health concerns related to excess fat deposition has spawned a proliferation of clinical and basic research efforts to understand variables including dietary uptake, metabolism, and lipid deposition by adipocytes. A full appreciation of these variables must also include a depot-specific understanding of content and location in order to elucidate mechanisms governing cellular development and regulation of fat deposition. Because adipose tissue depots contain various cell types, differences in the cellularity among and within adipose depots are presently being documented to ascertain functional differences. This has led to the possibility of there being, within any one adipose depot, cellular distinctions that essentially result in adipose depots within depots. The papers comprising this issue will underscore numerous differences in cellularity (development, histogenesis, growth, metabolic function, regulation) of different adipose depots. Such information is useful in deciphering adipose depot involvement both in normal physiology and in pathology. Obesity, diabetes, metabolic syndrome, carcass composition of meat animals, performance of elite athletes, physiology/pathophysiology of aging, and numerous other diseases might be altered with a greater understanding of adipose depots and the cells that comprise them—including stem cells—during initial development and subsequent periods of normal/abnormal growth into senescence. Once thought to be dormant and innocuous, the adipocyte is emerging as a dynamic and influential cell and research will continue to identify complex physiologic regulation of processes involved in adipose depot physiology. PMID:26317047

  9. A Short Hairpin RNA Screen of Interferon-Stimulated Genes Identifies a Novel Negative Regulator of the Cellular Antiviral Response

    PubMed Central

    Li, Jianqing; Ding, Steve C.; Cho, Hyelim; Chung, Brian C.; Gale, Michael; Chanda, Sumit K.; Diamond, Michael S.

    2013-01-01

    ABSTRACT The type I interferon (IFN) signaling pathway restricts infection of many divergent families of RNA and DNA viruses by inducing hundreds of IFN-stimulated genes (ISGs), some of which have direct antiviral activity. We screened 813 short hairpin RNA (shRNA) constructs targeting 245 human ISGs using a flow cytometry approach to identify genes that modulated infection of West Nile virus (WNV) in IFN-?-treated human cells. Thirty ISGs with inhibitory effects against WNV were identified, including several novel genes that had antiviral activity against related and unrelated positive-strand RNA viruses. We also defined one ISG, activating signal cointegrator complex 3 (ASCC3), which functioned as a negative regulator of the host defense response. Silencing of ASCC3 resulted in upregulation of multiple antiviral ISGs, which correlated with inhibition of infection of several positive-strand RNA viruses. Reciprocally, ectopic expression of human ASCC3 or mouse Ascc3 resulted in downregulation of ISGs and increased viral infection. Mechanism-of-action and RNA sequencing studies revealed that ASCC3 functions to modulate ISG expression in an IRF-3- and IRF-7-dependent manner. Compared to prior ectopic ISG expression studies, our shRNA screen identified novel ISGs that restrict infection of WNV and other viruses and defined a new counterregulatory ISG, ASCC3, which tempers cell-intrinsic immunity. PMID:23781071

  10. Lymphocytes as cellular vehicles for gene therapy in mouse and man

    SciTech Connect

    Culver, K.; Cornetta, K.; Morgan, R.; Morecki, S.; Aebersold, P.; Kasid, A.; Lotze, M.; Rosenberg, S.A.; Anderson, W.F.; Blaese, R.M. )

    1991-04-15

    The application of bone marrow gene therapy has been stalled by the inability to achieve stable high-level gene transfer and expression in the totipotent stem cells. The authors that retroviral vectors can stably introduce genes into antigen-specific murine and human T lymphocytes in culture. Murine helper T cells were transduced with the retroviral vector SAX to express both neomycin-resistance and human adenosine deaminase genes. To determine if cultured T cells might be used for gene therapy, their persistence and continued expression of the introduced genes was evaluated in nude mice transplanted with the SAX-transduced T cells. They studied cultured human tumor-infiltrating lymphocytes as a candidate cell for a trial of gene transfer in man. Gene insertion and subsequent G418 selection did not substantially alter the growth characteristics, interleukin 2 dependence, membrane phenotype, or cytotoxicity profile of the transduced T cells. These studies provided a portion of the experimental evidence supporting the feasibility of the presently ongoing clinical trials of lymphocyte gene therapy in cancer as well as in patients with adenosine deaminase deficiency.

  11. A gene pathway analysis highlights the role of cellular adhesion molecules in multiple sclerosis susceptibility.

    PubMed

    Damotte, V; Guillot-Noel, L; Patsopoulos, N A; Madireddy, L; El Behi, M; De Jager, P L; Baranzini, S E; Cournu-Rebeix, I; Fontaine, B

    2014-03-01

    Genome-wide association studies (GWASs) perform per-SNP association tests to identify variants involved in disease or trait susceptibility. However, such an approach is not powerful enough to unravel genes that are not individually contributing to the disease/trait, but that may have a role in interaction with other genes as a group. Pathway analysis is an alternative way to highlight such group of genes. Using SNP association P-values from eight multiple sclerosis (MS) GWAS data sets, we performed a candidate pathway analysis for MS susceptibility by considering genes interacting in the cell adhesion molecule (CAMs) biological pathway using Cytoscape software. This network is a strong candidate, as it is involved in the crossing of the blood-brain barrier by the T cells, an early event in MS pathophysiology, and is used as an efficient therapeutic target. We drew up a list of 76 genes belonging to the CAM network. We highlighted 64 networks enriched with CAM genes with low P-values. Filtering by a percentage of CAM genes up to 50% and rejecting enriched signals mainly driven by transcription factors, we highlighted five networks associated with MS susceptibility. One of them, constituted of ITGAL, ICAM1 and ICAM3 genes, could be of interest to develop novel therapeutic targets. PMID:24430173

  12. Promoter specific sensitivity to inhibition of histone deacetylases: implications for hormonal gene control, cellular differentiation and cancer.

    PubMed

    Dressel, U; Renkawitz, R; Baniahmad, A

    2000-01-01

    Alterations in histone acetylation status appear to play a central role in the regulation of neoplasia, tumor suppression, cell cycle control, hormone responsiveness and senescence. These alterations of chromatin control gene transcription. The histone acetylation status is regulated by the equilibrium of histone acetyl-transferase activity (HAT) and the histone deacetylase activity (HDAC). Commonly, DNA-transfection assays are used to measure the effect of histone acetylation and deacetylation on gene transcription. Here we have analyzed the response of various viral long terminal repeats and vertebrate promoters to the specific histone deacetylase inhibitor trichostatin A (TSA). We show that the activity of many, but not all, promoters is increased upon TSA treatment. Interestingly, the lysozyme promoter exhibited TSA resistance, while the activity of metallothionine, the human growth hormone, and the thymidine kinase promoters was increased. Furthermore, we found that all tested viral promoters are induced by TSA. Analysis of the transcriptional behaviour of the thyroid hormone receptor (TR), the cellular homologue of the v-erbA oncogene, revealed that TSA reduced the gene silencing function but had no influence on the hormone-induced gene activation function of the receptor. These results on gene specific effects, together with the HDAC structural data (1), may be a basis for the development of HDAC inhibitors as antitumor agents. PMID:10810390

  13. Systematic analysis of multiwalled carbon nanotube-induced cellular signaling and gene expression in human small airway epithelial cells.

    PubMed

    Snyder-Talkington, Brandi N; Pacurari, Maricica; Dong, Chunlin; Leonard, Stephen S; Schwegler-Berry, Diane; Castranova, Vincent; Qian, Yong; Guo, Nancy L

    2013-05-01

    Multiwalled carbon nanotubes (MWCNT) are one of the most commonly produced nanomaterials, and pulmonary exposure during production, use, and disposal is a concern for the developing nanotechnology field. The airway epithelium is the first line of defense against inhaled particles. In a mouse model, MWCNT were reported to reach the alveolar space of the lung after in vivo exposure, penetrate the epithelial lining, and result in inflammation and progressive fibrosis. This study sought to determine the cellular and gene expression changes in small airway epithelial cells (SAEC) after in vitro exposure to MWCNT in an effort to elucidate potential toxicity mechanisms and signaling pathways. A direct interaction between SAEC and MWCNT was confirmed by both internalization of MWCNT and interaction at the cell periphery. Following exposure, SAEC showed time-dependent increases in reactive oxygen species production, total protein phosphotyrosine and phosphothreonine levels, and migratory behavior. Analysis of gene and protein expression suggested altered regulation of multiple biomarkers of lung damage, carcinogenesis, and tumor progression, as well as genes involved in related signaling pathways. These results demonstrate that MWCNT exposure resulted in the activation of SAEC. Gene expression data derived from MWCNT exposure provide information that may be used to elucidate the underlying mode of action of MWCNT in the small airway and suggest potential prognostic gene signatures for risk assessment. PMID:23377615

  14. Up-regulation of A20 gene expression in peripheral blood mononuclear cells is associated with acute-on-chronic hepatitis B liver failure.

