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Sample records for hipk2 overexpression leads

  1. Hyperglycemia triggers HIPK2 protein degradation

    PubMed Central

    Granato, Marisa; Cuomo, Laura; Pistritto, Giuseppa; Cirone, Mara; D'Orazi, Gabriella

    2017-01-01

    Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies. PMID:27901482

  2. HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation

    PubMed Central

    De Nicola, F; Catena, V; Rinaldo, C; Bruno, T; Iezzi, S; Sorino, C; Desantis, A; Camerini, S; Crescenzi, M; Floridi, A; Passananti, C; Soddu, S; Fanciulli, M

    2014-01-01

    Che-1/AATF is an RNA polymerase II-binding protein that is involved in the regulation of gene transcription, which undergoes stabilization and accumulation in response to DNA damage. We have previously demonstrated that following apoptotic induction, Che-1 protein levels are downregulated through its interaction with the E3 ligase HDM2, which leads to Che-1 degradation by ubiquitylation. This interaction is mediated by Pin1, which determines a phosphorylation-dependent conformational change. Here we demonstrate that HIPK2, a proapoptotic kinase, is involved in Che-1 degradation. HIPK2 interacts with Che-1 and, upon genotoxic stress, phosphorylates it at specific residues. This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system. In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation. Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis. Our results establish Che-1 as a new HIPK2 target and confirm its important role in the cellular response to DNA damage. PMID:25210797

  3. Heat shock modulates the subcellular localization, stability, and activity of HIPK2

    SciTech Connect

    Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam

    2016-04-15

    The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress – such as hypoxia, oxidative stress, or UV damage – is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.

  4. Heat shock modulates the subcellular localization, stability, and activity of HIPK2.

    PubMed

    Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam

    2016-04-15

    The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress - such as hypoxia, oxidative stress, or UV damage - is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Essential function of HIPK2 in TGFβ-dependent survival of midbrain dopamine neurons

    PubMed Central

    Zhang, Jiasheng; Pho, Vanee; Bonasera, Stephen J; Holzmann, Jed; Tang, Amy T; Hellmuth, Joanna; Tang, Siuwah; Janak, Patricia H; Tecott, Laurence H; Huang, Eric J

    2013-01-01

    Transforming growth factor beta (TGFβ) is a potent trophic factor for midbrain dopamine (DA) neurons, but its in vivo function and signaling mechanisms are not entirely understood. We show that the transcriptional cofactor homeodomain interacting protein kinase 2 (HIPK2) is required for the TGFβ-mediated survival of mouse DA neurons. The targeted deletion of Hipk2 has no deleterious effect on the neurogenesis of DA neurons, but leads to a selective loss of these neurons that is due to increased apoptosis during programmed cell death. As a consequence, Hipk2−/− mutants show an array of psychomotor abnormalities. The function of HIPK2 depends on its interaction with receptor-regulated Smads to activate TGFβ target genes. In support of this notion, DA neurons from Hipk2−/− mutants fail to survive in the presence of TGFβ3 and Tgfβ3−/− mutants show DA neuron abnormalities similar to those seen in Hipk2−/− mutants. These data underscore the importance of the TGFβ-Smad-HIPK2 pathway in the survival of DA neurons and its potential as a therapeutic target for promoting DA neuron survival during neurodegeneration. PMID:17159989

  6. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  7. Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

    PubMed

    Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

    2015-07-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. HIPK2 deficiency causes chromosomal instability by cytokinesis failure and increases tumorigenicity.

    PubMed

    Valente, Davide; Bossi, Gianluca; Moncada, Alice; Tornincasa, Mara; Indelicato, Stefania; Piscuoglio, Salvatore; Karamitopoulou, Eva Diamantis; Bartolazzi, Armando; Pierantoni, Giovanna Maria; Fusco, Alfredo; Soddu, Silvia; Rinaldo, Cinzia

    2015-04-30

    HIPK2, a cell fate decision kinase inactivated in several human cancers, is thought to exert its oncosuppressing activity through its p53-dependent and -independent apoptotic function. However, a HIPK2 role in cell proliferation has also been described. In particular, HIPK2 is required to complete cytokinesis and impaired HIPK2 expression results in cytokinesis failure and tetraploidization. Since tetraploidy may yield to aneuploidy and chromosomal instability (CIN), we asked whether unscheduled tetraploidy caused by loss of HIPK2 might contribute to tumorigenicity. Here, we show that, compared to Hipk2+/+ mouse embryo fibroblasts (MEFs), hipk2-null MEFs accumulate subtetraploid karyotypes and develop CIN. Accumulation of these defects inhibits proliferation and spontaneous immortalization of primary MEFs whereas increases tumorigenicity when MEFs are transformed by E1A and Harvey-Ras oncogenes. Upon mouse injection, E1A/Ras-transformed hipk2-null MEFs generate tumors with genetic alterations resembling those of human cancers derived by initial tetraploidization events, such as pancreatic adenocarcinoma. Thus, we evaluated HIPK2 expression in different stages of pancreatic transformation. Importantly, we found a significant correlation among reduced HIPK2 expression, high grade of malignancy, and high nuclear size, a marker of increased ploidy. Overall, these results indicate that HIPK2 acts as a caretaker gene, whose inactivation increases tumorigenicity and causes CIN by cytokinesis failure.

  9. FHL2 mediates p53-induced transcriptional activation through a direct association with HIPK2

    SciTech Connect

    Lee, Sang-Wang . E-mail: umsj@sejong.ac.kr

    2006-01-27

    To understand the molecular mechanism underlying HIPK2 regulation of the transcriptional activation by p53, we sought to identify the protein that interacts with HIPK2. From our yeast two-hybrid screen, we found that four and a half LIM domains 2 (FHL2) could bind to the C-terminal half of HIPK2. Further assays in yeast mapped the minimal interaction domain to amino acids 812-907 in HIPK2. The interaction was confirmed using a GST pull-down assay in vitro, and an immunoprecipitation (IP) assay and fluorescence microscopy in vivo. FHL2 alone spread throughout both the cytoplasm and nucleus but was redistributed to dot-like structures in the nucleus when HIPK2 was coexpressed in HEK293 cells. When tethered to the Gal4-responsive promoter through the Gal4 DBD fusion, FHL2 showed autonomous transcriptional activity that was enhanced by wild-type HIPK2, but not by the kinase-defective mutant. In addition, FHL2 increased the p53-dependent transcriptional activation and had an additive effect on the activation when coexpressed with HIPK2, which was again not observed with the kinase-defective mutant of HIPK2. Finally, we found a ternary complex of p53, HIPK2, and FHL2 using IP, and their recruitment to the p53-responsive p21Waf1 promoter in chromatin IP assays. Overall, our findings indicate that FHL2 can also regulate p53 via a direct association with HIPK2.

  10. Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR.

    PubMed

    Winter, Melanie; Sombroek, Dirk; Dauth, Ilka; Moehlenbrink, Jutta; Scheuermann, Karin; Crone, Johanna; Hofmann, Thomas G

    2008-07-01

    The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells.

  11. PARP1 regulates the protein stability and proapoptotic function of HIPK2

    PubMed Central

    Choi, Jong-Ryoul; Shin, Ki Soon; Choi, Cheol Yong; Kang, Shin Jung

    2016-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in DNA damage response and development. In the present study, we propose that the protein stability and proapoptotic function of HIPK2 are regulated by poly(ADP-ribose) polymerase 1 (PARP1). We present evidence indicating that PARP1 promotes the proteasomal degradation of HIPK2. The tryptophan-glycine-arginine (WGR) domain of PARP1 was necessary and sufficient for the promotion of HIPK2 degradation independently of the PARP1 enzymatic activity. The WGR domain mediated the interaction between HIPK2 and C-terminus of HSP70-interacting protein (CHIP) via HSP70. We found that CHIP can function as a ubiquitin ligase for HIPK2. The interaction between PAPR1 and HIPK2 was weakened following DNA damage. Importantly, PARP1 reduced the HIPK2-mediated p53 phosphorylation, proapoptotic transcriptional activity and cell death. These results suggest that PARP1 can modulate the tumor-suppressing function of HIPK2 by regulating the protein stability of HIPK2. PMID:27787517

  12. HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation

    PubMed Central

    Siepi, Francesca; Gatti, Veronica; Camerini, Serena; Crescenzi, Marco; Soddu, Silvia

    2013-01-01

    HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354. PMID:23485397

  13. HIPK2 controls cytokinesis and prevents tetraploidization by phosphorylating histone H2B at the midbody.

    PubMed

    Rinaldo, Cinzia; Moncada, Alice; Gradi, Alessandra; Ciuffini, Laura; D'Eliseo, Donatella; Siepi, Francesca; Prodosmo, Andrea; Giorgi, Alessandra; Pierantoni, Giovanna M; Trapasso, Francesco; Guarguaglini, Giulia; Bartolazzi, Armando; Cundari, Enrico; Schininà, M Eugenia; Fusco, Alfredo; Soddu, Silvia

    2012-07-13

    Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Apoptosis induced by a HIPK2 full-length-specific siRNA is due to off-target effects rather than prevalence of HIPK2-Δe8 isoform

    PubMed Central

    Di Rocco, Giuliana; Verdina, Alessandra; Gatti, Veronica; Virdia, Ilaria; Toietta, Gabriele; Todaro, Matilde; Stassi, Giorgio; Soddu, Silvia

    2016-01-01

    Small interfering RNAs (siRNAs) are widely used to study gene function and extensively exploited for their potential therapeutic applications. HIPK2 is an evolutionary conserved kinase that binds and phosphorylates several proteins directly or indirectly related to apoptosis. Recently, an alternatively spliced isoform skipping 81 nucleotides of exon 8 (Hipk2-Δe8) has been described. Selective depletion of Hipk2 full-length (Hipk2-FL) with a specific siRNA that spares the Hipk2-Δe8 isoform has been shown to strongly induce apoptosis, suggesting an unpredicted dominant-negative effect of Hipk2-FL over the Δe8 isoform. From this observation, we sought to take advantage and assessed the therapeutic potential of generating Hipk2 isoform unbalance in tumor-initiating cells derived from colorectal cancer patients. Strong reduction of cell viability was induced in vitro and in vivo by the originally described exon 8-specific siRNA, supporting a potential therapeutic application. However, validation analyses performed with additional exon8-specific siRNAs with different stabilities showed that all exon8-targeting siRNAs can induce comparable Hipk2 isoform unbalance but only the originally reported e8-siRNA promotes cell death. These data show that loss of viability does not depend on the prevalence of Hipk2-Δe8 isoform but it is rather due to microRNA-like off-target effects. PMID:26625198

  15. Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53

    SciTech Connect

    Sung, Ki Sa; Lee, Yun-Ah; Kim, Eui Tae; Lee, Seung-Rock; Ahn, Jin-Hyun; Choi, Cheol Yong

    2011-04-15

    Homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of various transcription factors including p53 and CtBP in the DNA damage signaling pathway. PML-nuclear body (NB) is required for HIPK2-mediated p53 phosphorylation at Ser46 and induction of apoptosis. Although PML-NB targeting of HIPK2 has been shown, much is not clear about the molecular mechanism of HIPK2 recruitment to PML-NBs. Here we show that HIPK2 colocalizes specifically with PML-I and PML-IV. Mutational analysis showed that HIPK2 recruitment to PML-IV-NBs is mediated by the SUMO-interaction motifs (SIMs) of both PML-IV and HIPK2. Wild-type HIPK2 associated with SUMO-conjugated PML-IV at a higher affinity than with un-conjugated PML-IV, while the association of a HIPK2 SIM mutant with SUMO-modified PML-IV was impaired. In colony formation assays, HIPK2 strongly suppressed cell proliferation, but HIPK2 SIM mutants did not. In addition, activation and phosphorylation of p53 at the Ser46 residue were impaired by HIPK2 SIM mutants. These results suggest that SIM-mediated HIPK2 targeting to PML-NBs is crucial for HIPK2-mediated p53 activation and induction of apoptosis.

  16. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

    PubMed Central

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  17. The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage.

    PubMed

    Reuven, Nina; Adler, Julia; Porat, Ziv; Polonio-Vallon, Tilman; Hofmann, Thomas G; Shaul, Yosef

    2015-07-03

    The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Inhibition of the processing of miR-25 by HIPK2-Phosphorylated-MeCP2 induces NOX4 in early diabetic nephropathy

    PubMed Central

    Oh, Hyung Jung; Kato, Mitsuo; Deshpande, Supriya; Zhang, Erli; Sadhan, Das; Lanting, Linda; Wang, Mei; Natarajan, Rama

    2016-01-01

    Phosphorylated methyl-CpG binding protein2 (p-MeCP2) suppresses the processing of several microRNAs (miRNAs). Homeo-domain interacting protein kinase2 (HIPK2) phosphorylates MeCP2, a known transcriptional repressor. However, it is not known if MeCP2 and HIPK2 are involved in processing of miRNAs implicated in diabetic nephropathy. p-MeCP2 and HIPK2 levels were significantly increased, but Seven in Absentia Homolog1 (SIAH1), which mediates proteasomal degradation of HIPK2, was decreased in the glomeruli of streptozotocin injected diabetic mice. Among several miRNAs, miR-25 and its precursor were significantly decreased in diabetic mice, whereas primary miR-25 levels were significantly increased. NADPH oxidase4 (NOX4), a target of miR-25, was significantly increased in diabetic mice. Protein levels of p-MeCP2, HIPK2, and NOX4 were increased in high glucose (HG)- or TGF-β-treated mouse glomerular mesangial cells (MMCs). miR-25 (primary, precursor, and mature) and mRNA levels of genes indicated in the in vivo study showed similar trends of regulation in MMCs treated with HG or TGF-β. The HG- or TGF-β-induced upregulation of p-MeCP2, NOX4 and primary miR-25, but downregulation of precursor and mature miR-25, were attenuated by Hipk2 siRNA. These results demonstrate a novel role for the SIAH1/HIPK2/MeCP2 axis in suppressing miR-25 processing and thereby upregulating NOX4 in early diabetic nephropathy. PMID:27941951

  19. Circular RNA HIPK2 regulates astrocyte activation via cooperation of autophagy and ER stress by targeting MIR124-2HG.

    PubMed

    Huang, Rongrong; Zhang, Yuan; Han, Bing; Bai, Ying; Zhou, Rongbin; Gan, Guangming; Chao, Jie; Hu, Gang; Yao, Honghong

    2017-10-03

    Circular RNAs are a subclass of noncoding RNAs in mammalian cells; however, whether these RNAs are involved in the regulation of astrocyte activation is largely unknown. Here, we have shown that the circular RNA HIPK2 (circHIPK2) functions as an endogenous microRNA-124 (MIR124-2HG) sponge to sequester MIR124-2HG and inhibit its activity, resulting in increased sigma non-opioid intracellular receptor 1 (SIGMAR1/OPRS1) expression. Knockdown of circHIPK2 expression significantly inhibited astrocyte activation via the regulation of autophagy and endoplasmic reticulum (ER) stress through the targeting of MIR124-2HG and SIGMAR1. These findings were confirmed in vivo in mouse models, as microinjection of a circHIPK2 siRNA lentivirus into mouse hippocampi inhibited astrocyte activation induced by methamphetamine or lipopolysaccharide (LPS). These findings provide novel insights regarding the specific contribution of circHIPK2 to astrocyte activation in the context of drug abuse as well as for the treatment of a broad range of neuroinflammatory disorders.

  20. Homeodomain-interacting Protein Kinase-2 (HIPK2) Phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and Modulates Its DNA Binding Affinity

    PubMed Central

    Zhang, Qingchun; Wang, Yinsheng

    2008-01-01

    The chromosomal high-mobility group A (HMGA) proteins, comprising of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line ε promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. Synopsis Here we report that both HIPK2 and cdc2 phosphorylate HMGA1a at Ser-35, Thr-52 and Thr-77, but the two kinases exhibit different site preferences. Moreover, we found that HIPK2-induced phosphorylation of HMGA1a reduced the binding affinity of HMGA1a to DNA, and the drop in binding affinity was lower than that introduced by cdc2 phosphorylation, confirming

  1. A natural product from Cannabis sativa subsp. sativa inhibits homeodomain-interacting protein kinase 2 (HIPK2), attenuating MPP(+)-induced apoptosis in human neuroblastoma SH-SY5Y cells.

    PubMed

    Wang, Guan; Zhu, Lingjuan; Zhao, Yuqian; Gao, Suyu; Sun, Dejuan; Yuan, Jingquan; Huang, Yuxin; Zhang, Xue; Yao, Xinsheng

    2017-03-30

    Homeodomain-interacting protein kinase 2 (HIPK2) is a conserved serine/threonine kinase, which regulate transcription, cell differentiation, proliferation and apoptosis. Previous evidences indicated that HIPK2 could be involved in the pathogenesis of neurodegenerative diseases, suggesting as a novel target for Parkinson's disease (PD) therapeutic development. Herein, gene microarray analysis was performed to verify the key regulatory function of HIPK2 in PD. (Z)-methylp-hydroxycinnamate (ZMHC, 7) with other eighteen compounds were isolated from Cannabis sativa subsp. sativa, growing in Bama Yao Autonomous County, one of the five largest longevity regions of the world. Intriguingly, ZMHC was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics (MD) simulations. Moreover, cell morphology, flow cytometry and western blot assay suggested that ZMHC inhibited HIPK2, which attenuated MPP(+)-induced apoptosis in SH-SY5Y cells. In conclusion, these findings discovered a natural product that inhibited HIPK2, and highlighted that ZMHC could be a potential precursor agent for future PD therapy.

  2. Phosphorylation of Krüppel-like Factor 3 (KLF3/BKLF) and C-terminal Binding Protein 2 (CtBP2) by Homeodomain-interacting Protein Kinase 2 (HIPK2) Modulates KLF3 DNA Binding and Activity*

    PubMed Central

    Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R.; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J.; Quinlan, Kate G. R.; Cordwell, Stuart J.; Funnell, Alister P. W.; Pearson, Richard C. M; Crossley, Merlin

    2015-01-01

    Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. PMID:25659434

  3. Targeted overexpression of calcitonin in gonadotrophs of transgenic mice leads to chronic hypoprolactinemia.

    PubMed

    Yuan, Ren; Kulkarni, Trupti; Wei, Fu; Shah, Girish V

    2005-01-14

    It was previously shown that calcitonin-like pituitary peptide (pit-CT) is synthesized and secreted by gonadotrophs, and pit-CT inhibits PRL gene transcription and lactotroph cell proliferation. Present studies examined long-term consequences of pit-CT overexpression on the functioning of mouse anterior pituitary (AP) gland. Targeted overexpression of pit-CT in gonadotrophs of mouse pituitaries was achieved by generating mice overexpressing bovine luteinizing hormone (LH)-alpha subunit promoter-pit-CT cDNA transgene. Transgenic (pit-CT+) mice displayed chronic but selective overexpression of pit-CT in gonadotrophs. The mice also displayed a dramatic decline in PRL gene expression as assessed by PRL mRNA abundance, PRL immunohistochemistry (IHC) and serum PRL levels. LH secretion in pit-CT+ mice was also reduced, without any change in FSH secretion. Reproductive abnormalities such as prolonged estrous cycles, reduced pregnancy rate, delivery of smaller litters, increased neonatal mortality and deficient lactation were also observed. Administration of PRL during early pregnancy significantly increased the pregnancy rate and neonatal survival of newborns. These results demonstrate that overexpression of pit-CT leads to chronic hypoprolactinemia and reproductive dysfunction in female mice, and reinforces the possibility that gonadotroph-derived pit-CT is an important paracrine regulator of lactotroph function.

  4. Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance

    PubMed Central

    Ludwig, David S.; Tritos, Nicholas A.; Mastaitis, Jason W.; Kulkarni, Rohit; Kokkotou, Efi; Elmquist, Joel; Lowell, Bradford; Flier, Jeffrey S.; Maratos-Flier, Eleftheria

    2001-01-01

    Several lines of investigation suggest that the hypothalamic neuropeptide melanin-concentrating hormone (MCH) regulates body weight in mammals. Obese mice lacking functional leptin overexpress the MCH message in the fed or fasted state. Acute intracerebroventricular injection of MCH increases energy intake in rats. Mice lacking the MCH gene are lean. To test the hypothesis that chronic overexpression of MCH in mice causes obesity, we produced transgenic mice that overexpress MCH (MCH-OE) in the lateral hypothalamus at approximately twofold higher levels than normal mice. On the FVB genetic background, homozygous transgenic animals fed a high-fat diet ate 10% more and were 12% heavier at 13 weeks of age than wild-type animals, and they had higher systemic leptin levels. Blood glucose levels were higher both preprandially and after an intraperitoneal glucose injection. MCH-OE animals were insulin-resistant, as demonstrated by markedly higher plasma insulin levels and a blunted response to insulin; MCH-OE animals had only a 5% decrease in blood glucose after insulin administration, compared with a 31% decrease in wild-type animals. MCH-OE animals also exhibited a twofold increase in islet size. To evaluate the contribution of genetic background to the predisposition to obesity seen in MCH-OE mice, the transgene was bred onto the C57BL/6J background. Heterozygote C57BL/6J mice expressing the transgene showed increased body weight on a standard diet, confirming that MCH overexpression can lead to obesity. PMID:11160162

  5. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

    PubMed

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K; Arias, Cintia L; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E; González, María de la Cruz; Hernández, Jose A; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-02-01

    Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the

  6. Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

    PubMed Central

    Cao, Hong; Saraf, Amit; Zweifel, Larry S.

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

  7. ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice

    PubMed Central

    Vaisman, Boris L.; Lambert, Gilles; Amar, Marcelo; Joyce, Charles; Ito, Toshimitsu; Shamburek, Robert D.; Cain, William J.; Fruchart-Najib, Jamila; Neufeld, Edward D.; Remaley, Alan T.; Brewer, H. Bryan; Santamarina-Fojo, Silvia

    2001-01-01

    The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans. PMID:11457883

  8. Overexpressed CacyBP/SIP leads to the suppression of growth in renal cell carcinoma.

    PubMed

    Sun, Shiren; Ning, Xiaoxuan; Liu, Jie; Liu, Lili; Chen, Yu; Han, Shuang; Zhang, Yanqi; Liang, Jie; Wu, Kaichun; Fan, Daiming

    2007-05-18

    Calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP), a target protein of S100, has been identified as a component of a novel ubiquitinylation complex leading to beta-catenin degradation, which was found to be related to the malignant phenotypes of gastric cancer. However, the roles of CacyBP/SIP in renal cell carcinoma still remain unclear. In the present study, we had analyzed the expression of the CacyBP/SIP protein in human renal cancer cells and clinical tissue samples. The possible roles of CacyBP/SIP in regulating the malignant phenotype of renal cancer cells were also investigated. The results demonstrated that the expression of CacyBP/SIP was markedly down-regulated in renal cell carcinoma tissues and cell lines. Ectopic overexpression of CacyBP/SIP in A498 cells inhibited the proliferation of this cell and delayed cell cycle progression significantly, which might be related to the down-regulation of Cyclin D1 through reducing beta-catenin protein. CacyBP/SIP also suppressed colony formation in soft agar and its tumorigenicity in nude mice. Taken together, our work showed that CacyBP/SIP, as a novel down-regulated gene in renal cell carcinoma, suppressed proliferation and tumorigenesis of renal cancer cells.

  9. Lead-induced iron overload and attenuated effects of ferroportin 1 overexpression in PC12 cells.

    PubMed

    Zhou, Fankun; Chen, Ying; Fan, Guangqin; Feng, Chang; Du, Guihua; Zhu, Gaochun; Li, Yanshu; Jiao, Huan; Guan, Linfu; Wang, Zhiping

    2014-12-01

    Lead (Pb) neurotoxicity has received renewed interest with the growing evidence that Pb contributes to Alzheimer's disease (AD). However, the mechanism is not clear. In our previous study of long-term Pb exposure in vivo, a brain iron (Fe) overload induced by Pb was observed in elderly rats. It is well known that brain Fe overload is the mechanism of AD. Therefore, we have reason to believe that Pb induced Fe overload and caused neurodegenerative disease. However, the mechanism or route of Pb-induced Fe overload is unknown. In the current study, the effect of Pb exposure on Fe homeostasis in PC12 cells was determined at different Pb-exposure concentrations and periods with differing Fe exposure, and the role of ferroportin 1 (FP1), the sole iron efflux protein, in Pb-induced Fe metabolic disorders was further investigated. The results showed a Pb-induced cellular increase in Fe accompanying a decrease in the expression of FP1 in a concentration- and time-dependent manner in Pb-exposed PC12 cells. Furthermore, FP1 overexpression could attenuate Fe accumulation in Pb-exposed PC12 cells. These results indicated that FP1 might be a novel target to prevent cellular Fe accumulation induced by Pb exposure and subsequent neurotoxic consequences. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Overexpressed CacyBP/SIP leads to the suppression of growth in renal cell carcinoma

    SciTech Connect

    Sun, Shiren; Ning, Xiaoxuan; Liu, Jie; Liu, Lili; Chen, Yu; Han, Shuang; Zhang, Yanqi; Liang, Jie; Wu, Kaichun; Fan, Daiming . E-mail: fandaim@fmmu.edu.cn

    2007-05-18

    Calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP), a target protein of S100, has been identified as a component of a novel ubiquitinylation complex leading to {beta}-catenin degradation, which was found to be related to the malignant phenotypes of gastric cancer. However, the roles of CacyBP/SIP in renal cell carcinoma still remain unclear. In the present study, we had analyzed the expression of the CacyBP/SIP protein in human renal cancer cells and clinical tissue samples. The possible roles of CacyBP/SIP in regulating the malignant phenotype of renal cancer cells were also investigated. The results demonstrated that the expression of CacyBP/SIP was markedly down-regulated in renal cell carcinoma tissues and cell lines. Ectopic overexpression of CacyBP/SIP in A498 cells inhibited the proliferation of this cell and delayed cell cycle progression significantly, which might be related to the down-regulation of Cyclin D1 through reducing {beta}-catenin protein. CacyBP/SIP also suppressed colony formation in soft agar and its tumorigenicity in nude mice. Taken together, our work showed that CacyBP/SIP, as a novel down-regulated gene in renal cell carcinoma, suppressed proliferation and tumorigenesis of renal cancer cells.

  11. Overexpression of Shox2 leads to congenital dysplasia of the temporomandibular joint in mice.

    PubMed

    Li, Xihai; Liang, Wenna; Ye, Hongzhi; Weng, Xiaping; Liu, Fayuan; Liu, Xianxiang

    2014-07-24

    Our previous study reported that inactivation of Shox2 led to dysplasia and ankylosis of the temporomandibular joint (TMJ), and that replacing Shox2 with human Shox partially rescued the phenotype with a prematurely worn out articular disc. However, the mechanisms of Shox2 activity in TMJ development remain to be elucidated. In this study, we investigated the molecular and cellular basis for the congenital dysplasia of TMJ in Wnt1-Cre; pMes-stop Shox2 mice. We found that condyle and glenoid fossa dysplasia occurs primarily in the second week after the birth. The dysplastic TMJ of Wnt1-Cre; pMes-stop Shox2 mice exhibits a loss of Collagen type I, Collagen type II, Ihh and Gli2. In situ zymography and immunohistochemistry further demonstrate an up-regulation of matrix metalloproteinases (MMPs), MMP9 and MMP13, accompanied by a significantly increased cell apoptosis. In addition, the cell proliferation and expressions of Sox9, Runx2 and Ihh are no different in the embryonic TMJ between the wild type and mutant mice. Our results show that overexpression of Shox2 leads to the loss of extracellular matrix and the increase of cell apoptosis in TMJ dysplasia by up-regulating MMPs and down-regulating the Ihh signaling pathway.

  12. Overexpression of AtSTO1 leads to improved salt tolerance in Populus tremula × P. alba

    Treesearch

    Shaneka S. Lawson; Charles H. Michler

    2014-01-01

    One of the major abiotic stress conditions limiting healthy growth of trees is salinity stress. The use of gene manipulation for increased tolerance to abiotic stress has been successful in many plant species. Overexpression of the Arabidopsis SALT TOLERANT1 (STO1) gene leads to increased concentrations of 9-cis-epoxycarotenoid dioxygenase3, a vital...

  13. MusaWRKY71 Overexpression in Banana Plants Leads to Altered Abiotic and Biotic Stress Responses

    PubMed Central

    Shekhawat, Upendra K. S.; Ganapathi, Thumballi R.

    2013-01-01

    WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing MusaWRKY71 displayed enhanced tolerance towards oxidative and salt stress as indicated by better photosynthesis efficiency (Fv/Fm) and lower membrane damage of the assayed leaves. Further, differential regulation of putative downstream genes of MusaWRKY71 was investigated using real-time RT-PCR expression analysis. Out of a total of 122 genes belonging to WRKY, pathogenesis-related (PR) protein genes, non-expressor of pathogenesis-related genes 1 (NPR1) and chitinase families analyzed, 10 genes (six belonging to WRKY family, three belonging to PR proteins family and one belonging to chitinase family) showed significant differential regulation in MusaWRKY71 overexpressing lines. These results indicate that MusaWRKY71 is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana. PMID:24116051

  14. Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells.

    PubMed

    Carpenter, R L; Paw, I; Dewhirst, M W; Lo, H-W

    2015-01-29

    Epithelial-mesenchymal transition (EMT) is an essential step for tumor progression, although the mechanisms driving EMT are still not fully understood. In an effort to investigate these mechanisms, we observed that heregulin (HRG)-mediated activation of HER2, or HER2 overexpression, resulted in EMT, which is accompanied with increased expression of a known EMT regulator Slug, but not TWIST or Snail. We then investigated how HER2 induced Slug expression and found, for the first time, that there are four consensus HSF sequence-binding elements (HSEs), the binding sites for heat shock factor-1 (HSF-1), located in the Slug promoter. HSF-1 bound to and transactivated the Slug promoter independent of heat shock, leading to Slug expression in breast cancer cells. Mutation of the putative HSEs ablated Slug transcriptional activation induced by HRG or HSF-1 overexpression. Knockdown of HSF-1 expression by siRNA reduced Slug expression and HRG-induced EMT. The positive association between HSF-1 and Slug was confirmed by immunohistochemical staining of a cohort of 100 invasive breast carcinoma specimens. While investigating how HER2 activated HSF-1 independent of heat shock, we observed that HER2 activation resulted in concurrent phosphorylation of Akt and HSF-1. We then observed, also for the first time, that Akt directly interacted with HSF-1 and phosphorylated HSF-1 at S326. Inhibition of Akt using siRNA, dominant-negative Akt mutant, or small molecule inhibitors prevented HRG-induced HSF-1 activation and Slug expression. Conversely, constitutively active Akt induced HSF-1 phosphorylation and Slug expression. HSF-1 knockdown reduced the ability of Akt to induce Slug expression, indicating an essential role that HSF-1 plays in Akt-induced Slug upregulation. Altogether, our study uncovered the existence of a novel Akt-HSF-1 signaling axis that leads to Slug upregulation and EMT, and potentially contributes to progression of HER2-positive breast cancer.

  15. Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition

    PubMed Central

    Deniaud, Emmanuelle; Baguet, Joël; Chalard, Roxane; Blanquier, Bariza; Brinza, Lilia; Meunier, Julien; Michallet, Marie-Cécile; Laugraud, Aurélie; Ah-Soon, Claudette; Wierinckx, Anne; Castellazzi, Marc; Lachuer, Joël; Gautier, Christian

    2009-01-01

    Background The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. Methodology and Principal Findings We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. Conclusion This study shows that the binding to DNA of overexpressed Sp1

  16. Overexpression of MHC Class I Heavy Chain Protein in Young Skeletal Muscle Leads to Severe Myositis

    PubMed Central

    Li, Charles Kwok-chong; Knopp, Paul; Moncrieffe, Halima; Singh, Bhanu; Shah, Sonia; Nagaraju, Kanneboyina; Varsani, Hemlata; Gao, Bin; Wedderburn, Lucy R.

    2009-01-01

    Folding and transport of proteins, such as major histocompatibility complex (MHC) class I, through the endoplasmic reticulum (ER) is tightly regulated in all cells, including muscle tissue, where the specialized ER sarcoplasmic reticulum is also critical to muscle fiber function. Overexpression of MHC class I protein is a common feature of many muscle pathologies including idiopathic myositis and can induce ER stress. However, there has been no comparison of the consequences of MHC overexpression in muscle at different ages. We have adapted a transgenic model of myositis induced by overexpression of MHC class I protein in skeletal muscle to investigate the effects of this protein overload on young muscle fibers, as compared with adult tissue. We find a markedly more severe disease phenotype in young mice, with rapid onset of muscle weakness and pathology. Gene expression profiling to compare the two models indicates rapid onset of ER stress in young muscle tissue but also that gene expression of key muscle structural proteins is affected more rapidly in young mice than adults after this insult. This novel model has important implications for our understanding of muscle pathology in dermatomyositis of both adults and children. PMID:19700752

  17. Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity.

    PubMed

    Li, Yujing; Dhankher, Om Parkash; Carreira, Laura; Lee, David; Chen, Alice; Schroeder, Julian I; Balish, Rebecca S; Meagher, Richard B

    2004-12-01

    Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.

  18. Myofibril degeneration caused by tropomodulin overexpression leads to dilated cardiomyopathy in juvenile mice.

    PubMed Central

    Sussman, M A; Welch, S; Cambon, N; Klevitsky, R; Hewett, T E; Price, R; Witt, S A; Kimball, T R

    1998-01-01

    Loss of myofibril organization is a common feature of chronic dilated and progressive cardiomyopathy. To study how the heart compensates for myofibril degeneration, transgenic mice were created that undergo progressive loss of myofibrils after birth. Myofibril degeneration was induced by overexpression of tropomodulin, a component of the thin filament complex which determines and maintains sarcomeric actin filament length. The tropomodulin cDNA was placed under control of the alpha-myosin heavy chain gene promoter to overexpress tropomodulin specifically in the myocardium. Offspring with the most severe phenotype showed cardiomyopathic changes between 2 and 4 wk after birth. Hearts from these mice present characteristics consistent with dilated cardiomyopathy and a failed hypertrophic response. Histological analysis showed widespread loss of myofibril organization. Confocal microscopy of isolated cardiomyocytes revealed intense tropomodulin immunoreactivity in transgenic mice together with abnormal coincidence of tropomodulin and alpha-actinin reactivity at Z discs. Contractile function was compromised severely as determined by echocardiographic analyses and isolated Langendorff heart preparations. This novel experimentally induced cardiomyopathy will be useful for understanding dilated cardiomyopathy and the effect of thin filament-based myofibril degeneration upon cardiac structure and function. PMID:9421465

  19. Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua

    PubMed Central

    Jiang, Weimin; Fu, Xueqing; Pan, Qifang; Tang, Yueli; Shen, Qian; Lv, Zongyou; Yan, Tingxiang; Shi, Pu; Li, Ling; Zhang, Lida; Wang, Guofeng; Sun, Xiaofen; Tang, Kexuan

    2016-01-01

    Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future. PMID:27064403

  20. Overexpression of ARGOS Genes Modifies Plant Sensitivity to Ethylene, Leading to Improved Drought Tolerance in Both Arabidopsis and Maize.

    PubMed

    Shi, Jinrui; Habben, Jeffrey E; Archibald, Rayeann L; Drummond, Bruce J; Chamberlin, Mark A; Williams, Robert W; Lafitte, H Renee; Weers, Ben P

    2015-09-01

    Lack of sufficient water is a major limiting factor to crop production worldwide, and the development of drought-tolerant germplasm is needed to improve crop productivity. The phytohormone ethylene modulates plant growth and development as well as plant response to abiotic stress. Recent research has shown that modifying ethylene biosynthesis and signaling can enhance plant drought tolerance. Here, we report novel negative regulators of ethylene signal transduction in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). These regulators are encoded by the ARGOS gene family. In Arabidopsis, overexpression of maize ARGOS1 (ZmARGOS1), ZmARGOS8, Arabidopsis ARGOS homolog ORGAN SIZE RELATED1 (AtOSR1), and AtOSR2 reduced plant sensitivity to ethylene, leading to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the ZmARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 in the ethylene signaling pathway, affecting ethylene perception or the early stages of ethylene signaling. Overexpressed ZmARGOS1 is localized to the endoplasmic reticulum and Golgi membrane, where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and REVERSION-TO-ETHYLENE SENSITIVITY1 reside. In transgenic maize plants, overexpression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed that UBIQUITIN1:ZmARGOS8 maize events had a greater grain yield than nontransgenic controls under both drought stress and well-watered conditions.

  1. NR2B overexpression leads to the enhancement of specific protein phosphorylation in the brain.

    PubMed

    Li, Chunxia; Zhang, Ning; Hu, Yinghe; Wang, Huimin

    2014-11-07

    n-methyl-d-aspartate receptors (NMDARs) are highly expressed in the central nervous system (CNS) including the cerebral cortex, and it has been found that they contribute significantly to the processes of learning and memory. Dysfunctions of NMDARs are implicated in many neurological disorders. To further investigate the specific role of the NR2B subunit of NMDARs in brain functions, we have examined differences in gene expression in the cerebral cortex between NR2B transgenic mice and their wild-type littermates using the DNA microarray. Total of 179 differentially expressed genes were identified, including genes involved in ion channel activity and/or neurotransmission, signal transduction, structure/cytoskeleton, transcription, and hormone/growth factor activity. Signal pathway analysis has indicated that multiple pathways were involved in this process, especially the Mitogen-activated protein kinases/Extracellular signal-regulated kinases (MAPK/ERK) pathway. The phosphorylation levels of ERK and cAMP response element-binding protein (CREB), and the mRNA levels of CREB target genes (C-Fos and Nr4a1) were significantly upregulated in the cerebral cortices of NR2B transgenic mice compared to their wild-type littermates. Our study suggested that a chronic increase of NMDARs activation by NR2B overexpression in the forebrain may enhance the protein serine/threonine phosphorylation levels of MAPK/ERK-CREB and thereby regulated their signaling pathway.

  2. Cardiomyocyte-specific perilipin 5 overexpression leads to myocardial steatosis and modest cardiac dysfunction1[S

    PubMed Central

    Wang, Hong; Sreenivasan, Urmila; Gong, Da-Wei; O'Connell, Kelly A.; Dabkowski, Erinne R.; Hecker, Peter A.; Ionica, Nicoleta; Konig, Manige; Mahurkar, Anup; Sun, Yezhou; Stanley, William C.; Sztalryd, Carole

    2013-01-01

    Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid β-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)α target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment. PMID:23345411

  3. Overexpression of SKI oncoprotein leads to p53 degradation through regulation of MDM2 protein sumoylation.

    PubMed

    Ding, Boxiao; Sun, Yin; Huang, Jiaoti

    2012-04-27

    Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer.

  4. Overexpression of LEAFY in apple leads to a columnar phenotype with shorter internodes.

    PubMed

    Flachowsky, Henryk; Hättasch, Conny; Höfer, Monika; Peil, Andreas; Hanke, Magda-Viola

    2010-01-01

    To break the juvenile stage of apple (Malus x domestica Borkh.) we transferred the LFY gene of Arabidopsis into the genome of the apple cv. 'Pinova'. A total of five transgenic clones constitutively overexpressing the LFY gene were obtained. Approximately, 20 shoots of each clone were rooted and transferred to the glasshouse. No flowers were obtained on transgenic plants during the first 2 years of cultivation. Evaluation of the expression of possible LFY targets revealed that no transcripts could be detected for MdAP1-1 and MdAP1-2. MdTFL1 was unaffected. Based on the absence of the LFY core-binding sequence within promoter sequences of MdAP1-1 and MdAP1-2, it was concluded that LFY was not able to induce these genes. The LFY genes of apple were unaffected in transgenic plants and sequence alignments of the C-terminal amino acid sequence showed a high conservation of these proteins. A change in binding ability to DNA can therefore be excluded. Instead of early flowering, the transgenic plants showed an altered phenotype, which is similar to the columnar phenotype of the 'McIntosh Wijcik' mutant of apple. The transgenic plants showed shortened internodes and a significantly reduced length of the regrowing shoot. A negative correlation was observed between the length of the regrowing shoot and the LFY mRNA transcript level. Furthermore, the LFY transgenic apple plants showed an increased shoot diameter at node 20, which was positively correlated with the LFY mRNA transcript level. Based on our results, we assume an alternative role of LFY in apple.

  5. Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide.

    PubMed

    Dabral, Neha; Jain-Gupta, Neeta; Seleem, Mohamed N; Sriranganathan, Nammalwar; Vemulapalli, Ramesh

    2015-01-01

    Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice.

  6. Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide

    PubMed Central

    Dabral, Neha; Jain-Gupta, Neeta; Seleem, Mohamed N.; Sriranganathan, Nammalwar; Vemulapalli, Ramesh

    2015-01-01

    Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice. PMID:26157707

  7. Overexpression of Galectin-7 in Mouse Epidermis Leads to Loss of Cell Junctions and Defective Skin Repair

    PubMed Central

    Dang, Tien; Deshayes, Frédérique; Delacour, Delphine; Pichard, Evelyne; Advedissian, Tamara; Sidhu, Sukhvinder S.; Viguier, Mireille; Magnaldo, Thierry; Poirier, Francoise

    2015-01-01

    Background The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. Methods We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. Results The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. Conclusion These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations. PMID:25741714

  8. Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

    PubMed

    Gendronneau, Gaëlle; Sanii, Sadaf; Dang, Tien; Deshayes, Frédérique; Delacour, Delphine; Pichard, Evelyne; Advedissian, Tamara; Sidhu, Sukhvinder S; Viguier, Mireille; Magnaldo, Thierry; Poirier, Francoise

    2015-01-01

    The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.

  9. Cdc14B depletion leads to centriole amplification and its overexpression prevents unscheduled centriole duplication

    SciTech Connect

    Wu, Jun; Plumley, Hyekyung; Rhee, David; Johnson, Dabney K; Dunlap, John; Liu, Yie; Wang, Yisong

    2008-01-01

    Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B's involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S-phase arrested cells and proteasome inhibitor Z-L3VS-treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.

  10. Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors.

    PubMed Central

    Risma, K A; Clay, C M; Nett, T M; Wagner, T; Yun, J; Nilson, J H

    1995-01-01

    Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We have recently generated transgenic mice expressing a chimeric LH beta subunit (LH beta) in gonadotropes. The LH beta chimera contains the C-terminal peptide of the human chorionic gonadotropin beta subunit. Addition of this peptide to bovine LH beta resulted in a hormone with a longer half-life. Furthermore, targeted expression of the LH beta chimera led to elevated LH levels and infertility in female transgenics. These mice ovulated infrequently, maintained a prolonged luteal phase, and developed pathologic ovarian changes such as cyst formation, marked enlargement of ovaries, and granulosa cell tumors. Testosterone and estradiol levels were increased compared to nontransgenic littermates. An unusual extragonadal phenotype was also observed: transgenic females developed hydronephropathy and pyelonephritis. The pathology observed demonstrates a direct association between abnormal secretion of LH and infertility and underscores the utility of the transgenic model for studying how excess LH leads to cyst formation, ovarian tumorigenesis, and infertility. Images Fig. 2 Fig. 3 Fig. 5 PMID:7877975

  11. Overexpression of progesterone receptor A isoform in mice leads to endometrial hyperproliferation, hyperplasia and atypia.

    PubMed

    Fleisch, M C; Chou, Y C; Cardiff, Robert D; Asaithambi, A; Shyamala, G

    2009-04-01

    A delicate balance in estrogen and progesterone signaling through their cognate receptors is characteristic for the physiologic state of the endometrium, and a shift in receptor isotype expression can be frequently found in human endometrial pathology. In this study, using a transgenic mouse model, we examined the mechanisms whereby alterations in progesterone receptor (PR) isotype expression leads to endometrial pathology. For an experimental model, we used transgenic mice (PR-A transgenics) carrying an imbalance in the native ratio of the two PR isoforms A and B (PR-A and PR-B) through the expression of additional A form and examined their uterine phenotype under different hormonal regimens, using various criteria. Uterine epithelial cell proliferation was augmented in PR-A transgenics and was abolished by PR antagonists. In particular, proliferative response to progesterone, independent of signaling through estrogen, was enhanced. Upon continuous exposure to estradiol and progesterone, the uteri in PR-A transgenics displayed gross enlargement, endometrial hyperplasia including atypical lesions, endometritis and pelvic inflammatory disease. Imbalanced expression of the two isoforms of PR in a transgenic model reveals multiple derangements in the regulation of uterine physiology, resulting in various pathologies including hyperplasias.