    PubMed

    Fan, Y-C; Sun, Y-Y; Wang, N; Xiao, X-Y; Wang, K

    2016-03-01

    Aberrant immunity contributes to the pathogenesis of acute-on-chronic hepatitis B liver failure (ACHBLF), and A20 is a newly identified negative regulatory molecule of the immune response. However, no data have been reported for the role of A20 in ACHBLF. This study aimed to investigate A20 mRNA expression in ACHBLF and to determine the potential of A20 as a biomarker for the prognosis of ACHBLF. Quantitative real-time polymerase chain reaction (qPCR) was used to measure the mRNA expression of A20 in peripheral blood mononuclear cells (PBMCs) from 137 ACHBLF patients, 105 chronic hepatitis B (CHB) and 35 healthy controls (HCs). A secondary cohort with 37 ACHBLF patients was set up as validation data set. The plasma levels of interleukin (IL)-1β, IL-6 and IL-10 were determined using enzyme-linked immunosorbent assay (ELISA). Receiver-operating characteristic (ROC) curves were used to determine the predictive value of A20 for the prognosis of ACHBLF patients. A20 mRNA expression in ACHBLF was significantly higher compared with CHB and HCs. In ACHBLF patients, A20 mRNA was closely associated with total bilirubin, albumin, international normalized ratio, prothrombin time activity and model for end-stage liver disease. Furthermore, A20 mRNA was significantly correlated with IL-6 and IL-10. An optimal cut-off value of 12.32 for A20 mRNA had significant power in discriminating survival or death in ACHBLF patients. In conclusion, our results suggest that the up-regulation of the A20 gene might contribute to the severity of ACHBLF and A20 mRNA level might be a potential predictor for the prognosis of ACHBLF. PMID:26400407

  15. Aging and chronic administration of serotonin-selective reuptake inhibitor citalopram upregulate Sirt4 gene expression in the preoptic area of male mice

    PubMed Central

    Wong, Dutt Way; Soga, Tomoko; Parhar, Ishwar S.

    2015-01-01

    Sexual dysfunction and cognitive deficits are markers of the aging process. Mammalian sirtuins (SIRT), encoded by sirt 1-7 genes, are known as aging molecules which are sensitive to serotonin (5-hydroxytryptamine, 5-HT). Whether the 5-HT system regulates SIRT in the preoptic area (POA), which could affect reproduction and cognition has not been examined. Therefore, this study was designed to examine the effects of citalopram (CIT, 10 mg/kg for 4 weeks), a potent selective-serotonin reuptake inhibitor and aging on SIRT expression in the POA of male mice using real-time PCR and immunocytochemistry. Age-related increases of sirt1, sirt4, sirt5, and sirt7 mRNA levels were observed in the POA of 52 weeks old mice. Furthermore, 4 weeks of chronic CIT treatment started at 8 weeks of age also increased sirt2 and sirt4 mRNA expression in the POA. Moreover, the number of SIRT4 immuno-reactive neurons increased with aging in the medial septum area (12 weeks = 1.00 ± 0.15 vs. 36 weeks = 1.68 ± 0.14 vs. 52 weeks = 1.54 ± 0.11, p < 0.05). In contrast, the number of sirt4-immunopositive cells did not show a statistically significant change with CIT treatment, suggesting that the increase in sirt4 mRNA levels may occur in cells in which sirt4 is already being expressed. Taken together, these studies suggest that CIT treatment and the process of aging utilize the serotonergic system to up-regulate SIRT4 in the POA as a common pathway to deregulate social cognitive and reproductive functions. PMID:26442099

  16. Mangosteen leaf extract increases melanogenesis in B16F1 melanoma cells by stimulating tyrosinase activity in vitro and by up-regulating tyrosinase gene expression.

    PubMed

    Hamid, Mariani Abdul; Sarmidi, Mohamad Roji; Park, Chang Seo

    2012-02-01

    Melanin synthesis is stimulated by various effectors, including ?-melanocyte stimulating hormone (?-MSH), cyclic AMP (cAMP)-elevating agents (forskolin, isobutylmethylxantine, glycyrrhizin) and ultraviolet light. Our investigation focused on the identification of the melanogenic efficacy of mangosteen (Garcinia mangostana) leaf extract with regard to its effects on melanogenesis in B16F1 melanoma cells, since it has been known to possess strong anti-oxidant activities. The mangosteen leaf extract was found to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner without any significant effects on cell proliferation. Cytotoxicity of the extract was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; the highest concentration of the extract that did not affect cell viability was 32 g/ml. Formation of melanin from cultured B16F1 melanoma induced by extract treatment was estimated using spectrophotometry. In order to clarify the subsequent mechanism of tyrosinase activation by the extract, the levels of tyrosinase expression in B16F1 melanoma were examined using an intracellular tyrosinase assay and tyrosinase zymography. Up-regulation of intracellular tyrosinase expression seemed to correlate with an increase in microphtalmia-associated transcription factor (MITF) protein levels since MITF is the key factor for genes involved in melanogenesis. Both of the results showed that tyrosinase activity was markedly enhanced from extract-treated cells. The overall results suggest that mangosteen leaf extract may be a promising candidate for the treatment of hypopigmentation disorder and useful for self-tanning cosmetic products. PMID:22089762

  17. Fibrates and fish oil, but not corn oil, up-regulate the expression of the cholesteryl ester transfer protein (CETP) gene.

    PubMed

    Raposo, Helena F; Patrício, Patrícia R; Simões, Mariana C; Oliveira, Helena C F

    2014-06-01

    Cholesteryl ester transfer protein (CETP) is a plasma protein that reduces high density lipoprotein (HDL)-cholesterol (chol) levels and may increase atherosclerosis risk. n-3 and n-6 polyunsaturated fatty acids (PUFAs) are natural ligands, and fibrates are synthetic ligands for peroxisome proliferator activated receptor-alpha (PPARα), a transcription factor that modulates lipid metabolism. In this study, we investigated the effects of PUFA oils and fibrates on CETP expression. Hypertriglyceridemic CETP transgenic mice were treated with gemfibrozil, fenofibrate, bezafibrate or vehicle (control), and normolipidemic CETP transgenic mice were treated with fenofibrate or with fish oil (FO; n-3 PUFA rich), corn oil (CO, n-6 PUFA rich) or saline. Compared with the control treatment, only fenofibrate significantly diminished triglyceridemia (50%), whereas all fibrates decreased the HDL-chol level. Elevation of the CETP liver mRNA levels and plasma activity was observed in the fenofibrate (53%) and gemfibrozil (75%) groups. Compared with saline, FO reduced the plasma levels of nonesterified fatty acid (26%), total chol (15%) and HDL-chol (20%). Neither of the oil treatments affected the plasma triglyceride levels. Compared with saline, FO increased the plasma adiponectin level and reduced plasma leptin levels, whereas CO increased the leptin levels. FO, but not CO, significantly increased the plasma CETP mass (90%) and activity (23%) as well as increased the liver level of CETP mRNA (28%). In conclusion, fibrates and FO, but not CO, up-regulated CETP expression at both the mRNA and protein levels. We propose that these effects are mediated by the activation of PPARα, which acts on a putative PPAR response element in the CETP gene. PMID:24746832

  18. Contribution of Viral Mimics of Cellular Genes to KSHV Infection and Disease

    PubMed Central

    Sakakibara, Shuhei; Tosato, Giovanna

    2014-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV, also named Human herpesvirus 8 HHV-8) is the cause of Kaposi sarcoma (KS), the most common malignancy in HIV-infected individuals worldwide, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). KSHV is a double-stranded DNA virus that encodes several homologues of cellular proteins. The structural similarity between viral and host proteins explains why some viral homologues function as their host counterparts, but sometimes at unusual anatomical sites and inappropriate times. In other cases, structural modification in the viral proteins can suppress or override the function of the host homologue, contributing to KSHV-related diseases. For example, viral IL-6 (vIL-6) is sufficiently different from human IL-6 to activate gp130 signaling independent of the α subunit. As a consequence, vIL-6 can activate many cell types that are unresponsive to cellular IL-6, contributing to MCD disease manifestations. Here, we discuss the molecular biology of KSHV homologues of cellular products as conduits of virus/host interaction with a focus on identifying new strategies for therapy of KS and other KSHV-related diseases. PMID:25243371

  19. The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster

    PubMed Central

    Kari, Beáta; Csordás, Gábor; Honti, Viktor; Cinege, Gyöngyi; Williams, Michael J.; Andó, István; Kurucz, Éva

    2016-01-01

    Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5'-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid. PMID:26942456

  20. Cloning, expression and cellular localization of the Doublesex gene in the water flea, Daphnia carinata, during different developmental stages.