  12. Geminin overexpression prevents the completion of topoisomerase IIα chromosome decatenation, leading to aneuploidy in human mammary epithelial cells

    PubMed Central

    2011-01-01

    localization and function. CKIε kinase overexpression or Cdc7 kinase silencing, which we show phosphorylates TopoIIα in vitro, restored DNA decatenation and chromosome segregation in geminin-silenced cells before triggering cell death. In vivo, at normal concentration, geminin recruits the deSUMOylating sentrin-specific proteases SENP1 and SENP2 enzymes to deSUMOylate chromosome-bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that geminin recruits more of these deSUMOylating enzymes, or recruits them earlier, to bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakage generated through this mechanism were not sensed and/or repaired and the cell cycle was not arrested. Expression of mitosis-inducing proteins such as cyclin A and cell division kinase 1 was also increased in these cells because of the overexpression of geminin. Conclusions TopoIIα recruitment and its chromosome decatenation function require a normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation and/or segregation function. Geminin overexpression prematurely deSUMOylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug-resistant cancer cells. On the basis of our findings, we propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoIIα agents. PMID:21595939

  13. Neuronal overexpression of insulin receptor substrate 2 leads to increased fat mass, insulin resistance, and glucose intolerance during aging.

    PubMed

    Zemva, J; Udelhoven, M; Moll, L; Freude, S; Stöhr, O; Brönneke, H S; Drake, R B; Krone, W; Schubert, M

    2013-10-01

    The insulin receptor substrates (IRS) are adapter proteins mediating insulin's and IGF1's intracellular effects. Recent data suggest that IRS2 in the central nervous system (CNS) is involved in regulating fuel metabolism as well as memory formation. The present study aims to specifically define the role of chronically increased IRS2-mediated signal transduction in the CNS. We generated transgenic mice overexpressing IRS2 specifically in neurons (nIRS2 (tg)) and analyzed these in respect to energy metabolism, learning, and memory. Western blot (WB) analysis of nIRS2 (tg) brain lysates revealed increased IRS2 downstream signaling. Histopathological investigation of nIRS2 (tg) mice proved unaltered brain development and structure. Interestingly, nIRS2 (tg) mice showed decreased voluntary locomotoric activity during dark phase accompanied with decreased energy expenditure (EE) leading to increased fat mass. Accordingly, nIRS2 (tg) mice develop insulin resistance and glucose intolerance during aging. Exploratory behavior, motor function as well as food and water intake were unchanged in nIRS2 (tg) mice. Surprisingly, increased IRS2-mediated signals did not change spatial working memory in the T-maze task. Since FoxO1 is a key mediator of IRS2-transmitted signals, we additionally generated mice expressing a dominant negative mutant of FoxO1 (FoxO1DN) specifically in neurons. This mutant mimics the effect of increased IRS2 signaling on FoxO-mediated transcription. Interestingly, the phenotype observed in nIRS2 (tg) mice was not present in FoxO1DN mice. Therefore, increased neuronal IRS2 signaling causes decreased locomotoric activity in the presence of unaltered exploratory behavior and motor coordination that might lead to increased fat mass, insulin resistance, and glucose intolerance during aging independent of FoxO1-mediated transcription.

  14. Genomic Basis of Aromatase Excess Syndrome: Recombination- and Replication-Mediated Rearrangements Leading to CYP19A1 Overexpression

    PubMed Central

    Tsuchiya, Takayoshi; Vollbach, Heike; Brown, Kristy A.; Abe, Shuji; Ohtsu, Shigeyuki; Wabitsch, Martin; Burger, Henry; Simpson, Evan R.; Umezawa, Akihiro; Shihara, Daizou; Nakabayashi, Kazuhiko; Bulun, Serdar E.; Shozu, Makio; Ogata, Tsutomu

    2013-01-01

    Context: Genomic rearrangements at 15q21 have been shown to cause overexpression of CYP19A1 and resultant aromatase excess syndrome (AEXS). However, mutation spectrum, clinical consequences, and underlying mechanisms of these rearrangements remain to be elucidated. Objective: The aim of the study was to clarify such unsolved matters. Design, Setting, and Methods: We characterized six new rearrangements and investigated clinical outcome and local genomic environments of these rearrangements and of three previously reported duplications/deletions. Results: Novel rearrangements included simple duplication involving exons 1–10 of CYP19A1 and simple and complex rearrangements that presumably generated chimeric genes consisting of the coding region of CYP19A1 and promoter-associated exons of neighboring genes. Clinical severities were primarily determined by the copy number of CYP19A1 and the property of the fused promoters. Sequences at the fusion junctions suggested nonallelic homologous recombination, nonhomologous end-joining, and replication-based errors as the underlying mechanisms. The breakpoint-flanking regions were not enriched with GC content, palindromes, noncanonical DNA structures, or known rearrangement-associated motifs. The rearrangements resided in early-replicating segments. Conclusions: These results indicate that AEXS is caused by duplications involving CYP19A1 and simple and complex rearrangements that presumably lead to the usage of cryptic promoters of several neighboring genes. Our data support the notion that phenotypes depend on the dosage of CYP19A1 and the characteristics of the fused promoters. Furthermore, we show that the rearrangements in AEXS are generated by both recombination- and replication-mediated mechanisms, independent of the known rearrangement-inducing DNA features or late-replication timing. Thus, AEXS represents a unique model for human genomic disorders. PMID:24064691

  15. Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants.

    PubMed

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2003-04-01

    Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

  16. Keratinocyte but Not Endothelial Cell-Specific Overexpression of Tie2 Leads to the Development of Psoriasis

    PubMed Central

    Wolfram, Julie A.; Diaconu, Doina; Hatala, Denise A.; Rastegar, Jessica; Knutsen, Dorothy A.; Lowther, Abigail; Askew, David; Gilliam, Anita C.; McCormick, Thomas S.; Ward, Nicole L.

    2009-01-01

    Psoriasis is initiated and maintained through a multifaceted interplay between keratinocytes, blood vessels, gene expression, and the immune system. One previous psoriasis model demonstrated that overexpression of the angiopoietin receptor Tie2 in endothelial cells and keratinocytes led to the development of a psoriasiform phenotype; however, the etiological significance of overexpression in each cell type alone was unclear. We have now engineered two new mouse models whereby Tie2 expression is confined to either endothelial cells or keratinocytes. Both lines of mice have significant increases in dermal vasculature but only the KC-Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype. These mice spontaneously developed characteristic hallmarks of human psoriasis, including extensive acanthosis, increases in dermal CD4+ T cells, infiltrating epidermal CD8+ T cells, dermal dendritic cells and macrophages, and increased expression of cytokines and chemokines associated with psoriasis, including interferon-γ, tumor necrosis factor-α, and interleukins 1α, 6, 12, 22, 23, and 17. Host-defense molecules, cathelicidin, β-defensin, and S100A8/A9, were also up-regulated in the hyperproliferative skin. All of the phenotypic traits were completely reversed without any scarring following repression of the transgene and were significantly improved following treatment with the anti-psoriasis systemic therapeutic, cyclosporin A. Therefore, confining Tie2 overexpression solely to keratinocytes results in a mouse model that meets the clinical, histological, immunophenotypic, biochemical, and pharmacological criteria required for an animal model of human psoriasis. PMID:19342373

  17. Overexpression of EVI1 interferes with cytokinesis and leads to accumulation of cells with supernumerary centrosomes in G0/1 phase

    PubMed Central

    Karakaya, Kadin; Herbst, Friederike; Ball, Claudia; Glimm, Hanno; Krämer, Alwin; Löffler, Harald

    2012-01-01

    Ectopic viral integration site 1 (EVI1), a transcription factor frequently overexpressed in myeloid neoplasias, has been implicated in the generation of malignancy-associated centrosomal aberrations and chromosomal instability. Here, we sought to investigate the underlying cause of centrosome amplification in EVI1-overexpressing cells. We found that overexpression of EVI1-HA in U2OS cells induced supernumerary centrosomes, which were consistently associated with enlarged nuclei or binuclear cells. Live cell imaging experiments identified cytokinesis failure as the underlying cause of this phenotype. In accordance with previous reports, EVI1 overexpression induced a partial cell cycle arrest in G0/1 phase, accompanied by elevated cyclin D1 and p21 levels, reduced Cdk2 activity and activation of the p53 pathway. Supernumerary centrosomes predominantly occurred in resting cells, as identified by low levels of the proliferation marker Ki-67, leading to the conclusion that they result from tetraploidization after cytokinesis failure and are confined to G0/1-arrested tetraploid cells. Depletion of p53 using siRNA revealed that further polyploidization of these cells was inhibited by the p53-dependent tetraploidy checkpoint. PMID:22894935

  18. Over-expression of AtPAP2 in Camelina sativa leads to faster plant growth and higher seed yield

    PubMed Central

    2012-01-01

    Background Lipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa. Results Under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and null-lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants. Conclusions Lipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels. The improved growth rate and seed yield of transgenic Camelina under controlled environmental conditions have the potential to boost oil yield on an area basis in field conditions and thus make Camelina-based biofuels more environmentally friendly and economically attractive. PMID:22472516

  19. Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy.

    PubMed

    Wagner, Kay-Dietrich; Vukolic, Ana; Baudouy, Delphine; Michiels, Jean-François; Wagner, Nicole

    2016-01-01

    Peroxisome proliferator-activated receptors are nuclear receptors which function as ligand-activated transcription factors. Among them, peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in the heart and thought to have cardioprotective functions due to its beneficial effects in metabolic syndrome. As we already showed that PPARβ/δ activation resulted in an enhanced cardiac angiogenesis and growth without impairment of heart function, we were interested to determine the effects of a specific activation of PPARβ/δ in the vasculature on cardiac performance under normal and in chronic ischemic heart disease conditions. We analyzed the effects of a specific PPARβ/δ overexpression in endothelial cells on the heart using an inducible conditional vascular-specific mouse model. We demonstrate that vessel-specific overexpression of PPARβ/δ induces rapid cardiac angiogenesis and growth with an increase in cardiomyocyte size. Upon myocardial infarction, vascular overexpression of PPARβ/δ, despite the enhanced cardiac vessel formation, does not protect against chronic ischemic injury. Our results suggest that the proper balance of PPARβ/δ activation in the different cardiac cell types is required to obtain beneficial effects on the outcome in chronic ischemic heart disease.

  20. Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy

    PubMed Central

    Wagner, Kay-Dietrich; Vukolic, Ana; Baudouy, Delphine; Michiels, Jean-François

    2016-01-01

    Peroxisome proliferator-activated receptors are nuclear receptors which function as ligand-activated transcription factors. Among them, peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in the heart and thought to have cardioprotective functions due to its beneficial effects in metabolic syndrome. As we already showed that PPARβ/δ activation resulted in an enhanced cardiac angiogenesis and growth without impairment of heart function, we were interested to determine the effects of a specific activation of PPARβ/δ in the vasculature on cardiac performance under normal and in chronic ischemic heart disease conditions. We analyzed the effects of a specific PPARβ/δ overexpression in endothelial cells on the heart using an inducible conditional vascular-specific mouse model. We demonstrate that vessel-specific overexpression of PPARβ/δ induces rapid cardiac angiogenesis and growth with an increase in cardiomyocyte size. Upon myocardial infarction, vascular overexpression of PPARβ/δ, despite the enhanced cardiac vessel formation, does not protect against chronic ischemic injury. Our results suggest that the proper balance of PPARβ/δ activation in the different cardiac cell types is required to obtain beneficial effects on the outcome in chronic ischemic heart disease. PMID:27057154

  1. Deficiency and Also Transgenic Overexpression of Timp-3 Both Lead to Compromised Bone Mass and Architecture In Vivo

    PubMed Central

    Hopkinson, Mark; Poulet, Blandine; Pollard, Andrea S.; Shefelbine, Sandra J.; Chang, Yu-Mei; Francis-West, Philippa; Bou-Gharios, George; Pitsillides, Andrew A.

    2016-01-01

    Tissue inhibitor of metalloproteinases-3 (TIMP-3) regulates extracellular matrix via its inhibition of matrix metalloproteinases and membrane-bound sheddases. Timp-3 is expressed at multiple sites of extensive tissue remodelling. This extends to bone where its role, however, remains largely unresolved. In this study, we have used Micro-CT to assess bone mass and architecture, histological and histochemical evaluation to characterise the skeletal phenotype of Timp-3 KO mice and have complemented this by also examining similar indices in mice harbouring a Timp-3 transgene driven via a Col-2a-driven promoter to specifically target overexpression to chondrocytes. Our data show that Timp-3 deficiency compromises tibial bone mass and structure in both cortical and trabecular compartments, with corresponding increases in osteoclasts. Transgenic overexpression also generates defects in tibial structure predominantly in the cortical bone along the entire shaft without significant increases in osteoclasts. These alterations in cortical mass significantly compromise predicted tibial load-bearing resistance to torsion in both genotypes. Neither Timp-3 KO nor transgenic mouse growth plates are significantly affected. The impact of Timp-3 deficiency and of transgenic overexpression extends to produce modification in craniofacial bones of both endochondral and intramembranous origins. These data indicate that the levels of Timp-3 are crucial in the attainment of functionally-appropriate bone mass and architecture and that this arises from chondrogenic and osteogenic lineages. PMID:27519049

  2. Overexpression of the laeA gene leads to increased production of cyclopiazonic acid in Aspergillus fumisynnematus.

    PubMed

    Hong, Eun Jin; Kim, Na Kyeong; Lee, Doyup; Kim, Won Gon; Lee, Inhyung

    2015-11-01

    To explore novel bioactive compounds produced via activation of secondary metabolite (SM) gene clusters, we overexpressed an ortholog of laeA, a gene that encodes a global positive regulator of secondary metabolism in Aspergillus fumisynnematus F746. Overexpression of the laeA gene under the alcA promoter resulted in the production of less pigment, shorter conidial head chains, and fewer conidia. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis revealed that SM production in OE::laeA was significantly increased, and included new metabolites that were not detected in the wild type. Among them, a compound named F1 was selected on the basis of its high production levels and antibacterial effects. F1 was purified by column chromatography and preparative TLC and identified as cyclopiazonic acid (CPA) by LC/MS, which had been previously known as mycotoxin. As A. fumisynnematus was not known to produce CPA, these results suggest that overexpression of the laeA gene can be used to explore the synthesis of useful bioactive compounds, even in a fungus for which the genome sequence is unavailable. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  3. Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology.

    PubMed

    Son, Marjatta; Puttaparthi, Krishna; Kawamata, Hibiki; Rajendran, Bhagya; Boyer, Philip J; Manfredi, Giovanni; Elliott, Jeffrey L

    2007-04-03

    Cu, Zn superoxide dismutase (SOD1) has been detected within spinal cord mitochondria of mutant SOD1 transgenic mice, a model of familial ALS. The copper chaperone for SOD1 (CCS) provides SOD1 with copper, facilitates the conversion of immature apo-SOD1 to a mature holoform, and influences in yeast the cytosolic/mitochondrial partitioning of SOD1. To determine how CCS affects G93A-SOD1-induced disease, we generated transgenic mice overexpressing CCS and crossed them to G93A-SOD1 or wild-type SOD1 transgenic mice. Both CCS transgenic mice and CCS/wild-type-SOD1 dual transgenic mice are neurologically normal. In contrast, CCS/G93A-SOD1 dual transgenic mice develop accelerated neurological deficits, with a mean survival of 36 days, compared with 242 days for G93A-SOD1 mice. Immuno-EM and subcellular fractionation studies on the spinal cord show that G93A-SOD1 is enriched within mitochondria in the presence of CCS overexpression. Our results indicate that CCS overexpression in G93A-SOD1 mice produces severe mitochondrial pathology and accelerates disease course.

  4. Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in β1-adrenoceptor overexpressing mice

    PubMed Central

    Keller, Kirsten; Maass, Martina; Dizayee, Sara; Leiss, Veronika; Annala, Suvi; Köth, Jessica; Seemann, Wiebke K.; Müller-Ehmsen, Jochen; Mohr, Klaus; Nürnberg, Bernd; Engelhardt, Stefan; Herzig, Stefan; Birnbaumer, Lutz; Matthes, Jan

    2015-01-01

    Aims Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac β1-adrenoceptor overexpression. Methods and results β1-transgenic mice lacking Gαi2 (β1-tg/Gαi2−/−) were compared with wild-type mice and littermates either overexpressing cardiac β1-adrenoceptors (β1-tg) or lacking Gαi2 (Gαi2−/−). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or β1-tg, but similar to β1-tg/Gαi2−/−, mice. Beyond 300 days, mortality of β1-tg/Gαi2−/− mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, β1-tg, and Gαi2−/− mice, but significant impairment for β1-tg/Gαi2−/− mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2−/− and 394 ± 80% in β1-tg/Gαi2−/−, respectively). Conclusions Gαi2 deficiency combined with cardiac β1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, β1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased β1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or

  5. Overexpression of an isoprenyl diphosphate synthase in spruce leads to unexpected terpene diversion products that function in plant defense.

    PubMed

    Nagel, Raimund; Berasategui, Aileen; Paetz, Christian; Gershenzon, Jonathan; Schmidt, Axel

    2014-02-01

    Spruce (Picea spp.) and other conifers employ terpenoid-based oleoresin as part of their defense against herbivores and pathogens. The short-chain isoprenyl diphosphate synthases (IDS) are situated at critical branch points in terpene biosynthesis, producing the precursors of the different terpenoid classes. To determine the role of IDS and to create altered terpene phenotypes for assessing the defensive role of terpenoids, we overexpressed a bifunctional spruce IDS, a geranyl diphosphate and geranylgeranyl diphosphate synthase in white spruce (Picea glauca) saplings. While transcript level (350-fold), enzyme activity level (7-fold), and in planta geranyl diphosphate and geranylgeranyl diphosphate levels (4- to 8-fold) were significantly increased in the needles of transgenic plants, there was no increase in the major monoterpenes and diterpene acids of the resin and no change in primary isoprenoids, such as sterols, chlorophylls, and carotenoids. Instead, large amounts of geranylgeranyl fatty acid esters, known from various gymnosperm and angiosperm plant species, accumulated in needles and were shown to act defensively in reducing the performance of larvae of the nun moth (Lymantria monacha), a conifer pest in Eurasia. These results show the impact of overexpression of an IDS and the defensive role of an unexpected accumulation product of terpenoid biosynthesis with the potential for a broader function in plant protection.

  6. Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato.

    PubMed

    Zhang, Chanjuan; Liu, Junxia; Zhang, Yuyang; Cai, Xiaofeng; Gong, Pengjuan; Zhang, Junhong; Wang, Taotao; Li, Hanxia; Ye, Zhibiao

    2011-03-01

    GDP-Mannose 3',5'-epimerase (GME; EC 5.1.3.18) catalyses the conversion of GDP-D-mannose to GDP-L-galactose, an important step in the ascorbic acid (AsA) biosynthesis pathway in higher plants. In this study, two members of the GME gene family were isolated from tomato (Solanum lycopersicum). Both SlGME genes encode 376 amino acids and share a 92% similarity with each other. Semi-quantitative RT-PCR indicated that SlGME1 was constantly expressed in various tissues, whereas SlGME2 was differentially expressed in different tissues. Transient expression of fused SlGME1-GFP (green fluorescent protein) and SlGME2-GFP in onion cells revealed the cytoplasmic localisation of the two proteins. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants. They also showed enhanced stress tolerance based on less chlorophyll content loss and membrane-lipid peroxidation under methyl viologen (paraquat) stress, higher survival rate under cold stress, and significantly higher seed germination rate, fresh weight, and root length under salt stress. The present study demonstrates that the overexpression of two members of the GME gene family resulted in increased ascorbate accumulation in tomato and improved tolerance to abiotic stresses.

  7. Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

    PubMed Central

    Buchberger, Elisabeth; El Harchi, Miriam; Payrhuber, Dietmar; Zommer, Anna; Schauer, Dominic; Simonitsch-Klupp, Ingrid; Bilban, Martin; Brostjan, Christine

    2013-01-01

    Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones. PMID:24146931

  8. Bcl-xL overexpression blocks bax-mediated mitochondrial contact site formation and apoptosis in rod photoreceptors of lead-exposed mice

    NASA Astrophysics Data System (ADS)

    He, Lihua; Perkins, Guy A.; Poblenz, Ann T.; Harris, Jeffrey B.; Hung, Michael; Ellisman, Mark H.; Fox, Donald A.

    2003-02-01

    Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited and disease-, injury-, and chemical-induced retinal degeneration. A clinically relevant mouse model of progressive rod photoreceptor-selective apoptosis was produced by low-level developmental lead exposure and studied in combination with transgenic mice overexpressing Bcl-xL only in the photoreceptors. A multiparametric analysis of rod apoptosis and mitochondrial structure-function was performed. Mitochondrial cristae topography and connectivity, matrix volume, and contact sites were examined by using 3D electron tomography. Lead-induced rod-selective apoptosis was accompanied by rod Ca2+ overload, rhodopsin loss, translocation of Bax from the cytosol to the mitochondria, decreased rod mitochondrial respiration and membrane potential, mitochondrial cytochrome c release, caspase-3 activation, and an increase in the number of mitochondrial contact sites. These effects occurred without mitochondrial matrix swelling, outer membrane rupture, caspase-8 activation, or Bid cleavage. Bcl-xL overexpression completely blocked all apoptotic events, except Ca2+ overload, and maintained normal rod mitochondrial function throughout adulthood. This study presents images of mitochondrial contact sites in an in vivo apoptosis model and shows that Bcl-xL overexpression blocks increased contact sites and apoptosis. These findings extend our in vitro retinal studies with Pb2+ and Ca2+ and suggest that developmental lead exposure produced rod-selective apoptosis without mitochondrial swelling by translocating cytosolic Bax to the mitochondria, which likely sensitized the Pb2+ and Ca2+ overloaded rod mitochondria to release cytochrome c. These results have relevance for therapies in a wide variety of progressive retinal and neuronal degenerations where Ca2+ overload, lead exposure, and/or mitochondrial dysfunction occur.

  9. Overexpression of poplar xylem sucrose synthase in tobacco leads to a thickened cell wall and increased height.

    PubMed

    Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

    2015-01-01

    Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the

  10. Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

    PubMed Central

    Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

    2015-01-01

    Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the

  11. Overexpression of ARGOS Genes Modifies Plant Sensitivity to Ethylene, Leading to Improved Drought Tolerance in Both Arabidopsis and Maize[OPEN

    PubMed Central

    Shi, Jinrui; Habben, Jeffrey E.; Archibald, Rayeann L.; Drummond, Bruce J.; Chamberlin, Mark A.; Williams, Robert W.; Lafitte, H. Renee; Weers, Ben P.

    2015-01-01

    Lack of sufficient water is a major limiting factor to crop production worldwide, and the development of drought-tolerant germplasm is needed to improve crop productivity. The phytohormone ethylene modulates plant growth and development as well as plant response to abiotic stress. Recent research has shown that modifying ethylene biosynthesis and signaling can enhance plant drought tolerance. Here, we report novel negative regulators of ethylene signal transduction in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). These regulators are encoded by the ARGOS gene family. In Arabidopsis, overexpression of maize ARGOS1 (ZmARGOS1), ZmARGOS8, Arabidopsis ARGOS homolog ORGAN SIZE RELATED1 (AtOSR1), and AtOSR2 reduced plant sensitivity to ethylene, leading to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the ZmARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 in the ethylene signaling pathway, affecting ethylene perception or the early stages of ethylene signaling. Overexpressed ZmARGOS1 is localized to the endoplasmic reticulum and Golgi membrane, where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and REVERSION-TO-ETHYLENE SENSITIVITY1 reside. In transgenic maize plants, overexpression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed that UBIQUITIN1:ZmARGOS8 maize events had a greater grain yield than nontransgenic controls under both drought stress and well-watered conditions. PMID:26220950

  12. Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

    PubMed

    Hernández, Martín A; Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo; Alvarez, Héctor M

    2015-03-01

    Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

  13. Overexpression of PDH45 or SUV3 helicases in rice leads to delayed leaf senescence-associated events.

    PubMed

    Macovei, Anca; Sahoo, Ranjan K; Faè, Matteo; Balestrazzi, Alma; Carbonera, Daniela; Tuteja, Narendra

    2017-03-01

    Senescence is a very complex process characterized by a highly regulated series of degenerative events which include changes in cell structure, metabolism and gene expression. In animals, one of the indicators of senescence is telomere shortening. In plants, this aspect is more puzzling because telomere shortening is not always correlated with senescence. In some cases, there were no differences in telomere length during plant developmental stages while in other cases both shortening and lengthening have been observed. Several genes involved in telomere homeostasis have been identified in plants, including some helicases. In the present study, the salinity stress-tolerant transgenic IR64 rice plants overexpressing the PDH45 (Pea DNA Helicase 45) or SUV3 (Suppressor of Var1-3) genes were used to test their performance during natural senescence at flowering (S2) and seed maturation (S4) developmental stages. Our results reveal that both PDH45 and SUV3 transgenic rice lines present decreased levels of necrosis/apoptosis as compared to wild type plants. Additionally, in these plants, some senescence-associated genes (SAGs) were downregulated at S2 and S4 stages, while genes involved in the maintenance of genome stability and DNA repair were upregulated. More interestingly, the telomeres were up to 3.8-fold longer in the SUV3 overexpressing lines as compared to wild type plants. This was associated with an increase (2.5-fold) in telomerase (OsTERT) transcript level. This is an interesting result reporting a possible involvement of SUV3 in telomere homeostasis in plants.

  14. Overexpression of PREP-1 in F9 teratocarcinoma cells leads to a functionally relevant increase of PBX-2 by preventing its degradation.

    PubMed

    Longobardi, Elena; Blasi, Francesco

    2003-10-03

    To bind DNA and to be retained in the nucleus, PBX proteins must form heterodimeric complexes with members of the MEINOX family. Therefore the balance between PBX and MEINOX must be an important regulatory feature. We show that overexpression of PREP-1 influences the level of PBX-2 protein maintaining the PREP-1-PBX balance. This effect has important functional consequences. F9 teratocarcinoma cells stably transfected with PREP-1 had an increased DNA binding activity to a PREP-PBX-responsive element. Because PREP-1 binds DNA efficiently only when dimerized to PBX, the increased DNA binding activity suggests that the level of PBX might also have increased. Indeed PREP-1-overexpressing cells had a higher level of PBX-2 and PBX-1b proteins. PBX-2 increase did not depend on increased mRNA level or a higher rate of translation but rather because of a protein stabilization process. Indeed, PBX-2 level drastically decreased after 3 h of cycloheximide treatment in control but not in PREP-1-overexpressing cells and the proteasome inhibitor MG132 prevented PBX-2 decay in control cells. Hence, dimerization with PREP-1 appears to decrease proteasomal degradation of PBX-2. Retinoic acid induces differentiation of F9 teratocarcinoma cells with a cascade synthesis of HOX proteins. In PREP-1-overexpressing cells, HOXb1 induction was more sustained (3 days versus 1 day) and the induced level of MEIS-1b, another TALE (three amino acid loop extension) protein involved in embryonal development, was higher. Thus an increase in PREP-1 leads to changes in the fate-determining HOXb1 and has therefore important functional consequences.

  15. Imbalance in liver homeostasis leading to hyperplasia by overexpressing either one of the Bcl-2-related genes, zfBLP1 and zfMcl-1a.

    PubMed

    Her, Guor Mour; Cheng, Chih-Hung; Hong, Jiann-Ruey; Sundaram, Gnanapackiam Sheela; Wu, Jen-Leih

    2006-02-01

    Apoptosis is an essential part of normal embryonic development in vertebrates, and it is involved in sculpturing organs and controlling cell populations. In previous studies, we identified two novel proteins, zfBLP1 and zfMcl-1a, which are similar to those of the Bcl-2 family as a group of evolutionarily conserved proteins that regulate cellular anti-apoptosis. To evaluate the effect of dysregulated hepatocyte apoptosis during zebrafish hepatogenesis, we demonstrate the transgenic overexpression of either zfBLP1 or zfMcl-1a in zebrafish larval liver. Results showed that 18%-43% of larvae overexpressed zfBLP1 and that 16%-37% of larvae overexpressed zfMc1-1a in the liver leading to liver hyperplasia in 5-day postfertilization (dpf) zebrafish larvae. Histologically, zebrafish larvae exhibiting liver hyperplasia displayed a normal type of hepatocyte and the same cell numbers in their two liver buds compared with only one liver bud of wild-type larvae. Of interest, the expression of cyclin genes (A2, B, D1, and E), hepatocyte nuclear factor genes (HNF-1alpha, beta, -3beta, and 4alpha), and oncogenic markers (P53, c-myc, beta-catenin, N-ras, and gankyrin) were up-regulated, while the expression of C/EBP-alpha was down-regulated in a zfMcl-1a-mediated anti-apoptotic process of the liver. Increased cell death and proliferation was found in both hepatic cells of zebrafish larvae overexpressing either zfBLP1 or zfMcl-1a. However, those zebrafish larvae with liver hyperplasia only lived approximately 10 days. (This finding may have been due to liver abnormalities that led to failure of liver function.) In conclusion, transgenic overexpression of zfBLP1 or zfMcl-1a in zebrafish larvae interrupts regulation of the homeostatic balance between cell proliferation and programmed cell death during hepatogenesis and leads to liver hyperplasia. Copyright 2005 Wiley-Liss, Inc.

  16. Overexpression of wild-type PKD2 leads to increased proliferation and invasion of BON endocrine cells

    SciTech Connect

    Jackson, Lindsey N.; Li Jing; Chen, L. Andy; Townsend, Courtney M.; Evers, B. Mark . E-mail: mevers@utmb.edu

    2006-09-29

    Carcinoid tumors are rare neuroendocrine tumors with a predilection for the gastrointestinal tract. Protein kinase D (PKD), a novel serine/threonine protein kinase, has been implicated in the regulation of transport processes in certain cell types. We have reported an important role for PKD in stimulated peptide secretion from a human (BON) carcinoid cell line; however, the role of PKD isoforms, including PKD2, in the proliferation and invasion of carcinoid tumors remains unclear. In the present study, we found that overexpression of PKD2 by stable transfection of BON cells with PKD2-wild type (PKD2{sub WT}) significantly increased proliferation and invasion compared to cells transfected with PKD2-kinase dead (PKD2{sub KD}) or pcDNA3 (control). Similarly, inhibition of PKD2 activity with small interfering RNA (siRNA) significantly decreased proliferation and invasion compared to cells transfected with non-targeting control (NTC) siRNA. These data support an important role for PKD2 in carcinoid tumor progression. Targeted inhibition of the PKD family may prove to be a novel treatment option for patients with carcinoid tumors.

  17. Genomic loss of EZH2 leads to epigenetic modifications and overexpression of the HOX gene clusters in myelodysplastic syndrome.

    PubMed

    Xu, Feng; Liu, Li; Chang, Chun-Kang; He, Qi; Wu, Ling-Yun; Zhang, Zheng; Shi, Wen-Hui; Guo, Juan; Zhu, Yang; Zhao, You-Shan; Gu, Shu-Cheng; Fei, Cheng-Ming; Li, Xiao

    2016-02-16

    The role of EZH2 in cancer is complex and may vary depending on cancer type or stage. We examined the effect of altered EZH2 levels on H3K27 methylation, HOX gene expression, and malignant phenotype in myelodysplastic syndrome (MDS) cell lines and an in vivo xenograft model. We also studied links between EZH2 expression and prognosis in MDS patients. Patients with high-grade MDS exhibited lower levels of EZH2 expression than those with low-grade MDS. Low EZH2 expression was associated with high percentages of blasts, shorter survival, and increased transformation of MDS into acute myeloid leukemia (AML). MDS patients frequently had reductions in EZH2 copy number. EZH2 knockdown increased tumor growth capacity and reduced H3K27me3 levels in both MDS-derived leukemia cells and in a xenograft model. H3K27me3 levels were reduced and HOX gene cluster expression was increased in MDS patients. EZH2 knockdown also increased HOX gene cluster expression by reducing H3K27me3, and H3K27 demethylating agents increased HOX gene cluster expression in MDS-derived cell lines. These findings suggest genomic loss of EZH2 contributes to overexpression of the HOX gene clusters in MDS through epigenetic modifications.

  18. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    PubMed

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

  19. Targeted over-expression of endothelin-1 in astrocytes leads to more severe brain damage and vasospasm after subarachnoid hemorrhage

    PubMed Central

    2013-01-01

    Background Endothelin-1 (ET-1) is a potent vasoconstrictor, and astrocytic ET-1 is reported to play a role in the pathogenesis of cerebral ischemic injury and cytotoxic edema. However, it is still unknown whether astrocytic ET-1 also contributes to vasogenic edema and vasospasm during subarachnoid hemorrhage (SAH). In the present study, transgenic mice with astrocytic endothelin-1 over-expression (GET-1 mice) were used to investigate the pathophysiological role of ET-1 in SAH pathogenesis. Results The GET-1 mice experienced a higher mortality rate and significantly more severe neurological deficits, blood–brain barrier breakdown and vasogenic edema compared to the non-transgenic (Ntg) mice following SAH. Oral administration of vasopressin V1a receptor antagonist, SR 49059, significantly reduced the cerebral water content in the GET-1 mice. Furthermore, the GET-1 mice showed significantly more pronounced middle cerebral arterial (MCA) constriction after SAH. Immunocytochemical analysis showed that the calcium-activated potassium channels and the phospho-eNOS were significantly downregulated, whereas PKC-α expression was significantly upregulated in the MCA of the GET-1 mice when compared to Ntg mice after SAH. Administration of ABT-627 (ETA receptor antagonist) significantly down-regulated PKC-α expression in the MCA of the GET-1 mice following SAH. Conclusions The present study suggests that astrocytic ET-1 involves in SAH-induced cerebral injury, edema and vasospasm, through ETA receptor and PKC-mediated potassium channel dysfunction. Administration of ABT-627 (ETA receptor antagonist) and SR 49059 (vasopressin V1a receptor antagonist) resulted in amelioration of edema and vasospasm in mice following SAH. These data provide a strong rationale to investigate SR 49059 and ABT-627 as therapeutic drugs for the treatment of SAH patients. PMID:24156724

  20. Lead uptake increases drought tolerance of wild type and transgenic poplar (Populus tremula x P. alba) overexpressing gsh 1.

    PubMed

    Samuilov, Sladjana; Lang, Friedericke; Djukic, Matilda; Djunisijevic-Bojovic, Danijela; Rennenberg, Heinz

    2016-09-01

    Growth and development of plants largely depends on their adaptation ability in a changing climate. This is particularly true on heavy metal contaminated soils, but the interaction of heavy metal stress and climate on plant performance has not been intensively investigated. The aim of the present study was to elucidate if transgenic poplars (Populus tremula x P. alba) with enhanced glutathione content possess an enhanced tolerance to drought and lead (Pb) exposure (single and in combination) and if they are good candidates for phytoremediation of Pb contaminated soil. Lead exposure reduced growth and biomass accumulation only in above-ground tissue of wild type poplar, although most of lead accumulated in the roots. Drought caused a decline of the water content rather than reduced biomass production, while Pb counteracted this decline in the combined exposure. Apparently, metals such as Pb possess a protective function against drought, because they interact with abscisic acid dependent stomatal closure. Lead exposure decreased while drought increased glutathione content in leaves of both plant types. Lead accumulation was higher in the roots of transgenic plants, presumably as a result of chelation by glutathione. Water deprivation enhanced Pb accumulation in the roots, but Pb was subject to leakage out of the roots after re-watering. Transgenic plants showed better adaptation under mild drought plus Pb exposure partially due to improved glutathione synthesis. However, the transgenic plants cannot be considered as a good candidate for phytoremediation of Pb, due to its small translocation to the shoots and its leakage out of the roots upon re-watering.

  1. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    SciTech Connect

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45{sup +} hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45{sup low}c-Kit{sup +} cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

  2. Dyrk1A overexpression leads to increase of 3R-tau expression and cognitive deficits in Ts65Dn Down syndrome mice.

    PubMed

    Yin, Xiaomin; Jin, Nana; Shi, Jianhua; Zhang, Yanchong; Wu, Yue; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2017-04-04

    Alternative splicing of tau exon 10 generates tau isoforms with three or four microtubule-binding repeats, 3R-tau and 4R-tau, which is equally expressed in adult human brain. Imbalanced expression in 3R-tau and 4R-tau has been found in several sporadic and inherited tauopathies, suggesting that dysregulation of tau exon 10 is sufficient to cause neurodegenerative diseases. We previously reported that Dyrk1A, which is overexpressed in Down syndrome brains, regulates alternative splicing of exogenous tau exon 10. In the present study, we investigated the regulation of endogenous tau exon 10 splicing by Dyrk1A. We found that inhibition of Dyrk1A enhanced tau exon 10 inclusion, leading to an increase in 4R-tau/3R-tau ratio in differentiated-human neuronal progenitors and in the neonatal rat brains. Accompanied with overexpression of Dyrk1A, 3R-tau was increased and 4R-tau was decreased in the neonatal brains of Ts65Dn mice, a model of Down syndrome. Treatment with Dyrk1A inhibitor, green tea flavonol epigallocatechin-gallate (EGCG), from gestation to adulthood suppressed 3R-tau expression and rescued anxiety and memory deficits in Ts65Dn mouse brains. Thus, Dyrk1A might be an ideal therapeutic target for Alzheimer's disease, especially for Down syndrome and EGCG which inhibits Dyrk1A may have potential effect on the treatment or prevention of this disease.

  3. Overexpression of Pa_1_10620 encoding a mitochondrial Podospora anserina protein with homology to superoxide dismutases and ribosomal proteins leads to lifespan extension.

    PubMed

    Grimm, Carolin; Böhl, Lena; Osiewacz, Heinz D

    2015-02-01

    In biological systems, reactive oxygen species (ROS) represent 'double edged swords': as signaling molecules they are essential for proper development, as reactive agents they cause molecular damage and adverse effects like degeneration and aging. A well-coordinated control of ROS is therefore of key importance. Superoxide dismutases (SODs) are enzymes active in the detoxification of superoxide. The number of isoforms of these proteins varies among species. Here we report the characterization of the putative protein encoded by Pa_1_10620 that has been previously annotated to code for a mitochondrial ribosomal protein but shares also sequence domains with SODs. We report that the gene is transcribed in P. anserina cultures of all ages and that the encoded protein localizes to mitochondria. In strains overexpressing Pa_1_10620 in a genetic background in which PaSod3, the mitochondrial MnSOD of P. anserina, is deleted, no SOD activity could be identified in isolated mitochondria. However, overexpression of the gene leads to lifespan extension suggesting a pro-survival function of the protein in P. anserina.

  4. Retraction: "Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells" by Bao et al.

    PubMed

    2016-08-01

    The above article, published online on April 18, 2011 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the second author that found Figures 1C and 4C to be inappropriately re-used and re-labeled. REFERENCE Bao B, Wang Z, Ali S, Kong D, Banerjee S, Ahmad A, Li Y, Azmi AS, Miele L, Sarkar FH. 2011. Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells. J Cell Biochem 112:2296-2306; doi: 10.1002/jcb.23150.

  5. Overexpression of a truncated CTF7 construct leads to pleiotropic defects in reproduction and vegetative growth in Arabidopsis.

    PubMed

    Liu, Desheng; Makaroff, Christopher A

    2015-03-05

    Eco1/Ctf7 is essential for the establishment of sister chromatid cohesion during S phase of the cell cycle. Inactivation of Ctf7/Eco1 leads to a lethal phenotype in most organisms. Altering Eco1/Ctf7 levels or point mutations in the gene can lead to alterations in nuclear division as well as a wide range of developmental defects. Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe defects in reproduction and vegetative growth. To further investigate the function(s) of AtCTF7, a tagged version of AtCTF7 and several AtCTF7 deletion constructs were created and transformed into wild type or ctf7 +/- plants. Transgenic plants expressing 35S:NTAP:AtCTF7∆299-345 (AtCTF7∆B) displayed a wide range of phenotypic alterations in reproduction and vegetative growth. Male meiocytes exhibited chromosome fragmentation and uneven chromosome segregation. Mutant ovules contained abnormal megasporocyte-like cells during pre-meiosis, megaspores experienced elongated meiosis and megagametogenesis, and defective megaspores/embryo sacs were produced at various stages. The transgenic plants also exhibited a broad range of vegetative defects, including meristem disruption and dwarfism that were inherited in a non-Mendelian fashion. Transcripts for epigenetically regulated transposable elements (TEs) were elevated in transgenic plants. Transgenic plants expressing 35S:AtCTF7∆B displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7∆B plants are caused by high-level expression of AtCTF7∆B. High level expression of AtCTF7∆B disrupts megasporogenesis, megagametogenesis and male meiosis, as well as causing a broad range of vegetative defects, including dwarfism that are inherited in a non-Mendelian fashion.

  6. Epigenetic alterations leading to TMPRSS4 promoter hypomethylation and protein overexpression predict poor prognosis in squamous lung cancer patients

    PubMed Central

    Villalba, Maria; Diaz-Lagares, Angel; Redrado, Miriam; de Aberasturi, Arrate L.; Segura, Victor; Bodegas, Maria Elena; Pajares, Maria J.; Pio, Ruben; Freire, Javier; Gomez-Roman, Javier; Montuenga, Luis M.; Esteller, Manel; Sandoval, Juan; Calvo, Alfonso

    2016-01-01

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which highlights the need of innovative therapeutic options. Although targeted therapies can be successfully used in a subset of patients with lung adenocarcinomas (ADC), they are not appropriate for patients with squamous cell carcinomas (SCC). In addition, there is an unmet need for the identification of prognostic biomarkers that can select patients at risk of relapse in early stages. Here, we have used several cohorts of NSCLC patients to analyze the prognostic value of both protein expression and DNA promoter methylation status of the prometastatic serine protease TMPRSS4. Moreover, expression and promoter methylation was evaluated in a panel of 46 lung cancer cell lines. We have demonstrated that a high TMPRSS4 expression is an independent prognostic factor in SCC. Similarly, aberrant hypomethylation in tumors, which correlates with high TMPRSS4 expression, is an independent prognostic predictor in SCC. The inverse correlation between expression and methylation status was also observed in cell lines. In vitro studies showed that treatment of cells lacking TMPRSS4 expression with a demethylating agent significantly increased TMPRSS4 levels. In conclusion, TMPRSS4 is a novel independent prognostic biomarker regulated by epigenetic changes in SCC and a potential therapeutic target in this tumor type, where targeted therapy is still underdeveloped. PMID:26989022

  7. Epigenetic alterations leading to TMPRSS4 promoter hypomethylation and protein overexpression predict poor prognosis in squamous lung cancer patients.

    PubMed

    Villalba, Maria; Diaz-Lagares, Angel; Redrado, Miriam; de Aberasturi, Arrate L; Segura, Victor; Bodegas, Maria Elena; Pajares, Maria J; Pio, Ruben; Freire, Javier; Gomez-Roman, Javier; Montuenga, Luis M; Esteller, Manel; Sandoval, Juan; Calvo, Alfonso

    2016-04-19

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which highlights the need of innovative therapeutic options. Although targeted therapies can be successfully used in a subset of patients with lung adenocarcinomas (ADC), they are not appropriate for patients with squamous cell carcinomas (SCC). In addition, there is an unmet need for the identification of prognostic biomarkers that can select patients at risk of relapse in early stages. Here, we have used several cohorts of NSCLC patients to analyze the prognostic value of both protein expression and DNA promoter methylation status of the prometastatic serine protease TMPRSS4. Moreover, expression and promoter methylation was evaluated in a panel of 46 lung cancer cell lines. We have demonstrated that a high TMPRSS4 expression is an independent prognostic factor in SCC. Similarly, aberrant hypomethylation in tumors, which correlates with high TMPRSS4 expression, is an independent prognostic predictor in SCC. The inverse correlation between expression and methylation status was also observed in cell lines. In vitro studies showed that treatment of cells lacking TMPRSS4 expression with a demethylating agent significantly increased TMPRSS4 levels. In conclusion, TMPRSS4 is a novel independent prognostic biomarker regulated by epigenetic changes in SCC and a potential therapeutic target in this tumor type, where targeted therapy is still underdeveloped.