    PubMed

    Zhang, Mingqing; Li, Haixia; Liu, Ajing; Wu, Donglei; Wang, Danli; Zhao, Yunlong

    2014-10-25

    In this study, one of Doublesex genes from the common freshwater cladoceran Daphnia carinata, designated DapcaDsx1, was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). qPCR was employed to quantify differences in DapcaDsx1 expression between the different sexual phases, with expression levels being higher in sexual females. The role of DapcaDsx1 in the reproductive transformation was further investigated in parthenogenetic-phase females and sexual-phase females using whole-mount in situ hybridization. This cellular localization study showed specific expression of DapcaDsx1 in the thoracic segments, second antenna and part of the ventral carapace. Higher expression levels were exhibited in sexual females compared to parthenogenetic females. This suggests that the DapcaDsx1 gene plays significant roles in switching modes of reproduction and during sexual differentiation. PMID:25130908

  1. Intercellular communication: relative importance of cellular adhesion and paracrine signaling to hormonal gene expression.

    PubMed

    Abraham, E J; Faught, W J; Frawley, L S

    1996-09-01

    Although there is a consensus that a cell's microenvironment can have a dramatic influence on its ability to express a particular gene, the relative contribution of physical interaction (cell to cell adhesion) and paracrine signaling to this phenomenon has been difficult to discern. Here, we addressed this problem in mammotropes by making "real-time" measurements of prolactin (PRL) gene expression followed by immunocytochemistry (for post facto identification of a neighbor's phenotype). Our results show that it is the nature (phenotype) rather than the physical presence of a neighboring cell that dictates the degree to which the PRL gene is expressed. PMID:8756583

  2. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

    PubMed Central

    Kowalewski-Nimmerfall, Elisabeth; Schähs, Philipp; Maresch, Daniel; Rendic, Dubravko; Krämer, Helmut; Mach, Lukas

    2014-01-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of > 95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development. PMID:25173815

  3. TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways

    PubMed Central

    De Conti, Laura; Akinyi, Maureen V.; Mendoza-Maldonado, Ramiro; Romano, Maurizio; Baralle, Marco; Buratti, Emanuele

    2015-01-01

    In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile. PMID:26261209

  4. Dysregulation of host cellular genes targeted by human papillomavirus (HPV) integration contributes to HPV-related cervical carcinogenesis.

    PubMed

    Zhang, Ruiyang; Shen, Congle; Zhao, Lijun; Wang, Jianliu; McCrae, Malcolm; Chen, Xiangmei; Lu, Fengmin

    2016-03-01

    Integration of human papillomavirus (HPV) viral DNA into the human genome has been postulated as an important etiological event during cervical carcinogenesis. Several recent reports suggested a possible role for such integration-targeted cellular genes (ITGs) in cervical carcinogenesis. Therefore, a comprehensive analysis of HPV integration events was undertaken using data collected from 14 publications, with 499 integration loci on human chromosomes included. It revealed that HPV DNA preferred to integrate into intragenic regions and gene-dense regions of human chromosomes. Intriguingly, the host cellular genes nearby the integration sites were found to be more transcriptionally active compared with control. Furthermore, analysis of the integration sites in the human genome revealed that there were several integration hotspots although all chromosomes were represented. The ITGs identified were found to be enriched in tumor-related terms and pathways using gene ontology and KEGG analysis. In line with this, three of six ITGs tested were found aberrantly expressed in cervical cancer tissues. Among them, it was demonstrated for the first time that MPPED2 could induce HeLa cell and SiHa cell G1/S transition block and cell proliferation retardation. Moreover, "knocking out" the integrated HPV fragment in HeLa cell line decreased expression of MYC located ∼500 kb downstream of the integration site, which provided the first experimental evidence supporting the hypothesis that integrated HPV fragment influence MYC expression via long distance chromatin interaction. Overall, the results of this comprehensive analysis implicated that dysregulation of ITGs caused by viral integration as possibly having an etiological involvement in cervical carcinogenesis. PMID:26417997

  5. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    PubMed Central

    Rallis, Charalampos; López-Maury, Luis; Georgescu, Teodora; Pancaldi, Vera; Bähler, Jürg

    2014-01-01

    Summary Target of rapamycin complex 1 (TORC1), which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not. PMID:24463365

  6. MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation.

    PubMed

    Siengdee, Puntita; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2. PMID:26010876

  7. MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

    PubMed Central

    Siengdee, Puntita; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2. PMID:26010876

  8. Expression of lipogenic genes is upregulated in the heart with exercise training-induced but not pressure overload-induced left ventricular hypertrophy.

    PubMed

    Dobrzyn, Pawel; Pyrkowska, Aleksandra; Duda, Monika K; Bednarski, Tomasz; Maczewski, Michal; Langfort, Jozef; Dobrzyn, Agnieszka

    2013-06-15

    Cardiac hypertrophy is accompanied by molecular remodeling that affects different cellular pathways, including fatty acid (FA) utilization. In the present study, we show that cardiac lipid metabolism is differentially regulated in response to physiological (endurance training) and pathological [abdominal aortic banding (AAB)] hypertrophic stimuli. Physiological hypertrophy was accompanied by an increased expression of lipogenic genes and the activation of sterol regulatory element-binding protein-1c and Akt signaling. Additionally, FA oxidation pathways regulated by AMP-activated protein kinase (AMPK) and peroxisome proliferator activated receptor-α (PPARα) were induced in trained hearts. Cardiac lipid content was not changed by physiological stimulation, underlining balanced lipid utilization in the trained heart. Moreover, pathological hypertrophy induced the AMPK-regulated oxidative pathway, whereas PPARα and expression of its downstream targets, i.e., acyl-CoA oxidase and carnitine palmitoyltransferase I, were not affected by AAB. In contrast, pathological hypertrophy leads to cardiac triglyceride (TG) and diacylglycerol (DAG) accumulation, although the expression of lipogenic genes and the levels of FA transport proteins (CD36 and FATP) were not changed or reduced compared with the sham group. A possible explanation for this phenomenon is a decrease in lipolysis, as evidenced by the increased content of adipose triglyceride lipase inhibitor G0S2, the increased phosphorylation of hormone-sensitive lipase at Ser(565), and the decreased protein levels of DAG lipase that attenuate TG and DAG contents. The increased TG and DAG accumulation observed in AAB-induced hypertrophy might have lipotoxic effects, thereby predisposing to cardiomyopathy and heart failure in the future. PMID:23632628

  9. PpCBF3 from Cold-Tolerant Kentucky Bluegrass Involved in Freezing Tolerance Associated with Up-Regulation of Cold-Related Genes in Transgenic Arabidopsis thaliana

    PubMed Central

    Chen, Yu; Xu, Bin; Yang, Zhimin; Huang, Bingru

    2015-01-01

    Dehydration-Responsive Element Binding proteins (DREB)/C-repeat (CRT) Binding Factors (CBF) have been identified as transcriptional activators during plant responses to cold stress. The objective of this study was to determine the physiological roles of a CBF gene isolated from a cold-tolerant perennial grass species, Kentucky bluegrass (Poa pratensis L.), which designated as PpCBF3, in regulating plant tolerance to freezing stress. Transient transformation of Arabidopsis thaliana mesophyll protoplast with PpCBF3-eGFP fused protein showed that PpCBF3 was localized to the nucleus. RT-PCR analysis showed that PpCBF3 was specifically induced by cold stress (4°C) but not by drought stress [induced by 20% polyethylene glycol 6000 solution (PEG-6000)] or salt stress (150 mM NaCl). Transgenic Arabidopsis overexpressing PpCBF3 showed significant improvement in freezing (-20°C) tolerance demonstrated by a lower percentage of chlorotic leaves, lower cellular electrolyte leakage (EL) and H2O2 and O2.- content, and higher chlorophyll content and photochemical efficiency compared to the wild type. Relative mRNA expression level analysis by qRT-PCR indicated that the improved freezing tolerance of transgenic Arabidopsis plants overexpressing PpCBF3 was conferred by sustained activation of downstream cold responsive (COR) genes. Other interesting phenotypic changes in the PpCBF3-transgenic Arabidopsis plants included late flowering and slow growth or ‘dwarfism’, both of which are desirable phenotypic traits for perennial turfgrasses. Therefore, PpCBF3 has potential to be used in genetic engineering for improvement of turfgrass freezing tolerance and other desirable traits. PMID:26177510

  10. Laf4/Aff3, a Gene Involved in Intellectual Disability, Is Required for Cellular Migration in the Mouse Cerebral Cortex

    PubMed Central

    Lee, Sheena; Lickiss, Tom; Molnr, Zoltn; Davies, Kay E.

    2014-01-01

    Members of the AFF (AF4/FMR2) family of putative transcription factors are involved in infant acute leukaemia and intellectual disability (ID), although very little is known about their transcriptional targets. For example, deletion of human lymphoid nuclear protein related to AF4/AFF member 3 (LAF4/AFF3) is known to cause severe neurodevelopmental defects, and silencing of the gene is also associated with ID at the folate-sensitive fragile site (FSFS) FRA2A; yet the normal function of this gene in the nervous system is unclear. The aim of this study was to further investigate the function of Laf4 in the brain by focusing on its role in the cortex. By manipulating expression levels in organotypic slices, we demonstrate here that Laf4 is required for normal cellular migration in the developing cortex and have subsequently identified Mdga2, an important structural protein in neurodevelopment, as a target of Laf4 transcriptional activity. Furthermore, we show that the migration deficit caused by loss of Laf4 can be partially rescued by Mdga2 over-expression, revealing an important functional relationship between these genes. Our study demonstrates the key transcriptional role of Laf4 during early brain development and reveals a novel function for the gene in the process of cortical cell migration relevant to the haploinsufficiency and silencing observed in human neurodevelopmental disorders. PMID:25162227

  11. Intradermal Gene Immunization: The Possible Role of DNA Uptake in the Induction of Cellular Immunity to Viruses

    NASA Astrophysics Data System (ADS)

    Raz, Eyal; Carson, Dennis A.; Parker, Suezanne E.; Parr, Tyler B.; Abai, Anna M.; Aichinger, Gerald; Gromkowski, Stanislaw H.; Singh, Malini; Lew, Denise; Yankauckas, Michelle A.; Baird, Stephen M.; Rhodes, Gary H.

    1994-09-01

    The skin and mucous membranes are the anatomical sites where most viruses are first encountered by the immune system. Previous experiments have suggested that striated muscle cells are unique among mammalian cell types in their capacity to take up and express free DNA in the absence of a viral vector or physical carrier. However, we have found that mice injected into the superficial skin with free (naked) plasmid DNA encoding the influenza nucleoprotein gene had discrete foci of epidermal and dermal cells, including cells with dendritic morphology, that contained immunoreactive nucleoprotein antigen. A single intradermal administration of 0.3-15 ? g of free plasmid DNA induced anti-nucleoprotein-specific antibody and cytotoxic T lymphocytes that persisted for at least 68-70 weeks after vaccination. Intradermal gene administration induced higher antibody titers than did direct gene injection into skeletal muscle and did not cause local inflammation or necrosis. Compared with control animals, the gene-injected mice were resistant to challenge with a heterologous strain of influenza virus. These results indicate that the cells of the skin can take up and express free foreign DNA and induce cellular and humoral immune responses against the encoded protein. We suggest that DNA uptake by the skin-associated lymphoid tissues may play a role in the induction of cytotoxic T cells against viruses and other intracellular pathogens.

  12. Mechanisms of action of acetaldehyde in the up-regulation of the human ?2(I) collagen gene in hepatic stellate cells: key roles of Ski, SMAD3, SMAD4, and SMAD7.

    PubMed

    Reyes-Gordillo, Karina; Shah, Ruchi; Arellanes-Robledo, Jaime; Hernndez-Nazara, Zamira; Rincn-Snchez, Ana Rosa; Inagaki, Yutaka; Rojkind, Marcos; Lakshman, M Raj

    2014-05-01

    Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human ?2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties. PMID:24641900

  13. The EHV-1 UL4 protein that tempers viral gene expression interacts with cellular transcription factors.

    PubMed

    Zhang, Yunfei; Charvat, Robert A; Kim, Seong K; O'Callaghan, Dennis J

    2014-01-20

    The UL4 gene is conserved within the genome of defective interfering particles of equine herpesvirus type 1 (EHV-1) that mediate persistent infection. Here, we show that the UL4 protein inhibits EHV-1 reporter gene expression by decreasing the level of transcribed mRNA. The UL4 protein did not bind any gene class of EHV-1 promoters in electromobility or chromatin immunoprecipitation assays, but directly interacted with the TATA box-binding protein (TBP) and the carboxy-terminal domain of RNA polymerase II both in vitro (GST-pulldown assays) and in infected cells (coimmunoprecipitation analyses). Microarray analyses of the expression of the 78 EHV-1 genes revealed that viral late genes important for virion assembly displayed enhanced expression in cells infected with UL4-null virus as compared to wild-type or UL4-restored EHV-1. Quantitative PCR analyses showed that viral DNA replication was not retarded in cells infected with the UL4-null virus as compared to wild-type EHV-1. PMID:24418534

  14. PROX1 Gene is Differentially Expressed in Oral Cancer and Reduces Cellular Proliferation

    PubMed Central

    Rodrigues, Maria F.S.D.; de Oliveira Rodini, Camila; de Aquino Xavier, Flvia C.; Paiva, Katicia B.; Severino, Patrcia; Moyses, Raquel A.; Lpez, Rossana M.; DeCicco, Rafael; Rocha, Llia A.; Carvalho, Marcos B.; Tajara, Eloiza H.; Nunes, Fabio D.

    2014-01-01

    Abstract Homeobox genes are a family of transcription factors that play a pivotal role in embryogenesis. Prospero homeobox 1 (PROX1) has been shown to function as a tumor suppressor gene or oncogene in various types of cancer, including oral squamous cell carcinoma (OSCC). We have previously identified PROX1 as a downregulated gene in OSCC. The aim of this study is to clarify the underlying mechanism by which PROX1 regulates tumorigenicity of OSCC cells. PROX1 mRNA and protein expression levels were first investigated in 40 samples of OSCC and in nontumor margins. Methylation and amplification analysis was also performed to assess the epigenetic and genetic mechanisms involved in controlling PROX1 expression. OSCC cell line SCC9 was also transfected to stably express the PROX1 gene. Next, SCC9-PROX1-overexpressing cells and controls were subjected to proliferation, differentiation, apoptosis, migration, and invasion assays in vitro. OSCC samples showed reduced PROX1 expression levels compared with nontumor margins. PROX1 amplification was associated with better overall survival. PROX1 overexpression reduces cell proliferation and downregulates cyclin D1. PROX1-overexpressing cells also exhibited reduced CK18 and CK19 expression and transcriptionally altered the expression of WISP3, GATA3, NOTCH1, and E2F1. Our results suggest that PROX1 functions as a tumor suppressor gene in oral carcinogenesis. PMID:25526434

  15. High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition (ionome) of yeast Saccharomyces cerevisiae. Using inductively coupled...

  16. A gene involved in control of human cellular senescence on human chromosome 1q

    SciTech Connect

    Hensler, P.J.; Pereira-Smith, O.M. ); Annab, L.A.; Barrett, J.C. )

    1994-04-01

    Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, the authors introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras[sup +]-transformed derivative of TE85 (143B TK[sup [minus

  17. Re-engineering cellular physiology by rewiring high-level global regulatory genes

    PubMed Central

    Fitzgerald, Stephen; Dillon, Shane C.; Chao, Tzu-Chiao; Wiencko, Heather L.; Hokamp, Karsten; Cameron, Andrew D. S.; Dorman, Charles J.