  8. Proliferative Vitreoretinopathy after Eye Injuries: An Overexpression of Growth Factors and Cytokines Leading to a Retinal Keloid

    PubMed Central

    Morescalchi, Francesco; Duse, Sarah; Gambicorti, Elena; Romano, Mario R.; Costagliola, Ciro; Semeraro, Francesco

    2013-01-01

    Eye injury is a significant disabling worldwide health problem. Proliferative Vitreoretinopathy (PVR) is a common complication that develops in up to 40–60% of patients with an open-globe injury. Our knowledge about the pathogenesis of PVR has improved in the last decades. It seems that the introduction of immune cells into the vitreous, like in penetrating ocular trauma, triggers the production of growth factors and cytokines that come in contact with intra-retinal cells, like Müller cells and RPE cells. Growth factors and cytokines drive the cellular responses leading to PVR's development. Knowledge of the pathobiological and pathophysiological mechanisms involved in posttraumatic PVR is increasing the possibilities of management, and it is hoped that in the future our treatment strategies will evolve, in particular adopting a multidrug approach, and become even more effective in vision recovery. This paper reviews the current literature and clinical trial data on the pathogenesis of PVR and its correlation with ocular trauma and describes the biochemical/molecular events that will be fundamental for the development of novel treatment strategies. This literature review included PubMed articles published from 1979 through 2013. Only studies written in English were included. PMID:24198445

  9. Overexpression of PrfA Leads to Growth Inhibition of Listeria monocytogenes in Glucose-Containing Culture Media by Interfering with Glucose Uptake

    PubMed Central

    Marr, A. K.; Joseph, B.; Mertins, S.; Ecke, R.; Müller-Altrock, S.; Goebel, W.

    2006-01-01

    Listeria monocytogenes strains expressing high levels of the virulence regulator PrfA (mutant PrfA* or wild-type PrfA) show strong growth inhibition in minimal media when they are supplemented with glucose but not when they are supplemented with glucose-6-phosphate compared to the growth of isogenic strains expressing low levels of PrfA. A significantly reduced rate of glucose uptake was observed in a PrfA*-overexpressing strain growing in LB supplemented with glucose. Comparative transcriptome analyses were performed with RNA isolated from a prfA mutant and an isogenic strain carrying multiple copies of prfA or prfA* on a plasmid. These analyses revealed that in addition to high transcriptional up-regulation of the known PrfA-regulated virulence genes (group I), there was less pronounced up-regulation of the expression of several phage and metabolic genes (group II) and there was strong down-regulation of several genes involved mainly in carbon and nitrogen metabolism in the PrfA*-overexpressing strain (group III). Among the latter genes are the nrgAB, gltAB, and glnRA operons (involved in nitrogen metabolism), the ilvB operon (involved in biosynthesis of the branched-chain amino acids), and genes for some ABC transporters. Most of the down-regulated genes have been shown previously to belong to a class of genes in Bacillus subtilis whose expression is negatively affected by impaired glucose uptake. Our results lead to the conclusion that excess PrfA (or PrfA*) interferes with a component(s) essential for phosphotransferase system-mediated glucose transport. PMID:16707681

  10. Unmanipulated native fat exposed to high-energy diet, but not autologous grafted fat by itself, may lead to overexpression of Ki67 and PAI-1.

    PubMed

    Claro, Francisco; Morari, Joseane; Moreira, Luciana R; Sarian, Luís O Z; Pinto, Glauce A; Velloso, Licio A; Pinto-Neto, Aarão M

    2015-01-01

    Although its unclear oncological risk, which led to more than 20 years of prohibition of its use, fat grafting to the breast is widely used nowadays even for aesthetic purposes. Thus, we proposed an experimental model in rats to analyze the inflammatory activity, cellular proliferation and levels of Plasminogen Activator Inhibitor (PAI-1) in grafted fat, and in native fat exposed to high-energy diet in order to study the oncological potential of fat tissue. Samples of grafted fat of rats on regular-energy diet were compared with paired samples of native fat from the same rat on regular-energy diet and on high-energy diet in a different time. Analysis involved microscopic comparisons using hematoxylin-eosin staining, immunohistochemistry with anti-CD68-labelled macrophages, and gene expression of Ki-67 and PAI-1. Hematoxylin-eosin staining analyses did not find any atypical cellular infiltration or unusual tissue types in the samples of grafted fat. The inflammatory status, assessed through immunohistochemical identification of CD68-labelled macrophages, was similar among samples of native fat and grafted fat of rat on regular-energy diet and of native fat of rats on high-energy diet. Real-time PCR revealed that high-energy diet, but not fat grafting, leads to proliferative status on adipose tissue (overexpression of ki-67, p = 0.046) and raised its PAI-1 levels, p < 0.001. While the native adipose tissue overexpressed PAI-1 and KI67 when exposed to high-energy diet, the grafted fat by itself was unable to induce cellular proliferation, chronic inflammatory activity and/or elevation of PAI-1 levels.

  11. Overexpression of a type-I isopentenyl pyrophosphate isomerase of Artemisia annua in the cytosol leads to high arteannuin B production and artemisinin increase.

    PubMed

    Ma, Dongming; Li, Gui; Alejos-Gonzalez, Fatima; Zhu, Yue; Xue, Zhen; Wang, Aimin; Zhang, Hui; Li, Xing; Ye, Hechun; Wang, Hong; Liu, Benye; Xie, De-Yu

    2017-08-01

    We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the network may improve the production of artemisinin and its precursors. In this study, we cloned an isopentenyl pyrophosphate isomerase (IPPI) cDNA, AaIPPI1, from Artemisia annua (Aa). The full-length cDNA encodes a type-I IPPI containing a plastid transit peptide (PTP) at its amino terminus. After the removal of the PTP, the recombinant truncated AaIPPI1 isomerized isopentenyl pyrophosphate (IPP) to dimethyl allyl pyrophosphate (DMAPP) and vice versa. The steady-state equilibrium ratio of IPP/DMAPP in the enzymatic reactions was approximately 1:7. The truncated AaIPPI1 was overexpressed in the cytosol of the SP A. annua variety. The leaves of transgenic plants produced approximately 4% arteannuin B (g g(-1) , dry weight, dw) and 0.17-0.25% artemisinin (g g(-1) , dw), the levels of which were significantly higher than those in the leaves of wild-type plants. In addition, transgenic plants showed an increase in artemisinic acid production of more than 1% (g g(-1) , dw). In contrast, isoprene formation was significantly reduced in transgenic plants. These results provide evidence that overexpression of AaIPPI1 in the cytosol can lead to metabolic alterations of terpenoid biosynthesis, and show that these transgenic plants have the potential to yield high production levels of arteannuin B as a new precursor source for artemisinin. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  12. Lead

    MedlinePlus

    ... EPA United States Environmental Protection Agency Search Search Lead Contact Us Share Lead Poisoning is Preventable If your home was built ... to protect people from harmful lead exposures. Less Lead in Drinking Water = Better Health Learn about the ...

  13. Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

    PubMed

    Akaike, Y; Kuwano, Y; Nishida, K; Kurokawa, K; Kajita, K; Kano, S; Masuda, K; Rokutan, K

    2015-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent

  14. KIT over-expression by p55PIK-PI3K leads to Imatinib-resistance in patients with gastrointestinal stromal tumors

    PubMed Central

    Cao, Xiaonian; Luo, Xuelai; Wang, Guoping; Xia, Xianmin; Hu, Junbo; Wang, Jing

    2016-01-01

    Imatinib is the first-line drug for gastrointestinal stromal tumors (GISTs), as mutated KIT is closely associated with the occurrence of GIST. However, Imatinib resistance (IMA-resistance) occurs inevitably in most GIST patients. Although the over-expression of KIT in GIST is one of the major factors contributing to IMA-resistance, the underlying mechanism is still unclear. In this study, we demonstrate that p55PIK, an isoform of phosphoinositide 3-kinase (PI3K), increases KIT expression, leading to IMA-resistance in GISTs by activating NF-κB signaling pathway. Furthermore, down-regulation of p55PIK significantly decreases KIT expression and re-sensitizes IMA-resistance-GIST cells to Imatinib in vitro and in vivo. Interestingly, the expression of both p55PIK and KIT proteins is significantly increased in tumor samples from IMA-resistance-GIST patients, suggesting that p55PIK up-regulation may be important for IMA-resistance in the clinical setting. Altogether, our data provide evidence that p55PIK-PI3K signaling can contribute to IMA-resistance in GIST by increasing KIT expression. Moreover, p55PIK may be a novel potential drug target for treating tumors that develop IMA-resistance. PMID:26587973

  15. Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.

    PubMed

    Elitt, Christopher M; McIlwrath, Sabrina L; Lawson, Jeffery J; Malin, Sacha A; Molliver, Derek C; Cornuet, Pamela K; Koerber, H Richard; Davis, Brian M; Albers, Kathryn M

    2006-08-16

    Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRalpha3 and the receptor tyrosine kinase Ret. Expression of the GFRalpha3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRalpha3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRalpha3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.

  16. Lead

    MedlinePlus

    ... are approximately half a million U.S. children ages 1-5 with blood lead levels above 5 micrograms per deciliter (µg/dL), the reference level at which CDC recommends public health actions be initiated. No safe blood lead level in children has been ...

  17. Nonsymbiotic Hemoglobin-2 Leads to an Elevated Energy State and to a Combined Increase in Polyunsaturated Fatty Acids and Total Oil Content When Overexpressed in Developing Seeds of Transgenic Arabidopsis Plants1[OA

    PubMed Central

    Vigeolas, Helene; Hühn, Daniela; Geigenberger, Peter

    2011-01-01

    Nonsymbiotic hemoglobins are ubiquitously expressed in plants and divided into two different classes based on gene expression pattern and oxygen-binding properties. Most of the published research has been on the function of class 1 hemoglobins. To investigate the role of class 2 hemoglobins, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated overexpressing Arabidopsis hemoglobin-2 (AHb2) under the control of a seed-specific promoter. Overexpression of AHb2 led to a 40% increase in the total fatty acid content of developing and mature seeds in three subsequent generations. This was mainly due to an increase in the polyunsaturated C18:2 (ω-6) linoleic and C18:3 (ω-3) α-linolenic acids. Moreover, AHb2 overexpression led to an increase in the C18:2/C18:1 and C18:3/C18:2 ratios as well as in the C18:3 content in mol % of total fatty acids and in the unsaturation/saturation index of total seed lipids. The increase in fatty acid content was mainly due to a stimulation of the rate of triacylglycerol synthesis, which was attributable to a 3-fold higher energy state and a 2-fold higher sucrose content of the seeds. Under low external oxygen, AHb2 overexpression maintained an up to 5-fold higher energy state and prevented fermentation. This is consistent with AHb2 overexpression results in improved oxygen availability within developing seeds. In contrast to this, overexpression of class 1 hemoglobin did not lead to any significant increase in the metabolic performance of the seeds. These results provide evidence for a specific function of class 2 hemoglobin in seed oil production and in promoting the accumulation of polyunsaturated fatty acids by facilitating oxygen supply in developing seeds. PMID:21205621

  18. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway.

    PubMed

    Ahn, Chang Sook; Ahn, Hee-Kyung; Pai, Hyun-Sook

    2015-02-01

    Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  19. Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production

    PubMed Central

    Fu, Chunxiang; Sunkar, Ramanjulu; Zhou, Chuanen; Shen, Hui; Zhang, Ji-Yi; Matts, Jessica; Wolf, Jennifer; Mann, David G J; Stewart, C Neal; Tang, Yuhong; Wang, Zeng-Yu

    2012-01-01

    Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were non-flowering. These two groups of plants produced 58%–101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment. PMID:22239253

  20. The overexpression of the pine transcription factor PpDof5 in Arabidopsis leads to increased lignin content and affects carbon and nitrogen metabolism.

    PubMed

    Rueda-López, Marina; Cañas, Rafael A; Canales, Javier; Cánovas, Francisco M; Ávila, Concepción

    2015-12-01

    PpDof 5 is a regulator of the expression of glutamine synthetase (GS; EC 6.3.1.2) genes in photosynthetic and non-photosynthetic tissues of maritime pine. We have used Arabidopsis thaliana as a model system to study PpDof 5 function in planta, generating transgenic lines overexpressing the pine transcription factor. The overexpression of PpDof 5 resulted in a substantial increase of lignin content with a simultaneous regulation of carbon and nitrogen key genes. In addition, partitioning in carbon and nitrogen compounds was spread via various secondary metabolic pathways. These results suggest pleiotropic effects of PpDof 5 expression on various metabolic pathways of carbon and nitrogen metabolism. Plants overexpressing PpDof 5 exhibited upregulation of genes encoding enzymes for sucrose and starch biosynthesis, with a parallel increase in the content of soluble sugars. When the plants were grown under nitrate as the sole nitrogen source, they exhibited a significant regulation of the expression of genes involved mainly in signaling, but similar growth rates to wild-type plants. However, plants grown under ammonium exhibited major induction of the expression of photosynthetic genes and differential expression of ammonium and nitrate transporters. All these data suggest that in addition to controlling ammonium assimilation, PpDof 5 could be also involved in the regulation of other pathways in carbon and nitrogen metabolism in pine trees.

  1. A dual-targeted purple acid phosphatase in Arabidopsis thaliana moderates carbon metabolism and its overexpression leads to faster plant growth and higher seed yield.

    PubMed

    Sun, Feng; Suen, Pui Kit; Zhang, Youjun; Liang, Chao; Carrie, Chris; Whelan, James; Ward, Jane L; Hawkins, Nathaniel D; Jiang, Liwen; Lim, Boon Leong

    2012-04-01

    Overexpression of AtPAP2, a purple acid phosphatase (PAP) with a unique C-terminal hydrophobic motif in Arabidopsis, resulted in earlier bolting and a higher seed yield. Metabolite analysis showed that the shoots of AtPAP2 overexpression lines contained higher levels of sugars and tricarboxylic acid (TCA) metabolites. Enzyme assays showed that sucrose phosphate synthase (SPS) activity was significantly upregulated in the overexpression lines. The higher SPS activity arose from a higher level of SPS protein, and was independent of SnRK1. • AtPAP2 was found to be targeted to both plastids and mitochondria via its C-terminal hydrophobic motif. Ectopic expression of a truncated AtPAP2 without this C-terminal motif in Arabidopsis indicated that the subcellular localization of AtPAP2 is essential for its biological actions. • Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution. AtPAP2 is the first PAP shown to modulate carbon metabolism and the first shown to be dual-targeted to both plastids and mitochondria by a C-terminal targeting signal. • One PAP-like sequence carrying a hydrophobic C-terminal motif could be identified in the genome of the smallest free-living photosynthetic eukaryote, Ostreococcus tauri. This might reflect a common ancestral function of AtPAP2-like sequences in the regulation of carbon metabolism. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  2. Overexpressions of xylA and xylB in Klebsiella pneumoniae Lead to Enhanced 1,3-Propanediol Production by Cofermentation of Glycerol and Xylose.

    PubMed

    Lu, Xinyao; Fu, Xiaomeng; Zong, Hong; Zhuge, Bin

    2016-07-28

    1,3-Propanediol (1,3-PD) is a valuable platform compound. Many studies have shown that the supplement of NADH plays a key role in the bioproduction of 1,3-PD from Klebsiella pneumoniae. In this study, the xylA and xylB genes from Escherichia coli were overexpressed individually or simultaneously in K. pneumoniae to improve the production of 1,3-PD by cofermentation of glycerol and xylose. Compared with the parent strain, the xylose consumption was significantly increased by the introduction of these two genes. The 1,3-PD titers were raised from 17.9 g/l to 23.5, 23.9, and 24.4 g/l, respectively, by the overexpression of xylA and xylB as well as their coexpression. The glycerol conversion rate (mol/mol) was enhanced from 54.1% to 73.8%. The concentration of 2,3-butanediol was increased by 50% at the middle stage but drastically decreased after that. The NADH and NADH/NAD(+) ratio were improved. This report suggests that overexpression of xylA or xylB is an effective strategy to improve the xylose assimilation rate to provide abundant reducing power for the biosynthesis of 1,3-PD in K. pneumoniae.

  3. The Over-Expression of an Arabidopsis B3 Transcription Factor, ABS2/NGAL1, Leads to the Loss of Flower Petals

    PubMed Central

    Wang, Rui; Zhang, Gaisheng; Yu, Fei

    2012-01-01

    Transcriptional regulations are involved in many aspects of plant development and are mainly achieved through the actions of transcription factors (TF). To investigate the mechanisms of plant development, we carried out genetic screens for mutants with abnormal shoot development. Taking an activation tagging approach, we isolated a gain-of-function mutant abs2-1D (abnormal shoot 2-1D). abs2-1D showed pleiotropic growth defects at both the vegetative and reproductive developmental stages. We cloned ABS2 and it encodes a RAV sub-family of plant B3 type of transcriptional factors. Phylogenetic analysis showed that ABS2 was closely related to NGATHA (NGA) genes that are involved in flower development and was previously named NGATHA-Like 1 (NGAL1). NGAL1 was expressed mainly in the root and the filament of the stamen in flower tissues and sub-cellular localization assay revealed that NGAL1 accumulated in the nucleus. Interestingly, over-expression of NGAL1 driven by the constitutive 35S promoter led to transgenic plants with conspicuous flower defects, particularly a loss-of-petal phenotype. A loss-of-function ngal1-1 mutant did not show obvious phenotype, suggesting the existence of redundant activities and also the utility of gain-of-function genetic screens. Our results show that the over-expression of NGAL1 is capable of altering flower petal development, as well as shoot development. PMID:23185464

  4. The over-expression of an Arabidopsis B3 transcription factor, ABS2/NGAL1, leads to the loss of flower petals.

    PubMed

    Shao, Jingxia; Liu, Xiayan; Wang, Rui; Zhang, Gaisheng; Yu, Fei

    2012-01-01

    Transcriptional regulations are involved in many aspects of plant development and are mainly achieved through the actions of transcription factors (TF). To investigate the mechanisms of plant development, we carried out genetic screens for mutants with abnormal shoot development. Taking an activation tagging approach, we isolated a gain-of-function mutant abs2-1D (abnormal shoot 2-1D). abs2-1D showed pleiotropic growth defects at both the vegetative and reproductive developmental stages. We cloned ABS2 and it encodes a RAV sub-family of plant B3 type of transcriptional factors. Phylogenetic analysis showed that ABS2 was closely related to NGATHA (NGA) genes that are involved in flower development and was previously named NGATHA-Like 1 (NGAL1). NGAL1 was expressed mainly in the root and the filament of the stamen in flower tissues and sub-cellular localization assay revealed that NGAL1 accumulated in the nucleus. Interestingly, over-expression of NGAL1 driven by the constitutive 35S promoter led to transgenic plants with conspicuous flower defects, particularly a loss-of-petal phenotype. A loss-of-function ngal1-1 mutant did not show obvious phenotype, suggesting the existence of redundant activities and also the utility of gain-of-function genetic screens. Our results show that the over-expression of NGAL1 is capable of altering flower petal development, as well as shoot development.

  5. Overexpression of an Isoprenyl Diphosphate Synthase in Spruce Leads to Unexpected Terpene Diversion Products That Function in Plant Defense1[W][OPEN

    PubMed Central

    Nagel, Raimund; Berasategui, Aileen; Paetz, Christian; Gershenzon, Jonathan; Schmidt, Axel

    2014-01-01

    Spruce (Picea spp.) and other conifers employ terpenoid-based oleoresin as part of their defense against herbivores and pathogens. The short-chain isoprenyl diphosphate synthases (IDS) are situated at critical branch points in terpene biosynthesis, producing the precursors of the different terpenoid classes. To determine the role of IDS and to create altered terpene phenotypes for assessing the defensive role of terpenoids, we overexpressed a bifunctional spruce IDS, a geranyl diphosphate and geranylgeranyl diphosphate synthase in white spruce (Picea glauca) saplings. While transcript level (350-fold), enzyme activity level (7-fold), and in planta geranyl diphosphate and geranylgeranyl diphosphate levels (4- to 8-fold) were significantly increased in the needles of transgenic plants, there was no increase in the major monoterpenes and diterpene acids of the resin and no change in primary isoprenoids, such as sterols, chlorophylls, and carotenoids. Instead, large amounts of geranylgeranyl fatty acid esters, known from various gymnosperm and angiosperm plant species, accumulated in needles and were shown to act defensively in reducing the performance of larvae of the nun moth (Lymantria monacha), a conifer pest in Eurasia. These results show the impact of overexpression of an IDS and the defensive role of an unexpected accumulation product of terpenoid biosynthesis with the potential for a broader function in plant protection. PMID:24346420

  6. Degradation of MDM2 by the interaction between berberine and DAXX leads to potent apoptosis in MDM2-overexpressing cancer cells.

    PubMed

    Zhang, Xiaoling; Gu, Lubing; Li, Jiansha; Shah, Noopur; He, Jing; Yang, Lin; Hu, Qun; Zhou, Muxiang

    2010-12-01

    Berberine, a natural product derived from a plant used in Chinese herbal medicine, is reported to exhibit anticancer effects; however, its mechanism of action is not clearly defined. Herein, we demonstrate that berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells by downregulating the MDM2 oncoprotein. The proapoptotic effects of berberine were closely associated with both the MDM2 expression levels and p53 status of a set of ALL cell lines. The most potent apoptosis was induced by berberine in ALL cells with both MDM2 overexpression and a wild-type (wt)-p53, whereas no proapoptotic effect was detected in ALL cells that were negative for MDM2 and wt-p53. In contrast to the conventional chemotherapeutic drug doxorubicin, which induces p53 activation and a subsequent upregulation of MDM2, berberine strongly induced persistent downregulation of MDM2 followed by a steady-state activation of p53. We discovered that downregulation of MDM2 in ALL cells by berberine occurred at a posttranslational level through modulation of death domain-associated protein (DAXX), which disrupted the MDM2-DAXX-HAUSP interactions and thereby promoted MDM2 self-ubiquitination and degradation. Given that MDM2-overexpressing cancer cells are commonly chemoresistant, our findings suggest that this naturally derived agent may have a highly useful role in the treatment of cancer patients with refractory disease.

  7. Degradation of MDM2 by the interaction between berberine and DAXX leads to potent apoptosis in MDM2-overexpressing cancer cells

    PubMed Central

    Zhang, Xiaoling; Gu, Lubing; Li, Jiansha; Shah, Noopur; He, Jing; Yang, Lin; Hu, Qun; Zhou, Muxiang

    2010-01-01

    Berberine, a natural product derived from a plant used in Chinese herbal medicine, is reported to exhibit anticancer effects; however, its mechanism of action is not clearly defined. Herein, we demonstrate that berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells by downregulating the MDM2 oncoprotein. The pro-apoptotic effects of berberine were closely associated with both the MDM2 expression levels and p53 status of a set of ALL cell lines. The most potent apoptosis was induced by berberine in ALL cells with both MDM2 overexpression and a wild-type (wt) p53, while no pro-apoptotic effect was detected in ALL cells that were negative for MDM2 and wt-p53. In contrast to the conventional chemotherapeutic drug doxorubicin, which induces p53 activation and a subsequent upregulation of MDM2, berberine strongly induced persistent downregulation of MDM2 followed by a steady-state activation of p53. We discovered that downregulation of MDM2 in ALL cells by berberine occurred at a post-translational level through modulation of DAXX, which disrupted the MDM2-DAXX-HAUSP interactions and thereby promoted MDM2 self-ubiquitination and degradation. Given that MDM2-overexpressing cancer cells are commonly chemoresistant, our findings suggest that this naturally-derived agent may have a highly useful role in the treatment of cancer patients with refractory disease. PMID:20935220

  8. Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose

    PubMed Central

    2014-01-01

    Pectin is a structural heteropolysaccharide of the primary cell walls of plants and as such is a significant fraction of agricultural waste residues that is currently insufficiently used. Its main component, D-galacturonic acid, is an attractive substrate for bioconversion. The complete metabolic pathway is present in the genome of Aspergillus niger, that is used in this study. The objective was to identify the D-galacturonic acid transporter in A. niger and to use this transporter to study D-galacturonic acid metabolism. We have functionally characterized the gene An14g04280 that encodes the D-galacturonic acid transporter in A. niger. In a mixed sugar fermentation it was found that the An14g04280 overexpression strain, in contrast to the parent control strain, has a preference for D-galacturonic acid over D-xylose as substrate. Overexpression of this transporter in A. niger resulted in a strong increase of D-galacturonic acid uptake and induction of the D-galacturonic acid reductase activity, suggesting a metabolite controlled regulation of the endogenous D-galacturonic acid catabolic pathway. PMID:25177540

  9. Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

    PubMed Central

    Yu, Wenzhen; Wu, Xi; Zhou, Peng; Li, Xiaoxin

    2012-01-01

    Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis-like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis. PMID:23056244

  10. Overexpression of HTRA1 leads to down-regulation of fibronectin and functional changes in RF/6A cells and HUVECs.

    PubMed

    Jiang, Jingjing; Huang, Lvzhen; Yu, Wenzhen; Wu, Xi; Zhou, Peng; Li, Xiaoxin

    2012-01-01

    Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis-like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis.

  11. Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

    PubMed

    Dalman, Kerstin; Wind, Julia Johanna; Nemesio-Gorriz, Miguel; Hammerbacher, Almuth; Lundén, Karl; Ezcurra, Ines; Elfstrand, Malin

    2017-01-06

    The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated

  12. Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin.

    PubMed

    Hurteau, Gregory J; Carlson, J Andrew; Spivack, Simon D; Brock, Graham J

    2007-09-01

    MicroRNAs are approximately 22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, deltaEF1, Nil-2-alpha) had inversely proportional expression levels to miR-200c. Knockout experiments using anti-microRNA oligonucleotides increased TCF8 levels but with nonspecific effects. Therefore, to investigate target predictions, we overexpressed miR-200c in select cells lines. Ordinarily, the expression level of miR-200c in non-small-cell lung cancer A549 cells is low in contrast to normal human bronchial epithelial cells. Stable overexpression of miR-200c in A549 cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin, and altered cell morphology. In MCF7 (estrogen receptor-positive breast cancer) cells, there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (estrogen receptor-negative) cells lack detectable miR-200c and E-cadherin (the latter reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line also reduced levels of TCF8, restored E-cadherin expression, and altered cell morphology. Because the down-regulation of E-cadherin is a crucial event in epithelial-to-mesenchymal transition, loss of miR-200c expression could play a significant role in the initiation of an invasive phenotype, and, equally, miR-200c overexpression holds potential for its reversal.

  13. Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites

    PubMed Central

    Voss, Christiane; Ehrenman, Karen; Mlambo, Godfree; Mishra, Satish; Kumar, Kota Arun; Sacci, John B.; Sinnis, Photini

    2016-01-01

    ABSTRACT Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity. PMID:27353755

  14. Overexpression of TaNAC69 leads to enhanced transcript levels of stress up-regulated genes and dehydration tolerance in bread wheat.

    PubMed

    Xue, Gang-Ping; Way, Heather M; Richardson, Terese; Drenth, Janneke; Joyce, Priya A; McIntyre, C Lynne

    2011-07-01

    NAC proteins are plant-specific transcription factors and enriched with members involved in plant response to drought stress. In this study, we analyzed the expression profiles of TaNAC69 in bread wheat using Affymetrix Wheat Genome Array datasets and quantitative RT-PCR. TaNAC69 expression was positively associated with wheat responses to both abiotic and biotic stresses and was closely correlated with a number of stress up-regulated genes. The functional analyses of TaNAC69 in transgenic wheat showed that TaNAC69 driven by a barley drought-inducible HvDhn4s promoter led to marked drought-inducible overexpression of TaNAC69 in the leaves and roots of transgenic lines. The HvDhn4s:TaNAC69 transgenic lines produced more shoot biomass under combined mild salt stress and water-limitation conditions, had longer root and more root biomass under polyethylene glycol-induced dehydration. Analysis of transgenic lines with constitutive overexpression of TaNAC69 showed the enhanced expression levels of several stress up-regulated genes. DNA-binding assays revealed that TaNAC69 and its rice homolog (ONAC131) were capable of binding to the promoter elements of three rice genes (chitinase, ZIM, and glyoxalase I) and an Arabidopsis glyoxalase I family gene, which are homologs of TaNAC69 up-regulated stress genes. These data suggest that TaNAC69 is involved in regulating stress up-regulated genes and wheat adaptation to drought stress.

  15. Overexpression of BDNF Increases Excitability of the Lumbar Spinal Network and Leads to Robust Early Locomotor Recovery in Completely Spinalized Rats

    PubMed Central

    Ziemlińska, Ewelina; Kügler, Sebastian; Schachner, Melitta; Wewiór, Iwona; Czarkowska-Bauch, Julita; Skup, Małgorzata

    2014-01-01

    Strategies to induce recovery from lesions of the spinal cord have not fully resulted in clinical applications. This is a consequence of a number of impediments that axons encounter when trying to regrow beyond the lesion site, and that intraspinal rearrangements are subjected to. In the present study we evaluated (1) the possibility to improve locomotor recovery after complete transection of the spinal cord by means of an adeno-associated (AAV) viral vector expressing the neurotrophin brain-derived neurotrophic factor (BDNF) in lumbar spinal neurons caudal to the lesion site and (2) how the spinal cord transection and BDNF treatment affected neurotransmission in the segments caudal to the lesion site. BDNF overexpression resulted in clear increases in expression levels of molecules involved in glutamatergic (VGluT2) and GABAergic (GABA, GAD65, GAD67) neurotransmission in parallel with a reduction of the potassium-chloride co-transporter (KCC2) which contributes to an inhibitory neurotransmission. BDNF treated animals showed significant improvements in assisted locomotor performance, and performed locomotor movements with body weight support and plantar foot placement on a moving treadmill. These positive effects of BDNF local overexpression were detectable as early as two weeks after spinal cord transection and viral vector application and lasted for at least 7 weeks. Gradually increasing frequencies of clonic movements at the end of the experiment attenuated the quality of treadmill walking. These data indicate that BDNF has the potential to enhance the functionality of isolated lumbar circuits, but also that BDNF levels have to be tightly controlled to prevent hyperexcitability. PMID:24551172

  16. Overexpression of Smooth Muscle Myosin Heavy Chain Leads to Activation of the Unfolded Protein Response and Autophagic Turnover of Thick Filament-associated Proteins in Vascular Smooth Muscle Cells*

    PubMed Central

    Kwartler, Callie S.; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V.; Walker, Lori; Hill, Joseph A.; Epstein, Henry F.; Taegtmeyer, Heinrich; Milewicz, Dianna M.

    2014-01-01

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. PMID:24711452

  17. Overexpression of smooth muscle myosin heavy chain leads to activation of the unfolded protein response and autophagic turnover of thick filament-associated proteins in vascular smooth muscle cells.

    PubMed

    Kwartler, Callie S; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V; Walker, Lori; Hill, Joseph A; Epstein, Henry F; Taegtmeyer, Heinrich; Milewicz, Dianna M

    2014-05-16

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice.

    PubMed

    Azizi, Parisa; Rafii, Mohd Y; Abdullah, Siti N A; Hanafi, Mohamed M; Maziah, M; Sahebi, Mahbod; Ashkani, Sadegh; Taheri, Sima; Jahromi, Mohammad F

    2016-01-01

    Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g(-1) in transgenic plants. The M. oryzae population was constant at 31, 48

  19. A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest

    SciTech Connect

    Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S; Bissell, Mina J

    1994-01-01

    We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene

  20. Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice

    PubMed Central

    Azizi, Parisa; Rafii, Mohd Y.; Abdullah, Siti N. A.; Hanafi, Mohamed M.; Maziah, M.; Sahebi, Mahbod; Ashkani, Sadegh; Taheri, Sima; Jahromi, Mohammad F.

    2016-01-01

    Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g−1 in transgenic plants. The M. oryzae population was constant at 31, 48

  1. Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet.

    PubMed

    Lo, Amy C Y; Chen, Ann Y S; Hung, Victor K L; Yaw, Lai Ping; Fung, Maggie K L; Ho, Maggie C Y; Tsang, Margaret C S; Chung, Stephen S M; Chung, Sookja K

    2005-08-01

    Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET(A) antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.

  2. Overexpression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo

    PubMed Central

    Mahmoud, Salma; Ibrahim, Mohammed; Hago, Ahmed; Huang, Yuhong; Wei, Yuanyi; Zhang, Jun; Zhang, Qingqing; Xiao, Yu; Wang, Jingwen; Adam, Munkaila; Guo, Yu; Wang, Li; Zhou, Shuting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

    2016-01-01

    Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that upregulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 downregulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its overexpression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy. PMID:27626699

  3. E2F7 overexpression leads to tamoxifen resistance in breast cancer cells by competing with E2F1 at miR-15a/16 promoter.

    PubMed

    Chu, Junjun; Zhu, Yinghua; Liu, Yujie; Sun, Lijuan; Lv, Xiaobin; Wu, Yanqin; Hu, Pengnan; Su, Fengxi; Gong, Chang; Song, Erwei; Liu, Bodu; Liu, Qiang

    2015-10-13

    About 50-70% of breast cancers are estrogen receptor α (ERα) positive and most of them are sensitive to endocrine therapy including tamoxifen. However, one third of these patients will eventually develop resistance and relapse. We found that the expression of miR-15a and miR-16 were significantly decreased in tamoxifen resistant ER positive breast cancer cell lines. Exogenous expression of miR-15a/16 mimics re-sensitized resistant cells to tamoxifen by inhibiting Cyclin E1 and B cell lymphoma-2 (Bcl-2) to induce cell growth arrest and apoptosis respectively. Further, we identified that a repressive member of E2F family, E2F7, was responsible for the suppression of miR-15a/16 cluster by competing with E2F1 for E2F binding site at the promoter of their host gene DLEU2. Moreover, high expression of E2F7 is correlated with high risk of relapse and poor prognosis in breast cancer patients receiving tamoxifen treatment. Together, our results suggest that overexpression of E2F7 represses miR-15a/16 and then increases Cyclin E1 and Bcl-2 that result in tamoxifen resistance. E2F7 may be a valuable prognostic marker and a therapeutic target of tamoxifen resistance in breast cancer.

  4. Overexpression of an endo-1,4-β-glucanase V gene (EGV) from Trichoderma reesei leads to the accumulation of cellulase activity in transgenic rice.

    PubMed

    Li, X Y; Liu, F; Hu, Y F; Xia, M; Cheng, B J; Zhu, S W; Ma, Q

    2015-12-21

    The ectopic expression of cellulase in biomass can reduce the cost of biofuel conversion. This trait modification technique is highly beneficial for biofuel production. In this study, we isolated an endo-1,4-beta-glucanase gene (EGV) from Trichoderma reesei and inserted this gene downstream of a fragment encoding the signal peptide Apo-SP in a modified pCAMBIA1301 vector to obtain an Apo-SP and AsRed fusion protein. Transient expression of this fusion protein in onion epidermal cells showed that the Apo-SP signal was localized to the plastids. EGV transgenic rice plants that did not carry screening marker genes were obtained through overexpression of the pDTB double T-DNA vector. Western blotting showed that EGV was expressed in the dry straw of T0 generation transgenic rice plants and in fresh leaves of the T1 generation. More importantly, our results also showed that the peptide product of EGV in the transgenic plants folded correctly and was capable of digesting the cellulase substrate CMC. Additionally, cellulase activity remained stable in the straw that had been dried at room temperature for three months. This study presents an important technical approach for the development of transgenic rice straw that has stable cellulase activity and can be used for biofuel conversion.

  5. Variant Rett syndrome in a girl with a pericentric X-chromosome inversion leading to epigenetic changes and overexpression of the MECP2 gene.

    PubMed

    Vieira, José Pedro; Lopes, Fátima; Silva-Fernandes, Anabela; Sousa, Maria Vânia; Moura, Sofia; Sousa, Susana; Costa, Bruno M; Barbosa, Mafalda; Ylstra, Bauke; Temudo, Teresa; Lourenço, Teresa; Maciel, Patrícia

    2015-11-01

    Rett syndrome is a neurodevelopmental disorder caused by mutations in the MECP2 gene. We investigated the genetic basis of disease in a female patient with a Rett-like clinical. Karyotype analysis revealed a pericentric inversion in the X chromosome -46,X,inv(X)(p22.1q28), with breakpoints in the cytobands where the MECP2 and CDKL5 genes are located. FISH analysis revealed that the MECP2 gene is not dislocated by the inversion. However, and in spite of a balanced pattern of X inactivation, this patient displayed hypomethylation and an overexpression of the MECP2 gene at the mRNA level in the lymphocytes (mean fold change: 2.55±0.38) in comparison to a group of control individuals; the expression of the CDKL5 gene was similar to that of controls (mean fold change: 0.98±0.10). No gains or losses were detected in the breakpoint regions encompassing known or suspected transcription regulatory elements. We propose that the de-regulation of MECP2 expression in this patient may be due to alterations in long-range genomic interactions caused by the inversion and hypothesize that this type of epigenetic de-regulation of the MECP2 may be present in other RTT-like patients.

  6. Hyperhomocysteinemia in ApoE-/- Mice Leads to Overexpression of Enhancer of Zeste Homolog 2 via miR-92a Regulation

    PubMed Central

    Shengchao, Ma; Anning, Yang; Ning, Ding; Nan, Li; Yuexia, Jia; Xiaoming, Yang; Guizhong, Li; Yideng, Jiang

    2016-01-01

    Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases, such as atherosclerosis. HHcy promotes atherogenesis by modifying the histone methylation patterns and miRNA regulation. In this study, we investigated the effects of homocysteine (Hcy) on the expression of enhancer of zeste homolog 2 (EZH2), and tested our hypothesis that Hcy-induced atherosclerosis is mediated by increased EZH2 expression, which is regulated by miR-92a. The levels of EZH2 and H3K27me3 were increased in the aorta of ApoE-/- mice fed a high-methionine diet for 16 weeks, whereas miR-92a expression was decreased. Over-expression of EZH2 increased H3K27me3 level and the accumulation of total cholesterol and triglycerides in the foam cells. Furthermore, upregulation of miR-92a reduced EZH2 expression in the foam cells. These data suggested that EZH2 plays a key role in Hcy-mediated lipid metabolism disorders, and that miR-92a may be a novel therapeutic target in Hcy-related atherosclerosis. PMID:27936205

  7. Cloning of zebrafish BAD, a BH3-only proapoptotic protein, whose overexpression leads to apoptosis in COS-1 cells and zebrafish embryos.

    PubMed

    Hsieh, Yueh-Chun; Chang, Mau-Sun; Chen, Jeou-Yuan; Yen, Jeffrey Jong-Young; Lu, I-Ching; Chou, Chih-Ming; Huang, Chang-Jen

    2003-05-16

    The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.

  8. E2F7 overexpression leads to tamoxifen resistance in breast cancer cells by competing with E2F1 at miR-15a/16 promoter

    PubMed Central

    Chu, Junjun; Zhu, Yinghua; Liu, Yujie; Sun, Lijuan; Lv, Xiaobin; Wu, Yanqin; Hu, Pengnan; Su, Fengxi; Gong, Chang; Song, Erwei; Liu, Bodu; Liu, Qiang

    2015-01-01

    About 50–70% of breast cancers are estrogen receptor α (ERα) positive and most of them are sensitive to endocrine therapy including tamoxifen. However, one third of these patients will eventually develop resistance and relapse. We found that the expression of miR-15a and miR-16 were significantly decreased in tamoxifen resistant ER positive breast cancer cell lines. Exogenous expression of miR-15a/16 mimics re-sensitized resistant cells to tamoxifen by inhibiting Cyclin E1 and B cell lymphoma-2 (Bcl-2) to induce cell growth arrest and apoptosis respectively. Further, we identified that a repressive member of E2F family, E2F7, was responsible for the suppression of miR-15a/16 cluster by competing with E2F1 for E2F binding site at the promoter of their host gene DLEU2. Moreover, high expression of E2F7 is correlated with high risk of relapse and poor prognosis in breast cancer patients receiving tamoxifen treatment. Together, our results suggest that overexpression of E2F7 represses miR-15a/16 and then increases Cyclin E1 and Bcl-2 that result in tamoxifen resistance. E2F7 may be a valuable prognostic marker and a therapeutic target of tamoxifen resistance in breast cancer. PMID:26397135

  9. ALOX5AP Overexpression in Adipose Tissue Leads to LXA4 Production and Protection Against Diet-Induced Obesity and Insulin Resistance.

    PubMed

    Elias, Ivet; Ferré, Tura; Vilà, Laia; Muñoz, Sergio; Casellas, Alba; Garcia, Miquel; Molas, Maria; Agudo, Judith; Roca, Carles; Ruberte, Jesús; Bosch, Fatima; Franckhauser, Sylvie

    2016-08-01

    Eicosanoids, such as leukotriene B4 (LTB4) and lipoxin A4 (LXA4), may play a key role during obesity. While LTB4 is involved in adipose tissue inflammation and insulin resistance, LXA4 may exert anti-inflammatory effects and alleviate hepatic steatosis. Both lipid mediators derive from the same pathway, in which arachidonate 5-lipoxygenase (ALOX5) and its partner, arachidonate 5-lipoxygenase-activating protein (ALOX5AP), are involved. ALOX5 and ALOX5AP expression is increased in humans and rodents with obesity and insulin resistance. We found that transgenic mice overexpressing ALOX5AP in adipose tissue had higher LXA4 rather than higher LTB4 levels, were leaner, and showed increased energy expenditure, partly due to browning of white adipose tissue (WAT). Upregulation of hepatic LXR and Cyp7a1 led to higher bile acid synthesis, which may have contributed to increased thermogenesis. In addition, transgenic mice were protected against diet-induced obesity, insulin resistance, and inflammation. Finally, treatment of C57BL/6J mice with LXA4, which showed browning of WAT, strongly suggests that LXA4 is responsible for the transgenic mice phenotype. Thus, our data support that LXA4 may hold great potential for the future development of therapeutic strategies for obesity and related diseases.

  10. Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

    PubMed

    Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

    2015-08-24

    Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

  11. Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress.

    PubMed

    Rey, P; Gillet, B; Römer, S; Eymery, F; Massimino, J; Peltier, G; Kuntz, M

    2000-03-01

    Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints.

  12. Combined exposure to protons and (56)Fe leads to overexpression of Il13 and reactivation of repetitive elements in the mouse lung.

    PubMed

    Nzabarushimana, Etienne; Prior, Sara; Miousse, Isabelle R; Pathak, Rupak; Allen, Antiño R; Latendresse, John; Olsen, Reid H J; Raber, Jacob; Hauer-Jensen, Martin; Nelson, Gregory A; Koturbash, Igor

    2015-11-01

    Interest in deep space exploration underlines the needs to investigate the effects of exposure to combined sources of space radiation. The lung is a target organ for radiation, and exposure to protons and heavy ions as radiation sources may lead to the development of degenerative disease and cancer. In this study, we evaluated the pro-fibrotic and epigenetic effects of exposure to protons (150 MeV/nucleon, 0.1 Gy) and heavy iron ions ((56)Fe, 600 MeV/nucleon, 0.5 Gy) alone or in combination (protons on Day 1 and (56)Fe on Day 2) in C57BL/6 male mice 4 weeks after irradiation. Exposure to (56)Fe, proton or in combination, did not result in histopathological changes in the murine lung. At the same time, combined exposure to protons and (56)Fe resulted in pronounced molecular alterations in comparison with either source of radiation alone. Specifically, we observed a substantial increase in the expression of cytokine Il13, loss of expression of DNA methyltransferase Dnmt1, and reactivation of LINE-1, SINE B1 retrotransposons, and major and minor satellites. Given the deleterious potential of the observed effects that may lead to development of chronic lung injury, pulmonary fibrosis, and cancer, future studies devoted to the investigation of the long-term effects of combined exposures to proton and heavy ions are clearly needed. Copyright © 2015 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

  13. Combined exposure to protons and 56Fe leads to overexpression of Il13 and reactivation of repetitive elements in the mouse lung

    PubMed Central

    Nzabarushimana, Etienne; Prior, Sara; Miousse, Isabelle R.; Pathak, Rupak; Allen, Antino R.; Latendresse, John; Olsen, Reid H.J.; Raber, Jacob; Hauer-Jensen, Martin; Nelson, Gregory A.; Koturbash, Igor

    2015-01-01

    Interest in deep space exploration underlines the needs to investigate the effects of exposure to combined sources of space radiation. The lung is a target organ for radiation, and exposure to protons and heavy ions as radiation sources may lead to the development of degenerative disease and cancer. In this study, we evaluated the pro-fibrotic and epigenetic effects of exposure to protons (150 MeV/nucleon, 0.1 Gy) and heavy iron ions (56Fe, 600 MeV/nucleon, 0.5 Gy) alone or in combination (protons on Day 1 and 56Fe on Day 2) in C57BL/6 male mice 4 weeks after irradiation). Exposure to 56Fe, proton or in combination, did not result in histopathological changes in the murine lung. At the same time, combined exposure to protons and 56Fe resulted in pronounced molecular alterations in comparison with either source of radiation alone. Specifically, we observed a substantial increase in the expression of cytokine Il13, loss of expression of DNA methyltransferase Dnmt1, and reactivation of LINE-1, SINE B1 retrotransposons, and major and minor satellites. Given the deleterious potential of the observed effects that may lead to development of chronic lung injury, pulmonary fibrosis, and cancer, future studies devoted to the investigation of the long-term effects of combined exposures to proton and heavy ions are clearly needed. PMID:26553631

  14. Combined exposure to protons and 56Fe leads to overexpression of Il13 and reactivation of repetitive elements in the mouse lung

    NASA Astrophysics Data System (ADS)

    Nzabarushimana, Etienne; Prior, Sara; Miousse, Isabelle R.; Pathak, Rupak; Allen, Antiño R.; Latendresse, John; Olsen, Reid H. J.; Raber, Jacob; Hauer-Jensen, Martin; Nelson, Gregory A.; Koturbash, Igor

    2015-11-01

    Interest in deep space exploration underlines the needs to investigate the effects of exposure to combined sources of space radiation. The lung is a target organ for radiation, and exposure to protons and heavy ions as radiation sources may lead to the development of degenerative disease and cancer. In this study, we evaluated the pro-fibrotic and epigenetic effects of exposure to protons (150 MeV/nucleon, 0.1 Gy) and heavy iron ions (56Fe, 600 MeV/nucleon, 0.5 Gy) alone or in combination (protons on Day 1 and 56Fe on Day 2) in C57BL/6 male mice 4 weeks after irradiation. Exposure to 56Fe, proton or in combination, did not result in histopathological changes in the murine lung. At the same time, combined exposure to protons and 56Fe resulted in pronounced molecular alterations in comparison with either source of radiation alone. Specifically, we observed a substantial increase in the expression of cytokine Il13, loss of expression of DNA methyltransferase Dnmt1, and reactivation of LINE-1, SINE B1 retrotransposons, and major and minor satellites. Given the deleterious potential of the observed effects that may lead to development of chronic lung injury, pulmonary fibrosis, and cancer, future studies devoted to the investigation of the long-term effects of combined exposures to proton and heavy ions are clearly needed.

  15. Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N¹-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells.

    PubMed

    Mandal, Swati; Mandal, Ajeet; Park, Myung Hee

    2015-06-15

    The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with an adenovirus encoding a key polyamine catabolic enzyme, spermidine N¹-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/propidium iodide FACS analyses, terminal uridine nucleotide end-labelling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1-transduced cells (at 24-72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in pro-apoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2 and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, TEM images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines spermidine and spermine, as the polyamine analogues α-methylspermidine (α-MeSpd) and N¹,N¹²-dimethylspermine (Me₂Spm) that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of the polyamines spermidine and spermine leads to mitochondria-mediated apoptosis. © The Authors Journal compilation © 2015 Biochemical Society.

  16. Characterization of mutations in regulatory genes of Tyl cluster leading to overexpression of tylosin in mutant γ-1 of Streptomyces fradiae NRRL-2702.

    PubMed

    Khaliq, Shazia; Ghauri, Muhammad A; Akhtar, Kalsoom

    2014-01-01

    Tylosin is a veterinary antibiotic and is commercially produced using Streptomyces fradiae. Previously, we developed a mutant γ-1 of S. fradiae NRRL-2702 with a 6.87-fold increase in tylosin yield as compared with the wild-type strain through irradiation mutagenesis. The present studies were conducted to explore mutational changes in regulatory genes (TylQ, TylP, TylS, TylR, and TylT) of Tyl cluster that may lead to an enhanced expression of tylosin. Expression analysis by RT-PCR revealed that TylQ was switched off earlier in mutant γ-1 while no change in expression pattern of TylP was observed between the wild-type and mutant γ-1 strains. However, a point mutation with a substitution of T to A was recorded at position 214 in the 420-bp product of TylP from mutant γ-1 that resulted in a change of one amino acid (serine to threonine) at position 72. Moreover, no mutation in the nucleotide sequence of TylS, TylR, and TylT genes was detected.

  17. Substitution of a highly conserved histidine in the Escherichia coli heat shock transcription factor, sigma32, affects promoter utilization in vitro and leads to overexpression of the biofilm-associated flu protein in vivo.

    PubMed

    Kourennaia, Olga V; Dehaseth, Pieter L

    2007-12-01

    The heat shock sigma factor (sigma(32) in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of sigma(32) is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, sigma(70). This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing sigma(32), implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in sigma(32) impedes recovery from heat shock (exposure to 42 degrees C), and it also leads to overexpression at lower temperatures (30 degrees C) of the Flu protein, which is associated with biofilm formation.

  18. Overexpression of Hyaluronan-binding Protein 1 (HABP1/p32/gC1qR) in HepG2 Cells Leads to Increased Hyaluronan Synthesis and Cell Proliferation by Up-regulation of Cyclin D1 in AKT-dependent Pathway*

    PubMed Central

    Kaul, Rachna; Saha, Paramita; Saradhi, Mallampati; Prasad, Ramachandra L. A.; Chatterjee, Soumya; Ghosh, Ilora; Tyagi, Rakesh K.; Datta, Kasturi

    2012-01-01

    Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased “HA pool,” formation of the “HA cable” structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways. PMID:22451658

  19. Overexpression of the Trehalase Gene AtTRE1 Leads to Increased Drought Stress Tolerance in Arabidopsis and Is Involved in Abscisic Acid-Induced Stomatal Closure1[W][OA

    PubMed Central

    Van Houtte, Hilde; Vandesteene, Lies; López-Galvis, Lorena; Lemmens, Liesbeth; Kissel, Ewaut; Carpentier, Sebastien; Feil, Regina; Avonce, Nelson; Beeckman, Tom; Lunn, John E.; Van Dijck, Patrick

    2013-01-01

    Introduction of microbial trehalose biosynthesis enzymes has been reported to enhance abiotic stress resistance in plants but also resulted in undesirable traits. Here, we present an approach for engineering drought stress tolerance by modifying the endogenous trehalase activity in Arabidopsis (Arabidopsis thaliana). AtTRE1 encodes the Arabidopsis trehalase, the only enzyme known in this species to specifically hydrolyze trehalose into glucose. AtTRE1-overexpressing and Attre1 mutant lines were constructed and tested for their performance in drought stress assays. AtTRE1-overexpressing plants had decreased trehalose levels and recovered better after drought stress, whereas Attre1 mutants had elevated trehalose contents and exhibited a drought-susceptible phenotype. Leaf detachment assays showed that Attre1 mutants lose water faster than wild-type plants, whereas AtTRE1-overexpressing plants have a better water-retaining capacity. In vitro studies revealed that abscisic acid-mediated closure of stomata is impaired in Attre1 lines, whereas the AtTRE1 overexpressors are more sensitive toward abscisic acid-dependent stomatal closure. This observation is further supported by the altered leaf temperatures seen in trehalase-modified plantlets during in vivo drought stress studies. Our results show that overexpression of plant trehalase improves drought stress tolerance in Arabidopsis and that trehalase plays a role in the regulation of stomatal closure in the plant drought stress response. PMID:23341362

  20. Ectopic over-expression of BhHsf1, a heat shock factor from the resurrection plant Boea hygrometrica, leads to increased thermotolerance and retarded growth in transgenic Arabidopsis and tobacco.

    PubMed

    Zhu, Yan; Wang, Zhi; Jing, Yanjun; Wang, Lili; Liu, Xia; Liu, Yongxiu; Deng, Xin

    2009-11-01

    Plant heat shock transcription factors (Hsfs) are commonly found to be involved in various stress responses. Several Hsfs displayed dwarf phenotype while conferred stress tolerance when over-expressed. However, the underlying mechanisms were not fully understood. Here we report the cloning and characterization of an Hsf (BhHsf1) from the resurrection plant Boea hygrometrica. Drought, heat and wound can induce BhHsf1 expression. The over-expression of BhHsf1 conferred growth retardation and stress tolerance in both Arabidopsis and tobacco. Evidence was presented to show that the growth retardation of aerial organs in the transgenic plants was resulted from the reduction of cell proliferation. Gene expression profiling using microarray hybridization and pathway analysis showed that Hsps and stress-associated genes were induced whereas the genes related to DNA replication and mitotic cell cycle were down-regulated in BhHsf1 over-expression Arabidopsis, which was in consistence with the observation of the impaired nuclear endoreduplication. Taking together, our results suggest that BhHsf1 may play dual roles in mediating the processes in heat stress tolerance and growth retardation via regulation of target genes related to stress protection and mitotic cell cycle.

  1. Overexpression of the transcription factor RAP2.6 leads to enhanced callose deposition in syncytia and enhanced resistance against the beet cyst nematode Heterodera schachtii in Arabidopsis roots

    PubMed Central

    2013-01-01

    Background Cyst nematodes invade the roots of their host plants as second stage juveniles and induce a syncytium which is their source of nutrients throughout their life. A transcriptome analysis of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots has shown that gene expression in the syncytium is different from that of the root with thousands of genes upregulated or downregulated. Among the downregulated genes are many which code for defense-related proteins. One gene which is strongly downregulated codes for the ethylene response transcription factor RAP2.6. The genome of Arabidopsis contains 122 ERF transcription factor genes which are involved in a variety of developmental and stress responses. Results Expression of RAP2.6 was studied with RT-PCR and a promoter::GUS line. During normal growth conditions the gene was expressed especially in roots and stems. It was inducible by Pseudomonas syringae but downregulated in syncytia from a very early time point on. Overexpression of the gene enhanced the resistance against H. schachtii which was seen by a lower number of nematodes developing on these plants as well as smaller syncytia and smaller female nematodes. A T-DNA mutant had a reduced RAP2.6 transcript level but this did not further increase the susceptibility against H. schachtii. Neither overexpression lines nor mutants had an effect on P. syringae. Overexpression of RAP2.6 led to an elevated expression of JA-responsive genes during early time points after infection by H. schachtii. Syncytia developing on overexpression lines showed enhanced deposition of callose. Conclusions Our results showed that H. schachtii infection is accompanied by a downregulation of RAP2.6. It seems likely that the nematodes use effectors to actively downregulate the expression of this and other defense-related genes to avoid resistance responses of the host plant. Enhanced resistance of RAP2.6 overexpression lines seemed to be due to enhanced

  2. let-7 Overexpression Leads to an Increased Fraction of Cells in G2/M, Direct Down-regulation of Cdc34, and Stabilization of Wee1 Kinase in Primary Fibroblasts*S⃞

    PubMed Central

    Legesse-Miller, Aster; Elemento, Olivier; Pfau, Sarah J.; Forman, Joshua J.; Tavazoie, Saeed; Coller, Hilary A.

    2009-01-01

    microRNAs play a critically important role in a wide array of biological processes including those implicated in cancer, neuro-degenerative and metabolic disorders, and viral infection. Although we have begun to understand microRNA biogenesis and function, experimental demonstration of their functional effects and the molecular mechanisms by which they function remains a challenge. Members of the let-7/miR-98 family play a critical role in cell cycle control with respect to differentiation and tumorigenesis. In this study, we show that exogenous addition of pre-let-7 in primary human fibroblasts results in a decrease in cell number and an increased fraction of cells in the G2/M cell cycle phase. Combining microarray techniques with DNA sequence analysis to identify potential let-7 targets, we discovered 838 genes with a let-7 binding site in their 3′-untranslated region that were down-regulated upon overexpression of let-7b. Among these genes is cdc34, the ubiquitin-conjugating enzyme of the Skp1/cullin/F-box (SCF) complex. Cdc34 protein levels are strongly down-regulated by let-7 overexpression. Reporter assays demonstrated direct regulation of the cdc34 3′-untranslated region by let-7. We hypothesized that low Cdc34 levels would result in decreased SCF activity, stabilization of the SCF target Wee1, and G2/M accumulation. Consistent with this hypothesis, small interfering RNA-mediated down-regulation of Wee1 reversed the G2/M phenotype induced by let-7 overexpression. We conclude that Cdc34 is a functional target of let-7 and that let-7 induces down-regulation of Cdc34, stabilization of the Wee1 kinase, and an increased fraction of cells in G2/M in primary fibroblasts. PMID:19126550

  3. Zinc supplementation augments in vivo antitumor effect of chemotherapy by restoring p53 function.

    PubMed

    Margalit, Ofer; Simon, Amos J; Yakubov, Eduard; Puca, Rosa; Yosepovich, Ady; Avivi, Camila; Jacob-Hirsch, Jasmine; Gelernter, Ilana; Harmelin, Alon; Barshack, Iris; Rechavi, Gideon; D'Orazi, Gabriella; Givol, David; Amariglio, Ninette

    2012-08-15

    Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness.

  4. Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance.

    PubMed

    Gachon, Claire; Baltz, Rachel; Saindrenan, Patrick

    2004-01-01

    Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.

  5. Constitutive activation of p38 and ERK1/2 MAPKs in epithelial cells of myasthenic thymus leads to IL-6 and RANTES overexpression: effects on survival and migration of peripheral T and B cells.

    PubMed

    Colombara, Michaela; Antonini, Valeria; Riviera, Anna Pia; Mainiero, Fabrizio; Strippoli, Raffaele; Merola, Marcello; Fracasso, Giulio; Poffe, Ornella; Brutti, Nadia; Tridente, Giuseppe; Colombatti, Marco; Ramarli, Dunia

    2005-11-15

    Myasthenia gravis (MG) is an autoimmune disease of neuromuscular junctions where thymus plays a pathogenetic role. Thymectomy benefits patients, and thymic hyperplasia, a lymphoid infiltration of perivascular spaces becoming site of autoantibody production, is recurrently observed. Cytokines and chemokines, produced by thymic epithelium and supporting survival and migration of T and B cells, are likely to be of great relevance in pathogenesis of thymic hyperplasia. In thymic epithelial cell (TEC) cultures derived "in vitro" from normal or hyperplastic age-matched MG thymuses, we demonstrate by gene profiling analysis that MG-TEC basally overexpress genes coding for p38 and ERK1/2 MAPKs and for components of their signaling pathways. Immunoblotting experiments confirmed that p38 and ERK1/2 proteins were overexpressed in MG-TEC and, in addition, constitutively activated. Pharmacological blockage with specific inhibitors confirmed their role in the control of IL-6 and RANTES gene expression. According to our results, IL-6 and RANTES levels were abnormally augmented in MG-TEC, either basally or upon induction by adhesion-related stimuli. The finding that IL-6 and RANTES modulate, respectively, survival and migration of peripheral lymphocytes of myasthenic patients point to MAPK transcriptional and posttranscriptional abnormalities of MG-TEC as a key step in the pathological remodelling of myasthenic thymus.

  6. Primary over-expression of AβPP in muscle does not lead to the development of inclusion body myositis in a new lineage of the MCK-AβPP transgenic mouse.

    PubMed

    Luo, Yue-Bei; Johnsen, Russell D; Griffiths, Lisa; Needham, Merrilee; Fabian, Victoria A; Fletcher, Sue; Wilton, Steve D; Mastaglia, Frank L

    2013-12-01

    The aim of this study is to determine whether primary over-expression of AβPP in skeletal muscle results in the development of features of inclusion body myositis (IBM) in a new lineage of the MCK-AβPP transgenic mouse. Quantitative histological, immunohistochemical and western blotting studies were performed on muscles from 3 to 18 month old transgenic and wild-type C57BL6/SJL mice. Electron microscopy was also performed on muscle sections from selected animals. Although western blotting confirmed that there was over-expression of full length AβPP in transgenic mouse muscles, deposition of amyloid-β and fibrillar amyloid could not be demonstrated histochemically or with electron microscopy. Additionally, other changes typical of IBM such as rimmed vacuoles, cytochrome C oxidase-deficient fibres, upregulation of MHC antigens, lymphocytic inflammatory infiltration and T cell fibre invasion were absent. The most prominent finding in both transgenic and wild-type animals was the presence of tubular aggregates which was age-related and largely restricted to male animals. Expression of full length AβPP in this MCK-AβPP mouse lineage did not reach the levels required for immunodetection or deposition of amyloid-β as in the original transgenic strains, and was not associated with the development of pathological features of IBM. These negative results emphasise the potential pitfalls of re-deriving transgenic mouse strains in different laboratories.

  7. Overexpression of an N-terminally truncated isoform of the nuclear receptor coactivator amplified in breast cancer 1 leads to altered proliferation of mammary epithelial cells in transgenic mice.

    PubMed

    Tilli, Maddalena T; Reiter, Ronald; Oh, Annabell S; Henke, Ralf T; McDonnell, Kevin; Gallicano, G Ian; Furth, Priscilla A; Riegel, Anna Tate

    2005-03-01

    Amplified in breast cancer 1 (AIB1, also known as ACTR, SRC-3, RAC-3, TRAM-1, p/CIP) is a member of the p160 nuclear receptor coactivator family involved in transcriptional regulation of genes activated through steroid receptors, such as estrogen receptor alpha (ER(alpha)). The AIB1 gene and a more active N-terminally deleted isoform (AIB1-Delta3) are overexpressed in breast cancer. To determine the role of AIB1-Delta3 in breast cancer pathogenesis, we generated transgenic mice with human cytomegalovirus immediate early gene 1 (hCMVIE1) promoter-driven over-expression of human AIB1/ACTR-Delta3 (CMVAIB1/ACTR-Delta3 mice). AIB1/ACTR-Delta3 transgene mRNA expression was confirmed in CMV-AIB1/ACTR-Delta3 mammary glands by in situ hybridization. These mice demonstrated significantly increased mammary epithelial cell proliferation (P < 0.003), cyclin D1 expression (P = 0.002), IGF-I receptor protein expression (P = 0.026), mammary gland mass (P < 0.05), and altered expression of CCAAT/enhancer binding protein isoforms (P = 0.029). At 13 months of age, mammary ductal ectasia was found in CMV-AIB1/ACTR-Delta3 mice, but secondary and tertiary branching patterns were normal. There were no changes in the expression patterns of either ER(alpha) or Stat5a, a downstream mediator of prolactin signaling. Serum IGF-I levels were not altered in the transgenic mice. These data indicate that overexpression of the AIB1/ACTR-Delta3 isoform resulted in altered mammary epithelial cell growth. The observed changes in cell proliferation and gene expression are consistent with alterations in growth factor signaling that are thought to contribute to either initiation or progression of breast cancer. These results are consistent with the hypothesis that the N-terminally deleted isoform of AIB1 can play a role in breast cancer development and/or progression.

  8. Decreased Expression of Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Leads to Reduced Glucuronidation of Flavonoids in UGT1A1-Overexpressing HeLa Cells: The Role of Futile Recycling.

    PubMed

    Sun, Hua; Zhou, Xiaotong; Zhang, Xingwang; Wu, Baojian

    2015-07-08

    In this study, the role of futile recycling (or deglucuronidation) in the disposition of two flavonoids (i.e., genistein and apigenin) was explored using UGT1A1-overexpressing HeLa cells (or HeLa1A1 cells). Glucuronidation of the flavonoids by HeLa1A1 cell lysate followed the substrate inhibition kinetics (Vmax = 0.10 nmol/min/mg, Km = 0.54 μM, and Ksi = 2.0 μM for genistein; Vmax = 0.19 nmol/min/mg, Km = 0.56 μM, and Ksi = 3.7 μM for apigenin). Glucuronide was efficiently generated and excreted after incubation of the cells with the aglycone (at doses of 1.25-20 nmol). The excretion rates were 0.40-0.69 and 0.84-1.1 nmol/min/mg protein for genistein glucuronide (GG) and apigenin glucuronide (AG), respectively. Furthermore, glucuronide excretion and total glucuronidation were significantly reduced in MRP4 knocked-down as compared to control cells. The alterations were well characterized by a two-compartment pharmacokinetic model incorporating the process of futile recycling (defined by a first-order rate constant, Kde). The derived Kde values were 15 and 25 h(-1) for GG and AG, respectively. This was well consistent with the in vitro observation that AG was subjected to more efficient futile recycling compared to GG. In conclusion, futile recycling was involved in cellular glucuronidation, accounting for transporter-dependent glucuronidation of flavonoids.

  9. Physiological Response to Membrane Protein Overexpression in E. coli*

    PubMed Central

    Gubellini, Francesca; Verdon, Grégory; Karpowich, Nathan K.; Luff, Jon D.; Boël, Grégory; Gauthier, Nils; Handelman, Samuel K.; Ades, Sarah E.; Hunt, John F.

    2011-01-01

    Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially

  10. Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2.

    PubMed

    Mustafa, Dana A M; Sieuwerts, Anieta M; Zheng, Ping Pin; Kros, Johan M

    2010-10-20

    In previous studies we found expression of the protein colligin 2 (heat shock protein 47 (HSP47), SERPINH1) in glioma neovasculature while not in normal brain tissue. Generally, the regulation of heat shock gene expression in eukaryotes is mediated by heat shock factors (HSF). In mammals, three heat shock transcription factors, HSF-1, -2, and -4, have been isolated. Here we investigated the relation between the expression of colligin 2 and these heat shock factors at the mRNA level using real-time reverse transcriptase PCR (qRT-PCR) in different grades of astrocytic tumorigenesis, viz., low-grade glioma and glioblastoma. Endometrium samples, representing physiological angiogenesis, were included as controls. Since colligin 2 is a chaperon for collagens, the gene expression of collagen I (COL1A1) was also investigated. The blood vessel density of the samples was monitored by expression of the endothelial marker CD31 (PECAM1). Because NG2-immunopositive pericytic cells are involved in glioma neovascularization, the expression of NG2 (CSPG4) was also measured.We demonstrate overexpression of HSF2 in both stages of glial tumorigenesis (reaching significance only in low-grade glioma) and also minor elevated levels of HSF1 as compared to normal brain. There were no differences in expression of HSF4 between low-grade glioma and normal brain while HSF4 was downregulated in glioblastoma. In the endometrium samples, none of the HSFs were upregulated. In the low-grade gliomas SERPINH appeared to be slightly overexpressed with a parallel 4-fold upregulation of COL1A1, while in glioblastoma there was over 5-fold overexpression of SERPINH1 and more than 150-fold overexpression of COL1A1. In both the lowgrade gliomas and the glioblastomas overexpression of CSPG4 was found and overexpression of PECAM1 was only found in the latter. Our data suggest that the upregulated expression of colligin 2 in glioma is accompanied by upregulation of COL1A1, CSPG4, HSF2 and to a lesser extent

  11. Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice

    PubMed Central

    LI, XIHAI; LIANG, WENNA; YE, HONGZHI; WENG, XIAPING; LIU, FAYUAN; LIN, PINGDONG; LIU, XIANXIANG

    2015-01-01

    The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild-type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression-associated congenital dysplasia of the TMJ in mice. PMID:26096903

  12. Geminin overexpression induces mammary tumors via suppressing cytokinesis.

    PubMed

    Blanchard, Zannel; Malik, Rohit; Mullins, Nicole; Maric, Christine; Luk, Hugh; Horio, David; Hernandez, Brenda; Killeen, Jeffrey; Elshamy, Wael M

    2011-12-01

    Aneuploidy plays an important role in the development of cancer. Here, we uncovered an oncogenic role for geminin in mitotic cells. In addition to chromatin, tyrosine phosphorylated geminin also localizes to centrosome, spindle, cleavage furrow and midbody during mitosis. Geminin binding to Aurora B prevents its binding to INCENP, and thus activation leading to lack of histone H3-(serine 10) phosphorylation, chromosome condensation failure, aborted cytokinesis and the formation of aneuploid, drug resistance cells. Geminin overexpressing human mammary epithelial cells form aneuploid, aggressive tumors in SCID mice. Geminin is overexpressed in more than half of all breast cancers analyzed. The current study reveals that geminin is a genuine oncogene that promotes cytokinesis failure and production of aneuploid, aggressive breast tumors when overexpressed and thus a worthy therapeutic target (oncotarget) for aggressive breast cancer.

  13. Modification of photosynthetic regulation in tomato overexpressing glutathione peroxidase.

    PubMed

    Herbette, Stephane; Menn, Aline Le; Rousselle, Patrick; Ameglio, Thierry; Faltin, Zehava; Branlard, Gérard; Eshdat, Yuval; Julien, Jean-Louis; Drevet, Joël R; Roeckel-Drevet, Patricia

    2005-06-20

    To investigate the function of glutathione peroxidase (GPX) in plants, we produced transgenic tomato plants overexpressing an eukaryotic selenium-independent GPX (GPX5). We show here that total GPX activity was increased by 50% in transgenic plants, when compared to control plants transformed with the binary vector without the insert (PZP111). A preliminary two-dimensional electrophoretic protein analysis of the GPX overexpressing plants showed notably a decrease in the accumulation of proteins identified as rubisco small subunit 1 and fructose-1,6-bisphosphate aldolase, two proteins involved in photosynthesis. These observations, together with the fact that in standard culture conditions, GPX-overexpressing plants were not phenotypically distinct from control plants prompted us to challenge the plants with a chilling treatment that is known to affect photosynthesis activity. We found that upon chilling treatment with low light level, photosynthesis was not affected in GPX-overexpressing plants while it was in control plants, as revealed by chlorophyll fluorescence parameters and fructose-1,6-biphosphatase activity. These results suggest that overexpression of a selenium-independent GPX in tomato plants modifies specifically gene expression and leads to modifications of photosynthetic regulation processes.

  14. Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice.

    PubMed

    Li, Xihai; Liang, Wenna; Ye, Hongzhi; Weng, Xiaping; Liu, Fayuan; Lin, Pingdong; Liu, Xianxiang

    2015-09-01

    The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild‑type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression‑associated congenital dysplasia of the TMJ in mice.

  15. LEADING WITH LEADING INDICATORS

    SciTech Connect

    PREVETTE, S.S.

    2005-01-27

    This paper documents Fluor Hanford's use of Leading Indicators, management leadership, and statistical methodology in order to improve safe performance of work. By applying these methods, Fluor Hanford achieved a significant reduction in injury rates in 2003 and 2004, and the improvement continues today. The integration of data, leadership, and teamwork pays off with improved safety performance and credibility with the customer. The use of Statistical Process Control, Pareto Charts, and Systems Thinking and their effect on management decisions and employee involvement are discussed. Included are practical examples of choosing leading indicators. A statistically based color coded dashboard presentation system methodology is provided. These tools, management theories and methods, coupled with involved leadership and employee efforts, directly led to significant improvements in worker safety and health, and environmental protection and restoration at one of the nation's largest nuclear cleanup sites.

  16. Phospholamban Overexpression in Transgenic Rabbits

    PubMed Central

    Pattison, J. Scott; Waggoner, Jason R.; James, Jeanne; Martin, Lisa; Gulick, James; Osinska, Hanna; Klevitsky, Raisa; Kranias, Evangelia G.; Robbins, Jeffrey

    2008-01-01

    There has been considerable interest in pursuing phospholamban as a putative therapeutic target for overcoming depressed calcium handling in human heart failure. Studies predominantly done in mice have shown that phospholamban is a key regulator of sarcoplasmic reticulum calcium cycling and cardiac function. However, mice differ significantly from humans in how they regulate calcium, whereas rabbits better recapitulate human cardiac function and calcium handling. To investigate phospholamban’s role in the rabbit heart, transgenic rabbits that overexpressed wild-type phospholamban in the ventricular cardiomyocytes and slow-twitch skeletal muscles were generated. Rabbits expressing high levels of phospholamban were not viable due to severe skeletal muscle wasting, the onset of cardiac pathology and early death. A viable transgenic line exhibited a 30% increase in PLN protein levels in the heart. These animals showed isolated foci of cardiac pathology, but cardiac function as well as the response to β-adrenergic stimulation were normal. SR-calcium uptake measurements showed that the transgenic hearts had the expected reduced affinity for calcium. The data show that phospholamban-overexpressing transgenic rabbits differ markedly in phenotype from analogous transgenic mice in that rabbits are quite sensitive to alterations in phospholamban levels. Exceeding a relatively narrow window of phospholamban expression results in significant morbidity and early death. PMID:17882530

  17. NUCKS overexpression in breast cancer

    PubMed Central

    Drosos, Yiannis; Kouloukoussa, Mirsini; Østvold, Anne Carine; Grundt, Kirsten; Goutas, Nikos; Vlachodimitropoulos, Dimitrios; Havaki, Sophia; Kollia, Panagoula; Kittas, Christos; Marinos, Evangelos; Aleporou-Marinou, Vassiliki

    2009-01-01

    Background NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies. Results The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures. Conclusion The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and

  18. Lead Toxicity

    MedlinePlus

    Agency for Toxic Substances and Disease Registry Case Studies in Environmental Medicine (CSEM) Patient Information Sheet Lead Toxicity What is lead? How are people exposed to lead? • Lead is a soft, blue- ...

  19. Lead Poisoning

    MedlinePlus

    ... high levels of lead in household dust. DRINKING WATER: Lead may get into drinking water when materials used in plumbing materials, such as ... and dishware. Lead may also be in contaminated water. Lead poisoning is harmful to human health and ...

  20. Abetalipoproteinemia induced by overexpression of ORP150 in mice.

    PubMed

    Kobayashi, Tomohiro; Iguchi, Taisen; Ohta, Yasuhiko

    2007-06-01

    ORP150 is an endoplasmic-resident, hypoxic stress-induced protein, but little is known about the effects of its systemic overexpression. We have produced a transgenic strain of mice that overexpress ORP150 (ORP-Tg mice). These mice exhibit severe growth retardation concomitant with vacuolar degeneration in the heart. To investigate the cause of the observed growth retardation in response to ORP150 overexpression, we conducted a clinical evaluation of the ORP-Tg mice. Blood analysis showed significantly lower concentrations of serum triglyceride, cholesterol, glucose and insulin. The triglyceride components that were reduced in ORP-Tg mice were localized mainly at the origin and in the pre-beta fraction on agarose gel electrophoresis, corresponding to chylomicrons and very low-density lipoproteins. A lipid-loading test of ORP-Tg mice revealed reduced triglyceride uptake, which mainly was due to suppressed uptake of very low-density lipoproteins. An intraperitoneal glucose tolerance test indicated that the ORP-Tg mice have a significantly higher rate of glucose degradation. These findings suggest that overexpression of ORP150 in mice leads to abetalipoproteinemia with alteration of glucose and lipid metabolism. These data could provide clues for a therapeutic target of dyslipidemia or diabetes.

  1. Reduced viability of neuronal cells after overexpression of protein histidine phosphatase.

    PubMed

    Krieglstein, Josef; Lehmann, Martina; Mäurer, Anette; Gudermann, Thomas; Pinkenburg, Olaf; Wieland, Thomas; Litterscheid, Sarah; Klumpp, Susanne

    2008-11-01

    Protein histidine phosphatase (PHP) has just recently been discovered in eukaryotes and ATP-citrate lyase (ACL) was shown to be one of its substrates. Since ACL is crucial for cellular energy and fat metabolism we made an attempt to study the influence of PHP on cell viability. Using an adenoviral vector PHP was overexpressed in SN56 cholinergic murine neuroblastoma cells and in primary cultures of hippocampal neurons obtained from embryonic rats (E18). Overexpression of PHP in these cells caused a decrease in ACL activity and consequently impaired viability. To be sure that the reduced cellular viability was achieved by overexpression of PHP we also downregulated ACL in SN56 cells using RNAi-technology. Downregulation of ACL was harmful to the cells similar to what was observed upon overexpression of PHP. Taken together, it is concluded that overexpression of PHP results in increased dephosphorylation with concomitant inactivation of ACL, thus finally leading to cell damage.

  2. DEK over-expression promotes mitotic defects and micronucleus formation.

    PubMed

    Matrka, Marie C; Hennigan, Robert F; Kappes, Ferdinand; DeLay, Monica L; Lambert, Paul F; Aronow, Bruce J; Wells, Susanne I

    2015-01-01

    The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation.

  3. The effect of aquaporin 5 overexpression on the Ras signaling pathway

    SciTech Connect

    Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

    2008-03-07

    Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells.

  4. Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1.

    PubMed

    Gabellini, Davide; D'Antona, Giuseppe; Moggio, Maurizio; Prelle, Alessandro; Zecca, Chiara; Adami, Raffaella; Angeletti, Barbara; Ciscato, Patrizia; Pellegrino, Maria Antonietta; Bottinelli, Roberto; Green, Michael R; Tupler, Rossella

    2006-02-23

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is not due to a classical mutation within a protein-coding gene. Instead, almost all FSHD patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed D4Z4, located on chromosome 4q35 (ref. 3). D4Z4 contains a transcriptional silencer whose deletion leads to inappropriate overexpression in FSHD skeletal muscle of 4q35 genes located upstream of D4Z4 (ref. 4). To identify the gene responsible for FSHD pathogenesis, we generated transgenic mice selectively overexpressing in skeletal muscle the 4q35 genes FRG1, FRG2 or ANT1. We find that FRG1 transgenic mice develop a muscular dystrophy with features characteristic of the human disease; by contrast, FRG2 and ANT1 transgenic mice seem normal. FRG1 is a nuclear protein and several lines of evidence suggest it is involved in pre-messenger RNA splicing. We find that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing. Collectively, our results suggest that FSHD results from inappropriate overexpression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.

  5. SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

    PubMed Central

    Amraoui, Sonia; di Nunzio, Francesca; Kieffer, Camille; Porrot, Françoise; Opp, Silvana; Diaz-Griffero, Felipe; Casartelli, Nicoletta

    2016-01-01

    ABSTRACT In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. IMPORTANCE Cells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain

  6. Lead Poisoning

    MedlinePlus

    Lead is a metal that occurs naturally in the earth's crust. Lead can be found in all parts of our ... from human activities such as mining and manufacturing. Lead used to be in paint; older houses may ...

  7. Tuning Escherichia coli for membrane protein overexpression.

    PubMed

    Wagner, Samuel; Klepsch, Mirjam M; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J; Slotboom, Dirk J; Persson, Jan O; de Gier, Jan-Willem

    2008-09-23

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

  8. Tuning Escherichia coli for membrane protein overexpression

    PubMed Central

    Wagner, Samuel; Klepsch, Mirjam M.; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J.; Slotboom, Dirk J.; Persson, Jan O.; de Gier, Jan-Willem

    2008-01-01

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications. PMID:18796603

  9. WDR62 overexpression is associated with a poor prognosis in patients with lung adenocarcinoma.

    PubMed

    Shinmura, Kazuya; Kato, Hisami; Kawanishi, Yuichi; Igarashi, Hisaki; Inoue, Yusuke; Yoshimura, Katsuhiro; Nakamura, Satoki; Fujita, Hidehiko; Funai, Kazuhito; Tanahashi, Masayuki; Niwa, Hiroshi; Ogawa, Hiroshi; Sugimura, Haruhiko

    2017-08-01

    Human WDR62, which is localized in the cytoplasm including the centrosome, is known to be responsible for primary microcephaly; however, the role of WDR62 abnormality in cancers remains largely unknown. In this study, we aimed to reveal the pathological role of WDR62 abnormality in lung adenocarcinoma (LAC). We first examined the WDR62 mRNA expression level of LAC (n = 64) using a QRT-PCR analysis and found that WDR62 mRNA transcripts were significantly overexpressed in LAC (P = 0.0432, Wilcoxon matched pairs test). An immunohistochemical analysis for LAC (n = 237) showed that WDR62 proteins were also significantly overexpressed in LAC (P < 0.0001, Mann-Whitney U test). A Kaplan-Meier analysis demonstrated that patients with LAC who exhibit WDR62 overexpression have a short overall survival (P = 0.0378, log-rank test), and a multivariate analysis revealed that WDR62 overexpression was an independent predictor of a poor survival outcome among LAC patients (hazard ratio, 2.032; 95% confidence interval, 1.071-3.777; P = 0.0305). Next, we examined the functional effect of WDR62 overexpression on the lung cancer cell line H1299. WDR62-overexpressing lung cancer cells exhibited an increase in cell growth. Moreover, the concurrent overexpression of WDR62 and TPX2, a WDR62-interacting protein that is also overexpressed in LAC, induced centrosome amplification in the lung cells. Finally, we disclosed that the concurrent overexpression of WDR62 and TPX2 is common in diverse human cancers, using data from the Cancer Genome Atlas. These results suggested that WDR62 overexpression is associated with a poor prognosis in patients with LAC and leads to an increase in the malignant potential of lung cells. © 2017 Wiley Periodicals, Inc.

  10. Lead poisoning

    SciTech Connect

    Rekus, J.F.

    1992-08-01

    Construction workers who weld, cut or blast structural steel coated with lead-based paint are at significant risk of lead poisoning. Although technology to control these exposures may not have existed when the lead standard was promulgated, it is available today. Employers who do not take steps to protect their employees from lead exposure may be cited and fined severely for their failure.

  11. Craniosynostosis in transgenic mice overexpressing Nell-1

    PubMed Central

    Zhang, Xinli; Kuroda, Shun’ichi; Carpenter, Dale; Nishimura, Ichiro; Soo, Chia; Moats, Rex; Iida, Keisuke; Wisner, Eric; Hu, Fei-Ya; Miao, Steve; Beanes, Steve; Dang, Catherine; Vastardis, Heleni; Longaker, Michael; Tanizawa, Katsuyuki; Kanayama, Norihiro; Saito, Naoaki; Ting, Kang

    2002-01-01

    Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS), one of the most common congenital craniofacial deformities. Here we describe the creation and analysis of transgenic mice overexpressing Nell-1. Nell-1 transgenic animals exhibited CS-like phenotypes that ranged from simple to compound synostoses. Histologically, the osteogenic fronts of abnormally closing/closed sutures in these animals revealed calvarial overgrowth and overlap along with increased osteoblast differentiation and reduced cell proliferation. Furthermore, anomalies were restricted to calvarial bone, despite generalized, non-tissue-specific overexpression of Nell-1. In vitro, Nell-1 overexpression accelerated calvarial osteoblast differentiation and mineralization under normal culture conditions. Moreover, Nell-1 overexpression in osteoblasts was sufficient to promote alkaline phosphatase expression and micronodule formation. Conversely, downregulation of Nell-1 inhibited osteoblast differentiation in vitro. In summary, Nell-1 overexpression induced calvarial overgrowth resulting in premature suture closure in a rodent model. Nell-1, therefore, has a novel role in CS development, perhaps as part of a complex chain of events resulting in premature suture closure. On a cellular level, Nell-1 expression may modulate and be both sufficient and required for osteoblast differentiation. PMID:12235118

  12. Skeletal overexpression of gremlin impairs bone formation and causes osteopenia.

    PubMed

    Gazzerro, Elisabetta; Pereira, Renata C; Jorgetti, Vanda; Olson, Sarah; Economides, Aris N; Canalis, Ernesto

    2005-02-01

    Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Gremlin, a BMP antagonist, is expressed in osteoblasts and opposes BMP effects on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. To investigate the actions of gremlin on bone remodeling in vivo, we generated transgenic mice overexpressing gremlin under the control of the osteocalcin promoter. Gremlin transgenics exhibited bone fractures and reduced bone mineral density by 20-30%, compared with controls. Static and dynamic histomorphometry of femurs revealed that gremlin overexpression caused reduced trabecular bone volume and the appearance of woven bone. Polarized light microscopy revealed disorganized collagen bundles at the endosteal cortical surface. Gremlin transgenic mice displayed a 70% decrease in the number of osteoblasts/trabecular area and reduced mineral apposition and bone formation rates. In vivo bromodeoxyuridine labeling and marrow stromal cell cultures demonstrated an inhibitory effect of gremlin on osteoblastic cell replication, but no change on apoptosis was detected. Marrow stromal cells from gremlin transgenics displayed a reduced response to BMP on phosphorylated mothers against decapentaplegic 1/5/8 phosphorylation and reduced free cytosolic beta-catenin levels. In conclusion, transgenic mice overexpressing gremlin in the bone microenvironment have decreased osteoblast number and function leading to osteopenia and spontaneous fractures.

  13. Lead Test

    MedlinePlus

    ... the condition. Children should also be assessed for iron deficiency and general nutrition consistent with AAP guidelines. BLLs ... raised blood lead concentrations should be tested for iron deficiency . Each person eliminates lead differently. Thus, laboratory tests ...

  14. Lead Poisoning

    MedlinePlus

    ... through deteriorating paint, household dust, bare soil, air, drinking water, food, ceramics, home remedies, hair dyes and other ... an elevated blood lead level can easily result. Drinking water can also sometimes contribute to elevated blood lead ...

  15. Lead Pencils

    NASA Technical Reports Server (NTRS)

    Gray, L. B.

    1971-01-01

    A study, undertaken to determine the lead content of paint on various pencils in the Goddard supply system, is reported. The survey found that lead content varied from .04 mg per pencil for carmine colored pencils to approximately 43 mg per pencil for yellow colored pencils. Results also show that yellow pencils had higher lead content than other colors analyzed. More detailed results are given in tabular form.

  16. Transgenic mice overexpressing desmocollin-2 (DSC2) develop cardiomyopathy associated with myocardial inflammation and fibrotic remodeling

    PubMed Central

    Garnett, Lauren; Martens, Kristina; Abdelfatah, Nelly; Rodriguez, Marcela; Diao, Catherine; Chen, Yong-Xiang; Gordon, Paul M. K.; Nygren, Anders; Gerull, Brenda

    2017-01-01

    Background Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure, mainly as a result of mutations in cardiac desmosomal genes. Desmosomes are cell-cell junctions mediating adhesion of cardiomyocytes; however, the molecular and cellular mechanisms underlying the disease remain widely unknown. Desmocollin-2 is a desmosomal cadherin serving as an anchor molecule required to reconstitute homeostatic intercellular adhesion with desmoglein-2. Cardiac specific lack of desmoglein-2 leads to severe cardiomyopathy, whereas overexpression does not. In contrast, the corresponding data for desmocollin-2 are incomplete, in particular from the view of protein overexpression. Therefore, we developed a mouse model overexpressing desmocollin-2 to determine its potential contribution to cardiomyopathy and intercellular adhesion pathology. Methods and results We generated transgenic mice overexpressing DSC2 in cardiac myocytes. Transgenic mice developed a severe cardiac dysfunction over 5 to 13 weeks as indicated by 2D-echocardiography measurements. Corresponding histology and immunohistochemistry demonstrated fibrosis, necrosis and calcification which were mainly localized in patches near the epi- and endocardium of both ventricles. Expressions of endogenous desmosomal proteins were markedly reduced in fibrotic areas but appear to be unchanged in non-fibrotic areas. Furthermore, gene expression data indicate an early up-regulation of inflammatory and fibrotic remodeling pathways between 2 to 3.5 weeks of age. Conclusion Cardiac specific overexpression of desmocollin-2 induces necrosis, acute inflammation and patchy cardiac fibrotic remodeling leading to fulminant biventricular cardiomyopathy. PMID:28339476

  17. Leading Democratically

    ERIC Educational Resources Information Center

    Brookfield, Stephen

    2010-01-01

    Democracy is the most venerated of American ideas, the one for which wars are fought and people die. So most people would probably agree that leaders should be able to lead well in a democratic society. Yet, genuinely democratic leadership is a relative rarity. Leading democratically means viewing leadership as a function or process, rather than…

  18. Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

    PubMed Central

    Faria, Márcia; Capinha, Liliana; Simões-Pereira, Joana; Bugalho, Maria João; Silva, Ana Luísa

    2016-01-01

    RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions. PMID:27127508

  19. Lead poisoning

    MedlinePlus

    ... help if this information is not immediately available. Poison Control If someone has severe symptoms from possible ... be caused by lead poisoning, call your local poison control center. Your local poison center can be ...

  20. Hand1 overexpression inhibits medulloblastoma metastasis

    SciTech Connect

    Asuthkar, Swapna; Guda, Maheedhara R.; Martin, Sarah E.; Antony, Reuben; Fernandez, Karen; Lin, Julian; Tsung, Andrew J.; Velpula, Kiran K.

    2016-08-19

    Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

  1. Overexpression of Protochlorophyllide Oxidoreductase C Regulates Oxidative Stress in Arabidopsis

    PubMed Central

    Pattanayak, Gopal K.; Tripathy, Baishnab C.

    2011-01-01

    Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen (1O2). As there is no enzymatic detoxification mechanism available in plants to destroy 1O2, its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the 1O2 generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of 1O2 and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to 1O2-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5

  2. Ecotoxicology: Lead

    USGS Publications Warehouse

    Scheuhammer, A.M.; Beyer, W.N.; Schmitt, C.J.; Jorgensen, Sven Erik; Fath, Brian D.

    2008-01-01

    Lead (Pb) is a naturally occurring metallic element; trace concentrations are found in all environmental media and in all living things. However, certain human activities, especially base metal mining and smelting; combustion of leaded gasoline; the use of Pb in hunting, target shooting, and recreational angling; the use of Pb-based paints; and the uncontrolled disposal of Pb-containing products such as old vehicle batteries and electronic devices have resulted in increased environmental levels of Pb, and have created risks for Pb exposure and toxicity in invertebrates, fish, and wildlife in some ecosystems.