    2015-01-01

    Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism. PMID:26631971

  18. Re-engineering cellular physiology by rewiring high-level global regulatory genes.

    PubMed

    Fitzgerald, Stephen; Dillon, Shane C; Chao, Tzu-Chiao; Wiencko, Heather L; Hokamp, Karsten; Cameron, Andrew D S; Dorman, Charles J

    2015-01-01

    Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism. PMID:26631971

  19. Cellular Localization of PRL-1 and PRL-2 Gene Expression in Normal Adult Human Tissues

    PubMed Central

    Dumaual, Carmen M.; Sandusky, George E.; Crowell, Pamela L.; Randall, Stephen K.

    2006-01-01

    Recent evidence suggests that the PRL-1 and ?2 phosphatases may be multifunctional enzymes with diverse roles in a variety of tissue and cell types. Northern blotting has previously shown widespread expression of both transcripts; however, little is known about the cell type-specific expression of either gene, especially in human tissues. Therefore, we investigated expression patterns for PRL-1 and ?2 genes in multiple normal, adult human tissues using in situ hybridization. Although both transcripts were ubiquitously expressed, they exhibited strikingly different patterns of expression. PRL-2 was expressed heavily in almost every tissue and cell type examined, whereas PRL-1 expression levels varied considerably both between tissue types and between individuals. Widespread expression of PRL-1 and ?2 in multiple organ systems suggests an important functional role for these enzymes in normal tissue homeostasis. In addition, the variable patterns of expression for these genes may provide distinct activities in each tissue or cell type. PMID:16957164

  20. A single gene change can extend yeast life span: the role of Ras in cellular senescence.

    PubMed

    Jazwinski, S M; Chen, J B; Sun, J

    1993-01-01

    The budding yeast Saccharomyces cerevisiae has a limited life span (reproductive capacity), which is measured by the number of times an individual cell divides. There is evidence for the involvement of a senescence factor that affects cell cycle traversal in older yeast cells. Distinct alterations in the abundance of a handful of transcripts have been identified during the life span of this organism, and the genes that specify these mRNAs have been cloned. This raises the question whether the activity of one or more genes can alter the yeast life span. Indeed, the controlled expression of the transforming gene of Harvey murine sarcoma virus (v-Ha-ras) was found to extend the life span nearly two-fold. The normal homologs of this oncogene, RAS1 and RAS2, play a central role in the integration of cell growth and the cell cycle in yeast. Expression of v-Ha-ras appears to impinge on this integration. We suggest that it is the relative levels of the senescence factor and the Ras protein that determine whether a cell ceases to divide and senesces. We liken the senescence factor to the product of an anti-oncogene or tumor suppressor gene that neutralizes Ras. PMID:8368142

  1. Expression of Novel Gene Products Upregulated by Disuse is Normalized by an Osteogenic Mechanical Stimulus: Evidence for the Molecular Basis of a Low Level Biomechanical Countermeasure for Osteoporosis?

    NASA Technical Reports Server (NTRS)

    Rubin, C.; Zhi, J.; Xu, G.; Cute, M.; McLeod, K.; Hadjiargyrou, M.

    1999-01-01

    The National Research Council's report entitled: A Strategy for Space Biology and Medical Science, highlighted several areas of fundamental scientific investigation which must be addressed to make long-term space exploration not only feasible, but safe. This "Goldberg Strategy," as well as several subsequent reports published by the NRC's Space Studies Board (e.g., Assessment of Programs in Space Biology and Medicine, Smith et. al., 1991), suggests that the principal hurdle to man's extended presence in space is the osteopenia which parallels reduced gravity. Ironically, the most significant risk to the skeleton may only be realized on return to normal gravitational fields, and full recovery of bone mass may never occur. Effective counter-measures to this microgravity induced bone loss are thus essential. Considering the similarities of space and aging induced osteopenia, an indisputable benefit of such a prophylaxis would be its potential as a treatment for the bone loss which plagues over 25 million people in the U.S. The osteogenic potential of mechanical strain is strongly frequency dependent, with sensitivity increasing up through at least 60 Hz (cycles per second). One hundred seconds per day of a 1 Hz cyclic loading will inhibit disuse osteopenia only if sufficient in magnitude to engender 1000 microstrain (mu(epsilon)) in the tissue. When loading is applied at 30 Hz, however, mechanical strains on the order of 5O mu(epsilon) (approx. 1% of the peak strains which occur in bone during vigorous functional activity), can stimulate bone formation in a duration dependent manner. In longer term animal studies, strains of less than 10 mu(epsilon), induced non-invasively via a whole body vibration, will stimulate bone formation on the surfaces of trabeculae, increase bone density, and improve strength. Finally, preliminary results from a double blind prospective clinical trial shows promise in inhibiting the bone loss which parallels the menopause. Based on these observations, we propose that these high frequency, low magnitude, mechanical strains effectively serve as a "surrogate" for musculoskeletal ground reaction forces, and thus represent an ideal countermeasure to the osteopenia which parallels microgravity conditions. The specific goal of this NASA funded work is to identify genes in bone upregulated by disuse, and to determine the efficacy of an osteogenic mechanical stimulus to downregulate their expression.

  2. Genomic mapping and cellular expression of human CPG2 transcripts in the SYNE1 gene.

    PubMed

    Loebrich, Sven; Rathje, Mette; Hager, Emily; Ataman, Bulent; Harmin, David A; Greenberg, Michael E; Nedivi, Elly

    2016-03-01

    Bipolar disorder (BD) is a prevalent and severe mood disorder characterized by recurrent episodes of mania and depression. Both genetic and environmental factors have been implicated in BD etiology, but the biological underpinnings remain elusive. Recent genome-wide association studies (GWAS) for identifying genes conferring risk for schizophrenia, BD, and major depression, identified an association between single-nucleotide polymorphisms (SNPs) in the SYNE1 gene and increased risk of BD. SYNE1 has also been identified as a risk locus for multiple other neurological or neuromuscular genetic disorders. The BD associated SNPs map within the gene region homologous to part of rat Syne1 encompassing the brain specific transcripts encoding CPG2, a postsynaptic neuronal protein localized to excitatory synapses and an important regulator of glutamate receptor internalization. Here, we use RNA-seq, ChIP-seq and RACE to map the human SYNE1 transcriptome, focusing on the CPG2 locus. We validate several CPG2 transcripts, including ones not previously annotated in public databases, and identify and clone a full-length CPG2 cDNA expressed in human neocortex, hippocampus and striatum. Using lenti-viral gene knock down/replacement and surface receptor internalization assays, we demonstrate that human CPG2 protein localizes to dendritic spines in rat hippocampal neurons and is functionally equivalent to rat CPG2 in regulating glutamate receptor internalization. This study provides a valuable gene-mapping framework for relating multiple genetic disease loci in SYNE1 with their transcripts, and for evaluating the effects of missense SNPs identified by patient genome sequencing on neuronal function. PMID:26704904

  3. Glucocorticoids Regulate Gene Expression and Repress Cellular Proliferation in Human Uterine Leiomyoma Cells

    PubMed Central

    Whirledge, Shannon; Dixon, Darlene

    2016-01-01

    Sex hormones and growth factors have been implicated in the pathogenesis of uterine leiomyomas. The uterus is also an abundant source of the glucocorticoid receptor but its role and function have been largely ignored. Human samples of uterine leiomyomas and matched myometrium retain expression of the glucocorticoid receptor (GR) suggesting a potential role for GR in leiomyoma function. However, hormone responsive gene expression varies between normal myometrium and leiomyoma cells. We now employ genome-wide microarray studies comparing glucocorticoid and estrogen-treated human uterine leiomyoma cells to those treated with both steroids to identify the potential role of glucocorticoids in uterine leiomyoma cells. Treatment with the synthetic glucocorticoid dexamethasone (Dex) regulated 3,128 probes. Estrogen (E2) treatment identified 2,094 probes, and in the presence of both hormones, 4,626 probes were regulated. Of the 552 probes identified, the majority of genes co-regulated by Dex, E2, and Dex+E2 exhibited co-downregulation. Interestingly, a small group of 17 genes displayed antagonistic regulation by Dex and E2, where all genes in this group, Dex reversed the E2 effect with. Ingenuity Pathway Analysis of the data identified cell growth, development, and differentiation as significant glucocorticoid regulated pathways. Flow cytometry confirmed that glucocorticoids regulated cell proliferation and significantly reduced the percentage of S-phase cells either in the presence or absence of estrogen in leiomyomas but not smooth muscle cells. Translation of our results suggest that glucocorticoids may play a significant role in regulating uterine leiomyoma gene expression and cell growth, and thus may have implications for therapeutic development of uterine leiomyoma treatment. PMID:22311344