  3. Leading Meetings.

    ERIC Educational Resources Information Center

    Lindelow, John; Heynderickx, James

    Chapter 13 of a revised volume on school leadership, this chapter offers suggestions to help educators improve their performance in meetings, both as group leaders and as participants. Well-run meetings can rejuvenate an organization, leading to improved teamwork, communication, and morale. A poor meeting, on the other hand, can have a…

  4. Tetraethyl lead

    Integrated Risk Information System (IRIS)

    Tetraethyl lead ; CASRN 78 - 00 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  5. An apple rootstock overexpressing a peach CBF gene alters growth and flowering in the scion but does not impact cold hardiness or dormancy

    USDA-ARS?s Scientific Manuscript database

    The C-repeat Binding Factor (CBF) transcription factor is involved in responses to low temperature and water deficit in many plant species. Overexpression of CBF genes leads to enhanced freezing tolerance and growth inhibition in many species. The overexpression of a peach CBF (PpCBF1) gene in a t...

  6. Overexpression of neurofilament H disrupts normal cell structure and function

    NASA Technical Reports Server (NTRS)

    Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

    2002-01-01

    Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

  7. Suppression of Hepatocellular Carcinoma by Inhibition of Overexpressed Ornithine Aminotransferase.

    PubMed

    Zigmond, Ehud; Ben Ya'acov, Ami; Lee, Hyunbeom; Lichtenstein, Yoav; Shalev, Zvi; Smith, Yoav; Zolotarov, Lidya; Ziv, Ehud; Kalman, Rony; Le, Hoang V; Lu, Hejun; Silverman, Richard B; Ilan, Yaron

    2015-08-13

    Hepatocellular carcinoma is the second leading cause of cancer death worldwide. DNA microarray analysis identified the ornithine aminotransferase (OAT) gene as a prominent gene overexpressed in hepatocellular carcinoma (HCC) from Psammomys obesus. In vitro studies demonstrated inactivation of OAT by gabaculine (1), a neurotoxic natural product, which suppressed in vitro proliferation of two HCC cell lines. Alpha-fetoprotein (AFP) secretion, a biomarker for HCC, was suppressed by gabaculine in both cell lines, but not significantly. Because of the active site similarity between GABA aminotransferase (GABA-AT) and OAT, a library of 24 GABA-AT inhibitors was screened to identify a more selective inhibitor of OAT. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidene)cyclopentane-1-carboxylic acid (2) was found to be an inactivator of OAT that only weakly inhibits GABA-AT, l-aspartate aminotransferase, and l-alanine aminotransferase. In vitro administration of 2 significantly suppressed AFP secretion in both Hep3B and HepG2 HCC cells; in vivo, 2 significantly suppressed AFP serum levels and tumor growth in HCC-harboring mice, even at 0.1 mg/kg. Overexpression of the OAT gene in HCC and the ability to block the growth of HCC by OAT inhibitors support the role of OAT as a potential therapeutic target to inhibit HCC growth. This is the first demonstration of suppression of HCC by an OAT inactivator.

  8. Overexpression of neurofilament H disrupts normal cell structure and function

    NASA Technical Reports Server (NTRS)

    Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

    2002-01-01

    Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

  9. Frequent Nek1 overexpression in human gliomas

    SciTech Connect

    Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

    2016-08-05

    Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

  10. Who Leads China's Leading Universities?

    ERIC Educational Resources Information Center

    Huang, Futao

    2017-01-01

    This study attempts to identify the major characteristics of two different groups of institutional leaders in China's leading universities. The study begins with a review of relevant literature and theory. Then, there is a brief introduction to the selection of party secretaries, deputy secretaries, presidents and vice presidents in leading…

  11. Who Leads China's Leading Universities?

    ERIC Educational Resources Information Center

    Huang, Futao

    2017-01-01

    This study attempts to identify the major characteristics of two different groups of institutional leaders in China's leading universities. The study begins with a review of relevant literature and theory. Then, there is a brief introduction to the selection of party secretaries, deputy secretaries, presidents and vice presidents in leading…

  12. An efficient method to isolate yeast genes causing overexpression-mediated growth arrest.

    PubMed

    Espinet, C; de la Torre, M A; Aldea, M; Herrero, E

    1995-01-01

    In order to characterize new yeast genes regulating cell proliferation, a number of overexpression-sensitive clones have been isolated from a Saccharomyces cerevisiae cDNA library in a multicopy vector under the control of the GAL1 promoter, on the basis of growth arrest phenotype under galactose-induction conditions. Thirteen of the independent clones isolated in this way correspond to previously known genes (predominantly coding for morphogenesis-related proteins or for multifunctional transcriptional factors), while the remaining 11 independent clones represent new genes with unknown functions. The more stringent conditions employed in this screening compared with previous ones that also employed a dominant genetics approach to isolate overexpression-sensitive genes has allowed us to extend the number of yeast genes that exhibit this phenotype. The effect of overexpression of MCM1 (whose product participates in the regulation of a number of apparently unrelated cellular functions) has been studied in more detail. Galactose-induced overexpression of MCM1 leads to rapid growth arrest at the G1 or S cell cycle stages, with many morphologically-abnormal cells. Several of the other clones also exhibit a G1 arrest terminal phenotype when overexpressed.

  13. Overexpression of MMP-7 increases collagen 1A2 in the aging kidney

    PubMed Central

    Ślusarz, Anna; Nichols, LaNita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-01-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis that leads to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however, it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, with over a 500-fold upregulation in 2-year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of protein kinase A (PKA), src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 upregulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src, and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  14. Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

    SciTech Connect

    Chuang, Jian-Ying; Hung, Jan-Jong

    2011-04-15

    Highlights: {yields} Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. {yields} Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. {yields} Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

  15. Nuclear overexpression of the overexpressed in lung cancer 1 predicts worse prognosis in gastric adenocarcinoma.

    PubMed

    Wang, Jue; Shen, Hongchang; Fu, Guobin; Zhao, Dandan; Wang, Weibo

    2017-02-07

    We have performed this retrospective study to elucidate whether elevated expression of the overexpressed in lung cancer 1 (OLC1) was related to the clinicopathological parameters and prognosis of patients with gastric adenocarcinoma. Additionally, different effects of various subcellular OLC1 expression on gastric adeno-carcinogenesis were focused on in our study. Both overall and subcellular expression of OLC1 was evaluated by immunohistochemistry(IHC) via tissue microarrays from total 393 samples. The Kaplan-Meier method and Cox's proportional hazard model were exerted to further explore the correlation between OLC1 and prognosis. Total overexpression of OLC1 was significantly associated with stage (P = 0.004) and differentiation (P = 0.009), and only the strong total expression could predict a poor prognosis (HR = 1.31, P = 0.04). There were significant associations found between nuclear overexpression and tumor invasion depth(P = 0.002), lymph node (P < 0.001), stage (P = 0.004), differentiation (P < 0.001) and smoking history (P = 0.045). Furthermore, over-expressed nuclear OLC1 protein could be an independent risk factor for gastric adenocarcinoma (univariate: HR = 1.43, P = 0.003; multivariate: HR = 1.39, P = 0.011). In general, both total and nuclear overexpression of OLC1 could be the signs of gastric adeno-carcinogenesis, which might be served as the biomarkers for diagnosis at an early stage, even at the onset of tumorigenesis. Rather than the total expression, nuclear overexpression of OLC1 was correlated with most clinicopathological parameters and could predict a poor overall survival as an independent factor for prognosis, which made it a more effective and sensitive biomarker for gastric adenocarcinoma.

  16. Overexpression of multisubunit replication factors in yeast.

    PubMed

    Burgers, P M

    1999-07-01

    Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible and inexpensive source of these factors in large quantities. The methodology is illustrated with the five-subunit replication factor C. Whole-cell extracts are prepared by blending yeast cells with glass beads or frozen yeast with dry ice. Procedures are described that maximize the yield of these factors while minimizing proteolytic degradation.

  17. Overexpression of facioscapulohumeral muscular dystrophy region gene 1 causes primary defects in myogenic stem cells.

    PubMed

    Xynos, Alexandros; Neguembor, Maria Victoria; Caccia, Roberta; Licastro, Danilo; Nonis, Alessandro; Di Serio, Clelia; Stupka, Elia; Gabellini, Davide

    2013-05-15

    Overexpression of facioscapulohumeral muscular dystrophy region gene 1 (FRG1) in mice, frogs and worms leads to muscular and vascular abnormalities. Nevertheless, the mechanism that follows FRG1 overexpression and finally leads to muscular defects is currently unknown. Here, we show that the earliest phenotype displayed by mice overexpressing FRG1 is a postnatal muscle-growth defect. Long before the development of muscular dystrophy, FRG1 mice also exhibit a muscle regeneration impairment. Ex vivo and in vivo experiments revealed that FRG1 overexpression causes myogenic stem cell activation and proliferative, clonogenic and differentiation defects. A comparative gene expression profiling of muscles from young pre-dystrophic wild-type and FRG1 mice identified differentially expressed genes in several gene categories and networks that could explain the emerging tissue and myogenic stem cell defects. Overall, our study provides new insights into the pathways regulated by FRG1 and suggests that muscle stem cell defects could contribute to the pathology of FRG1 mice.

  18. Genetic manipulation of the γ-aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ-aminobutyric acid transaminase (GABA-T) lead to sustained and high levels of GABA accumulation in rice kernels.

    PubMed

    Shimajiri, Yasuka; Oonishi, Takayuki; Ozaki, Kae; Kainou, Kumiko; Akama, Kazuhito

    2013-06-01

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA-transaminase, GABA-T), we attempted seed-specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB-1) or rice embryo globulin promoters (REG) and GABA-T-based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2-100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA-T expression was relatively weak. In these two lines both with two T-DNA copies, their starch, amylose, and protein levels were slightly lower than non-transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75-350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  19. Tumorigenesis promotes Mdm4-S overexpression

    PubMed Central

    Pant, Vinod; Larsson, Connie A.; Aryal, Neeraj; Xiong, Shunbin; You, M. James; Quintas-Cardama, Alfonso; Lozano, Guillermina

    2017-01-01

    Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution. PMID:28460439

  20. Tumorigenesis promotes Mdm4-S overexpression.

    PubMed

    Pant, Vinod; Larsson, Connie A; Aryal, Neeraj; Xiong, Shunbin; You, M James; Quintas-Cardama, Alfonso; Lozano, Guillermina

    2017-04-18

    Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.

  1. Nucleophosmin is overexpressed in thyroid tumors

    SciTech Connect

    Pianta, Annalisa; Puppin, Cinzia; Franzoni, Alessandra; Fabbro, Dora; Di Loreto, Carla; Bulotta, Stefania; Deganuto, Marta; Paron, Igor; Tell, Gianluca; Puxeddu, Efisio; Filetti, Sebastiano; Russo, Diego; Damante, Giuseppe

    2010-07-02

    Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

  2. VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms.

    PubMed

    Latil, A; Bièche, I; Pesche, S; Valéri, A; Fournier, G; Cussenot, O; Lidereau, R

    2000-03-20

    Studies comparing tumor neovascularity with pathological findings suggest that angiogenesis contributes to the pathogenesis of prostate cancer. We have examined 42 primary sporadic prostate tumors at different clinical stages, together with 3 prostate cancer cell lines (DU145, PC3 and LNCaP), for expression of VEGF and the gene encoding the recently identified VEGF165 isoform-specific receptor neuropilin-1, by using a quantitative reverse transcription (RT)-PCR method. We also evaluated the VEGF transcription pattern. Upregulation of VEGF and neuropilin-1 was observed in 12 and 14 tumors, respectively. The VEGF165 isoform was slighly overrepresented in tumors that overexpressed VEGF. VEGF overexpression correlated with stage II disease (p < 0.05); neuropilin-1 overexpression correlated with advanced disease (p < 0. 01) and a high Gleason grade (p < 0.02). Our observations suggest that VEGF expression could be used as a prognostic marker in early-stage prostate tumors, whereas neuropilin-1 overexpression might be a marker of aggressiveness. Copyright 2000 Wiley-Liss, Inc.

  3. Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction.

    PubMed

    Weiss, N; Zhang, Y Y; Heydrick, S; Bierl, C; Loscalzo, J

    2001-10-23

    Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine beta-synthase-deficient (CBS((-/+))) mice and their wild-type littermates (CBS((+/+))) were crossbred with mice that overexpress GPx-1 [GPx-1((tg+)) mice]. GPx-1 activity was 28% lower in CBS((-/+))/GPx-1((tg-)) compared with CBS((+/+))/GPx-1((tg-)) mice (P < 0.05), and CBS((-/+)) and CBS((+/+)) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS((-/+))/GPx-1((tg-)) mice showed vasoconstriction to superfusion with beta-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS((+/+))/GPx-1((tg-)) and CBS((+/+))/GPx-1((tg+)) mice] demonstrated dose-dependent vasodilation in response to both agonists. Overexpression of GPx-1 in hyperhomocyst(e)inemic mice restored the normal endothelium-dependent vasodilator response. Bovine aortic endothelial cells (BAEC) were transiently transfected with GPx-1 and incubated with dl-homocysteine (HcyH) or l-cysteine. HcyH incubation decreased GPx-1 activity in sham-transfected BAEC (P < 0.005) but not in GPx-1-transfected cells. Nitric oxide release from BAEC was significantly decreased by HcyH but not cysteine, and GPx-1 overexpression attenuated this decrease. These findings demonstrate that overexpression of GPx-1 can compensate for the adverse effects of Hcy on endothelial function and suggest that the adverse vascular effects of Hcy are at least partly mediated by oxidative inactivation of NO.

  4. Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction

    PubMed Central

    Weiss, Norbert; Zhang, Ying-Yi; Heydrick, Stanley; Bierl, Charlene; Loscalzo, Joseph

    2001-01-01

    Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine β-synthase-deficient (CBS(−/+)) mice and their wild-type littermates (CBS(+/+)) were crossbred with mice that overexpress GPx-1 [GPx-1(tg+) mice]. GPx-1 activity was 28% lower in CBS(−/+)/GPx-1(tg−) compared with CBS(+/+)/GPx-1(tg−) mice (P < 0.05), and CBS(−/+) and CBS(+/+) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS(−/+)/GPx-1(tg−) mice showed vasoconstriction to superfusion with β-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS(+/+)/GPx-1(tg−) and CBS(+/+)/GPx-1(tg+) mice] demonstrated dose-dependent vasodilation in response to both agonists. Overexpression of GPx-1 in hyperhomocyst(e)inemic mice restored the normal endothelium-dependent vasodilator response. Bovine aortic endothelial cells (BAEC) were transiently transfected with GPx-1 and incubated with dl-homocysteine (HcyH) or l-cysteine. HcyH incubation decreased GPx-1 activity in sham-transfected BAEC (P < 0.005) but not in GPx-1-transfected cells. Nitric oxide release from BAEC was significantly decreased by HcyH but not cysteine, and GPx-1 overexpression attenuated this decrease. These findings demonstrate that overexpression of GPx-1 can compensate for the adverse effects of Hcy on endothelial function and suggest that the adverse vascular effects of Hcy are at least partly mediated by oxidative inactivation of NO. PMID:11606774

  5. In vivo clonal overexpression of neuroligin 3 and neuroligin 2 in neurons of the rat cerebral cortex. Differential effects on GABAergic synapses and neuronal migration

    PubMed Central

    Fekete, Christopher D.; Chiou, Tzu-Ting; Miralles, Celia P.; Harris, Rachel S.; Fiondella, Christopher G.; LoTurco, Joseph J.; De Blas, Angel L.

    2015-01-01

    We have studied the effect of clonal overexpression of neuroligin 3 (NL3) or neuroligin 2 (NL2) in the adult rat cerebral cortex following in utero electroporation (IUEP) at embryonic stage E14. Overexpression of NL3 leads to a large increase in vGAT and GAD65 in the GABAergic contacts that the overexpressing neurons receive. Overexpression of NL2 produced a similar effect but to a lesser extent. In contrast, overexpression of NL3 or NL2 after IUEP, does not affect vGlut1 in the glutamatergic contacts that the NL3 or NL2 overexpressing neurons receive. The NL3 or NL2 overexpressing neurons do not show increased innervation by parvalbumin-containing GABAergic terminals or increased parvalbumin in the same terminals that show increased vGAT. These results indicate that the observed increase in vGAT and GAD65 is not due to increased GABAergic innervation but to increased expression of vGAT and GAD65 in the GABAergic contacts that NL3 or NL2 overexpressing neurons receive. The majority of bright vGAT puncta contacting the NL3 overexpressing neurons have no gephyrin juxtaposed to them indicating that many of these contacts are non-synaptic. This contrasts with the majority of the NL2 overexpressing neurons, which show plenty of synaptic gephyrin clusters juxtaposed to vGAT. Besides having an effect on GABAergic contacts, overexpression of NL3 interferes with the neuronal radial migration, in the cerebral cortex, of the neurons overexpressing NL3. PMID:25565602

  6. CD3-epsilon overexpressed in prothymocytes acts as an oncogene.

    PubMed Central

    Wang, B.; She, J.; Salio, M.; Allen, D.; Lacy, E.; Lonberg, N.; Terhorst, C.

    1997-01-01

    BACKGROUND: Upon engagement of the T cell receptor for antigen, its associated CD3 proteins recruit signal transduction molecules, which in turn regulate T lymphocyte proliferation, apoptosis, and thymocyte development. Because some signal transducing molecules recruited by CD3-epsilon, i.e., p56lck and p59fyn, are oncogenic and since we previously found that overexpression of CD3-epsilon transgenes causes a block in T lymphocyte and NK cell development, we tested the hypothesis that aberrant CD3-epsilon signaling leads both to abnormal T lymphocyte death and lymphomagenesis. MATERIALS AND METHODS: Ten independently derived transgenic mouse lines were generated with four different genomic CD3-epsilon constructs. Mice either homozygous or hemizygous for each transgene were analyzed for an arrest in T lymphocyte development and for the occurrence of T cell lymphomas. RESULTS: Aggressive clonal T cell lymphomas developed at very high frequencies in seven mouse lines with intermediate levels of copies of CD3-epsilon derived transgenes. However, these lymphomas were not found when high copy numbers of CD3-epsilon transgenes caused a complete block in early thymic development or when a transgene was used in which the exons coding for the CD3-epsilon protein were deleted. Analyses of a series of double mutant mice, tgCD3-epsilon x RAG-2null, indicated that lymphomagenesis was initiated in lineage-committed prothymocytes, i.e., before rearrangement of the T cell receptor genes. In addition, the transgene coding for the CD3-epsilon cytoplasmic domain and its transmembrane region induced a T cell differentiation signal in premalignant tgCD3-epsilon x RAG-2null mice. CONCLUSION: The nonenzymatic CD3-epsilon protein acted as a potent oncogene when overexpressed early in T lymphocyte development. Lymphomagenesis was dependent on signal transduction events initiated by the cytoplasmic domain of CD3-epsilon. Images FIG. 2 FIG. 4 FIG. 5 PMID:9132282

  7. Statins Reduce Melanoma Development and Metastasis through MICA Overexpression.

    PubMed

    Pich, Christine; Teiti, Iotefa; Rochaix, Philippe; Mariamé, Bernard; Couderc, Bettina; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2013-01-01

    Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments.

  8. Statins Reduce Melanoma Development and Metastasis through MICA Overexpression

    PubMed Central

    Pich, Christine; Teiti, Iotefa; Rochaix, Philippe; Mariamé, Bernard; Couderc, Bettina; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2013-01-01

    Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments. PMID:23493799

  9. The influence of follistatin on mechanical properties of bone tissue in growing mice with overexpression of follistatin.

    PubMed

    Gajos-Michniewicz, Anna; Pawlowska, Elzbieta; Ochedalski, Tomasz; Piastowska-Ciesielska, Agnieszka

    2012-07-01

    Mechanical competence of bones is mainly associated with tissue quality that depends on proper bone metabolism processes. An imbalance in the regulation of bone metabolism leads to pathological changes in bone tissue leading to susceptibility to bone fractures and bone deterioration processes. Bone metabolism is regulated to a large extent by the members of the transforming growth factor-β superfamily, i.e., activins and bone morphogenetic proteins. However, their function is regulated by a single-chain protein called follistatin (FS). The aim of this study was to test the hypothesis that overexpression of FS in growing mice results in impairments in bone morphology and mechanical properties. Moreover, we wanted to investigate how geometrical, structural and material properties of bone tissue change with age. The experiment was performed on growing C57BL/6 TgNK14-mFst/6J mice, overexpressing FS (F mice) versus C57BL/6J mice used as controls (C mice). To establish how overexpression of FS influences bone tissue quality, we studied mice femurs to determine geometrical, structural and material properties of the skeleton. To determine mechanical resistance of bone tissue, femurs were loaded to failure in a three-point bending test. Obtained results indicated that overexpression of FS negatively influences bone metabolism. It was found that mutation results with a significant decrease of all measured biomechanical strength variables in F mice in comparison to C mice. Overexpression of FS leads to decreased quality of skeleton, increasing susceptibility to bone fractures.

  10. Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy.

    PubMed

    Li, Qiaoling; Xie, Jun; Wang, Bingjian; Li, Ran; Bai, Jian; Ding, Liang; Gu, Rong; Wang, Lian; Xu, Biao

    2016-01-01

    Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.

  11. Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

    PubMed Central

    Li, Ran; Bai, Jian; Ding, Liang; Gu, Rong; Wang, Lian; Xu, Biao

    2016-01-01

    Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure. PMID:26914935

  12. Photosynthesis of root chloroplasts developed in Arabidopsis lines overexpressing GOLDEN2-LIKE transcription factors.

    PubMed

    Kobayashi, Koichi; Sasaki, Daichi; Noguchi, Ko; Fujinuma, Daiki; Komatsu, Hirohisa; Kobayashi, Masami; Sato, Mayuko; Toyooka, Kiminori; Sugimoto, Keiko; Niyogi, Krishna K; Wada, Hajime; Masuda, Tatsuru

    2013-08-01

    In plants, genes involved in photosynthesis are encoded separately in nuclei and plastids, and tight cooperation between these two genomes is therefore required for the development of functional chloroplasts. Golden2-like (GLK) transcription factors are involved in chloroplast development, directly targeting photosynthesis-associated nuclear genes for up-regulation. Although overexpression of GLKs leads to chloroplast development in non-photosynthetic organs, the mechanisms of coordination between the nuclear gene expression influenced by GLKs and the photosynthetic processes inside chloroplasts are largely unknown. To elucidate the impact of GLK-induced expression of photosynthesis-associated nuclear genes on the construction of photosynthetic systems, chloroplast morphology and photosynthetic characteristics in greenish roots of Arabidopsis thaliana lines overexpressing GLKs were compared with those in wild-type roots and leaves. Overexpression of GLKs caused up-regulation of not only their direct targets but also non-target nuclear and plastid genes, leading to global induction of chloroplast biogenesis in the root. Large antennae relative to reaction centers were observed in wild-type roots and were further enhanced by GLK overexpression due to the increased expression of target genes associated with peripheral light-harvesting antennae. Photochemical efficiency was lower in the root chloroplasts than in leaf chloroplasts, suggesting that the imbalance in the photosynthetic machinery decreases the efficiency of light utilization in root chloroplasts. Despite the low photochemical efficiency, root photosynthesis contributed to carbon assimilation in Arabidopsis. Moreover, GLK overexpression increased CO₂ fixation and promoted phototrophic performance of the root, showing the potential of root photosynthesis to improve effective carbon utilization in plants.

  13. Photosynthesis of Root Chloroplasts Developed in Arabidopsis Lines Overexpressing GOLDEN2-LIKE Transcription Factors

    PubMed Central

    Kobayashi, Koichi; Sasaki, Daichi; Noguchi, Ko; Fujinuma, Daiki; Komatsu, Hirohisa; Kobayashi, Masami; Sato, Mayuko; Toyooka, Kiminori; Sugimoto, Keiko; Niyogi, Krishna K.; Wada, Hajime; Masuda, Tatsuru

    2013-01-01

    In plants, genes involved in photosynthesis are encoded separately in nuclei and plastids, and tight cooperation between these two genomes is therefore required for the development of functional chloroplasts. Golden2-like (GLK) transcription factors are involved in chloroplast development, directly targeting photosynthesis-associated nuclear genes for up-regulation. Although overexpression of GLKs leads to chloroplast development in non-photosynthetic organs, the mechanisms of coordination between the nuclear gene expression influenced by GLKs and the photosynthetic processes inside chloroplasts are largely unknown. To elucidate the impact of GLK-induced expression of photosynthesis-associated nuclear genes on the construction of photosynthetic systems, chloroplast morphology and photosynthetic characteristics in greenish roots of Arabidopsis thaliana lines overexpressing GLKs were compared with those in wild-type roots and leaves. Overexpression of GLKs caused up-regulation of not only their direct targets but also non-target nuclear and plastid genes, leading to global induction of chloroplast biogenesis in the root. Large antennae relative to reaction centers were observed in wild-type roots and were further enhanced by GLK overexpression due to the increased expression of target genes associated with peripheral light-harvesting antennae. Photochemical efficiency was lower in the root chloroplasts than in leaf chloroplasts, suggesting that the imbalance in the photosynthetic machinery decreases the efficiency of light utilization in root chloroplasts. Despite the low photochemical efficiency, root photosynthesis contributed to carbon assimilation in Arabidopsis. Moreover, GLK overexpression increased CO2 fixation and promoted phototrophic performance of the root, showing the potential of root photosynthesis to improve effective carbon utilization in plants. PMID:23749810

  14. Linear correlation between bacterial overexpression of recombinant peptides and cell light scatter.

    PubMed Central

    Lavergne-Mazeau, F; Maftah, A; Cenatiempo, Y; Julien, R

    1996-01-01

    Fusion of multiple copies of a test peptide leads to insoluble inclusion bodies. Their presence within bacteria increases either forward-angle light scattering or, to a lesser extent, right-angle light scattering. A linear correlation has been established between cell forward-angle scattering and the level of overexpression of atrial natriuretic peptide. The correlation is valid only for unlysed cells and is protein product specific. PMID:8702299

  15. Overexpression of membrane proteins using Pichia pastoris.

    PubMed

    Bornert, Olivier; Alkhalfioui, Fatima; Logez, Christel; Wagner, Renaud

    2012-02-01

    Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.

  16. MARCKS protein overexpression in inflammatory breast cancer

    PubMed Central

    Manai, Maroua; Lopez, Marc; Eghozzi, Radhia; Ayadi, Sinda; Lamine, Olfa Ben; Manai, Mohamed; Rahal, Khaled; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Viens, Patrice; Birnbaum, Daniel; Boussen, Hamouda; Chaffanet, Max; Bertucci, François

    2017-01-01

    Background Inflammatory breast cancer (IBC) is the most aggressive form of locally-advanced breast cancer. Identification of new therapeutic targets is crucial. We previously reported MARCKS mRNA overexpression in IBC in the largest transcriptomics study reported to date. Here, we compared MARCKS protein expression in IBC and non-IBC samples, and searched for correlations between protein expression and clinicopathological features. Results Tumor samples showed heterogeneity with respect to MARCKS staining: 18% were scored as MARCKS-positive (stained cells ≥ 1%) and 82% as MARCKS-negative. MARCKS expression was more frequent in IBC (36%) than in non-IBC (11%; p = 1.4E−09), independently from molecular subtypes and other clinicopathological variables. We found a positive correlation between protein and mRNA expression in the 148/502 samples previously analyzed for MARCKS mRNA expression. MARCKS protein expression was associated with other poor-prognosis features in the whole series of samples such as clinical axillary lymph node or metastatic extension, high pathological grade, ER-negativity, PR-negativity, HER2-positivity, and triple-negative and HER2+ statutes. In IBC, MARCKS expression was the sole tested variable associated with poor MFS. Materials and Methods We retrospectively analyzed MARCKS protein expression by immunohistochemistry in 502 tumors, including 133 IBC and 369 non-IBC, from Tunisian and French patients. All samples were pre-therapeutic clinical samples. We searched for correlations between MARCKS expression and clinicopathological features including the IBC versus non-IBC phenotype and metastasis-free survival (MFS). Conclusions MARCKS overexpression might in part explain the poor prognosis of IBC. As an oncogene associated with poor MFS, MARCKS might represent a new potential therapeutic target in IBC. PMID:28009981

  17. Targeted anticancer therapy: overexpressed receptors and nanotechnology.

    PubMed

    Akhtar, Mohd Javed; Ahamed, Maqusood; Alhadlaq, Hisham A; Alrokayan, Salman A; Kumar, Sudhir

    2014-09-25

    Targeted delivery of anticancer drugs to cancer cells and tissues is a promising field due to its potential to spare unaffected cells and tissues, but it has been a major challenge to achieve success in these therapeutic approaches. Several innovative approaches to targeted drug delivery have been devised based on available knowledge in cancer biology and on technological advancements. To achieve the desired selectivity of drug delivery, nanotechnology has enabled researchers to design nanoparticles (NPs) to incorporate anticancer drugs and act as nanocarriers. Recently, many receptor molecules known to be overexpressed in cancer have been explored as docking sites for the targeting of anticancer drugs. In principle, anticancer drugs can be concentrated specifically in cancer cells and tissues by conjugating drug-containing nanocarriers with ligands against these receptors. Several mechanisms can be employed to induce triggered drug release in response to either endogenous trigger or exogenous trigger so that the anticancer drug is only released upon reaching and preferentially accumulating in the tumor tissue. This review focuses on overexpressed receptors exploited in targeting drugs to cancerous tissues and the tumor microenvironment. We briefly evaluate the structure and function of these receptor molecules, emphasizing the elegant mechanisms by which certain characteristics of cancer can be exploited in cancer treatment. After this discussion of receptors, we review their respective ligands and then the anticancer drugs delivered by nanotechnology in preclinical models of cancer. Ligand-functionalized nanocarriers have delivered significantly higher amounts of anticancer drugs in many in vitro and in vivo models of cancer compared to cancer models lacking such receptors or drug carrying nanocarriers devoid of ligand. This increased concentration of anticancer drug in the tumor site enabled by nanotechnology could have a major impact on the efficiency of cancer

  18. Over-Expression of Catalase in Myeloid Cells Confers Acute Protection Following Myocardial Infarction

    PubMed Central

    Cabigas, E. Bernadette; Somasuntharam, Inthirai; Brown, Milton E.; Che, Pao Lin; Pendergrass, Karl D.; Chiang, Bryce; Taylor, W. Robert; Davis, Michael E.

    2014-01-01

    Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H2O2), has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H2O2 levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option. PMID:24853285

  19. Over-expression of catalase in myeloid cells confers acute protection following myocardial infarction.

    PubMed

    Cabigas, E Bernadette; Somasuntharam, Inthirai; Brown, Milton E; Che, Pao Lin; Pendergrass, Karl D; Chiang, Bryce; Taylor, W Robert; Davis, Michael E

    2014-05-21

    Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H2O2), has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H2O2 levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option.

  20. Overexpression of isocitrate lyase-glyoxylate bypass influence on metabolism in Aspergillus niger.

    PubMed

    Meijer, S; Otero, J; Olivares, R; Andersen, M R; Olsson, L; Nielsen, J

    2009-03-01

    In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase (icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However,metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed. The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Further more, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium. Overall,the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and interesting production of fumarate and malate was found.

  1. Creating drought- and salt-tolerant cotton by overexpressing a vacuolar pyrophosphatase gene.

    PubMed

    Zhang, Hong; Shen, Guoxin; Kuppu, Sundaram; Gaxiola, Roberto; Payton, Paxton

    2011-06-01

    Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proton pump activity of the vacuolar pyrophosphatase, which generates a higher proton electrochemical gradient across the vacuolar membrane, leading to lower water potential in the plant vacuole and higher secondary transporter activities that prevent ion accumulation to toxic levels in the cytoplasm. Additionally, overexpression of AVP1 appears to stimulate auxin polar transport, which in turn stimulates root development. The larger root system allows AVP1-overexpressing plants to absorb water more efficiently under drought and saline conditions, resulting in stress tolerance and increased yields. Multi-year field-trial data indicate that overexpression of AVP1 in cotton leads to at least 20% more fiber yield than wild-type control plants in dry-land conditions, which highlights the potential use of AVP1 in improving drought tolerance in crops in arid and semiarid areas of the world. 

  2. Virus-induced over-expression of protein phosphatase 2A inhibits insulin signalling in chronic hepatitis C.

    PubMed

    Bernsmeier, Christine; Duong, Francois H T; Christen, Verena; Pugnale, Paolo; Negro, Francesco; Terracciano, Luigi; Heim, Markus H

    2008-09-01

    Hepatitis C virus (HCV) infection disturbs glucose and lipid metabolism contributing to the development of liver steatosis, insulin resistance and type 2 diabetes mellitus. On the other hand, insulin resistance and steatosis have been found to be associated with increased rates of fibrosis progression and lower rates of response to interferon therapy in chronic hepatitis C (CHC). The molecular mechanisms contributing to insulin resistance in CHC are not well understood. We have shown previously that protein phosphatase 2A (PP2A) is over-expressed in biopsies from patients with CHC. In this study, we tested if PP2A over-expression leads to insulin resistance. We studied insulin signalling in cell lines that allow the regulated over-expression of HCV proteins and of the PP2A catalytic subunit (PP2Ac). Insulin signalling and PP2Ac expression were also studied in HCV transgenic mice and in liver biopsies from patients with CHC. Over-expression of PP2Ac in cells inhibited insulin signalling by dephosphorylation of PKB/Akt. PP2Ac over-expression and impaired insulin signalling were found in the liver of HCV transgenic mice and in liver biopsies of patients with CHC. HCV-induced over-expression of PP2A in the liver contributes to the pathogenesis of insulin resistance in patients with CHC.

  3. N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

    PubMed Central

    Rinne, M. L.; He, Y.; Pachkowski, B. F.; Nakamura, J.; Kelley, M. R.

    2005-01-01

    Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents. PMID:15905475

  4. SIRT4 overexpression protects against diabetic nephropathy by inhibiting podocyte apoptosis

    PubMed Central

    Shi, Jian-Xia; Wang, Qi-Jin; Li, Hui; Huang, Qin

    2017-01-01

    Diabetic nephropathy is a diabetic complication associated with capillary damage and increased mortality. Sirtuin 4 (SIRT4) plays an important role in mitochondrial function and the pathogenesis of metabolic diseases, including aging kidneys. The aim of the present study was to investigate the association between SIRT4 and diabetic nephropathy in a glucose-induced mouse podocyte model. A CCK-8 assay showed that glucose simulation significantly inhibited podocyte proliferation in a time- and concentration-dependent manner. Reverse transcription-quantitative polymerase chain reaction and western blot analysis showed that the mRNA and protein levels of SIRT4 were notably decreased in a concentration-dependent manner in glucose-simulated podocytes. However, SIRT4 overexpression increased proliferation and suppressed apoptosis, which was accompanied by increases in mitochondrial membrane potential and reduced production of reactive oxygen species (ROS). Notably, SIRT4 overexpression downregulated the expression of apoptosis-related proteins NOX1, Bax and phosphorylated p38 and upregulated the expression of Bcl-2 in glucose-simulated podocytes. In addition, SIRT4 overexpression significantly attenuated the inflammatory response, indicated by reductions in the levels of TNF-α, IL-1β and IL-6. These results demonstrate for the first time that the overexpression of SIRT4 prevents glucose-induced podocyte apoptosis and ROS production and suggest that podocyte apoptosis represents an early pathological mechanism leading to diabetic nephropathy. PMID:28123512

  5. EphB6 overexpression and Apc mutation together promote colorectal cancer.

    PubMed

    Xu, Dan; Yuan, Liang; Liu, Xin; Li, Mingqi; Zhang, Fubin; Gu, Xin Yue; Zhang, Dongwei; Yang, Youlin; Cui, Binbin; Tong, Jinxue; Zhou, Jin; Yu, Zhiwei

    2016-05-24

    The erythropoietin-producing hepatocyte (Eph) family tyrosine kinases play important roles in tumorigenesis and cancer aggression. In this study, we investigated the role of EphB6 in oncogenic transformation of colorectal epithelial cells in vitro and in vivo. EphB6 is upregulated in human colorectal cancer (CRC) tissues as compared to normal tissues, and its overexpression promotes proliferation, migration and invasion by IMCE colorectal adenoma cells, in which one Apc allele is mutated. EphB6 overexpression together with Apc mutation leads to the development of colorectal tumors in vivo. Expression microarrays using mRNAs and lncRNAs isolated from EphB6-overexpresssing IMCE and control cells revealed a large number of dysregulated genes involved in cancer-related functions and pathways. The present study is the first to demonstrate that EphB6 overexpression together with Apc gene mutations may enhance proliferation, invasion and metastasis by colorectal epithelial cells. Microarray data and pathway analysis of differentially expressed genes provided insight into possible EphB6-regulated mechanisms promoting tumorigenesis and cancer progression. EphB6 overexpression may represent a novel, effective biomarker predictive of cell proliferation, invasion and metastasis patterns in CRC tumors.

  6. Overexpression of Interleukin-18 Aggravates Cardiac Fibrosis and Diastolic Dysfunction in Fructose-Fed Rats

    PubMed Central

    Xing, Shan-Shan; Bi, Xiu-Ping; Tan, Hong-Wei; Zhang, Yun; Xing, Qi-Chong; Zhang, Wei

    2010-01-01

    Inflammation plays an important role in the pathophysiology of the metabolic syndrome (MS). We determined whether the overexpression of interleukin (IL)-18 could aggravate left ventricular (LV) remodeling and diastolic dysfunction in fructose-fed rats (FFRs). To create an animal model for MS, male Wistar rats received 10% fructose in water for 8 months. We used an adenovirus encoding rat IL-18 to overexpress IL-18 in FFRs by intravenous administration. IL-18 overexpression led to increases in collagen volume fraction and collagen deposition. LV systolic function was unaltered. But the LV end-diastolic pressure and the time constant of isovolumic relaxation (tau) were increased. Peak negative value of time derivative of LV pressure (−dp/dt) was decreased. Isovolumic relaxation time and myocardial index, as assessed by echocardiography, were increased. Overexpression of IL-18 leads to aggravated LV remodeling and dysfunction in FFRs. Attenuation of the inflammatory process may provide a novel therapeutic strategy in treating metabolic cardiomyopathy. PMID:20644901

  7. The relation between xyr1 overexpression in Trichoderma harzianum and sugarcane bagasse saccharification performance.

    PubMed

    da Silva Delabona, Priscila; Rodrigues, Gisele Nunes; Zubieta, Mariane Paludetti; Ramoni, Jonas; Codima, Carla Aloia; Lima, Deise Juliana; Farinas, Cristiane Sanchez; da Cruz Pradella, José Geraldo; Seiboth, Bernhard

    2017-03-20

    This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and β-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum.

  8. Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco

    PubMed Central

    John, Riffat; Ganeshan, Uma; Singh, Badri N.; Kaul, Tanushri; Reddy, Malireddy K.; Sopory, Sudhir K.; Rajam, Manchikatla V.

    2016-01-01

    Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle, and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth, and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants. PMID:27630644

  9. Overexpression of a novel regulator of p120 catenin, NLBP, promotes lung adenocarcinoma proliferation

    PubMed Central

    Kim, Chang Hee; Nam, Hae-Seong; Lee, Eun Hee; Han, Seung Hun; Cho, Hyun Jung; Chung, Hee Jin; Lee, Nam Soo; Choi, Suk Jin; Kim, Hojoong; Ryu, Jeong Seon; Kwon, Junhye; Kim, Hongtae

    2013-01-01

    NLBP (novel LZAP-binding protein) was recently shown to function as a tumor suppressor capable of inhibiting the NFκB signaling pathway. NLBP is also known as a negative regulator of cell invasion, and its expression is reduced in several cancer cell lines that have little invasive activity. Although these phenomena suggest that NLBP may be a potential tumor suppressor, its role as a tumor suppressor in human lung cancer is not well established. In contrast to our expectation, NLBP was highly expressed in the early stage of lung adenocarcinoma tissues, and overexpression of NLBP promoted proliferation of H1299 lung adenocarcinoma cells. We also found that p120 catenin (p120ctn) was a novel binding partner of NLBP, and that NLBP binds to the regulatory domain of p120ctn, and p120ctn associates with N-terminal region of NLBP, respectively. This binding leads to p120ctn stability to inhibit proteasomal degradation of p120ctn by inhibiting its ubiqutination. In addition, we also found that overexpression of NLBP and p120ctn in human lung cancer are closely related with adenocarcinoma compared with squamous cell carcinoma. Taken together, our findings reveal that NLBP is highly overexpressed in human lung adenocarcinoma, and that overexpression of NLBP promotes the cell proliferation of lung adenocarcinoma through interacting with p120ctn and suggest that NLBP may function as an oncogene in early stage carcinogenesis of lung adenocarcinoma. PMID:23839039

  10. Significance of Pathways Leading to RhoC Overexpression in Breast Cancer

    DTIC Science & Technology

    2006-04-01

    13. SUPPLEMENTARY NOTES 14. ABSTRACT: Tumor biology is a recognized determinant of tumor behavior , including growth rate, motility...recurrence data, will be combined with gene expression data. Molecular and statistical analysis will occur in collaboration with the University of...begin the analysis of the data for key results. Although case identification and key patient level information had already been collected, it was

  11. Significance of Pathways Leading to RhoC Overexpression in Breast Cancer

    DTIC Science & Technology

    2008-04-01

    likely that RhoC, which is recognized as an important marker of breast cancer, aggressiveness is also regulated by NF-kappa B. The tumor...Awarded: (number pending) Hornbrook (PI), Hensley Alford (Site PI) 8/1/2008-4/30/2010 NCI Building a Pharmacovigilance Population-Based...categories. Marker status Estrogen-receptor (ER) and progesterone-receptor (PR) status at diagnosis is an important prognostic factor and robust

  12. Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

    PubMed Central

    Rajendran, R. Samuel; Shen, Chia-Ning; Chen, Te-Hao; Yen, Chueh-Chuan; Chuang, Chih-Kuang; Lin, Dar-Shong; Hsiao, Chung-Der

    2012-01-01

    Background Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. Methodology/Principal Findings Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1)cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. Conclusion/Significance Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity. PMID:22623957

  13. Diet high in fructose leads to an overexpression of lipocalin-2 in rat fatty liver.

    PubMed

    Alwahsh, Salamah Mohammad; Xu, Min; Seyhan, Hatice Ali; Ahmad, Shakil; Mihm, Sabine; Ramadori, Giuliano; Schultze, Frank Christian

    2014-02-21

    To explore lipocalin-2 (LCN-2) expression and its possible role and mechanism(s) of production in rat models of diet-inducible fatty liver. Fatty liver was triggered in male Sprague-Dawley rats fed either with liquid Lieber-DeCarli (LDC) or LDC + 70% cal fructose (L-HFr) diet for 4 or 8 wk. Chow-nourished animals served as controls. Hepatic expression of LCN-2 and other metabolic and inflammatory mediators was assessed by quantitative reverse transcription polymerase chain reaction and Western blotting. Serum LCN-2, fasting leptin, and lipid profile were evaluated via Enzyme-Linked Immunosorbent Assay, Radioimmunoassay, and colorimetric assays, respectively. The localization of LCN-2 in the liver was detected by using immunofluorescence staining. Furthermore, HE stain was used to evaluate hepatic fat degeneration and inflammation. Both LDC-fed and L-HFr-fed rat histologically featured fatty liver. In the liver, mRNA transcriptions of Mcp-1, a2-m, Il-8 and Glut5 were increased in the L-HFr group at both time points (P < 0.001), while the transcription of Tlr4, Inos, and Tnf-α was significantly up-regulated at week 4. Interestingly, hepatic Lcn-2 expression was 90-fold at week 4 and 507-fold at week 8 higher in L-HFr-subjected rats vs control (P < 0.001). In contrast to HDL-cholesterol, systemic levels of LCN-2, fasting leptin and triglycerides were elevated in the L-HFr regimen (P < 0.001). Moreover, protein expression of hepatic LCN-2, CD14, phospho-MAPK, caspase-9, cytochrome c and 4-hydroxynonenal was increased in the L-HFr group. Conversely, the hepatic expression of PGC-1α (a mitochondrial-biogenic protein) was reduced in the L-HFr category at week 8. The localization of LCN-2 in the liver was predominantly restricted to MPO⁺ granulocytes. Fructose diet up-regulates hepatic LCN-2 expression, which correlates with the increased indicators of oxidative stress and mitochondrial dysfunction. The LCN-2 may be involved in liver protection.

  14. Impact of pr-10a overexpression on the cryopreservation success of Solanum tuberosum suspension cultures.

    PubMed

    Vaas, Lea A I; Marheine, Maja; Seufert, Stephanie; Schumacher, Heinz Martin; Kiesecker, Heiko; Heine-Dobbernack, Elke

    2012-06-01

    Although many genes are supposed to be a part of plant cell tolerance mechanisms against osmotic or salt stress, their influence on tolerance towards stress during cryopreservation procedures has rarely been investigated. For instance, the overexpression of the pathogenesis-related gene 10a (pr-10a) leads to improved osmotic tolerance in a transgenic cell culture of Solanum tuberosum cv. Désirée. In this study, a cryopreservation method, consisting of osmotic pretreatment, cryoprotection with DMSO and controlled-rate freezing, was used to characterize the relation between cryopreservation success and pr-10a expression in suspension cultures of S. tuberosum wild-type cells and cells overexpressing pathogenesis-related protein 10a (Pr-10a). By varying the sorbitol concentration, thus modifying the strength of the osmotic stress during the pretreatment phase, it can be shown that the wild type can successfully be cryopreserved only in a relatively narrow range of sorbitol concentrations, while the pr-10a overexpression leads to an enhanced cryopreservation success over the whole range of applied sorbitol concentrations. Together with transcription data we show that the pr-10a overexpression causes an enhanced osmotic tolerance, which in turn leads to enhanced cryopreservability, but also indicates a role of pr-10a in signal transduction. An increased cryopreservability of the transgenic cell line occurs for pretreatments longer than 24 h. Since both genotypes, characterized by distinct baseline levels of expression, exhibited similar patterns of expression induction, the induction of pr-10a appears to be a key step in the stress signal transduction of plant cells under osmotic stress.

  15. Vldlr overexpression causes hyperactivity in rats

    PubMed Central

    2012-01-01

    Background Reelin regulates neuronal positioning in cortical brain structures and neuronal migration via binding to the lipoprotein receptors Vldlr and Lrp8. Reeler mutant mice display severe brain morphological defects and behavioral abnormalities. Several reports have implicated reelin signaling in the etiology of neurodevelopmental and psychiatric disorders, including autism, schizophrenia, bipolar disorder, and depression. Moreover, it has been reported that VLDLR mRNA levels are increased in the post-mortem brain of autistic patients. Methods We generated transgenic (Tg) rats overexpressing Vldlr, and examined their histological and behavioral features. Results Spontaneous locomotor activity was significantly increased in Tg rats, without detectable changes in brain histology. Additionally, Tg rats tended to show performance deficits in the radial maze task, suggesting that their spatial working memory was slightly impaired. Thus, Vldlr levels may be involved in determining locomotor activity and memory function. Conclusions Unlike reeler mice, patients with neurodevelopmental or psychiatric disorders do not show striking neuroanatomical aberrations. Therefore, it is notable, from a clinical point of view, that we observed behavioral phenotypes in Vldlr-Tg rats in the absence of neuroanatomical abnormalities. PMID:23110844

  16. Vldlr overexpression causes hyperactivity in rats.

    PubMed

    Iwata, Keiko; Izumo, Nobuo; Matsuzaki, Hideo; Manabe, Takayuki; Ishibashi, Yukiko; Ichitani, Yukio; Yamada, Kazuo; Thanseem, Ismail; Anitha, Ayyappan; Vasu, Mahesh Mundalil; Shimmura, Chie; Wakuda, Tomoyasu; Kameno, Yosuke; Takahashi, Taro; Iwata, Yasuhide; Suzuki, Katsuaki; Nakamura, Kazuhiko; Mori, Norio

    2012-10-30

    Reelin regulates neuronal positioning in cortical brain structures and neuronal migration via binding to the lipoprotein receptors Vldlr and Lrp8. Reeler mutant mice display severe brain morphological defects and behavioral abnormalities. Several reports have implicated reelin signaling in the etiology of neurodevelopmental and psychiatric disorders, including autism, schizophrenia, bipolar disorder, and depression. Moreover, it has been reported that VLDLR mRNA levels are increased in the post-mortem brain of autistic patients. We generated transgenic (Tg) rats overexpressing Vldlr, and examined their histological and behavioral features. Spontaneous locomotor activity was significantly increased in Tg rats, without detectable changes in brain histology. Additionally, Tg rats tended to show performance deficits in the radial maze task, suggesting that their spatial working memory was slightly impaired. Thus, Vldlr levels may be involved in determining locomotor activity and memory function. Unlike reeler mice, patients with neurodevelopmental or psychiatric disorders do not show striking neuroanatomical aberrations. Therefore, it is notable, from a clinical point of view, that we observed behavioral phenotypes in Vldlr-Tg rats in the absence of neuroanatomical abnormalities.

  17. Effect of overexpression of PPARgamma on the healing process of corneal alkali burn in mice.

    PubMed

    Saika, Shizuya; Yamanaka, Osamu; Okada, Yuka; Miyamoto, Takeshi; Kitano, Ai; Flanders, Kathleen C; Ohnishi, Yoshitaka; Nakajima, Yuji; Kao, Winston W-Y; Ikeda, Kazuo

    2007-07-01

    Wound healing involves both local cells and inflammatory cells. Alkali burn of ocular surface tissue is a serious clinical problem often leading to permanent visual impairment resulting from ulceration, scarring and neovascularization during healing. Behaviors of corneal cells and inflammatory cells are orchestrated by growth factor signaling networks that have not been fully uncovered. Here we showed that adenoviral gene introduction of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits activation of ocular fibroblasts and macrophages in vitro and also induced anti-inflammatory and anti-fibrogenic responses in an alkali-burned mouse cornea. PPARgamma overexpression suppressed upregulation of inflammation/scarring-related growth factors and matrix metalloproteinases (MMPs) in macrophages. It also suppressed expression of such growth factors and collagen Ialpha2 and myofibroblast generation upon exposure to TGFbeta1. Exogenous PPARgamma did not alter phosphorylation of Smad2, but inhibited its nuclear translocation. PPARgamma overexpression enhanced proliferation of corneal epithelial cells, but not of fibroblasts in vitro. Epithelial cell expression of MMP-2/-9 and TGFbeta1 and its migration were suppressed by PPARgamma overexpression. In vivo experiments showed that PPARgamma gene introduction suppressed monocytes/macrophages invasion and suppressed the generation of myofibroblasts, as well as upregulation of cytokines/growth factors and MMPs in a healing cornea. In vivo re-epitheliazation with basement membrane reconstruction in the healing, burned, cornea was accelerated by PPARgamma-Ad expression, although PPARgamma overexpression was considered to be unfavorable for cell migration. Together, these data suggest that overexpression of PPARgamma may represent an effective new strategy for treatment of ocular surface burns.

  18. Heterologous Overexpression of Poplar SnRK2 Genes Enhanced Salt Stress Tolerance in Arabidopsis thaliana

    PubMed Central

    Song, Xueqing; Yu, Xiang; Hori, Chiaki; Demura, Taku; Ohtani, Misato; Zhuge, Qiang

    2016-01-01

    Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and wheat SnRK2.8 overexpressors were not changed by PtSnRK2.7 overexpression. Furthermore, PtSnRK2.7 overexpression widely and largely influenced the transcriptome in response to salt stress; genes related to transport activity, including anion transport-related genes, were characteristically upregulated, and a variety of metabolic genes were specifically downregulated. We also found that the salt stress response genes were greatly upregulated in the PtSnRK2.7 overexpressor. Taken together, poplar subclass 2 PtSnRK2 genes can modulate salt stress tolerance in Arabidopsis, through the activation of cellular signaling pathways in a different manner from that by herbal subclass 2 SnRK2 genes. PMID:27242819

  19. Overexpression of Glycolate Oxidase Confers Improved Photosynthesis under High Light and High Temperature in Rice.

    PubMed

    Cui, Li-Li; Lu, Yu-Sheng; Li, Yong; Yang, Chengwei; Peng, Xin-Xiang

    2016-01-01

    While glycolate oxidase (GLO) is well known as a key enzyme for the photorespiratory metabolism in plants, its physiological function and mechanism remains to be further clarified. Our previous studies have shown that suppression of GLO in rice leads to stunted growth and inhibited photosynthesis (Pn) which is positively and linearly correlated with decreased GLO activities. It is, therefore, of interest to further understand whether Pn can be improved when GLO is up-regulated? In this study, four independent overexpression rice lines, with gradient increases in GLO activity, were generated and functionally analyzed. Phenotypic observations showed that the growth could be improved when GLO activities were increased by 60 or 100%, whereas reduced growth was noticed when the activity was further increased by 150 or 210%. As compared with WT plants, all the overexpression plants exhibited significantly improved Pn under conditions of high light and high temperature, but not under normal conditions. In addition, the overexpression plants were more resistant to the MV-induced photooxidative stress. It was further demonstrated that the antioxidant enzymes, and the antioxidant metabolite glutathione was not significantly altered in the overexpression plants. In contrast, H2O2 and salicylic acid (SA) were correspondingly induced upon the GLO overexpression. Taken together, the results suggest that GLO may play an important role for plants to cope with high light and high temperature, and that H2O2 and SA may serve as signaling molecules to trigger stress defense responses but antioxidant reactions appear not to be involved in the defense.

  20. Overexpression of Glycolate Oxidase Confers Improved Photosynthesis under High Light and High Temperature in Rice

    PubMed Central

    Cui, Li-Li; Lu, Yu-sheng; Li, Yong; Yang, Chengwei; Peng, Xin-Xiang

    2016-01-01

    While glycolate oxidase (GLO) is well known as a key enzyme for the photorespiratory metabolism in plants, its physiological function and mechanism remains to be further clarified. Our previous studies have shown that suppression of GLO in rice leads to stunted growth and inhibited photosynthesis (Pn) which is positively and linearly correlated with decreased GLO activities. It is, therefore, of interest to further understand whether Pn can be improved when GLO is up-regulated? In this study, four independent overexpression rice lines, with gradient increases in GLO activity, were generated and functionally analyzed. Phenotypic observations showed that the growth could be improved when GLO activities were increased by 60 or 100%, whereas reduced growth was noticed when the activity was further increased by 150 or 210%. As compared with WT plants, all the overexpression plants exhibited significantly improved Pn under conditions of high light and high temperature, but not under normal conditions. In addition, the overexpression plants were more resistant to the MV-induced photooxidative stress. It was further demonstrated that the antioxidant enzymes, and the antioxidant metabolite glutathione was not significantly altered in the overexpression plants. In contrast, H2O2 and salicylic acid (SA) were correspondingly induced upon the GLO overexpression. Taken together, the results suggest that GLO may play an important role for plants to cope with high light and high temperature, and that H2O2 and SA may serve as signaling molecules to trigger stress defense responses but antioxidant reactions appear not to be involved in the defense. PMID:27540387

  1. Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome

    PubMed Central

    Livolsi, Angelo; Niederhoffer, Nathalie; Dali-Youcef, Nassim; Rambaud, Caroline; Olexa, Catherine; Mokni, Walid; Gies, Jean-Pierre; Bousquet, Pascal

    2010-01-01

    Background Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. Methodology/Principal Findings Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [3H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled Bmax value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). Conclusions In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism. PMID:20209124

  2. Overexpression of calmodulin in pancreatic beta cells induces diabetic nephropathy.

    PubMed

    Yuzawa, Yukio; Niki, Ichiro; Kosugi, Tomoki; Maruyama, Shoichi; Yoshida, Futoshi; Takeda, Motohiro; Tagawa, Yoshiaki; Kaneko, Yukiko; Kimura, Toshihide; Kato, Noritoshi; Yamamoto, Jyunichiro; Sato, Waichi; Nakagawa, Takahiko; Matsuo, Seiichi

    2008-09-01

    Recently, endothelial dysfunction induced by an uncoupling of vascular endothelial growth factor (VEGF) and nitric oxide has been implicated in the pathogenesis of diabetic nephropathy (DN). Investigating the pathogenesis of DN has been limited, however, because of the lack of animal models that mimic the human disease. In this report, pancreatic beta cell-specific calmodulin-overexpressing transgenic (CaMTg) mice, a potential new model of DN, are characterized with particular emphasis on VEGF and related molecules. CaMTg mice developed hyperglycemia at 3 wk and persistent proteinuria by 3 mo. Morphometric analysis showed considerable increases in the glomerular and mesangial areas with deposition of type IV collagen. Moreover, the pathologic hallmarks of human DN (mesangiolysis, Kimmelstiel-Wilson-like nodular lesions, exudative lesions, and hyalinosis of afferent and efferent arteries with neovascularization) were observed. In addition, increased VEGF expression was associated with an increased number of peritubular capillaries. Expression of endothelial nitric oxidase synthase was reduced and that of VEGF was markedly elevated in CaMTg mice kidney compared with nontransgenic mice. No differences in VEGF receptor-1 or VEGF receptor-2 expression were observed between CaMTg mice and nontransgenic kidneys. In summary, CaMTg mice develop most of the distinguishing lesions of human DN, and the elevated VEGF expression in the setting of diminished endothelial nitric oxide synthase expression may lead to endothelial proliferation and dysfunction. This model may prove useful in the study of the pathogenesis and treatment of DN.

  3. Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

    PubMed

    Pourahmad Jaktaji, Razieh; Pasand, Shirin

    2016-01-15

    Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Ovarian cancer stem cell like side populations are enriched following chemotherapy and overexpress EZH2

    PubMed Central

    Rizzo, Siân; Hersey, Jenny M.; Mellor, Paul; Dai, Wei; Santos-Silva, Alessandra; Liber, Daniel; Luk, Louisa; Titley, Ian; Carden, Craig P; Box, Garry; Hudson, David L.; Kaye, Stanley B.; Brown, Robert

    2010-01-01

    Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer, however acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumour cells with stem-cell like properties, such as so-called side populations (SP) which over-express ABC drug-transporters, can sustain the growth of drug resistant tumour cells, leading to tumour recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the Polycomb Repressive Complex 2 (PRC2) required for maintenance of a stem cell state and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observe higher percentage SP in ascites from patients that have relapsed following chemotherapy compared to chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently over-expressed in SP compared to non-SP from patients’ tumour cells. SiRNA knockdown of EZH2 leads to loss of SP in ovarian tumour models, reduced anchorage-independent growth and reduced tumour growth in vivo. Together these data support a key role for EZH2 in the maintenance of a drug-resistant tumour-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development. PMID:21216927

  5. Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins.

    PubMed

    Cheng, Chung-Hsien; Lee, Wen-Chien

    2010-08-28

    Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R). Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could

  6. Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins

    PubMed Central

    2010-01-01

    Background Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R). Results Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Conclusions Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of

  7. An immunological renal disease in transgenic mice that overexpress Fli-1, a member of the ets family of transcription factor genes

    SciTech Connect

    Zhang, Liqun; Teng, Yen-Tung; Melet, F.

    1995-12-01

    This report describes how expression of the proto-oncogene Fli-1 is involved in the regulation of lymphopoiesis. Transgenic mice were generated which overexpressed the oncogene, leading to a high incidence of immunological renal diseases and death. The data suggests that overexpression of Fli-1 perturbs normal lymphoid cell function and programmed cell death, making these transgenic mice suitable as a biological model for autoimmune disease in humans. 35 refs., 6 figs., 4 tabs.

  8. Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

    SciTech Connect

    Wang, Jiying; Rao, Qing; Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang

    2009-09-04

    Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

  9. Beclin1 overexpression inhibitis proliferation, invasion and migration of CaSki cervical cancer cells.

    PubMed

    Sun, Yang; Liu, Jia-hua; Sui, Yu-xia; Jin, Long; Yang, Yin; Lin, Sai-mei; Shi, Hong

    2011-01-01

    The influence of the autophagy-related gene Beclin1 on proliferation, invasion and metastasis of the cervical cancer CaSki cells and its possible mechanism in vitro were here targeted. After the overexpression vector pcDNA3.1-Beclin1 and RNA interference vector pSUPER-Beclin1 were transfected into CaSki cells in vitro, stable expression cell lines demonstration Beclin1 expression was upregulated, and VEGF and MMP-9 expression were decreased, leading to cell arrest in the G0/G1 phase of the cell cycle. MTT assays further revealed proliferation of cells was significantly inhibited in Beclin1-overexpressing transfectant cells, with invasion and metastasis also being inhibited in Transwell chamber assays. The present results suggest that Beclin1 inhibits invasion and metastasis of cervical cancer CaSki cells in vitro. Mechanisms probably involve Beclin1 inhibition of cell proliferation, and decreased expression of VEGF and MMP-9 proteins.

  10. Ras1(CA) overexpression in the posterior silk gland improves silk yield.

    PubMed

    Ma, Li; Xu, Hanfu; Zhu, Jinqi; Ma, Sanyuan; Liu, Yan; Jiang, Rong-Jing; Xia, Qingyou; Li, Sheng

    2011-06-01

    Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.

  11. Enhanced Energetic State and Protection from Oxidative Stress in Human Myoblasts Overexpressing BMI1.

    PubMed

    Dibenedetto, Silvia; Niklison-Chirou, Maria; Cabrera, Claudia P; Ellis, Matthew; Robson, Lesley G; Knopp, Paul; Tedesco, Francesco Saverio; Ragazzi, Martina; Di Foggia, Valentina; Barnes, Michael R; Radunovic, Aleksandar; Marino, Silvia

    2017-08-08

    The Polycomb group gene BMI1 is essential for efficient muscle regeneration in a mouse model of Duchenne muscular dystrophy, and its enhanced expression in adult skeletal muscle satellite cells ameliorates the muscle strength in this model. Here, we show that the impact of mild BMI1 overexpression observed in mouse models is translatable to human cells. In human myoblasts, BMI1 overexpression increases mitochondrial activity, leading to an enhanced energetic state with increased ATP production and concomitant protection against DNA damage both in vitro and upon xenografting in a severe dystrophic mouse model. These preclinical data in mouse models and human cells provide a strong rationale for the development of pharmacological approaches to target BMI1-mediated mitochondrial regulation and protection from DNA damage to sustain the regenerative potential of the skeletal muscle in conditions of chronic muscle wasting. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

    PubMed Central

    Delic, Marizela; Graf, Alexandra B.; Koellensperger, Gunda; Haberhauer-Troyer, Christina; Hann, Stephan; Mattanovich, Diethard; Gasser, Brigitte

    2014-01-01

    Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG) in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant) proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity. PMID:28357216

  13. Individual Amino Acid Supplementation Can Improve Energy Metabolism and Decrease ROS Production in Neuronal Cells Overexpressing Alpha-Synuclein.

    PubMed

    Delic, Vedad; Griffin, Jeddidiah W D; Zivkovic, Sandra; Zhang, Yumeng; Phan, Tam-Anh; Gong, Henry; Chaput, Dale; Reynes, Christian; Dinh, Vinh B; Cruz, Josean; Cvitkovic, Eni; Placides, Devon; Frederic, Ernide; Mirzaei, Hamed; Stevens, Stanley M; Jinwal, Umesh; Lee, Daniel C; Bradshaw, Patrick C

    2017-06-15

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein accumulation and loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Increased levels of alpha-synuclein have been shown to result in loss of mitochondrial electron transport chain complex I activity leading to increased reactive oxygen species (ROS) production. WT alpha-synuclein was stably overexpressed in human BE(2)-M17 neuroblastoma cells resulting in increased levels of an alpha-synuclein multimer, but no increase in alpha-synuclein monomer levels. Oxygen consumption was decreased by alpha-synuclein overexpression, but ATP levels did not decrease and ROS levels did not increase. Treatment with ferrous sulfate, a ROS generator, resulted in decreased oxygen consumption in both control and alpha-synuclein overexpressing cells. However, this treatment only decreased ATP levels and increased ROS production in the cells overexpressing alpha-synuclein. Similarly, paraquat, another ROS generator, decreased ATP levels in the alpha-synuclein overexpressing cells, but not in the control cells, further demonstrating how alpha-synuclein sensitized the cells to oxidative insult. Proteomic analysis yielded molecular insights into the cellular adaptations to alpha-synuclein overexpression, such as the increased abundance of many mitochondrial proteins. Many amino acids and citric acid cycle intermediates and their ester forms were individually supplemented to the cells with L-serine, L-proline, L-aspartate, or L-glutamine decreasing ROS production in oxidatively stressed alpha-synuclein overexpressing cells, while diethyl oxaloacetate or L-valine supplementation increased ATP levels. These results suggest that dietary supplementation with individual metabolites could yield bioenergetic improvements in PD patients to delay loss of dopaminergic neurons.

  14. Influence of protein histidine phosphatase overexpression and down-regulation on human umbilical-vein endothelial cell viability.

    PubMed

    Seeger, Anna; Rose, Karsten; Ma, Nien Tze; Kremmer, Elisabeth; Klumpp, Susanne; Krieglstein, Josef

    2012-03-01

    PHP (protein histidine phosphatase) is expressed by mammalian tissues, particularly in blood vessel walls. We investigated whether PHP plays a significant role in endothelial cells. By Western blot and immunofluorescence analysis PHP was found in HUVEC (human umbilical-vein endothelial cells). Overexpression of PHP by the use of a plasmid vector, pIRES2-AcGFP1-PHP, induced apoptosis in HUVEC. To exclude the possibility that increased cellular protein alone unspecifically caused cell damage, the inactive H53A mutant of PHP was also overexpressed as a control; it did not lead to apoptosis. Down-regulation of PHP by the RNAi (RNA interference) technique did not affect cell viability. In conclusion, HUVEC are damaged by overexpression, but not down-regulation, of PHP, suggesting a pronounced impact of the enzyme on the cells when its activity is increased.

  15. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy

    PubMed Central

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  16. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  17. Overexpression of vsr in Escherichia coli is mutagenic.

    PubMed

    Doiron, K M; Viau, S; Koutroumanis, M; Cupples, C G

    1996-07-01

    Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.

  18. [Overexpression of FKS1 to improve yeast autolysis-stress].

    PubMed

    Li, Jia; Wang, Jinjing; Li, Qi

    2015-09-01

    With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing.

  19. Lead - nutritional considerations

    MedlinePlus

    Lead poisoning - nutritional considerations; Toxic metal - nutritional considerations ... utensils . Old paint poses the greatest danger for lead poisoning , especially in young children. Tap water from lead ...

  20. Overexpression of c-Myc alters G(1)/S arrest following ionizing radiation.

    PubMed

    Sheen, Joon-Ho; Dickson, Robert B

    2002-03-01

    Study of the mechanism(s) of genomic instability induced by the c-myc proto-oncogene has the potential to shed new light on its well-known oncogenic activity. However, an underlying mechanism(s) for this phenotype is largely unknown. In the present study, we investigated the effects of c-Myc overexpression on the DNA damage-induced G(1)/S checkpoint, in order to obtain mechanistic insights into how deregulated c-Myc destabilizes the cellular genome. The DNA damage-induced checkpoints are among the primary safeguard mechanisms for genomic stability, and alterations of cell cycle checkpoints are known to be crucial for certain types of genomic instability, such as gene amplification. The effects of c-Myc overexpression were studied in human mammary epithelial cells (HMEC) as one approach to understanding the c-Myc-induced genomic instability in the context of mammary tumorigenesis. Initially, flow-cytometric analyses were used with two c-Myc-overexpressing, nontransformed immortal lines (184A1N4 and MCF10A) to determine whether c-Myc overexpression leads to alteration of cell cycle arrest following ionizing radiation (IR). Inappropriate entry into S phase was then confirmed with a bromodeoxyuridine incorporation assay measuring de novo DNA synthesis following IR. Direct involvement of c-Myc overexpression in alteration of the G(1)/S checkpoint was then confirmed by utilizing the MycER construct, a regulatable c-Myc. A transient excess of c-Myc activity, provided by the activated MycER, was similarly able to induce the inappropriate de novo DNA synthesis following IR. Significantly, the transient expression of full-length c-Myc in normal mortal HMECs also facilitated entry into S phase and the inappropriate de novo DNA synthesis following IR. Furthermore, irradiated, c-Myc-infected, normal HMECs developed a sub-G(1) population and a >4N population of cells. The c-Myc-induced alteration of the G(1)/S checkpoint was also compared to the effects of expression of MycS (N

  1. Decreased proliferation kinetics of mouse myoblasts overexpressing FRG1.

    PubMed

    Chen, Steven C; Frett, Ellie; Marx, Joseph; Bosnakovski, Darko; Reed, Xylena; Kyba, Michael; Kennedy, Brian K

    2011-01-01

    Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding increased doubling time and G1-phase cells in mass culture after induction of FRG1 and decreased levels of pRb phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice overexpressing FRG1 and may play a part in FSHD.

  2. N-Myc overexpression increases cisplatin resistance in neuroblastoma via deregulation of mitochondrial dynamics

    PubMed Central

    Casinelli, Gabriella; LaRosa, Jeff; Sharma, Manika; Cherok, Edward; Banerjee, Swati; Branca, Maria; Edmunds, Lia; Wang, Yudong; Sims-Lucas, Sunder; Churley, Luke; Kelly, Samantha; Sun, Ming; Stolz, Donna; Graves, J Anthony

    2016-01-01

    N-Myc is a global transcription factor that regulates the expression of genes involved in a number of essential cellular processes including: ribosome biogenesis, cell cycle and apoptosis. Upon deregulation, N-Myc can drive pathologic expression of many of these genes, which ultimately defines its oncogenic potential. Overexpression of N-Myc has been demonstrated to contribute to tumorigenesis, most notably for the pediatric tumor, neuroblastoma. Herein, we provide evidence that deregulated N-Myc alters the expression of proteins involved in mitochondrial dynamics. We found that N-Myc overexpression leads to increased fusion of the mitochondrial reticulum secondary to changes in protein expression due to aberrant transcriptional and post-translational regulation. We believe the structural changes in the mitochondrial network in response to N-Myc amplification in neuroblastoma contributes to two important aspects of tumor development and maintenance—bioenergetic alterations and apoptotic resistance. Specifically, we found that N-Myc overexpressing cells are resistant to programmed cell death in response to exposure to low doses of cisplatin, and demonstrated that this was dependent on increased mitochondrial fusion. We speculate that these changes in mitochondrial structure and function may contribute significantly to the aggressive clinical ph9enotype of N-Myc amplified neuroblastoma. PMID:28028439

  3. Overexpression of the RieskeFeS Protein Increases Electron Transport Rates and Biomass Yield.

    PubMed

    Simkin, Andrew J; McAusland, Lorna; Lawson, Tracy; Raines, Christine A

    2017-09-01

    In this study, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing the Rieske FeS protein (PetC), a component of the cytochrome b6f (cyt b6f) complex. Increasing the levels of this protein resulted in concomitant increases in the levels of cyt f (PetA) and cyt b6 (PetB), core proteins of the cyt b6f complex. Interestingly, an increase in the levels of proteins in both the photosystem I (PSI) and PSII complexes also was seen in the Rieske FeS overexpression plants. Although the mechanisms leading to these changes remain to be identified, the transgenic plants presented here provide novel tools to explore this. Importantly, overexpression of the Rieske FeS protein resulted in substantial and significant impacts on the quantum efficiency of PSI and PSII, electron transport, biomass, and seed yield in Arabidopsis plants. These results demonstrate the potential for manipulating electron transport processes to increase crop productivity. © 2017 The author(s). All Rights Reserved.

  4. Overexpressed homeobox B9 regulates oncogenic activities by transforming growth factor-β1 in gliomas

    SciTech Connect

    Fang, Liping; Xu, Yinghui; Zou, Lijuan

    2014-03-28

    Highlights: • HOXB9 is overexpressed in gliomas. • HOXB9 over expression had shorter survival time than down expression in gliomas. • HOXB9 stimulated the proliferation, migration and sphere formation of glioma cells. • Activation of TGF-β1 contributed to HOXB9-induced oncogenic activities. - Abstract: Glioma is the leading cause of deaths related to tumors in the central nervous system. The mechanisms of gliomagenesis remain elusive to date. Homeobox B9 (HOXB9) has a crucial function in the regulation of gene expression and cell survival, but its functions in glioma formation and development have yet to be elucidated. This study showed that HOXB9 expression in glioma tissues was significantly higher than that in nontumor tissues. Higher HOXB9 expression was also significantly associated with advanced clinical stage in glioma patients. HOXB9 overexpression stimulated the proliferation, migration, and sphere formation of glioma cells, whereas HOXB9 knockdown elicited an opposite effect. HOXB9 overexpression also increased the tumorigenicity of glioma cells in vivo. Moreover, the activation of transforming growth factor-β1 contributed to HOXB9-induced oncogenic activities. HOXB9 could be used as a predictable biomarker to be detected in different pathological and histological subtypes in glioma for diagnosis or prognosis.

  5. A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.

    PubMed

    Hu, Jian; Qin, Huajun; Gao, Fei Philip; Cross, Timothy A

    2011-11-01

    Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.

  6. Glucose Limitation Alters Glutamine Metabolism in MUC1-Overexpressing Pancreatic Cancer Cells.

    PubMed

    Gebregiworgis, Teklab; Purohit, Vinee; Shukla, Surendra K; Tadros, Saber; Chaika, Nina V; Abrego, Jaime; Mulder, Scott E; Gunda, Venugopal; Singh, Pankaj K; Powers, Robert

    2017-10-06

    Pancreatic cancer cells overexpressing Mucin 1 (MUC1) rely on aerobic glycolysis and, correspondingly, are dependent on glucose for survival. Our NMR metabolomics comparative analysis of control (S2-013.Neo) and MUC1-overexpressing (S2-013.MUC1) cells demonstrates that MUC1 reprograms glutamine metabolism upon glucose limitation. The observed alteration in glutamine metabolism under glucose limitation was accompanied by a relative decrease in the proliferation of MUC1-overexpressing cells compared with steady-state conditions. Moreover, glucose limitation induces G1 phase arrest where S2-013.MUC1 cells fail to enter S phase and synthesize DNA because of a significant disruption in pyrimidine nucleotide biosynthesis. Our metabolomics analysis indicates that glutamine is the major source of oxaloacetate in S2-013.Neo and S2-013.MUC1 cells, where oxaloacetate is converted to aspartate, an important metabolite for pyrimidine nucleotide biosynthesis. However, glucose limitation impedes the flow of glutamine carbons into the pyrimidine nucleotide rings and instead leads to a significant accumulation of glutamine-derived aspartate in S2-013.MUC1 cells.

  7. NDRG1 overexpressing gliomas are characterized by reduced tumor vascularization and resistance to antiangiogenic treatment.

    PubMed

    Broggini, Thomas; Wüstner, Marie; Harms, Christoph; Stange, Lena; Blaes, Jonas; Thomé, Carina; Harms, Ulrike; Mueller, Susanne; Weiler, Markus; Wick, Wolfgang; Vajkoczy, Peter; Czabanka, Marcus

    2016-10-01

    Hypoxia-regulated molecules play an important role in vascular resistance to antiangiogenic treatment. N-myc downstream-regulated-gene 1 (NDRG1) is significantly upregulated during hypoxia in glioma. It was the aim of the present study to analyze the role of NDRG1 on glioma angiogenesis and on antiangiogenic treatment. Orthotopically implanted NDRG1 glioma showed reduced tumor growth and vessel density compared to controls. RT-PCR gene array analysis revealed a 30-fold TNFSF15 increase in NDRG1 tumors. Consequently, the supernatant from NDRG1 transfected U87MG glioma cells resulted in reduced HUVEC proliferation, migration and angiogenic response in tube formation assays in vitro. This effect was provoked by increased TNFSF15 promoter activity in NDRG1 cells. Mutations in NF-κB and AP-1 promoter response elements suppressed TNFSF15 promoter activity. Moreover, U87MG glioma NDRG1 knockdown supernatant contained multiple proangiogenic proteins and increased HUVEC spheroid sprouting. Sunitinib treatment of orhotopically implanted mice reduced tumor volume and vessel density in controls; in NDRG1 overexpressing cells no reduction of tumor volume or vessel density was observed. NDRG1 overexpression leads to reduced tumor growth and angiogenesis in experimental glioma via upregulation of TNFSF15. In NDRG1 overexpressing glioma antiangiogenic treatment does not yield a therapeutic response.

  8. Calpastatin overexpression limits calpain-mediated proteolysis and behavioral deficits following traumatic brain injury

    PubMed Central

    Schoch, Kathleen M.; Evans, Heather N.; Brelsfoard, Jennifer M.; Madathil, Sindhu K.; Takano, Jiro; Saido, Takaomi C.; Saatman, Kathryn E.

    2012-01-01

    Traumatic brain injury (TBI) results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Although the specific inhibitor of calpains, calpastatin, is present within neurons, normal levels of calpastatin are unable to fully prevent the damaging proteolytic activity of calpains after injury. In this study, increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a calcium-calmodulin dependent kinase II α promoter. Naïve hCAST transgenic mice exhibited enhanced neuronal calpastatin expression and significantly reduced protease activity. Acute calpain-mediated spectrin proteolysis in the cortex and hippocampus induced by controlled cortical impact brain injury was significantly attenuated in calpastatin overexpressing mice. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points. Partial hippocampal neuroprotection observed at a moderate injury severity was lost after severe TBI. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. In addition, the reduction in spectrin proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury. PMID:22572592

  9. Metabolic effects of transgenic melanocyte-stimulating hormone overexpression in lean and obese mice.

    PubMed

    Savontaus, Eriika; Breen, Tracy L; Kim, Andrea; Yang, Lucy M; Chua, Streamson C; Wardlaw, Sharon L

    2004-08-01

    The proopiomelanocortin-derived peptide, alpha-MSH, inhibits feeding via melanocortin receptors in the hypothalamus and genetic defects inactivating the melanocortin system have been shown to lead to obesity in experimental animals and humans. To determine whether long-term melanocortinergic activation has significant effects on body weight and composition and insulin sensitivity, transgenic mice overexpressing N-terminal proopiomelanocortin, including alpha- and gamma(3)-MSH, under the control of the cytomegalovirus-promoter were generated. The transgene was expressed in multiple tissues including the hypothalamus, in which both alpha-MSH and gamma(3)-MSH levels were increased approximately 2-fold, compared with wild-type controls. Transgene homozygous mice were also crossed with obese leptin receptor-deficient db(3J) and obese yellow A(y) mice. MSH overexpression led to uniform, dose- dependent darkening of coat color. MSH overexpression reduced weight gain and adiposity and improved glucose tolerance in lean male mice. In female transgenic mice, there was no significant effect on body weight, but there was a significant decrease in insulin levels. Obesity was attenuated in obese db(3J)/db(3J) male and female mice, but there was no improvement in glucose metabolism. In contrast, the MSH transgene improved glucose tolerance in male A(y) mice. These results support the hypothesis that long-term melanocortinergic activation could serve as a potential strategy for anti-obesity and/or antidiabetic therapy.

  10. Wnt-11 overexpression promoting the invasion of cervical cancer cells.

    PubMed

    Wei, Heng; Wang, Ning; Zhang, Yao; Wang, Shizhuo; Pang, Xiaoao; Zhang, Shulan

    2016-09-01

    Wnt-11 is a positive regulator of the Wnt signaling pathway, which plays a crucial role in carcinogenesis. However, Wnt-11 expression in cervical cancer has not been well investigated. The aim of this study was to investigate the role of Wnt-11 in cervical tumor proliferation and invasion. This study examined 24 normal cervical squamous epithelia, 29 cervical intraepithelial neoplasia (CIN), and 78 cervical cancer samples. The expression of Wnt-11 was investigated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction analysis. The expression of the high-risk human papilloma virus (HR-HPV) E6 oncoprotein was also investigated by immunohistochemistry. In addition, the expression of Wnt-11, HR-HPV E6, JNK-1, phosphorylated JNK-1(P-JNK1), and β-catenin was examined by western blot analysis following Wnt-11 knockdown or overexpression in HeLa or SiHa cells, respectively. The promotion of cervical cancer cell proliferation and invasion was investigated using the cell counting kit-8 and Matrigel invasion assay, respectively. Wnt-11 and HR-HPV E6 expression increased in a manner that corresponded with the progression of cervical cancer and was significantly correlated with the International Federation of Gynecology and Obstetrics cancer stage, lymph node metastasis, tumor size, and HPV infection. Wnt-11 protein expression was positively associated with HR-HPV E6 protein expression in all 78 cervical cancer samples (P < 0.001). Furthermore, Wnt-11 was positively associated with P-JNK1 expression and promoted cervical cancer cell proliferation and invasion. These observations suggest that the increased Wnt-11 expression observed in cervical cancer cells may lead to the phosphorylation and activation of JNK-1 and significantly promote tumor cell proliferation and cell migration/invasion through activation of the Wnt/JNK pathway. Consequently, Wnt-11 may serve as a novel target for cervical cancer therapy.

  11. Lead aVR--the neglected lead.

    PubMed

    Chenniappan, M; Sankar, R Uday; Saravanan, K; Karthikeyan

    2013-09-01

    The aVR is often neglected lead. It is an unipolar lead facing the right superior surface. As all the depolarisations are going away from lead aVR, all waves are negative in aVR (P, QRS, T) in normal sinus rhythm. In dextrocardia, (True and technical) the p is upright in aVR. The lead aVR is a very important lead in localisation of Coronary Artery Disease. In the presence of anterior ST elevation, ST elevation in lead aVR and V1 denotes proximal LAD obstruction where ST elevation is more in lead V1, than in aVR. In the presence of anterior ST depression, ST elevation in lead aVR indicates Left Main Coronary Artery (LMCA) Disease where ST elevation is more in aVR than in V1. In wide QRS tachycardia, tall R wave in aVR indicates Ventricular Tachycardia rather than SVT with aberrancy. In the presence of QS complexes in inferior leads, the lead aVR helps to differentiate between inferior wall MI (IWMI) and left anterior fascicular block (LAFB). Initial R in aVR is suggestive of IWMI and terminal R is suggestive of LAFB. In pericarditis, lead aVR is most often the only lead which shows reciprocal ST depression where as in Acute Infarction, usually a group of leads shows reciprocal depression. In the presence of persistent ST elevation in anterior chest leads, the R in aVR is suggestive of left ventricular aneurysm (Goldburger's sign). In acute pulmonary embolism, ST elevation in lead aVR is a bad prognostic sign. In Tricyclic antidepressant toxicity, R in aVR more than 3 mm is an adverse prognostic sign. So in variety of conditions, the aVR is proved to be a valuable lead not only in diagnosis but also in predicting the prognosis.

  12. Lead levels - blood

    MedlinePlus

    Blood lead levels ... is used to screen people at risk for lead poisoning. This may include industrial workers and children ... also used to measure how well treatment for lead poisoning is working. Lead is common in the ...

  13. Lead (Pb) Air Pollution

    MedlinePlus

    ... States Environmental Protection Agency Search Search Lead (Pb) Air Pollution Share Facebook Twitter Google+ Pinterest Contact Us As ... and protect aquatic and terrestrial ecosystems. Lead (Pb) Air Pollution Lead Air Pollution Basics How does lead get ...

  14. Learn about Lead

    MedlinePlus

    ... the Environment . Top of page What are the Health Effects of Lead? Lead can affect almost every organ ... both men and women) Read more on the health effects of lead EPA’s Integrated Science Assessment for Lead ...

  15. Immunohistochemical COX-2 overexpression correlates with HER-2/neu overexpression in invasive breast carcinomas: a pilot study.

    PubMed

    Çiriş, Ibrahim Metin; Bozkurt, Kemal Kürşat; Başpinar, Sirin; Kapucuoğlu, Fatma Nilgün

    2011-03-15

    Cyclooxygenase-2 (COX-2) is a prostaglandin synthase that catalyzes the synthesis of prostaglandin G2 and H2. It has been shown that COX-2 plays an important role in tumorigenesis of different tumor types and it is thought to take part in breast carcinogenesis. In the present study, we aimed to investigate the relationship of immunohistochemical COX-2 expression with clinicopathological parameters, including HER-2/neu overexpression in invasive breast carcinoma (IBC). Our study population comprised 10 normal breasts, 25 ductal carcinomas in situ (DCIS), and 51 invasive breast carcinomas. Immunohistochemical overexpressions of COX-2 and HER-2/neu were investigated in sections of formalin-fixed, paraffin-embedded blocks by 3 observers. In normal breast, DCIS and IBC, the COX-2 overexpression rate was 0%, 84%, and 58.8%, respectively. In IBC, COX-2 overexpression had a significant relationship with HER-2/neu overexpression (p=0.026) and a high histological grade (p=0.026). COX-2 expression in both DCIS (n=25) and IBC (n=51) was significantly higher than in normal breast tissue (p<0.0001). In addition, the COX-2 expression rate was significantly higher in DCIS than in IBC (p=0.042). Our results indicated that COX-2 overexpression correlates with aggressive phenotypic features, such as HER-2/neu overexpression and high histological grade in IBC. Increased expression of COX-2 in both DCIS and IBC in comparison to normal breast could indicate a role in breast carcinogenesis. COX-2 overexpression may provide a clinically useful biomarker for estimating tumor aggressiveness.

  16. Overexpression of Serpinb1 in Chinese hamster ovary cells increases recombinant IgG productivity.

    PubMed

    Lin, Nan; Brooks, Jeanne; Sealover, Natalie; George, Henry J; Kayser, Kevin J

    2015-01-10

    We report the discovery and validation of a novel CHO cell engineering target for improving IgG expression, serpin peptidase inhibitor, clade B, member 1 (Serpinb1). Transcriptomic studies using microarrays revealed that Serpinb1 was up-regulated in cultures with IgG heavy and light chain transcription transiently repressed compared with cultures treated with non-targeting siRNA. As proof of concept, a lentiviral vector was employed to overexpress the Chinese Hamster Serpinb1 in a CHOZN(®) Glutamine Synthetase (-/-) recombinant IgG producing CHO line. The lentiviral stable pool demonstrated 4.2-fold SERPINB1 overexpression compared with the non-transduced control. The peak viable cell density (VCD) and peak IgG volumetric productivity of the lentiviral stable pool increased 1.3 and 2.0 fold, respectively, compared with the non-transduced control. For host cell engineering, a plasmid encoding SERPINB1 was transfected into the CHOZN(®) GS (-/-) host cell line to create several stable pools. Single-cell clones isolated from the pools were characterized for their SERPINB1 expression levels and growth. The clone (SERPINB1_OE_27) with the highest SERPINB1 expression had decreased peak viable cell density and exponential phase growth rate. Selected SERPINB1 OE clones were subsequently evaluated for their IgG expression capabilities using GS selection. Clone SERPINB1_OE_42 with moderate SERPINB1 overexpression demonstrated increased IgG productivity in "bulk" selection. We conclude that manipulating Serpinb1 expression can lead to increased recombinant IgG productivity, but the effect in host cell lines may vary by clone and by overexpression level. This work represents the ongoing effort in applying "-omics" findings to novel CHO host cell line engineering. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Loss of tolerance of anti-dsDNA B cells in mice overexpressing CD19.

    PubMed

    Taylor, Devon K; Ito, Emi; Thorn, Mitchell; Sundar, Krishnan; Tedder, Thomas; Spatz, Linda A

    2006-04-01

    Mice transgenic for the R4A-Cmu heavy chain of an anti-dsDNA antibody, maintain tolerance by anergy and deletion. In C57BL/6 mice overexpressing CD19, a molecule, which lowers the threshold for B cell activation, elevated levels of serum autoantibodies have been observed. In the present study, we wished to determine whether CD19 overexpression could alter the induction of tolerance in R4A-Cmu mice and lead to the secretion of transgenic anti-dsDNA antibodies. We, therefore, bred R4A-Cmu transgenic mice-to-mice transgenic for human CD19 (hCD19) and generated R4A-Cmu mice heterozygous and homozygous for hCD19. We, now report the spontaneous secretion of transgenic IgM anti-dsDNA antibody in the sera of R4A-Cmu mice overexpressing CD19, indicative of a loss of B cell tolerance. We observe that transgenic B cells secreting anti-dsDNA antibody in these mice are T independent and display a marginal zone like phenotype althought they do not reside in the MZ. In addition, they appear to be derived from the conventional B2 subset rather than the B1 subset. Interestingly, a subset of the anti-dsDNA B cells in these mice still display the phenotype and functional characteristics of anergic B cells. These B cells cannot be activated to secrete antibody following BCR crosslinking, however, they are hyper-responsive to activation by innate signaling mechanisms. This suggests that CD19 overexpression may promote anergic B cells to escape tolerance by converging with BCR independent pathways, thereby rendering these B cells hyper-responsive to innate signaling.