  4. Upregulation of orphan nuclear receptor Nur77 following PGF2?, Bimatoprost, and Butaprost treatments. Essential role of a protein kinase C pathway involved in EP2 receptor activated Nur77 gene transcription

    PubMed Central

    Liang, Yanbin; Li, Chen; Guzman, Victor M; Chang, William W; Evinger, Albert J; Pablo, Jozelyn V; Woodward, David F

    2004-01-01

    Using gene chip technology, we first identified that PGF2? (FP agonist) and Butaprost (EP2 agonist) induced about a five-fold upregulation of Nur77 mRNA expression in hFP-HEK 293/EBNA and hEP2-HEK293/EBNA cells. Northern Blot analysis revealed that PGF2?- and Butaprost-induced upregulation of Nur77 expression are dose- and time-dependent. Both PGF2? and Butaprost upregulated Nur77 gene expression through the protein kinase C (PKC) pathway. These data are the first showing a link between EP2 receptor stimulation and protein kinase C activation. Calcineurin was found to be involved downstream of the PKC pathway in PGF2?-induced Nur77 expression, but not in Butaprost-induced Nur77 expression. We also used Nur77 as a marker gene to compare the effects of PGF2?, Butaprost, and Bimatoprost (a prostamide) on Nur77 expression in human primary trabecular meshwork and ciliary smooth muscle (SM) cells, which are target cells for antiglaucoma drugs. The results showed that PGF2? and Butaprost, but not Bimatoprost, induced upregulation of Nur77 expression in human TM cells. PGF2?, but not Bimatoprost, dramatically induced upregulation of Nur77 mRNA expression in human ciliary SM cells, whereas Butaprost slightly upregulated Nur77 mRNA expression in SM cells. Nur77 promoter deletion analysis indicated that PGF2?, but not Bimatoprost, activated Nur77 promoter-luciferase reporter in hFP-HEK 293/EBNA cells. Butaprost was less efficacious in inducing Nur77 promoter-luciferase reporter activity in hEP2-HEK293/EBNA cells relative to PGF2? in the comparable assay. The data for Nur77 promoter functional analysis were matched to the Northern blot analysis. It appears that PGF2? and Butaprost activate Nur77 transcription mechanisms through the activation of FP and EP2 receptor-coupled signaling pathways, whereas Bimatoprost stimulates neither FP nor EP2 receptors. PMID:15159280

  5. fra-1: a serum-inducible, cellular immediate-early gene that encodes a fos-related antigen.

    PubMed Central

    Cohen, D R; Curran, T

    1988-01-01

    A set of proteins antigenically related to the c-fos protein (Fos) are induced by serum in fibroblasts. To isolate cDNA clones of genes encoding such proteins, a lambda gt11 expression cDNA library constructed from serum-stimulated rat fibroblasts was screened with antibodies raised against a hydrophilic region (amino acids 127 to 152) of Fos. One of the positive clones identified, termed fra-1 (Fos-related antigen) was characterized. It encoded a protein that shared several regions of extensive amino acid homology with Fos (including the region that showed similarity to both the yeast GCN4 regulatory protein and the protein encoded by the jun oncogene), although its nucleotide sequence was considerably diverged from that of the c-fos gene. Only a subset of the agents and conditions that activated c-fos also induced fra-1. Induction of fra-1 expression following serum stimulation was delayed compared with that of c-fos. However, like c-fos, fra-1 was induced rapidly by serum in the presence of protein synthesis inhibitors. Thus, a family of Fos-related, inducible genes are involved in the cellular immediate-early transcriptional response to extracellular stimuli. Images PMID:3133553

  6. Digital Encoding of Cellular mRNAs Enabling Precise and Absolute Gene Expression Measurement by Single-Molecule Counting

    PubMed Central

    2014-01-01

    We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification. PMID:24579851

  7. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease.

    PubMed

    lvarez, Enrique; Castell, Alfredo; Carrasco, Luis; Izquierdo, Jos M

    2011-10-14

    Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A(pro) modulating the alternative splicing of pre-mRNAs. Expression of 2A(pro) potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A(pro) abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A(pro), leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A(pro) on splicing is to selectively block the second catalytic step. PMID:21945619

  8. Important step in radiation carcinogenesis may be inactivation of cellular genes

    SciTech Connect

    Weichselbaum, R.R.; Beckett, M.A.; Diamond, A.A.

    1989-01-01

    The loss of genetic material may result in a predisposition to malignant disease. The best studied example is retinoblastoma where deletion or transcriptional inactivation of a specific gene is associated with the development of the tumor. When hereditary retinoblastoma patients are treated with radiation, the incidence of osteosarcoma within the treatment field is extremely high compared to other cancer patients treated with radiotherapy. These data, together with cytogenetic and molecular data on the development of acute non-lymphocytic leukemia secondary to radiotherapy and chemotherapy treatment suggest that radiation-induced deletions of critical DNA sequences may be an important event in radiation carcinogenesis. Therefore, we propose that radiation-induced tumors may result from deletion of tissue specific regulatory genes. Base alterations caused by radiation in dominantly transforming oncogenes may also contribute to radiation carcinogenesis.62 references.

  9. Effect of passage number on cellular response DNA-damaging agents: cell survival and gene expression

    SciTech Connect

    Chang-Liu, Chin-Mei; Wolschak, G.E.

    1996-03-01

    The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or {gamma}-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to {sup 60}Co {gamma} rays or 254-m UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a ``crisis`` period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant- than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of {gamma}-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following {gamma}-ray exposure of the intermediate (passage 45) epithelial cells. Differential display, however, revealed changes in expression of several transcripts following exposure to ionizing and ultraviolet radiations. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

  10. Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington's disease

    PubMed Central

    Czeredys, Magdalena; Gruszczynska-Biegala, Joanna; Schacht, Teresa; Methner, Axel; Kuznicki, Jacek

    2013-01-01

    Huntington's disease (HD) is a hereditary neurodegenerative disease caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein and characterized by dysregulated calcium homeostasis. We investigated whether these disturbances are correlated with changes in the mRNA level of the genes that encode proteins involved in calcium homeostasis and signaling (i.e., the calciosome). Using custom-made TaqMan low-density arrays containing probes for 96 genes, we quantified mRNA in the striatum in YAC128 mice, a model of HD, and wildtype mice. HTT mutation caused the increased expression of some components of the calcium signalosome, including calretinin, presenilin 2, and calmyrin 1, and the increased expression of genes indirectly involved in calcium homeostasis, such as huntingtin-associated protein 1 and calcyclin-binding protein. To verify these findings in a different model, we used PC12 cells with an inducible expression of mutated full-length HTT. Using single-cell imaging with Fura-2AM, we found that store-operated Ca2+ entry but not endoplasmic reticulum (ER) store content was changed as a result of the expression of mutant HTT. Statistically significant downregulation of the Orai calcium channel subunit 2, calmodulin, and septin 4 was detected in cells that expressed mutated HTT. Our data indicate that the dysregulation of calcium homeostasis correlates with changes in the gene expression of members of the calciosome. These changes, however, differed in the two models of HD used in this study. Our results indicate that each HD model exhibits distinct features that may only partially resemble the human disease. PMID:24324398