  18. Dendrosomal nanocurcumin and p53 overexpression synergistically trigger apoptosis in glioblastoma cells

    PubMed Central

    Keshavarz, Reihaneh; Bakhshinejad, Babak; Babashah, Sadegh; Baghi, Narges; Sadeghizadeh, Majid

    2016-01-01

    Objective(s): Glioblastoma is the most lethal tumor of the central nervous system. Here, we aimed to evaluate the effects of exogenous delivery of p53 and a nanoformulation of curcumin called dendrosomal curcumin (DNC), alone and in combination, on glioblastoma tumor cells. Materials and Methods: MTT assay was exploited to measure the viability of U87-MG cells against DNC treatment. Cells were separately subjected to DNC treatment and transfected with p53-containing vector and then were co-exposed to DNC and p53 overexpression[A GA1][B2]. Annexin-V-FLUOS staining followed by flow cytometry and real-time PCR were applied to examine apoptosis and analyze the expression levels of the genes involved in cell cycle and oncogenesis, respectively. Results: The results of cell viability assay through MTT indicated that DNC inhibits the proliferation of U87-MG cells in a time- and dose-dependent manner. Apoptosis evaluation revealed that p53 overexpression accompanied by DNC treatment can act in a synergistic manner to significantly enhance the number of apoptotic cells (90%) compared with their application alone (15% and 38% for p53 overexpression and DNC, respectively). Also, real-time PCR data showed that the concomitant exposure of cells to both DNC and p53 overexpression leads to an enhanced expression of GADD45 and a reduced expression of NF-κB and c-Myc. Conclusion: The findings of the current study suggest that our combination strategy, which merges two detached gene (p53) and drug (curcumin) delivery systems into an integrated platform, may represent huge potential as a novel and efficient modality for glioblastoma treatment. PMID:28096969

  19. Compensation of the AKT signaling by ERK signaling in transgenic mice hearts overexpressing TRIM72

    SciTech Connect

    Ham, Young-Mi; Mahoney, Sarah Jane

    2013-06-10

    The AKT and ERK signaling pathways are known to be involved in cell hypertrophy, proliferation, survival and differentiation. Although there is evidence for crosstalk between these two signaling pathways in cellulo, there is less evidence for cross talk in vivo. Here, we show that crosstalk between AKT and ERK signaling in the hearts of TRIM72-overexpressing transgenic mice (TRIM72-Tg) with alpha-MHC promoter regulates and maintains their heart size. TRIM72, a heart- and skeletal muscle-specific protein, downregulates AKT-mTOR signaling via IRS-1 degradation and reduces the size of rat cardiomyocytes and the size of postnatal TRIM72-Tg hearts. TRIM72 expression was upregulated by hypertrophic inducers in cardiomyocytes, while IRS-1 was downregulated by IGF-1. TRIM72 specifically regulated IGF-1-dependent AKT-mTOR signaling, resulting in a reduction of the size of cardiomyocytes. Postnatal TRIM72-Tg hearts were smaller than control-treated hearts with inhibition of AKT-mTOR signaling. However, adult TRIM72-Tg hearts were larger than of control despite the suppression of AKT-mTOR signaling. Activation of ERK, PKC-α, and JNK were observed to be elevated in adult TRIM72-Tg, and these signals were mediated by ET-1 via the ET receptors A and B. Altogether, these results suggest that AKT signaling regulates cardiac hypertrophy in physiological conditions, and ERK signaling compensates for the absence of AKT signaling during TRIM72 overexpression, leading to pathological hypertrophy. -- Highlights: • TRIM72 inhibits AKT signaling through ubiquitination of IRS-1 in cardiac cells. • TRIM72 regulates the size of cardiac cells. • TRIM72 regulates size of postnatal TRIM72-overexpressing transgenic mice hearts. • Adult TRIM72-overexpressing transgenic mice hearts showed cardiac dysfunction. • Adult TRIM72 transgenic mice hearts showed higher expression of endothelin receptors.

  20. Lead in petrol. The isotopic lead experiment

    SciTech Connect

    Facchetti, S. )

    1989-10-01

    Many studies were dedicated to the evaluation of the impact of automotive lead on the environment and to the assessment of its absorption in the human population. They can be subdivided into two groups, those based on changes of air and blood lead concentrations and those based on changes of air and blood lead isotopic compositions. According to various authors, 50-66% of the lead added to petrol is mobilized in the atmosphere, while most of the remainder adheres to the walls of the exhaust system from which it is expelled by mechanical and thermal shocks in the forms of easily sedimented particles. The fraction directly emitted by engine exhaust fumes is found in the form of fine particles, which can be transferred a long way from the emitting sources. However important the contribution of petrol lead to the total airborne lead may be, our knowledge does not permit a straightforward calculation of the percentage of petrol lead in total blood lead, which of course can also originate from other sources (e.g., industrial, natural). To evaluate this percentage in 1973, the idea of the Isotopic Lead Experiment (ILE project) was conceived to label, on a regional scale, petrol with a nonradioactive lead of an isotopic composition sufficiently different from that of background lead and sufficiently stable in time. This Account summarizes the main results obtained by the ILE project.

  1. Bone lead, hypertension, and lead nephropathy

    SciTech Connect

    Wedeen, R.P.

    1988-06-01

    There is considerable clinical evidence that excessive lead absorption causes renal failure with hypertension and predisposes individuals to hypertension even in the absence of detectable renal failure. Recent analyses of transiliac bone biopsies indicate that unsuspected elevated bone leads may reflect the cause (or contributing cause) of end-stage renal disease in 5% of the European dialysis population. In these patients, bone lead levels were four times higher than in unexposed cadavers (6 micrograms/g wet weight) and approximated levels found in lead workers (30 micrograms/g). At present, the most reliable index of the body lead burden is the CaNa2 EDTA lead mobilization test. In vivo tibial X-ray-induced X-ray fluorescence (XRF) is a more practical noninvasive technique for assessing bone lead, which should find widespread application as a diagnostic tool and for epidemiologic studies.

  2. Lead Aprons Are a Lead Exposure Hazard.

    PubMed

    Burns, Kevin M; Shoag, Jamie M; Kahlon, Sukhraj S; Parsons, Patrick J; Bijur, Polly E; Taragin, Benjamin H; Markowitz, Morri

    2017-05-01

    To determine whether lead-containing shields have lead dust on the external surface. Institutional review board approval was obtained for this descriptive study of a convenience sample of 172 shields. Each shield was tested for external lead dust via a qualitative rapid on-site test and a laboratory-based quantitative dust wipe analysis, flame atomic absorption spectrometry (FAAS). The χ(2) test was used to test the association with age, type of shield, lead sheet thickness, storage method, and visual and radiographic appearance. Sixty-three percent (95% confidence interval [CI]: 56%-70%) of the shields had detectable surface lead by FAAS and 50% (95% CI: 43%-57%) by the qualitative method. Lead dust by FAAS ranged from undetectable to 998 μg/ft(2). The quantitative detection of lead was significantly associated with the following: (1) visual appearance of the shield (1 = best, 3 = worst): 88% of shields that scored 3 had detectable dust lead; (2) type of shield: a greater proportion of the pediatric patient, full-body, and thyroid shields were positive than vests and skirts; (3) use of a hanger for storage: 27% of shields on a hanger were positive versus 67% not on hangers. Radiographic determination of shield intactness, thickness of interior lead sheets, and age of shield were unrelated to presence of surface dust lead. Sixty-three percent of shields had detectable surface lead that was associated with visual appearance, type of shield, and storage method. Lead-containing shields are a newly identified, potentially widespread source of lead exposure in the health industry. Copyright © 2016 American College of Radiology. Published by Elsevier Inc. All rights reserved.

  3. Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps

    PubMed Central

    Olivares Pacheco, Jorge; Alvarez-Ortega, Carolina; Alcalde Rico, Manuel

    2017-01-01

    ABSTRACT It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H+ accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic “reaccommodation” might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations. PMID:28743808

  4. Targeted Overexpression of Amelotin Disrupts the Microstructure of Dental Enamel

    PubMed Central

    Lacruz, Rodrigo S.; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L.; White, Shane N.; Paine, Michael L.; Ganss, Bernhard

    2012-01-01

    We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms. PMID:22539960

  5. Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

    PubMed

    Lacruz, Rodrigo S; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L; White, Shane N; Paine, Michael L; Ganss, Bernhard

    2012-01-01

    We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

  6. RECK overexpression reduces invasive ability in ameloblastoma cells.

    PubMed

    Liang, Qi-xiang; Liang, Yan-can; Xu, Zhi-ying; Chen, Wei-liang; Xie, Hong-liang; Zhang, Bin

    2014-09-01

    Ameloblastoma is a frequent odontogenic neoplasm characterized by local invasiveness and high risk of recurrence. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a tumor suppressor that inhibits metastasis and angiogenesis. The aim of this study was to investigate effects of RECK overexpression on invasive potential in ameloblastoma cells. Lentiviral vectors containing human RECK gene were created and subsequently stably transfected into immortalized ameloblastoma cell line hTERT(+) -AM. Functional characteristics of hTERT(+) -AM cells with stable RECK overexpression included proliferation, migration, invasion, and regulation of matrix metalloproteinases (MMP)-2, MMP-9 measured by zymography or commercially available assays. The stable and higher expression of RECK mRNA and protein (P < 0.01) was detected in RECK-transfected hTERT(+) -AM cells. RECK overexpression caused a decrease in migration and invasion (P < 0.01) for hTERT(+) -AM cells and a decrease in activity of MMP-2, MMP-9 (P < 0.01). Proliferation was not affected by RECK overexpression (P > 0.05). Overexpression of RECK gene significantly inhibited cell invasive ability of hTERT(+) -AM cells, suggesting RECK may be a new target for ameloblastoma treatment. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Temporal Effects of Catalase Overexpression on Healing Following Myocardial Infarction

    PubMed Central

    Pendergrass, Karl D.; Varghese, Susan T.; Maiellaro-Rafferty, Kathryn; Brown, Milton E.; Taylor, W. Robert; Davis, Michael E.

    2011-01-01

    Background Reactive oxygen species, such as hydrogen peroxide (H2O2), contribute to progression of dysfunction following myocardial infarction (MI). However, chronic overexpression studies do not agree with acute protein delivery studies. The purpose of the present study was to assess the temporal role of cardiomyocyte-derived H2O2 scavenging on cardiac function after infarction using an inducible system. Methods and Results We developed a tamoxifen-inducible, cardiomyocyte-specific catalase overexpressing mouse. Catalase overexpression was induced either 5 days pre or post-MI. Mice exhibited a 3-fold increase in cardiac catalase activity that was associated with a significant decrease in H2O2 levels at both 7 and 21 days. However, cardiac function improved only at the later time point. Pro-inflammatory and fibrotic genes were acutely upregulated after MI, but catalase overexpression abolished the increase, despite no acute change in function. This led to reduced overall scar formation, with lower levels of Collagen 1A and increased contractile Collagen 3A expression at 21 days. Conclusions In contrast to prior studies, there were no acute functional improvements with physiological catalase overexpression prior to MI. Scavenging of H2O2 however, reduced pro-inflammatory cytokines and altered cardiac collagen isoforms, associated with an improvement in cardiac function after 21 days. Our results suggest that sustained H2O2 levels, rather than acute levels immediately following MI, may be critical in directing remodeling and cardiac function at later time points. PMID:20971939

  8. Genetic basis for p53 overexpression in human breast cancer.

    PubMed Central

    Davidoff, A M; Humphrey, P A; Iglehart, J D; Marks, J R

    1991-01-01

    Overexpression of an activated form of the p53 protein may be involved in neoplastic transformation. We found widespread overexpression of p53 by immunohistochemical staining in 11 (22%) of 49 primary invasive human breast cancers. Northern blot analysis showed that this overexpression was not due to an increase in the steady-state level of p53 mRNA. The p53 gene was directly sequenced in 7 of these tumors with elevated levels of the protein and, in each case, a mutation that altered the coding sequence for p53 was found in a highly conserved region of the gene. Whereas 4 of these tumors contained only a mutant p53 allele, the other 3 tumors exhibited coding sequences from both a mutant and a wild-type allele. p53 mutations have previously been correlated with allelic loss of part of chromosome 17p that contains the p53 locus. Examination of all 49 breast tumors revealed a 61% frequency of deletion at or near the p53 locus. However, the presence of allelic deletion did not correlate with overexpression of the protein. Six tumors that were deleted but did not express high levels of the protein were sequenced and all retained a wild-type p53 allele. In this series of human breast cancers, overexpression of the p53 protein, not allelic loss on chromosome 17p, was always associated with mutation of the p53 gene. Images PMID:2052583

  9. PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. Results In the present study we show that overexpression of PGM2 under control of the HXT7'promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. PGM2 overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When PGM2 was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying PGM2 on the integrative plasmid. Conclusion PGM2 overexpression in S. cerevisiae from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity. PMID:20507616

  10. Phytochrome a overexpression inhibits hypocotyl elongation in transgenic Arabidopsis.

    PubMed Central

    Boylan, M T; Quail, P H

    1991-01-01

    To develop a model plant system for efficient functional analysis of mutagenized phytochrome polypeptides, we have overexpressed oat phytochrome A in Arabidopsis thaliana. R1 seedlings from selfed primary transformants segregated for hypocotyl length, when grown in the light, with a ratio of 3 short to 1 of normal length. When homozygous lines were established from these two size classes, accumulation of immunologically detectable oat phytochrome cosegregated with the short-hypocotyl trait. The short-hypocotyl seedlings contained substantially more spectrally active phytochrome than their normal-sized siblings, indicating that the introduced oat protein was photoreversible. The short-hypocotyl phenotype was strictly light-dependent, since no morphological effects of phytochrome overexpression could be seen in etiolated seedlings. Overexpression of only the chromophore-bearing, N-terminal domain of phytochrome A did not induce short hypocotyls in light-grown seedlings, indicating that additional sequence is essential for photoreceptor function. Similarly, overexpression of a full-length sequence mutated at the chromophore attachment site had no effect on phenotype, indicating the absence of any detectable dominant negative effect of the chromophoreless polypeptide on the activity of endogenous Arabidopsis phytochrome. Thus, the readily scorable short-hypocotyl phenotype of Arabidopsis seedlings overexpressing phytochrome A provides a simple visual assay for rapidly monitoring the biological activity of mutagenized phytochrome A polypeptides. Images PMID:11607244

  11. Clinical significance of Her2/neu overexpression in urothelial carcinomas.

    PubMed

    Alexa, Aurora; Baderca, Flavia; Zăhoi, Delia Elena; Lighezan, Rodica; Izvernariu, D; Raica, M

    2010-01-01

    HER2/neu is a defective transmembrane tyrosine kinase receptor, homologue to the epidermal growth factor receptor, showing overexpression in a large variety of tumor cells. There are no studies published so far regarding HER2/neu overexpression and sensitivity of the urothelial tumors of the urinary bladder to anti-HER2/neu therapy. There are a relatively high number of articles in the literature referring to HER2/neu expression in urothelial tumors of the urinary bladder, but only two of them had investigated HER2/neu expression in patients with urothelial tumors of the upper urinary tract. We have studied HER2/neu overexpression in 59 patients with urothelial carcinomas of the urinary tract by immunohistochemistry. Normal urothelium and the elements of the neighboring renal parenchyma were negative. Out of the 59 cases of urothelial carcinomas, 38 were negative (0 and +1) and 21 were positive: eight were moderately and 13 were intensely positive (+2 and +3). The percentage of positive cases was 35.59%. The negative cases were mostly well-differentiated, G1 tumors, no matter the T-tumor stage. Most of the cases were diagnosed as papillary or, rarely, infiltrative. There is no correlation between HER2/neu overexpression and the tumor stage. The same was true for the lymph node status. The expression intensity, however, was significantly correlated with the differentiation grade. Overexpression was most likely present in tumors with high differentiation grade (p<0.05).

  12. Lead and the Romans

    ERIC Educational Resources Information Center

    Reddy, Aravind; Braun, Charles L.

    2010-01-01

    Lead poisoning has been a problem since early history and continues into modern times. An appealing characteristic of lead is that many lead salts are sweet. In the absence of cane and beet sugars, early Romans used "sugar of lead" (lead acetate) to sweeten desserts, fruits, and sour wine. People most at risk would have been those who…

  13. Lead and the Romans

    ERIC Educational Resources Information Center

    Reddy, Aravind; Braun, Charles L.

    2010-01-01

    Lead poisoning has been a problem since early history and continues into modern times. An appealing characteristic of lead is that many lead salts are sweet. In the absence of cane and beet sugars, early Romans used "sugar of lead" (lead acetate) to sweeten desserts, fruits, and sour wine. People most at risk would have been those who…

  14. Expanded Terminal Fields of Gustatory Nerves Accompany Embryonic BDNF Overexpression in Mouse Oral Epithelia

    PubMed Central

    Sun, Chengsan; Dayal, Arjun

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) is expressed in gustatory epithelia and is required for gustatory neurons to locate and innervate their correct target during development. When BDNF is overexpressed throughout the lingual epithelium, beginning embryonically, chorda tympani fibers are misdirected and innervate inappropriate targets, leading to a loss of taste buds. The remaining taste buds are hyperinnervated, demonstrating a disruption of nerve/target matching in the tongue. We tested the hypothesis here that overexpression of BDNF peripherally leads to a disrupted terminal field organization of nerves that carry taste information to the brainstem. The chorda tympani, greater superficial petrosal, and glossopharyngeal nerves were labeled in adult wild-type (WT) mice and in adult mice in which BDNF was overexpressed (OE) to examine the volume and density of their central projections in the nucleus of the solitary tract. We found that the terminal fields of the chorda tympani and greater superficial petrosal nerves and overlapping fields that included these nerves in OE mice were at least 80% greater than the respective field volumes in WT mice. The shapes of terminal fields were similar between the two groups; however, the density and spread of labels were greater in OE mice. Unexpectedly, there were also group-related differences in chorda tympani nerve function, with OE mice showing a greater relative taste response to a concentration series of sucrose. Overall, our results show that disruption in peripheral innervation patterns of sensory neurons have significant effects on peripheral nerve function and central organization of their terminal fields. PMID:25568132

  15. Expanded terminal fields of gustatory nerves accompany embryonic BDNF overexpression in mouse oral epithelia.

    PubMed

    Sun, Chengsan; Dayal, Arjun; Hill, David L

    2015-01-07

    Brain-derived neurotrophic factor (BDNF) is expressed in gustatory epithelia and is required for gustatory neurons to locate and innervate their correct target during development. When BDNF is overexpressed throughout the lingual epithelium, beginning embryonically, chorda tympani fibers are misdirected and innervate inappropriate targets, leading to a loss of taste buds. The remaining taste buds are hyperinnervated, demonstrating a disruption of nerve/target matching in the tongue. We tested the hypothesis here that overexpression of BDNF peripherally leads to a disrupted terminal field organization of nerves that carry taste information to the brainstem. The chorda tympani, greater superficial petrosal, and glossopharyngeal nerves were labeled in adult wild-type (WT) mice and in adult mice in which BDNF was overexpressed (OE) to examine the volume and density of their central projections in the nucleus of the solitary tract. We found that the terminal fields of the chorda tympani and greater superficial petrosal nerves and overlapping fields that included these nerves in OE mice were at least 80% greater than the respective field volumes in WT mice. The shapes of terminal fields were similar between the two groups; however, the density and spread of labels were greater in OE mice. Unexpectedly, there were also group-related differences in chorda tympani nerve function, with OE mice showing a greater relative taste response to a concentration series of sucrose. Overall, our results show that disruption in peripheral innervation patterns of sensory neurons have significant effects on peripheral nerve function and central organization of their terminal fields. Copyright © 2015 the authors 0270-6474/15/350409-13$15.00/0.

  16. Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli*

    PubMed Central

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-01-01

    Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters. PMID:20525689

  17. Combinatorial method for overexpression of membrane proteins in Escherichia coli.

    PubMed

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-07-30

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

  18. Biglycan Overexpression on Tooth Enamel Formation in Transgenic Mice

    PubMed Central

    Wen, Xin; Zou, YanMing; Luo, Wen; Goldberg, Michel; Moats, Rex; Conti, Peter S.; Snead, Malcolm L.; Paine, Michael L.

    2008-01-01

    Previously it was shown that the volume of forming enamel of molar teeth in biglycan-null mice was greater than in genetically matched wild-type mice. This phenotypic change appeared to result from an increase in amelogenin expression, implying that biglycan directly influences amelogenin synthesis. To determine whether biglycan over-expression resulted in decreased amelogenin expression, we engineered transgenic mice to over-express biglycan in the enamel organ epithelium. Biglycan over-expression did not significantly affect the amelogenin expression in incisor and molar teeth in 3-day transgenic mice. In the transgenic animals we observed that the immature and mature enamel appeared normal. These results suggested that increasing the biglycan expression, in the cells that synthesize the precursor protein matrix for enamel, has a negligible influence on amelogenesis. PMID:18727043

  19. CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

    PubMed

    Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

    2016-04-01

    CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells.

  20. Overexpression of TFAM protects 3T3-L1 adipocytes from NYGGF4 (PID1) overexpression-induced insulin resistance and mitochondrial dysfunction.

    PubMed

    Shi, Chun-Mei; Xu, Guang-Feng; Yang, Lei; Fu, Zi-Yi; Chen, Ling; Fu, Hai-Long; Shen, Ya-Hui; Zhu, Lu; Ji, Chen-Bo; Guo, Xi-Rong

    2013-07-01

    NYGGF4, also known as phosphotyrosine interaction domain containing 1(PID1), is a recently discovered gene which is involved in obesity-related insulin resistance (IR) and mitochondrial dysfunction. We aimed to further elucidate the effects and mechanisms underlying NYGGF4-induced IR by investigating the effect of overexpressing mitochondrial transcription factor A (TFAM), which is essential for mitochondrial DNA transcription and replication, on NYGGF4-induced IR and mitochondrial abnormalities in 3T3-L1 adipocytes. Overexpression of TFAM increased the mitochondrial copy number and ATP content in both control 3T3-L1 adipocytes and NYGGF4-overexpressing adipocytes. Reactive oxygen species (ROS) production was enhanced in NYGGF4-overexpressing adipocytes and reduced in TFAM-overexpressing adipocytes; co-overexpression of TFAM significantly attenuated ROS production in NYGGF4-overexpressing adipocytes. However, overexpression of TFAM did not affect the mitochondrial transmembrane potential (ΔΨm) in control 3T3-L1 adipocytes or NYGGF4-overexpressing adipocytes. In addition, co-overexpression of TFAM-enhanced insulin-stimulated glucose uptake by increasing Glucose transporter type 4 (GLUT4) translocation to the PM in NYGGF4-overexpressing adipocytes. Overexpression of NYGGF4 significantly inhibited tyrosine phosphorylation of Insulin receptor substrate 1 (IRS-1) and serine phosphorylation of Akt, whereas overexpression of TFAM strongly induced phosphorylation of IRS-1 and Akt in NYGGF4-overexpressing adipocytes. This study demonstrates that NYGGF4 plays a role in IR by impairing mitochondrial function, and that overexpression of TFAM can restore mitochondrial function to normal levels in NYGGF4-overexpressing adipocytes via activation of the IRS-1/PI3K/Akt signaling pathway.

  1. Functional assay using lectin gene targeting technologies (over-expression).

    PubMed

    Nonaka, Motohiro; Kawasaki, Toshisuke

    2014-01-01

    Function of lectin depends on its amino acid sequence of carbohydrate-recognition domain (CRD), conformation, and extracellular/intracellular localization. Altering lectin gene expression by over-expression or knockdown is a powerful tool for analyzing its cellular function. Here, we describe a method of lectin gene over-expression, taking a C-type lectin, mannan-binding protein (MBP), as an example. Carbohydrate-binding ability of MBP, its subcellular localization, and functional co-localization with ligand glycoprotein are assayed comparing with an inactive mutant MBP.

  2. Overexpression of Wild-Type Murine Tau Results in Progressive Tauopathy and Neurodegeneration

    PubMed Central

    Adams, Stephanie J.; Crook, Richard J.P.; DeTure, Michael; Randle, Suzanne J.; Innes, Amy E.; Yu, Xin Z.; Lin, Wen-Lang; Dugger, Brittany N.; McBride, Melinda; Hutton, Mike; Dickson, Dennis W.; McGowan, Eileen

    2009-01-01

    Here, we describe the generation and characterization of a novel tau transgenic mouse model (mTau) that overexpresses wild-type murine tau protein by twofold compared with endogenous levels. Transgenic tau expression was driven by a BAC transgene containing the entire wild-type mouse tau locus, including the endogenous promoter and the regulatory elements associated with the tau gene. The mTau model therefore differs from other tau models in that regulation of the genomic mouse transgene mimics that of the endogenous gene, including normal exon splicing regulation. Biochemical data from the mTau mice demonstrated that modest elevation of mouse tau leads to tau hyperphosphorylation at multiple pathologically relevant epitopes and accumulation of sarkosyl-insoluble tau. The mTau mice show a progressive increase in hyperphosphorylated tau pathology with age up to 15 to 18 months, which is accompanied by gliosis and vacuolization. In contrast, older mice show a decrease in tau pathology levels, which may represent hippocampal neuronal loss occurring in this wild-type model. Collectively, these results describe a novel model of tauopathy that develops pathological changes reminiscent of early stage Alzheimer’s disease and other related neurodegenerative diseases, achieved without overexpression of a mutant human tau transgene. This model will provide an important tool for understanding the early events leading to the development of tau pathology and a model for analysis of potential therapeutic targets for sporadic tauopathies. PMID:19717642

  3. Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    SciTech Connect

    Schild, David; Wiese, Claudia

    2009-10-15

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

  4. Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    PubMed Central

    Schild, David; Wiese, Claudia

    2010-01-01

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the ‘recombination mediators’. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as ‘recombination co-mediators’. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression. PMID:19942681

  5. Accelerated Telomere Shortening and Replicative Senescence in Human Fibroblasts Overexpressing Mutant and Wild Type Lamin A

    PubMed Central

    Huang, Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectible WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes. PMID:17870066

  6. Mannose 6-Phosphate Receptor Is Reduced in -Synuclein Overexpressing Models of Parkinsons Disease

    PubMed Central

    Matrone, Carmela; Dzamko, Nicolas; Madsen, Peder; Nyegaard, Mette; Pohlmann, Regina; Søndergaard, Rikke V.; Lassen, Louise B.; Andresen, Thomas L.; Halliday, Glenda M.; Jensen, Poul Henning

    2016-01-01

    Increasing evidence points to defects in autophagy as a common denominator in most neurodegenerative conditions. Progressive functional decline in the autophagy-lysosomal pathway (ALP) occurs with age, and the consequent impairment in protein processing capacity has been associated with a higher risk of neurodegeneration. Defects in cathepsin D (CD) processing and α-synuclein degradation causing its accumulation in lysosomes are particularly relevant for the development of Parkinson's disease (PD). However, the mechanism by which alterations in CD maturation and α-synuclein degradation leads to autophagy defects in PD neurons is still uncertain. Here we demonstrate that MPR300 shuttling between endosomes and the trans Golgi network is altered in α-synuclein overexpressing neurons. Consequently, CD is not correctly trafficked to lysosomes and cannot be processed to generate its mature active form, leading to a reduced CD-mediated α-synuclein degradation and α-synuclein accumulation in neurons. MPR300 is downregulated in brain from α-synuclein overexpressing animal models and in PD patients with early diagnosis. These data indicate MPR300 as crucial player in the autophagy-lysosomal dysfunctions reported in PD and pinpoint MRP300 as a potential biomarker for PD. PMID:27509067

  7. Striatal overexpression of ΔFosB reproduces chronic levodopa-induced involuntary movements

    PubMed Central

    Cao, Xuebing; Yasuda, Toru; Uthayathas, Subramaniam; Watts, Ray L.; Mouradian, M. Maral; Mochizuki, Hideki; Papa, Stella M.

    2010-01-01

    Long-term dopamine replacement therapy in Parkinson’s disease leads to the development of disabling involuntary movements named dyskinesias that are related to adaptive changes in striatal signaling pathways. The chronic transcription factor ΔFosB, which is overexpressed in striatal neurons after chronic dopaminergic drug exposure, is suspected to mediate these adaptive changes. Here, we sought to demonstrate the ability of ΔFosB to lead directly to the abnormal motor responses associated with chronic dopaminergic therapy. Using rAAV viral vectors, high levels of ΔFosB expression were induced in the striatum of dopamine-denervated rats naïve of chronic drug administration. Transgenic ΔFosB overexpression reproduced the entire spectrum of altered motor behaviors in response to acute levodopa tests, including different types of abnormal involuntary movements and hypersensitivity of rotational responses that are typically associated with chronic levodopa treatment. JunD, the usual protein partner of ΔFosB binding to AP-1 sites of genes, remained unchanged in rats with high ΔFosB expression induced by viral vectors. These findings demonstrate that the increase of striatal ΔFosB in the evolution of chronically treated Parkinson’s disease may be a trigger for the development of abnormal responsiveness to dopamine and the emergence of involuntary movements. PMID:20505100

  8. Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis

    SciTech Connect

    Ji, Jiayao; Li, Qinggang; Xie, Yuansheng; Zhang, Xueguang; Cui, Shaoyuan; Shi, Suozhu; Chen, Xiangmei

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Overexpression of Robo2 caused reduced UB branching and glomerular number. Black-Right-Pointing-Pointer Fewer MM cells surrounding the UB after overexpression of Robo2 in vitro. Black-Right-Pointing-Pointer No abnormal Epithelial Morphology of UB or apoptosis of mm cells in the kidney. Black-Right-Pointing-Pointer Overexpression of Robo2 affected MM cells migration and caused UB deficit. Black-Right-Pointing-Pointer The reduced glomerular number can also be caused by fewer MM cells. -- Abstract: Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that

  9. Perspectives on lead toxicity.

    PubMed

    Lockitch, G

    1993-10-01

    Lead toxicity causes hematological, gastrointestinal, and neurological dysfunction in adults and children. Symptoms are usually noted with blood lead greater than 1.93 mumol/L. Severe or prolonged exposure may also cause chronic nephropathy, hypertension, and reproductive impairment. Lead inhibits enzymes; alters cellular calcium metabolism; stimulates synthesis of binding proteins in kidney, brain, and bone; and slows nerve conduction. Less severe exposure to lead, designated by blood lead levels of 0.48-0.96 mumol/L, has been implicated in poor pregnancy outcome, impaired neurobehavioral development, reduced stature in young children, and higher blood pressure in adults. Biochemical and systemic effects of high and low level lead toxicity are described. Dust, water, and paint chips are still major sources of lead but lead from folk remedies, cosmetics, food supplements, food preparation utensils, and improperly prepared infant formula has caused epidemic and sporadic severe lead toxicity. Screening for pediatric low level lead exposure requires measurement of blood lead.

  10. Transplantation of PDGF-AA-Overexpressing Oligodendrocyte Precursor Cells Promotes Recovery in Rat Following Spinal Cord Injury.

    PubMed

    Yao, Zong-Feng; Wang, Ying; Lin, Yu-Hong; Wu, Yan; Zhu, An-You; Wang, Rui; Shen, Lin; Xi, Jin; Qi, Qi; Jiang, Zhi-Quan; Lü, He-Zuo; Hu, Jian-Guo

    2017-01-01

    Our previous study showed that Schwann cells (SCs) promote survival, proliferation and migration of co-transplanted oligodendrocyte progenitor cells (OPCs) and neurological recovery in rats with spinal cord injury (SCI). A subsequent in vitro study confirmed that SCs modulated OPC proliferation and migration by secreting platelet-derived growth factor (PDGF)-AA and fibroblast growth factor-2 (FGF)-2. We also found that PDGF-AA stimulated OPC proliferation and their differentiation into oligodendrocytes (OLs) at later stages. We therefore speculated that PDGF-AA administration can exert the same effect as SC co-transplantation in SCI repair. To test this hypothesis, in this study we investigated the effect of transplanting PDGF-AA-overexpressing OPCs in a rat model of SCI. We found that PDGF-AA overexpression in OPCs promoted their survival, proliferation, and migration and differentiation into OLs in vivo. OPCs overexpressing PDGF-AA were also associated with increased myelination and tissue repair after SCI, leading to the recovery of neurological function. These results indicate that PDGF-AA-overexpressing OPCs may be an effective treatment for SCI.

  11. Aquaporin 1 and aquaporin 4 overexpression in bovine spongiform encephalopathy in a transgenic murine model and in cattle field cases.

    PubMed

    Costa, Carme; Tortosa, Raül; Rodríguez, Agustín; Ferrer, Isidre; Torres, Juan Maria; Bassols, Anna; Pumarola, Martí

    2007-10-17

    Aquaporins (AQP) are a family of transmembrane proteins that act as water selective channels. AQP1 and AQP4 are widely expressed in the central nervous system where they play several roles. Overexpression of AQP has been reported in some human and animal transmissible spongiform encephalopathies, but information is scanty about their distribution in the central nervous system in bovine spongiform encephalopathy (BSE). Double immunohistochemistry for AQP1, AQP4 and GFAP was developed in a transgenic mouse line overexpressing the bovine cellular prion protein (BoTg110), intracerebrally infected with cattle BSE. Western blot for AQP1 and AQP4, and immunohistochemistry for both AQP and GFAP were carried out in cases of BSE-diagnosed cattle as part of surveillance plan in Catalonia (Spain). A marked increase in AQP1 and AQP4 was observed in mice at the terminal stage of the disease, when they had a wide range of clinical signs, whereas no increase could be observed in the early stage before the onset of the clinical signs. In cattle which did not show evidence of clinical signs, both AQP already showed a great increase. The AQP overexpression correlated with GFAP-immunoreactive astrocytes and PrPres deposition in both cases. The results of this study suggest that AQP overexpression in glial cells could lead to an imbalance in water and ion homeostasis which could contribute to triggering the typical histopathological changes of BSE.

  12. Transplantation of PDGF-AA-Overexpressing Oligodendrocyte Precursor Cells Promotes Recovery in Rat Following Spinal Cord Injury

    PubMed Central

    Yao, Zong-Feng; Wang, Ying; Lin, Yu-Hong; Wu, Yan; Zhu, An-You; Wang, Rui; Shen, Lin; Xi, Jin; Qi, Qi; Jiang, Zhi-Quan; Lü, He-Zuo; Hu, Jian-Guo

    2017-01-01

    Our previous study showed that Schwann cells (SCs) promote survival, proliferation and migration of co-transplanted oligodendrocyte progenitor cells (OPCs) and neurological recovery in rats with spinal cord injury (SCI). A subsequent in vitro study confirmed that SCs modulated OPC proliferation and migration by secreting platelet-derived growth factor (PDGF)-AA and fibroblast growth factor-2 (FGF)-2. We also found that PDGF-AA stimulated OPC proliferation and their differentiation into oligodendrocytes (OLs) at later stages. We therefore speculated that PDGF-AA administration can exert the same effect as SC co-transplantation in SCI repair. To test this hypothesis, in this study we investigated the effect of transplanting PDGF-AA-overexpressing OPCs in a rat model of SCI. We found that PDGF-AA overexpression in OPCs promoted their survival, proliferation, and migration and differentiation into OLs in vivo. OPCs overexpressing PDGF-AA were also associated with increased myelination and tissue repair after SCI, leading to the recovery of neurological function. These results indicate that PDGF-AA-overexpressing OPCs may be an effective treatment for SCI. PMID:28377695

  13. Lead Surveillance Program

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Background on lead exposure is presented including forms of lead, sources, hematologic effects, neurologic effects, endocrine effects, renal effects, and reproductive and developmental effects. The purpose of the Lead Surveillance Program at LeRC is outlined, and the specifics of the Medical Surveillance Program for Lead Exposure at LeRC are discussed.

  14. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer.

    PubMed

    Mansilla, Francisco; da Costa, Kerry-Ann; Wang, Shuli; Kruhøffer, Mogens; Lewin, Tal M; Orntoft, Torben F; Coleman, Rosalind A; Birkenkamp-Demtröder, Karin

    2009-01-01

    The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p < 10(-8)) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [(14)C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p < 10(-5)) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.

  15. Laboratory and field studies of guayule modified to overexpress HMGR

    USDA-ARS?s Scientific Manuscript database

    We report the genetic modification of guayule to overexpress the isoprenoid pathway enzyme HMGR. The rubber content of two-month old in vitro transformed plantlets showed a 65% increase in rubber over the control for one line (HMGR6), and lower resin for another (HMGR2). In field evaluations HMGR6...

  16. Macrophages overexpressing Aire induce CD4+Foxp3+ T cells.

    PubMed

    Sun, Jitong; Fu, Haiying; Wu, Jing; Zhu, Wufei; Li, Yi; Yang, Wei

    2013-01-01

    Aire plays an important role in central immune tolerance by regulating the transcription of thousands of genes. However, the role of Aire in the peripheral immune system is poorly understood. Regulatory T (Treg) cells are considered essential for the maintenance of peripheral tolerance, but the effect of Aire on Treg cells in the peripheral immune system is currently unknown. In this study, we investigated the effects of macrophages overexpressing Aire on CD4+Foxp3+ Treg cells by co-culturing Aire-overexpressing RAW264.7 cells or their supernatant with splenocytes. The results show that macrophages overexpressing Aire enhanced the expression of Foxp3 mRNA and induced different subsets of Treg cells in splenocytes through cell-cell contact or a co-culture supernatants. TGF-β is a key molecule in the increases of CD4+CD45RA+Foxp3hi T cell and activating Treg (aTreg) levels observed following cell‑supernatant co-culturing. Subsets of Treg cells were induced by Aire-overexpressing macrophages, and the manipulation of Treg cells by the targeting of Aire may provide a method for the treatment of inflammatory or autoimmune diseases.

  17. POMC overexpression in the ventral tegmental area ameliorates dietary obesity.

    PubMed

    Andino, Lourdes M; Ryder, Daniel J; Shapiro, Alexandra; Matheny, Michael K; Zhang, Yi; Judge, Melanie K; Cheng, K Y; Tümer, Nihal; Scarpace, Philip J

    2011-08-01

    The activation of proopiomelanocortin (POMC) neurons in different regions of the brain, including the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract curtails feeding and attenuates body weight. In this study, we compared the effects of delivery of a recombinant adeno-associated viral (rAAV) construct encoding POMC to the ARC with delivery to the ventral tegmental area (VTA). F344×Brown Norway rats were high-fat (HF) fed for 14 days after which self-complementary rAAV constructs expressing either green fluorescent protein or the POMC gene were injected using coordinates targeting either the VTA or the ARC. Corresponding increased POMC levels were found at the predicted injection sites and subsequent α-melanocyte-stimulating hormone levels were observed. Food intake and body weight were measured for 4 months. Although caloric intake was unaltered by POMC overexpression, weight gain was tempered with POMC overexpression in either the VTA or the ARC compared with controls. There were parallel decreases in adipose tissue reserves. In addition, levels of oxygen consumption and brown adipose tissue uncoupling protein 1 were significantly elevated with POMC treatment in the VTA. Interestingly, tyrosine hydroxylase levels were increased in both the ARC and VTA with POMC overexpression in either the ARC or the VTA. In conclusion, these data indicate a role for POMC overexpression within the VTA reward center to combat HF-induced obesity.

  18. Control of cellulose biosynthesis by overexpression of a transcription factor

    DOEpatents

    Han, Kyung-Hwan; Ko, Jae-Heung; Kim, Won-Chan; Kim; , Joo-Yeol

    2017-05-16

    The invention relates to the over-expression of a transcription factor selected from the group consisting of MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, and any combination thereof in a plant, which can modulate and thereby modulating the cellulose content of the plant.

  19. Moesin overexpression is a unique biomarker of adenomyosis.

    PubMed

    Ohara, Rena; Michikami, Hiroo; Nakamura, Yuko; Sakata, Akiko; Sakashita, Shingo; Satomi, Kaishi; Shiba-Ishii, Aya; Kano, Junko; Yoshikawa, Hiroyuki; Noguchi, Masayuki

    2014-03-01

    Adenomyosis is characterized by extension of endometrial glands and stromal cells into the myometrium. Here we proved that 'moesin' is a unique biomarker of adenomyosis. We selected two cases of adenomyosis that had been surgically resected and fixed with formalin. Proteins were extracted from the infiltrating adenomyosis lesions and normal endometrium by tissue microdissection. The extracted proteins were examined using a LC-MS/MS system and the expression profiles of each region were compared. Two hundred and sixty proteins were detected, among which 73 were expressed more in adenomyosis than in normal endometrium. Among these proteins, we focused on overexpression of moesin in adenomyosis. Expression of moesin estimated semiquantitatively using an immunohistochemistry score was higher in adenomyosis than in normal endometrium. In particular, moesin was significanly overexpressed in stromal cells of adenomyosis than in those of normal endometrium. Relative to normal endometrium, moesin was also overexpressed at the RNA level in 9 of 14 cases of adenomyosis and at the protein level in all 14 cases. We also detected activated (phosphorylated) moesin in adenomyosis lesions. The present findings suggest that moesin is characteristically overexpressed and activated in adenomyosis, and that moesin activation may be related to extension of adenomyosis in the myometrium.

  20. Tumor Necrosis Factor–α Overexpression in Lung Disease

    PubMed Central

    Lundblad, Lennart K. A.; Thompson-Figueroa, John; Leclair, Timothy; Sullivan, Michael J.; Poynter, Matthew E.; Irvin, Charles G.; Bates, Jason H. T.

    2005-01-01

    Rationale: Tumor necrosis factor α (TNF-α) has been implicated as a key cytokine in many inflammatory lung diseases. These effects are currently unclear, because a transgenic mouse overexpressing TNF-α in the lung has been shown in separate studies to produce elements of both emphysema and pulmonary fibrosis. Objectives: We sought to elucidate the phenotypic effects of TNF-α overexpression in a mouse model. Measurements: We established the phenotype by measuring lung impedance and thoracic gas volume, and using micro–computed tomography and histology. Main Results: We found that airways resistance in this mouse was not different to control mice, but that lung tissue dampening, elastance, and hysteresivity were significantly elevated. Major heterogeneous abnormalities of the parenchyma were also apparent in histologic sections and in micro–computed tomography images of the lung. These changes included airspace enlargement, loss of small airspaces, increased collagen, and thickened pleural septa. We also found significant increases in lung and chest cavity volumes in the TNF-α–overexpressing mice. Conclusions: We conclude that TNF-α overexpression causes pathologic changes consistent with both emphysema and pulmonary fibrosis combined with a general lung inflammation, and consequently does not model any single human disease. Our study thus confirms the pleiotropic effects of TNF-α, which has been implicated in multiple inflammatory disorders, and underscores the necessity of using a wide range of investigative techniques to link gene expression and phenotype in animal models of disease. PMID:15805183

  1. AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation.

    PubMed

    Nazari, Mahmood; Salabi, Fatemeh; Zhang, Li; Zhao, Fuping; Wei, Caihong; Du, Lixin

    2014-10-21

    Follistatin (FST) has been shown to bind to some TGF-β family members and can function as a potent myostatin (MSTN) antagonist. Recent studies have revealed that over-expression of FST by adeno-associated viruses increases muscle growth in mice, humans and nonhuman primates. In the present study, to determine the effect of FST on ovine primary myoblast (OPM) proliferation, FST was over-expressed using an adeno-associated virus serotype 2 (AAV 2) vector. Western blot results showed that AAV induced the expression of FST protein in transduced OPM cells. Real-time quantitative PCR results indicated that over-expression of FST resulted in a dramatic increase in Akt I and CDK2 expression and a decrease in p21 expression. Moreover, cell cycle analysis confirmed that FST down-regulated p21, a CDK inhibitor, and increased the level of CDK2 expression in OPM cells. Hence, follistatin positively regulated the G1 to S progression. Our results showed that FST induced proliferation through a down-regulation of p21, as only the p21 expression level was down-regulated as a result of FST over-expression in myoblasts, whereas no change was observed in the level of p57 expression. These results expanded our understanding of the regulation mechanism of FST in ovine primary myoblasts. Our results provide the first evidence that the AAV viral system can be used for gene transfer in ovine myoblast cells. Moreover, the results showed that an AAV vector can successfully induce the expression of FST in OPM cells in vitro. These findings demonstrated that FST over-expression induces proliferation through a down-regulation of the p21 gene under proliferating conditions.

  2. Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis.

    PubMed

    Lang, Kuan Chih; Lin, I Hsuan; Teng, Han Fang; Huang, Yi Cheng; Li, Chung Leung; Tang, Kam Tsun; Chen, Shen Liang

    2009-07-01

    Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.

  3. Overexpression of Reelin prevents the manifestation of behavioral phenotypes related to schizophrenia and bipolar disorder.