  11. Convergence of genes and cellular pathways dysregulated in autism spectrum disorders.

    PubMed

    Pinto, Dalila; Delaby, Elsa; Merico, Daniele; Barbosa, Mafalda; Merikangas, Alison; Klei, Lambertus; Thiruvahindrapuram, Bhooma; Xu, Xiao; Ziman, Robert; Wang, Zhuozhi; Vorstman, Jacob A S; Thompson, Ann; Regan, Regina; Pilorge, Marion; Pellecchia, Giovanna; Pagnamenta, Alistair T; Oliveira, Bárbara; Marshall, Christian R; Magalhaes, Tiago R; Lowe, Jennifer K; Howe, Jennifer L; Griswold, Anthony J; Gilbert, John; Duketis, Eftichia; Dombroski, Beth A; De Jonge, Maretha V; Cuccaro, Michael; Crawford, Emily L; Correia, Catarina T; Conroy, Judith; Conceição, Inês C; Chiocchetti, Andreas G; Casey, Jillian P; Cai, Guiqing; Cabrol, Christelle; Bolshakova, Nadia; Bacchelli, Elena; Anney, Richard; Gallinger, Steven; Cotterchio, Michelle; Casey, Graham; Zwaigenbaum, Lonnie; Wittemeyer, Kerstin; Wing, Kirsty; Wallace, Simon; van Engeland, Herman; Tryfon, Ana; Thomson, Susanne; Soorya, Latha; Rogé, Bernadette; Roberts, Wendy; Poustka, Fritz; Mouga, Susana; Minshew, Nancy; McInnes, L Alison; McGrew, Susan G; Lord, Catherine; Leboyer, Marion; Le Couteur, Ann S; Kolevzon, Alexander; Jiménez González, Patricia; Jacob, Suma; Holt, Richard; Guter, Stephen; Green, Jonathan; Green, Andrew; Gillberg, Christopher; Fernandez, Bridget A; Duque, Frederico; Delorme, Richard; Dawson, Geraldine; Chaste, Pauline; Café, Cátia; Brennan, Sean; Bourgeron, Thomas; Bolton, Patrick F; Bölte, Sven; Bernier, Raphael; Baird, Gillian; Bailey, Anthony J; Anagnostou, Evdokia; Almeida, Joana; Wijsman, Ellen M; Vieland, Veronica J; Vicente, Astrid M; Schellenberg, Gerard D; Pericak-Vance, Margaret; Paterson, Andrew D; Parr, Jeremy R; Oliveira, Guiomar; Nurnberger, John I; Monaco, Anthony P; Maestrini, Elena; Klauck, Sabine M; Hakonarson, Hakon; Haines, Jonathan L; Geschwind, Daniel H; Freitag, Christine M; Folstein, Susan E; Ennis, Sean; Coon, Hilary; Battaglia, Agatino; Szatmari, Peter; Sutcliffe, James S; Hallmayer, Joachim; Gill, Michael; Cook, Edwin H; Buxbaum, Joseph D; Devlin, Bernie; Gallagher, Louise; Betancur, Catalina; Scherer, Stephen W

    2014-05-01

    Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10(-5)) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10(-15), ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation. PMID:24768552

  12. Convergence of Genes and Cellular Pathways Dysregulated in Autism Spectrum Disorders

    PubMed Central

    Pinto, Dalila; Delaby, Elsa; Merico, Daniele; Barbosa, Mafalda; Merikangas, Alison; Klei, Lambertus; Thiruvahindrapuram, Bhooma; Xu, Xiao; Ziman, Robert; Wang, Zhuozhi; Vorstman, Jacob A.S.; Thompson, Ann; Regan, Regina; Pilorge, Marion; Pellecchia, Giovanna; Pagnamenta, Alistair T.; Oliveira, Bárbara; Marshall, Christian R.; Magalhaes, Tiago R.; Lowe, Jennifer K.; Howe, Jennifer L.; Griswold, Anthony J.; Gilbert, John; Duketis, Eftichia; Dombroski, Beth A.; De Jonge, Maretha V.; Cuccaro, Michael; Crawford, Emily L.; Correia, Catarina T.; Conroy, Judith; Conceição, Inês C.; Chiocchetti, Andreas G.; Casey, Jillian P.; Cai, Guiqing; Cabrol, Christelle; Bolshakova, Nadia; Bacchelli, Elena; Anney, Richard; Gallinger, Steven; Cotterchio, Michelle; Casey, Graham; Zwaigenbaum, Lonnie; Wittemeyer, Kerstin; Wing, Kirsty; Wallace, Simon; van Engeland, Herman; Tryfon, Ana; Thomson, Susanne; Soorya, Latha; Rogé, Bernadette; Roberts, Wendy; Poustka, Fritz; Mouga, Susana; Minshew, Nancy; McInnes, L. Alison; McGrew, Susan G.; Lord, Catherine; Leboyer, Marion; Le Couteur, Ann S.; Kolevzon, Alexander; Jiménez González, Patricia; Jacob, Suma; Holt, Richard; Guter, Stephen; Green, Jonathan; Green, Andrew; Gillberg, Christopher; Fernandez, Bridget A.; Duque, Frederico; Delorme, Richard; Dawson, Geraldine; Chaste, Pauline; Café, Cátia; Brennan, Sean; Bourgeron, Thomas; Bolton, Patrick F.; Bölte, Sven; Bernier, Raphael; Baird, Gillian; Bailey, Anthony J.; Anagnostou, Evdokia; Almeida, Joana; Wijsman, Ellen M.; Vieland, Veronica J.; Vicente, Astrid M.; Schellenberg, Gerard D.; Pericak-Vance, Margaret; Paterson, Andrew D.; Parr, Jeremy R.; Oliveira, Guiomar; Nurnberger, John I.; Monaco, Anthony P.; Maestrini, Elena; Klauck, Sabine M.; Hakonarson, Hakon; Haines, Jonathan L.; Geschwind, Daniel H.; Freitag, Christine M.; Folstein, Susan E.; Ennis, Sean; Coon, Hilary; Battaglia, Agatino; Szatmari, Peter; Sutcliffe, James S.; Hallmayer, Joachim; Gill, Michael; Cook, Edwin H.; Buxbaum, Joseph D.; Devlin, Bernie; Gallagher, Louise; Betancur, Catalina; Scherer, Stephen W.

    2014-01-01

    Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10−5) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10−15, ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation. PMID:24768552

  13. Minireview: Long Noncoding RNAs: New Links Between Gene Expression and Cellular Outcomes in Endocrinology

    PubMed Central

    Sun, Miao

    2013-01-01

    Recent advances in sequencing technologies have revealed that the genome is extensively transcribed, yielding a large repertoire of noncoding RNAs. These include long noncoding RNAs (lncRNAs), mRNA-like molecules that do not code for proteins, which are emerging as a new class of RNAs that play important roles in a variety of cellular processes. Ongoing studies are revealing new insights about lncRNAs, including their physiological functions, disease relationships, and molecular mechanisms of action. Characterized lncRNAs have been shown to interact with and modulate the activity of other RNAs and protein partners, leading to alterations in transcriptional and posttranscriptional regulatory processes. In this review, we summarize the key features of lncRNAs, their molecular mechanisms of action, biological functions, and therapeutic implications, particularly as they apply to the field of molecular endocrinology. In addition, we provide a brief overview of how molecular biologists are beginning to probe the identity, mechanisms, and functions of this emerging class of RNA molecules. PMID:23885095

  14. Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

    PubMed

    Wasfi, Reham; Elkhatib, Walid F; Khairalla, Ahmed S

    2016-01-01

    The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

  15. Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli

    PubMed Central

    Elkhatib, Walid F.

    2016-01-01

    The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed “ghost” cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

  16. Association of Angiotensin-Converting Enzyme (ACE) Gene Polymorphism with Inflammation and Cellular Cytotoxicity in Vitiligo Patients

    PubMed Central

    Hasan, Nermeen; Zahra, Amr; Fayez, Salwa

    2015-01-01

    Background Vitiligo is a disorder with profound heterogeneity in its aetio-pathophysiology. Angiotensin converting enzyme (ACE) plays an important role in the physiology of the vasculature, blood pressure and inflammation. An insertion/deletion (I/D) polymorphism of the ACE gene was reported be associated with the development of vitiligo. Objective Our aim was to evaluate the ACE I/D polymorphism in vitiligo patients and controls. Our second aim was to find a possible association between ACE gene polymorphism and inflammatory mediators (as interleukin (IL)-6) and/or cellular cytotoxicity induced by serum nitrite (as a breakdown product of the cytotoxic nitric oxide) in vitiligo patients. Methods This case-control study included 74 vitiligo patients and 75 apparently healthy controls. The distribution of ACE gene I/D genotype was investigated using PCR. Serum ACE, IL-6 and nitrite were measured by colorimetric method, ELISA and Griess assay respectively. Results The ACE allele frequency was significantly different between vitiligo patients and healthy controls (P = 0.026). However there was no significant difference between the ACE genotyping frequency in both groups (P = 0.115). There were statistically significant higher VIDA score (P = 0.007), and serum IL-6 (P < 0.001) in patients with the DD genotype when compared to other genotypes. Serum nitrite in patients with the DD genotype was significantly higher (P = 0.007) when compared to patients with II genotype. Serum levels of ACE, IL-6 and nitrite in vitiligo patients were statistically significantly higher than those in controls. Conclusion As a conclusion, ACE gene polymorphism might grant susceptibility to develop vitiligo. Serum IL-6 and nitrite levels might have an important role in the pathogenesis of vitiligo. Targeting these two factors might have an implication in the treatment of some resistant cases. PMID:26177100

  17. A Single Argonaute Gene Participates in Exogenous and Endogenous RNAi and Controls Cellular Functions in the Basal Fungus Mucor circinelloides

    PubMed Central

    Nicols, Francisco E.; Moxon, Simon; de Haro, Juan P.; Dalmay, Tamas; Torres-Martnez, Santiago; Ruiz-Vzquez, Rosa M

    2013-01-01

    The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1? M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi. PMID:23935973

  18. Glutathione S-Transferase (GST) Gene Diversity in the Crustacean Calanus finmarchicus – Contributors to Cellular Detoxification

    PubMed Central

    Roncalli, Vittoria; Cieslak, Matthew C.; Passamaneck, Yale; Christie, Andrew E.; Lenz, Petra H.

    2015-01-01

    Detoxification is a fundamental cellular stress defense mechanism, which allows an organism to survive or even thrive in the presence of environmental toxins and/or pollutants. The glutathione S-transferase (GST) superfamily is a set of enzymes involved in the detoxification process. This highly diverse protein superfamily is characterized by multiple gene duplications, with over 40 GST genes reported in some insects. However, less is known about the GST superfamily in marine organisms, including crustaceans. The availability of two de novo transcriptomes for the copepod, Calanus finmarchicus, provided an opportunity for an in depth study of the GST superfamily in a marine crustacean. The transcriptomes were searched for putative GST-encoding transcripts using known GST proteins from three arthropods as queries. The identified transcripts were then translated into proteins, analyzed for structural domains, and annotated using reciprocal BLAST analysis. Mining the two transcriptomes yielded a total of 41 predicted GST proteins belonging to the cytosolic, mitochondrial or microsomal classes. Phylogenetic analysis of the cytosolic GSTs validated their annotation into six different subclasses. The predicted proteins are likely to represent the products of distinct genes, suggesting that the diversity of GSTs in C. finmarchicus exceeds or rivals that described for insects. Analysis of relative gene expression in different developmental stages indicated low levels of GST expression in embryos, and relatively high expression in late copepodites and adult females for several cytosolic GSTs. A diverse diet and complex life history are factors that might be driving the multiplicity of GSTs in C. finmarchicus, as this copepod is commonly exposed to a variety of natural toxins. Hence, diversity in detoxification pathway proteins may well be key to their survival. PMID:25945801

  19. Cellular Extract Facilitates Nuclear Reprogramming by Altering DNA Methylation and Pluripotency Gene Expression

    PubMed Central

    Xiong, Xian-Rong; Lan, Dao-Liang; Zi, Xiang-Dong; Ma, Li; Wang, Yong

    2014-01-01

    Abstract The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in disease mechanisms and regenerative medicine. Epigenetic modifications enable differentiated cells to perpetuate molecular memory to retain their identity. Therefore, the aim of this study was to investigate the reprogramming modification of yak fibroblast cells that were permeabilized and incubated in the extracts of mesenchymal stem cells derived from mice adipose tissue [adipose-derived stem cells (ADSCs)]. According to the results, the treatment of ADSC extracts promoted colony formation. Moreover, pluripotent gene expression was associated with the loss of repressive histone modifications and increased global demethylation. The genes Col1a1 and Col1a2, which are typically found in differentiated cells only, demonstrated decreased expression and increased methylation in the 5′-flanking regulatory regions. Moreover, yak fibroblast cells that were exposed to ADSC extracts resulted in significantly different eight-cell and blastocyst formation rates of cloned embryos compared with their untreated counterparts. This investigation provides the first evidence that nuclear reprogramming of yak fibroblast cells is modified after the ADSC extract treatment. This research also presents a methodology for studying the dedifferentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells toward a pluripotent state without genetic alteration. PMID:24738992

  20. Tertiary-Amine Functionalized Polyplexes Enhanced Cellular Uptake and Prolonged Gene Expression

    PubMed Central

    Lo, Chia-Wen; Chang, Yung; Lee, Jyun-Lin; Tsai, Wei-Bor; Chen, Wen-Shiang

    2014-01-01

    Ultrasound (US) has been found to facilitate the transport of DNA across cell membranes. However, the transfection efficiency is generally low, and the expression duration of the transfected gene is brief. In this study, a tertiary polycation, Poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA), was used as a carrier for US-mediated gene transfection. Its in-vitro and in-vivo effects on the transfection efficiency and the expression duration were evaluated. A mixture of pCI-neo-luc and PDMAEMA was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulse US. A strong expression of luciferase was found 10 days after the transfection in vitro regardless of US exposure. However, effective transfection only occurred in the US groups in vivo. The transfection ability depended on the weight ratio of PDMAEMA to DNA, and was different for the in-vitro and in-vivo conditions. Lower weight ratios, e.g., 0.25, exhibited better in-vivo expression for at least 45 days. PMID:24827929

  1. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  2. Cellular Immune Response Against Firefly Luciferase After Sleeping BeautyMediated Gene Transfer In Vivo

    PubMed Central

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

    2014-01-01

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

  3. Reprogramming the genetic code: The emerging role of ribosomal frameshifting in regulating cellular gene expression.

    PubMed

    Advani, Vivek M; Dinman, Jonathan D

    2016-01-01

    Reading frame maintenance is a critical property of ribosomes. However, a number of genetic elements have been described that can induce ribosomes to shift on mRNAs, the most well understood of which are a class that directs ribosomal slippage by one base in 5' (-1) direction. This is referred to as programmed -1 ribosomal frameshifting (-1 PRF). Recently, a new -1 PRF promoting element was serendipitously discovered in a study examining the effects of stretches of adenosines in the coding sequences of mRNAs. Here, we discuss this finding, recent studies describing how -1 PRF is used to control gene expression in eukaryotes, and how -1 PRF is itself regulated. The implications of dysregulation of -1 PRF on human health are examined, as are possible new areas in which novel -1 PRF promoting elements might be discovered. Also watch the Video Abstract. PMID:26661048

  4. Morphogenesis in sea urchin embryos: linking cellular events to gene regulatory network states

    PubMed Central

    Lyons, Deidre; Kaltenbach, Stacy; McClay, David R.

    2013-01-01

    Gastrulation in the sea urchin begins with ingression of the primary mesenchyme cells (PMCs) at the vegetal pole of the embryo. After entering the blastocoel the PMCs migrate, form a syncitium, and synthesize the skeleton of the embryo. Several hours after the PMCs ingress the vegetal plate buckles to initiate invagination of the archenteron. That morphogenetic process occurs in several steps. The non-skeletogenic cells produce the initial inbending of the vegetal plate. Endoderm cells then rearrange and extend the length of the gut across the blastocoel to a target near the animal pole. Finally, cells that will form part of the midgut and hindgut are added to complete gastrulation. Later, the stomodeum invaginates from the oral ectoderm and fuses with the foregut to complete the archenteron. In advance of, and during these morphogenetic events an increasingly complex gene regulatory network controls the specification and the cell biological events that conduct the gastrulation movements. PMID:23801438

  5. Snai2 and Snai3 transcriptionally regulate cellular fitness and functionality of T cell lineages through distinct gene programs.

    PubMed

    Pioli, Peter D; Whiteside, Sarah K; Weis, Janis J; Weis, John H

    2016-05-01

    T lymphocytes are essential contributors to the adaptive immune system and consist of multiple lineages that serve various effector and regulatory roles. As such, precise control of gene expression is essential to the proper development and function of these cells. Previously, we identified Snai2 and Snai3 as being essential regulators of immune tolerance partly due to the impaired function of CD4(+) regulatory T cells in Snai2/3 conditional double knockout mice. Here we extend those previous findings using a bone marrow transplantation model to provide an environmentally unbiased view of the molecular changes imparted onto various T lymphocyte populations once Snai2 and Snai3 are deleted. The data presented here demonstrate that Snai2 and Snai3 transcriptionally regulate the cellular fitness and func