    PubMed

    Teixeira, Cátia M; Martín, Eduardo D; Sahún, Ignasi; Masachs, Nuria; Pujadas, Lluís; Corvelo, André; Bosch, Carles; Rossi, Daniela; Martinez, Albert; Maldonado, Rafael; Dierssen, Mara; Soriano, Eduardo

    2011-11-01

    Despite the impact of schizophrenia and mood disorders, which in extreme cases can lead to death, recent decades have brought little progress in the development of new treatments. Recent studies have shown that Reelin, an extracellular protein that is critical for neuronal development, is reduced in schizophrenia and bipolar disorder patients. However, data on a causal or protective role of Reelin in psychiatric diseases is scarce. In order to study the direct influence of Reelin's levels on behavior, we subjected two mouse lines, in which Reelin levels are either reduced (Reelin heterozygous mice) or increased (Reelin overexpressing mice), to a battery of behavioral tests: open-field, black-white box, novelty-suppressed-feeding, forced-swim-test, chronic corticosterone treatment followed by forced-swim-test, cocaine sensitization and pre-pulse inhibition (PPI) deficits induced by N-methyl-D-aspartate (NMDA) antagonists. These tests were designed to model some aspects of psychiatric disorders such as schizophrenia, mood, and anxiety disorders. We found no differences between Reeler heterozygous mice and their wild-type littermates. However, Reelin overexpression in the mouse forebrain reduced the time spent floating in the forced-swim-test in mice subjected to chronic corticosterone treatment, reduced behavioral sensitization to cocaine, and reduced PPI deficits induced by a NMDA antagonist. In addition, we demonstrate that while stress increased NMDA NR2B-mediated synaptic transmission, known to be implicated in depression, Reelin overexpression significantly reduced it. Together, these results point to the Reelin signaling pathway as a relevant drug target for the treatment of a range of psychiatric disorders.

  4. Field Evaluation of Transgenic Switchgrass Plants Overexpressing PvMYB4 for Reduced Biomass Recalcitrance

    DOE PAGES

    Baxter, Holly L.; Poovaiah, Charleson R.; Yee, Kelsey L.; ...

    2015-01-07

    High biomass yields and minimal agronomic input requirements have made switchgrass, Panicum virgatum L., a leading candidate lignocellulosic bioenergy crop. Large-scale lignocellulosic biofuel production from such crops is limited by the difficulty to deconstruct cell walls into fermentable sugars: the recalcitrance problem. In this study, we assessed the field performance of switchgrass plants overexpressing the switchgrass MYB4 (PvMYB4) transcription factor gene. PvMYB4 transgenic switchgrass can have great lignin reduction, which commensurately increases sugar release and biofuel production. Our results over two growing seasons showed that one transgenic event (out of eight) had important gains in both biofuel (32% more) andmore » biomass (63% more) at the end of the second growing season relative to non-transgenic controls. These gains represent a doubling of biofuel production per hectare, which is the highest gain reported from any field-grown modified feedstock. In contrast to this transgenic event, which had relatively low ectopic overexpression of the transgene, five of the eight transgenic events planted did not survive the first field winter. The dead plants were all high-overexpressing events that performed well in the earlier greenhouse studies. Disease susceptibility was not compromised in any transgenic events over the field experiments. These results demonstrate the power of modifying the expression of an endogenous transcription factor to improve biofuel and biomass simultaneously, and also highlight the importance of field studies for "sorting" transgenic events. In conclusion, further research is needed to develop strategies for fine-tuning temporal-spatial transgene expression in feedstocks to optimize desired phenotypes.« less

  5. Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

    PubMed

    Tsai, Steven C; Chang, David F; Hong, Chang-Mu; Xia, Ping; Senadheera, Dinithi; Trump, Lisa; Mishra, Suparna; Lutzko, Carolyn

    2014-06-15

    Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

  6. Increase in Cellulose Accumulation and Improvement of Saccharification by Overexpression of Arabinofuranosidase in Rice

    PubMed Central

    Nakamura, Hidemitsu; Hakata, Makoto; Ichikawa, Hiroaki; Hirochika, Hirohiko; Ishii, Tadashi; Satoh, Shinobu; Iwai, Hiroaki

    2013-01-01

    Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5)-linked l-Ara (LM6) and β-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol. PMID:24223786

  7. Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced myocardial contractile dysfunction: role of autophagy.

    PubMed

    Turdi, Subat; Han, Xuefeng; Huff, Anna F; Roe, Nathan D; Hu, Nan; Gao, Feng; Ren, Jun

    2012-09-15

    Lipopolysaccharide (LPS) from gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complications in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6 mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis, and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity, and carbonyl formation. A Kaplan-Meier curve was constructed for survival after LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice after LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O(2)(-), and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury after LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by the antioxidant N-acetylcysteine and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy.

  8. Cardiac-Specific Overexpression of Catalase Attenuates Lipopolysaccharide-Induced Myocardial Contractile Dysfunction: Role of Autophagy

    PubMed Central

    Turdi, Subat; Han, Xuefeng; Huff, Anna F.; Roe, Nathan D.; Hu, Nan; Gao, Feng; Ren, Jun

    2012-01-01

    Lipopolysaccharide (LPS) from Gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complication in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6 mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity and carbonyl formation. Kaplan-Meier curve was constructed for survival following LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice following LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O2−, and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury following LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by antioxidant NAC and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy. PMID:22902401

  9. A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor

    PubMed Central

    Fujimoto, Kumiko; Araki, Kiyomi; McCarthy, Deirdre M.; Sims, John R.; Ren, Jia-Qian; Zhang, Xuan; Bhide, Pradeep G.

    2010-01-01

    Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence than the lateral ganglionic eminence or cerebral wall. Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by crossbreeding

  10. Small heterodimer partner overexpression partially protects against liver tumor development in farnesoid X receptor knockout mice

    SciTech Connect

    Li, Guodong; Kong, Bo; Zhu, Yan; Zhan, Le; Williams, Jessica A.; Tawfik, Ossama; Kassel, Karen M.; Luyendyk, James P.; Wang, Li; Guo, Grace L.

    2013-10-15

    Farnesoid X receptor (FXR, Nr1h4) and small heterodimer partner (SHP, Nr0b2) are nuclear receptors that are critical to liver homeostasis. Induction of SHP serves as a major mechanism of FXR in suppressing gene expression. Both FXR{sup −/−} and SHP{sup −/−} mice develop spontaneous hepatocellular carcinoma (HCC). SHP is one of the most strongly induced genes by FXR in the liver and is a tumor suppressor, therefore, we hypothesized that deficiency of SHP contributes to HCC development in the livers of FXR{sup −/−} mice and therefore, increased SHP expression in FXR{sup −/−} mice reduces liver tumorigenesis. To test this hypothesis, we generated FXR{sup −/−} mice with overexpression of SHP in hepatocytes (FXR{sup −/−}/SHP{sup Tg}) and determined the contribution of SHP in HCC development in FXR{sup −/−} mice. Hepatocyte-specific SHP overexpression did not affect liver tumor incidence or size in FXR{sup −/−} mice. However, SHP overexpression led to a lower grade of dysplasia, reduced indicator cell proliferation and increased apoptosis. All tumor-bearing mice had increased serum bile acid levels and IL-6 levels, which was associated with activation of hepatic STAT3. In conclusion, SHP partially protects FXR{sup −/−} mice from HCC formation by reducing tumor malignancy. However, disrupted bile acid homeostasis by FXR deficiency leads to inflammation and injury, which ultimately results in uncontrolled cell proliferation and tumorigenesis in the liver. - Highlights: • SHP does not prevent HCC incidence nor size in FXR KO mice but reduces malignancy. • Increased SHP promotes apoptosis. • Bile acids and inflammation maybe critical for HCC formation with FXR deficiency.

  11. Cardiac overexpression of myotrophin triggers myocardial hypertrophy and heart failure in transgenic mice.

    PubMed

    Sarkar, Sagartirtha; Leaman, Douglas W; Gupta, Sudhiranjan; Sil, Parames; Young, David; Morehead, Annitta; Mukherjee, Debabrata; Ratliff, Norman; Sun, Yaping; Rayborn, Mary; Hollyfield, Joe; Sen, Subha

    2004-05-07

    Cardiac hypertrophy and heart failure remain leading causes of death in the United States. Many studies have suggested that, under stress, myocardium releases factors triggering protein synthesis and stimulating myocyte growth. We identified and cloned myotrophin, a 12-kDa protein from hypertrophied human and rat hearts. Myotrophin (whose gene is localized on human chromosome 7q33) stimulates myocyte growth and participates in cellular interaction that initiates cardiac hypertrophy in vitro. In this report, we present data on the pathophysiological significance of myotrophin in vivo, showing the effects of overexpression of cardio-specific myotrophin in transgenic mice in which cardiac hypertrophy occurred by 4 weeks of age and progressed to heart failure by 9-12 months. This hypertrophy was associated with increased expression of proto-oncogenes, hypertrophy marker genes, growth factors, and cytokines, with symptoms that mimicked those of human cardiomyopathy, functionally and morphologically. This model provided a unique opportunity to analyze gene clusters that are differentially up-regulated during initiation of hypertrophy versus transition of hypertrophy to heart failure. Importantly, changes in gene expression observed during initiation of hypertrophy were significantly different from those seen during its transition to heart failure. Our data show that overexpression of myotrophin results in initiation of cardiac hypertrophy that progresses to heart failure, similar to changes in human heart failure. Knowledge of the changes that take place as a result of overexpression of myotrophin at both the cellular and molecular levels will suggest novel strategies for treatment to prevent hypertrophy and its progression to heart failure.

  12. Transgenic overexpression of mitofilin attenuates diabetes mellitus-associated cardiac and mitochondria dysfunction

    PubMed Central

    Thapa, Dharendra; Nichols, Cody E.; Lewis, Sara E.; Shepherd, Danielle L.; Jagannathan, Rajaganapathi; Croston, Tara L.; Tveter, Kevin J.; Holden, Anthony A.; Baseler, Walter A.; Hollander, John M.

    2014-01-01

    Mitofilin, also known as heart muscle protein, is an inner mitochondrial membrane structural protein that plays a central role in maintaining cristae morphology and structure. It is a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex which is important for mitochondrial architecture and cristae morphology. Our laboratory has previously reported alterations in mitochondrial morphology and proteomic make-up during type 1 diabetes mellitus, with mitofilin being significantly down-regulated in interfibrillar mitochondria (IFM). The goal of this study was to investigate whether overexpression of mitofilin can limit mitochondrial disruption associated with the diabetic heart through restoration of mitochondrial morphology and function. A transgenic mouse line overexpressing mitofilin was generated and mice injected intraperitoneally with streptozotocin using a multi low-dose approach. Five weeks following diabetes mellitus onset, cardiac contractile function was assessed. Restoration of ejection fraction and fractional shortening was observed in mitofilin diabetic mice as compared to wild-type controls (P<0.05 for both). Decrements observed in electron transport chain (ETC) complexes I, III, IV and V activities, state 3 respiration, lipid peroxidation as well as mitochondria membrane potential in type 1 diabetic IFM were restored in mitofilin diabetic mice (P<0.05 for all). Qualitative analyses of electron micrographs revealed restoration of mitochondrial cristae structure in mitofilin diabetic mice as compared to wild-type controls. Furthermore measurement of mitochondrial internal complexity using flow cytometry displayed significant reduction in internal complexity in diabetic IFM which was restored in mitofilin diabetic IFM (P<0.05). Taken together these results suggest that transgenic overexpression of mitofilin preserves mitochondrial structure, leading to restoration of mitochondrial function and attenuation of

  13. Astrocyte-Dependent Vulnerability to Excitotoxicity in Spermine Oxidase-Overexpressing Mouse.

    PubMed

    Cervetto, Chiara; Vergani, Laura; Passalacqua, Mario; Ragazzoni, Milena; Venturini, Arianna; Cecconi, Francesco; Berretta, Nicola; Mercuri, Nicola; D'Amelio, Marcello; Maura, Guido; Mariottini, Paolo; Voci, Adriana; Marcoli, Manuela; Cervelli, Manuela

    2016-03-01

    Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

  14. Centrosome amplification and overexpression of aurora A are early events in rat mammary carcinogenesis.

    PubMed

    Goepfert, Thea M; Adigun, Yetunde E; Zhong, Ling; Gay, Jason; Medina, Daniel; Brinkley, William R

    2002-07-15

    The cells of many solid tumors have been found to contain supernumerary centrosomes, a condition known as centrosome amplification. Centrosome amplification, accompanied by the overexpression of an associated kinase, Aurora A (AurA), has been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Using a well-established rat mammary model favorable for experimental carcinogenesis, we analyzed centrosome amplification as a cellular marker for early stages of transformation and its regulation by the kinase ratAurA. Parity or treatment with estrogen and progesterone conferred resistance to tumorigenesis, as well as to overexpression of ratAurA and to centrosome amplification. ratAurA, cloned from a rat mammary gland cDNA library, is a bona fide Ser/Thr kinase, and sequence comparison demonstrated high homology to members of the entire AurA kinase family. Using immunocytochemical localization with confocal microscopy, we found ratAurA to be localized at the centrosome in normal and neoplastic tissues of the rat mammary gland. Normal ductal epithelium and stromal cells displayed an expected complement of one to two centrosomes/cell, whereas comparable cells in methylnitrosourea-treated animals displayed significantly elevated centrosome numbers. In tumors, 46% of cells showed more than two centrosomes/cell, and ratAurA expression levels coincided with higher centrosome numbers. Both centrosome numbers and ratAurA expression were permanently elevated. Centrosome amplification was found to occur at a very early, premalignant stage prior to detectable lesions after treatment with methylnitrosourea, a condition that was not detected in mammary glands of rats made refractory to the carcinogen via pregnancy or estrogen and progesterone treatment. Our results indicate that hormones influence kinase expression, and progesterone had the major effect on ratAurA expression levels. Cumulatively, these results suggest that rat

  15. Overexpression of maelstrom promotes bladder urothelial carcinoma cell aggressiveness by epigenetically downregulating MTSS1 through DNMT3B.

    PubMed

    Li, X-D; Zhang, J-X; Jiang, L-J; Wang, F-W; Liu, L-L; Liao, Y-J; Jin, X-H; Chen, W-H; Chen, X; Guo, S-J; Zhou, F-J; Zeng, Y-X; Guan, X-Y; Liu, Z-W; Xie, D

    2016-12-08

    We have recently identified and characterized a novel oncogene, maelstrom (MAEL) from 1q24, in the pathogenesis of hepatocellular carcinoma. In this study, MAEL was investigated for its oncogenic role in urothelial carcinoma of the bladder (UCB) tumorigenesis/aggressiveness and underlying molecular mechanisms. Here, we report that overexpression of MAEL in UCB is important in the acquisition of an aggressive and/or poor prognostic phenotype. In UCB cell lines, knockdown of MAEL by short hairpin RNA is sufficient to inhibit cell growth, invasiveness/metastasis and suppressed epithelial-mesenchymal transition (EMT), whereas ectopic overexpression of MAEL promoted cell growth, invasive and/or metastatic capacity and enhanced EMT both in vitro and in vivo. We further demonstrate that MAEL could induce UCB cell EMT by downregulating a critical downstream target, the metastasis suppressor 1 (MTSS1) gene, ultimately leading to an increased invasiveness of cancer cells. Notably, overexpression of MAEL in UCB cells substantially enhanced the enrichment of DNA methyltrans-ferase (DNMT)3B and histone deacetylase (HDAC)1/2 on the promoter of the MTSS1, and thereby epigenetically suppressing the MTSS1 transcription. Downregulation of MTSS1 by MAEL in UCB cells is partially dependent on DNMT3B. Furthermore, we identify that beside the gene amplification of MAEL, miR-186 is a key negative regulator of MAEL and downregulation of miR-186 is another important mechanism for MAEL overexpression in UCBs. These data suggest that overexpression of MAEL, caused by gene amplification and/or decreased miR-186, has a critical oncogenic role in UCB pathogenesis by downregulation of MTSS1, and MAEL could be used as a novel prognostic marker and/or effective therapeutic target for human UCB.

  16. Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.).

    PubMed

    Yue, Erkui; Liu, Zhen; Li, Chao; Li, Yu; Liu, Qiuxiang; Xu, Jian-Hong

    2017-07-01

    Overexpressing miR529a can enhance oxidative stress resistance by targeting OsSPL2 and OsSPL14 genes that can regulate the expression of their downstream SOD and POD related genes. MicroRNAs are involved in the regulation of plant developmental and physiological processes, and their expression can be altered when plants suffered environment stresses, including salt, oxidative, drought and Cadmium. The expression of microRNA529 (miR529) can be induced under oxidative stress. However, its biological function under abiotic stress responses is still unclear. In this study, miR529a was overexpressed to investigate the function of miR529a under oxidative stress in rice. Our results demonstrated that the expression of miR529a can be induced by exogenous H2O2, and overexpressing miR529a can increase plant tolerance to high level of H2O2, resulting in increased seed germination rate, root tip cell viability, reduced leaf rolling rate and chlorophyll retention. The expression of oxidative stress responsive genes and the activities of superoxide dismutase (SOD) and peroxidase (POD) were increased in miR529a overexpression plant, which could help to reduce redundant reactive oxygen species (ROS). Furthermore, only OsSPL2 and OsSPL14 were targeted by miR529a in rice seedlings, repressing their expression in miR529aOE plants could lead to strengthen plant tolerance to oxidation stress. Our study provided the evidence that overexpression of miR529a could strengthen oxidation resistance, and its target genes OsSPL2 and OsSPL14 were responsible for oxidative tolerance, implied the manipulation of miR529a and its target genes regulation on H2O2 related response genes could improve oxidative stress tolerance in rice.

  17. Overexpression of Serum Response Factor in Neurons Restores Ocular Dominance Plasticity in a Model of Fetal Alcohol Spectrum Disorders.

    PubMed

    Foxworthy, W Alex; Medina, Alexandre E

    2015-10-01

    Deficits in neuronal plasticity underlie many neurobehavioral and cognitive problems presented in fetal alcohol spectrum disorder (FASD). Our laboratory has developed a ferret model showing that early alcohol exposure leads to a persistent disruption in ocular dominance plasticity (ODP). For instance, a few days of monocular deprivation results in a robust reduction of visual cortex neurons' responsiveness to stimulation of the deprived eye in normal animals, but not in ferrets with early alcohol exposure. Previously our laboratory demonstrated that overexpression of serum response factor (SRF) exclusively in astrocytes can improve neuronal plasticity in FASD. Here, we test whether neuronal overexpression of SRF can achieve similar effects. Ferrets received 3.5 g/kg alcohol intraperitoneally (25% in saline) or saline as control every other day between postnatal day 10 to 30, which is roughly equivalent to the third trimester of human gestation. Animals were given intracortical injections of a Herpes Simplex Virus-based vector to express either green fluorescent protein or a constitutively active form of SRF in infected neurons. They were then monocularly deprived by eyelid suture for 4 to 5 days after which single-unit recordings were conducted to determine whether changes in ocular dominance had occurred. Overexpression of a constitutively active form of SRF by neurons restored ODP in alcohol-treated animals. This effect was observed only in areas near the site of viral infection. Overexpression of SRF in neurons can restore plasticity in the ferret model of FASD, but only in areas near the site of infection. This contrasts with SRF overexpression in astrocytes which restored plasticity throughout the visual cortex. Copyright © 2015 by the Research Society on Alcoholism.

  18. Transboundary atmospheric lead pollution.

    PubMed

    Erel, Yigal; Axelrod, Tamar; Veron, Alain; Mahrer, Yitzak; Katsafados, Petros; Dayan, Uri

    2002-08-01

    A high-temporal resolution collection technique was applied to refine aerosol sampling in Jerusalem, Israel. Using stable lead isotopes, lead concentrations, synoptic data, and atmospheric modeling, we demonstrate that lead detected in the atmosphere of Jerusalem is not only anthropogenic lead of local origin but also lead emitted in other countries. Fifty-seven percent of the collected samples contained a nontrivial fraction of foreign atmospheric lead and had 206Pb/207Pb values which deviated from the local petrol-lead value (206Pb/207Pb = 1.113) by more than two standard deviations (0.016). Foreign 206Pb/207Pb values were recorded in Jerusalem on several occasions. The synoptic conditions on these dates and reported values of the isotopic composition of lead emitted in various countries around Israel suggest that the foreign lead was transported to Jerusalem from Egypt, Turkey, and East Europe. The average concentration of foreign atmospheric lead in Jerusalem was 23 +/- 17 ng/m3, similar to the average concentration of local atmospheric lead, 21 +/- 18 ng/ m3. Hence, the load of foreign atmospheric lead is similar to the load of local atmospheric lead in Jerusalem.

  19. Lead Content of Foodstuffs

    PubMed Central

    Mitchell, Douglas G.; Aldous, Kenneth M.

    1974-01-01

    The lead content of a number of foodstuffs, particularly baby fruit juices and milk, is reported. Samples were analyzed in quadruplicate by using an automated Delves cup atomic absorption procedure. A large proportion of the products examined contained significant amounts of lead. Of 256 metal can examined, the contents of 62% contained a lead level of 100 μg/l. or more, 37% contained 200 μg/l. or more and 12% contained 400 μg/l. lead or more. Of products in glass and aluminum containers, only 1% had lead levels in excess of 200 μg/l. Lead levels of contents also correlate with the seam length/volume ratio of the leaded seam can. A survey of bulk milk showed a mean lead level of 40 μg/l. for 270 samples; for canned evaporated milk the mean level was 202 μg/l. These data indicate a potential health hazard. PMID:4406645

  20. Lead Poisoning (For Parents)

    MedlinePlus

    ... metal used in everything from construction materials to batteries, can cause serious health problems, particularly in young ... introduce lead dust into the home. water that flows through old lead pipes or faucets, if the ...

  1. Aquatic Life Criteria - Lead

    EPA Pesticide Factsheets

    References and documents pertaining to Acute and Chronic Ambient Aquatic Life Water Quality Criteria for Lead. These documents include the safe levels of Lead in water that should protect the majority of species.

  2. Lead and tap water

    MedlinePlus

    Water contaminated with lead ... The Environmental Protection Agency (EPA) monitors drinking water in the United States. It requires water suppliers to produce annual water quality reports. These reports include information about lead amounts, and they ...

  3. VOLUMETRIC LEAD ASSAY

    SciTech Connect

    M.A. Ebadian, Ph.D.; S.K. Dua; David Roelant; Sachin Kumar

    2001-01-01

    This report describes a system for handling and radioassay of lead, consisting of a robot, a conveyor, and a gamma spectrometer. The report also presents a cost-benefit analysis of options: radioassay and recycling lead vs. disposal as waste.

  4. Developmental Neurotoxicity of Lead.

    PubMed

    Caito, Samuel; Aschner, Michael

    2017-01-01

    Lead exposure is a major concern for the developing nervous system. Environmental exposures to lead, predominantly from contaminated water or lead paint chips, account for the majority of exposures to children. In utero and early life exposures to lead have been associated with lower IQ, antisocial and delinquent behaviors, and attention-deficit hyperactivity disorder. In this review, we will discuss sources of developmental lead exposure and mechanisms of lead neurotoxicity. We will highlight both human epidemiological studies showing associations between lead exposure and behavioral abnormalities as well as experimental data from animal studies. Finally, we will discuss the effects of lead on neurological endpoint past childhood, namely, development of Alzheimer's disease in old age.

  5. PHOTOEMISSION PROPERTIES OF LEAD.

    SciTech Connect

    SMEDLEY,J.; RAO,T.; WARREN,J.; SEKUTOWICZ,J.; LEFFERTS,R.; LIPSKI,A.

    2004-07-05

    In this paper we present a study of the photoemission properties of lead at several UV wavelengths, including a study of the damage threshold of electroplated lead under laser cleaning. A quantum efficiency in excess of 0.1% has been achieved for a laser cleaned, electroplated lead sample with a laser wavelength of 193 nm. Niobium cathodes have been measured for comparison, and lead is found to be a superior photoemitter for all measured wavelengths.

  6. Transplacental transport of lead

    SciTech Connect

    Goyer, R.A. )

    1990-11-01

    Neurotoxicity is the major health effect from exposure to lead for infants and young children, and there is current concern regarding possible toxic effects of lead on the child while in utero. there is no placental-fetal barrier to lead transport. Maternal and fetal blood lead levels are nearly identical, so lead passes through the placenta unencumbered. Lead has been measured in the fetal brain as early as the end of the first trimester (13 weeks). There is a similar rate of increase in brain size and lead content throughout pregnancy in the fetus of mothers in the general population, so concentration of lead probably does not differ greatly during gestation unless exposure of the mother changes. Cell-specific sensitivity to the toxic effects of lead, however, may be greater the younger the fetus. Lead toxicity to the nervous system is characterized by edema or swelling of the brain due to altered permeability of capillary endothelial cells. Experimental studies suggest that immature endothelial cells forming the capillaries of the developing brain are less resistant to the effects of lead, permitting fluid and cations including lead to reach newly formed components of the brain, particularly astrocytes and neurons. Also, the ability of astrocytes and neurons to sequester lead in the form of lead protein complexes occurs only in the later stages of fetal development, permitting lead in maturing brain cells to interact with vital subcellular organelles, particularly mitochondria, which are the major cellular energy source. Intracellular lead also affects binding sites for calcium which, in turn, may affect numerous cell functions including neurotransmitter release.

  7. [Lead content in alginates].

    PubMed

    Castagnola, L; Wirz, J

    1977-03-01

    Alginates containing a high level of lead may lead to health damages in dentists and their personnel. Walter and Söremark have pointed out these hazards. The author's investigations with the Perkin-Elmer absorption-photospectrometer shall show how high the lead content of the 25 brands of alginate sold in this country is. Ca 37, Protex and Algihard S contain sizeable amounts of lead. Recommendations are given towards the protection of dentist and assistant.

  8. Lead poisoning: An overview

    NASA Technical Reports Server (NTRS)

    Gendel, Neil

    1993-01-01

    A problem that should be of great concern to all of us is the lead poisoning of children. First, I would like to present a short overview concerning the reasons everyone should care about lead poisoning, then discuss the history of lead poisoning, what is happening today across the country, and the future.

  9. Lead Poisoning in Schools.

    ERIC Educational Resources Information Center

    Guyaux, Susan

    1990-01-01

    Overexposure to lead can permanently impair a child's mental and physical development. This article discusses sources of lead paint, survey and testing methods, management and abatement plans, drinking water contamination, and associated federal standards. Although lead is present in soil and in art, theater, and vocational programs, no federal…

  10. Lead Poisoning in Childhood.

    ERIC Educational Resources Information Center

    Pueschel, Siegfried M., Ed.; Linakis, James G., Ed.; Anderson, Angela C., Ed.

    The magnitude of childhood lead poisoning has been inexplicably neglected by modern medicine and by legislators. However, since the 1970s, increased attention has been focused on lead poisoning, and advances have been made in several areas, including understanding of the neurodevelopmental and behavioral ramifications of lead poisoning, and…

  11. Lead Poisoning in Children.

    ERIC Educational Resources Information Center

    Drummond, A. H., Jr.

    1981-01-01

    Early symptoms of lead poisoning in children are often overlooked. Lead poisoning has its greatest effects on the brain and nervous system. The obvious long-term solution to the lead poisoning problem is removal of harmful forms of the metal from the environment. (JN)

  12. Lead Poisoning in Children.

    ERIC Educational Resources Information Center

    Boeckx, Roger L.

    1986-01-01

    Urban children are exposed to lead through the air they breathe, the water they drink, and the food and nonfood substances they ingest. The history, diagnosis, and treatment of lead poisoning in these children are discussed. Includes information on the toxicology of lead and the various risk classes. (JN)

  13. Lead Poisoning in Schools.

    ERIC Educational Resources Information Center

    Guyaux, Susan

    1990-01-01

    Overexposure to lead can permanently impair a child's mental and physical development. This article discusses sources of lead paint, survey and testing methods, management and abatement plans, drinking water contamination, and associated federal standards. Although lead is present in soil and in art, theater, and vocational programs, no federal…

  14. Lead and children

    PubMed Central

    Abelsohn, Alan R.; Sanborn, Margaret

    2010-01-01

    Abstract OBJECTIVE To provide family physicians with a practical, evidence-based approach to screening for and preventing children’s exposure to lead. SOURCES OF INFORMATION MEDLINE was searched using terms relevant to lead exposure and poisoning. We reviewed English-language articles published in 2003 to 2008. Most cited studies provide level 2 or 3 evidence. MAIN MESSAGE Lead is a developmental neurotoxin. Children are most commonly exposed and they are most vulnerable. Lead exposure has been associated with many cognitive and motor deficits, as well as distractibility and other characteristics of attention deficit hyperactivity disorder. Although children’s blood lead levels have declined considerably over the past 3 decades with removal of lead from gasoline and paint, children can still be exposed to lead from lead paint in older homes, toys, and other sources. Because post-exposure treatment cannot reverse the cognitive effects of lead exposure, preventing lead exposure is essential. CONCLUSION Family physicians have an important role in screening for children at high risk of lead exposure, and in educating families to prevent the exposure of children to lead. PMID:20547517

  15. Lead Poisoning in Childhood.

    ERIC Educational Resources Information Center

    Pueschel, Siegfried M., Ed.; Linakis, James G., Ed.; Anderson, Angela C., Ed.

    The magnitude of childhood lead poisoning has been inexplicably neglected by modern medicine and by legislators. However, since the 1970s, increased attention has been focused on lead poisoning, and advances have been made in several areas, including understanding of the neurodevelopmental and behavioral ramifications of lead poisoning, and…

  16. Proteomic insight into reduced cell elongation resulting from overexpression of patatin-related phospholipase pPLAIIIδ in Arabidopsis thaliana

    PubMed Central

    Zheng, Yong; Li, Maoyin; Wang, Xuemin

    2014-01-01

    Patatin-containing phospholipase A (pPLA) hydrolyzes membrane glycerolipids, producing free fatty acids and lysoglycerolipids. Ten pPLAs in the Arabidopsis thaliana genome are grouped into 3 subfamilies, and pPLAIIIs differ from pPLAI and IIs in their catalytic motifs and overexpression (OE) of pPLAIIIs reduces cell elongation and cellulose content. To probe the question of how pPLAIII overexpression results in the changes, comparative proteomic analyses were conducted between pPLAIIIδ-OE and WT seedlings. The data indicate a change in the microtubule-associated protein, MAP18. MAP18 is involved in destabilizing cortical microtubules and modulating directional cell growth. The result suggests that pPLAIII and their derived products may regulate cytoskeletal dynamics leading to retardation in anisotropic growth. PMID:24705037

  17. Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

    PubMed

    Yamazaki, Ken; Takamura, Masaaki; Masugi, Yohei; Mori, Taisuke; Du, Wenlin; Hibi, Taizo; Hiraoka, Nobuyoshi; Ohta, Tsutomu; Ohki, Misao; Hirohashi, Setsuo; Sakamoto, Michiie

    2009-04-01

    Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

  18. Muscle-specific Drp1 overexpression impairs skeletal muscle growth via translational attenuation

    PubMed Central

    Touvier, T; De Palma, C; Rigamonti, E; Scagliola, A; Incerti, E; Mazelin, L; Thomas, J-L; D'Antonio, M; Politi, L; Schaeffer, L; Clementi, E; Brunelli, S

    2015-01-01

    Mitochondrial fission and fusion are essential processes in the maintenance of the skeletal muscle function. The contribution of these processes to muscle development has not been properly investigated in vivo because of the early lethality of the models generated so far. To define the role of mitochondrial fission in muscle development and repair, we have generated a transgenic mouse line that overexpresses the fission-inducing protein Drp1 specifically in skeletal muscle. These mice displayed a drastic impairment in postnatal muscle growth, with reorganisation of the mitochondrial network and reduction of mtDNA quantity, without the deficiency of mitochondrial bioenergetics. Importantly we found that Drp1 overexpression activates the stress-induced PKR/eIF2α/Fgf21 pathway thus leading to an attenuated protein synthesis and downregulation of the growth hormone pathway. These results reveal for the first time how mitochondrial network dynamics influence muscle growth and shed light on aspects of muscle physiology relevant in human muscle pathologies. PMID:25719247

  19. Effects of ADH2 overexpression in Saccharomyces bayanus during alcoholic fermentation.

    PubMed

    Maestre, Oscar; García-Martínez, Teresa; Peinado, Rafael A; Mauricio, Juan C

    2008-02-01

    The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and L-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.

  20. Viral-mediated Ntf3 overexpression disrupts innervation and hearing in nondeafened guinea pig cochleae.

    PubMed

    Lee, Min Young; Kurioka, Takaomi; Nelson, Megan M; Prieskorn, Diane M; Swiderski, Donald L; Takada, Yohei; Beyer, Lisa A; Raphael, Yehoash

    2016-01-01

    Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing.

  1. Overexpression of Notch1 ectodomain in myeloid cells induces vascular malformations through a paracrine pathway.

    PubMed

    Li, Xiujie; Calvo, Ezequiel; Cool, Marc; Chrobak, Pavel; Kay, Denis G; Jolicoeur, Paul

    2007-01-01

    We previously reported that truncation of Notch1 (N1) by provirus insertion leads to overexpression of both the intracellular (N1(IC)) and the extracellular (N1(EC)) domains. We produced transgenic (Tg) mice expressing N1(EC) in T cells and in cells of the myeloid lineage under the regulation of the CD4 gene. These CD4C/N1(EC) Tg mice developed vascular disease, predominantly in the liver: superficial distorted vessels, cavernae, lower branching of parenchymal vessels, capillarized sinusoids, and aberrant smooth muscle/endothelial cell topography. The disease developed in lethally irradiated normal mice transplanted with Tg bone marrow or fetal liver cells as well as in Rag-/- Tg mice. In nude mice transplanted with fetal liver cells from (ROSA26 x CD4C/N1(EC)) F1 Tg mice, abnormal vessels were of recipient origin. Transplantation of Tg peritoneal macrophages into normal recipients also induced abnormal vessels. These Tg macrophages showed impaired functions, and their conditioned medium inhibited the proliferation of liver sinusoid endothelial cells in vitro. The Egr-1 gene and some of its targets (Jag1, FIII, FXIII-A, MCP-1, and MCP-5), previously implicated in hemangioma or vascular malformations, were overexpressed in Tg macrophages. These results show that myeloid cells can be reprogrammed by N1(EC) to induce vascular malformations through a paracrine pathway.

  2. Genome-wide overexpression screen for sodium acetate resistance in Saccharomyces cerevisiae.

    PubMed

    Peña, Pedro V; Glasker, Steven; Srienc, Friedrich

    2013-03-10

    The production of biofuels from cellulosic biomass is a promising technology for developing a renewable source of energy. Efforts to produce ethanol from cellulosic biomass using microbes, such as the yeast Saccharomyces cerevisiae, face major challenges, including the need for detoxification. Here, we apply a strategy to discover genetic alterations that lead to improved robustness of S. cerevisiae in the presence of acetate, which is present at toxic concentrations in hemicellulose hydrolysates. Acetate in its protonated form (acetic acid) enters the cell through passive diffusion and dissociates into a proton and acetate, acidifying the cytosol and inhibiting cell function, an effect that is exacerbated in the presence of sodium. Through flow cytometry analysis, implemented as part of a novel cell culture technique, the Cytostat, we characterized the deleterious effects of sodium acetate on growth and on cell size homeostasis. Further, using the Cytostat to screen a genome-wide, gene overexpression library, we identified that overexpressing the ENA2 gene, a P-type sodium pump ATPase, provides a significant growth improvement in the presence of sodium acetate. Together, our data support the proposed mechanism for the synergistic growth inhibition exerted by acetate and sodium, as well as the mechanism that develops resistance. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. The GRK2 Overexpression Is a Primary Hallmark of Mitochondrial Lesions during Early Alzheimer Disease

    PubMed Central

    Obrenovich, Mark E.; Palacios, Hector H.; Gasimov, Eldar; Leszek, Jerzy; Aliev, Gjumrakch

    2009-01-01

    Increasing evidence points to vascular damage as an early contributor to the development of two leading causes of age-associated dementia, namely Alzheimer disease (AD) and AD-like pathology such as stroke. This review focuses on the role of G protein-coupled receptor kinases (GRKs) as they relate to dementia and how the cardio and cerebrovasculature is involved in AD pathogenesis. The exploration of GRKs in AD pathogenesis may help bridge gaps in our understanding of the heart-brain connection in relation to neurovisceral damage and vascular complications of AD. The a priori basis for this inquiry stems from the fact that kinases of this family regulate numerous receptor functions in the brain, myocardium and elsewhere. The aim of this review is to discuss the finding of GRK2 overexpression in the context of early AD pathogenesis. Also, we consider the consequences for this overexpression as a loss of G-protein coupled receptor (GPCR) regulation, as well as suggest a potential role for GPCRs and GRKs in a unifying theory of AD pathogenesis through the cerebrovasculature. Finally, we synthesize this newer information in an attempt to put it into context with GRKs as regulators of cellular function, which makes these proteins potential diagnostic and therapeutic targets for future pharmacological intervention. PMID:20204079

  4. Viral-mediated Ntf3 overexpression disrupts innervation and hearing in nondeafened guinea pig cochleae

    PubMed Central

    Lee, Min Young; Kurioka, Takaomi; Nelson, Megan M; Prieskorn, Diane M; Swiderski, Donald L; Takada, Yohei; Beyer, Lisa A; Raphael, Yehoash

    2016-01-01

    Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing. PMID:27525291

  5. Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury

    PubMed Central

    Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae

    2016-01-01

    Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745

  6. Leucine-rich α2-glycoprotein overexpression in the brain contributes to memory impairment.

    PubMed

    Akiba, Chihiro; Nakajima, Madoka; Miyajima, Masakazu; Ogino, Ikuko; Miura, Masami; Inoue, Ritsuko; Nakamura, Eri; Kanai, Fumio; Tada, Norihiro; Kunichika, Miyuki; Yoshida, Mitsutaka; Nishimura, Kinya; Kondo, Akihide; Sugano, Hidenori; Arai, Hajime

    2017-08-24

    We previously reported increase in leucine-rich α2-glycoprotein (LRG) concentration in cerebrospinal fluid is associated with cognitive decline in humans. To investigate relationship between LRG expression in the brain and memory impairment, we analyzed transgenic mice overexpressing LRG in the brain (LRG-Tg) focusing on hippocampus. Immunostaining and Western blotting revealed age-related increase in LRG expression in hippocampal neurons in 8-, 24-, and 48-week-old controls and LRG-Tg. Y-maze and Morris water maze tests indicated retained spatial memory in 8- and 24-week-old LRG-Tg, while deteriorated in 48-week-old LRG-Tg compared with age-matched controls. Field excitatory postsynaptic potentials declined with age in LRG-Tg compared with controls at 8, 24, and 48 weeks. Paired-pulse ratio decreased with age in LRG-Tg, while increased in controls. As a result, long-term potentiation was retained in 8- and 24-week-old LRG-Tg, whereas diminished in 48-week-old LRG-Tg compared with age-matched controls. Electron microscopy observations revealed fewer synaptic vesicles and junctions in LRG-Tg compared with age-matched controls, which became significant with age. Hippocampal LRG overexpression contributes to synaptic dysfunction, which leads to memory impairment with advance of age. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Magmas Overexpression Inhibits Staurosporine Induced Apoptosis in Rat Pituitary Adenoma Cell Lines

    PubMed Central

    Gentilin, Erica; Minoia, Mariella; Molè, Daniela; delgi Uberti, Ettore C.; Zatelli, Maria Chiara

    2013-01-01

    Magmas is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. Magmas is overexpressed in the majority of human pituitary adenomas and in a mouse ACTH-secreting pituitary adenoma cell line. Here we report that Magmas is highly expressed in two out of four rat pituitary adenoma cell lines and its expression levels inversely correlate to the extent of cellular response to staurosporine in terms of apoptosis activation and cell viability. Magmas over-expression in rat GH/PRL-secreting pituitary adenoma GH4C1 cells leads to an increase in cell viability and to a reduction in staurosporine-induced apoptosis and DNA fragmentation, in parallel with the increase in Magmas protein expression. These results indicate that Magmas plays a pivotal role in response to pro-apoptotic stimuli and confirm and extend the finding that Magmas protects pituitary cells from staurosporine-induced apoptosis, suggesting its possible involvement in pituitary adenoma development. PMID:24069394

  8. Overexpression of SbMyb60 in Sorghum bicolor impacts both primary and secondary metabolism.

    PubMed

    Scully, Erin D; Gries, Tammy; Palmer, Nathan A; Sarath, Gautam; Funnell-Harris, Deanna L; Baird, Lisa; Twigg, Paul; Seravalli, Javier; Clemente, Thomas E; Sattler, Scott E

    2017-09-25

    Few transcription factors have been identified in C4 grasses that either positively or negatively regulate monolignol biosynthesis. Previously, the overexpression of SbMyb60 in sorghum (Sorghum bicolor) has been shown to induce monolignol biosynthesis, which leads to elevated lignin deposition and altered cell wall composition. To determine how SbMyb60 overexpression impacts other metabolic pathways, RNA-Seq and metabolite profiling were performed on stalks and leaves. 35S::SbMyb60 was associated with the transcriptional activation of genes involved in aromatic amino acid, S-adenosyl methionine (SAM) and folate biosynthetic pathways. The high coexpression values between SbMyb60 and genes assigned to these pathways indicate that SbMyb60 may directly induce their expression. In addition, 35S::SbMyb60 altered the expression of genes involved in nitrogen (N) assimilation and carbon (C) metabolism, which may redirect C and N towards monolignol biosynthesis. Genes linked to UDP-sugar biosynthesis and cellulose synthesis were also induced, which is consistent with the observed increase in cellulose deposition in the internodes of 35S::SbMyb60 plants. However, SbMyb60 showed low coexpression values with these genes and is not likely to be a direct regulator of cell wall polysaccharide biosynthesis. These findings indicate that SbMyb60 can activate pathways beyond monolignol biosynthesis, including those that synthesize the substrates and cofactors required for lignin biosynthesis. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  9. OsLBD3-7 Overexpression Induced Adaxially Rolled Leaves in Rice

    PubMed Central

    Zhang, Chunyu; Shao, Qinghao; Liu, Jun; Liu, Bin; Li, Hongyu

    2016-01-01

    Appropriate leaf rolling enhances erect-leaf habits and photosynthetic efficiency, which consequently improves grain yield. Here, we reported the novel lateral organ boundaries domain (LBD) gene OsLBD3-7, which is involved in the regulation of leaf rolling. OsLBD3-7 works as a transcription activator and its protein is located on the plasma membrane and in the nucleus. Overexpression of OsLBD3-7 leads to narrow and adaxially rolled leaves. Microscopy of flag leaf cross-sections indicated that overexpression of OsLBD3-7 led to a decrease in both bulliform cell size and number. Transcriptional analysis showed that key genes that had been reported to be negative regulators of bulliform cell development were up-regulated in transgenic plants. These results indicated that OsLBD3-7 might acts as an upstream regulatory gene of bulliform cell development to regulate leaf rolling, which will give more insights on the leaf rolling regulation mechanism. PMID:27258066

  10. Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination.

    PubMed

    Klemens, Patrick A W; Patzke, Kathrin; Trentmann, Oliver; Poschet, Gernot; Büttner, Michael; Schulz, Alexander; Marten, Irene; Hedrich, Rainer; Neuhaus, H Ekkehard

    2014-04-01

    Arabidopsis vacuoles harbor, besides sugar transporter of the TMT-type, an early response to dehydration like 6 (ERDL6) protein involved in glucose export into the cytosol. However, the mode of transport of ERDL6 and the plant's feedback to overexpression of its activity on essential properties such as, for example, seed germination or freezing tolerance, remain unexplored. Using patch-clamp studies on vacuoles expressing AtERDL6 we demonstrated directly that this carrier operates as a proton-driven glucose exporter. Overexpression of BvIMP, the closest sugar beet (Beta vulgaris) homolog to AtERDL6, in Arabidopsis leads surprisingly to impaired seed germination under both conditions, sugar application and low environmental temperatures, but not under standard conditions. Upon cold treatment, BvIMP overexpressor plants accumulated lower quantities of monosaccharides than the wild-type, a response in line with the reduced frost tolerance of the transgenic Arabidopsis plants, and the fact that cold temperatures inhibits BvIMP transcription in sugar beet leaves. With these findings we show that the tight control of vacuolar sugar import and export is a key requisite for cold tolerance and seed germination of plants.

  11. Overexpression of gamma-sarcoglycan induces severe muscular dystrophy. Implications for the regulation of Sarcoglycan assembly.

    PubMed

    Zhu, X; Hadhazy, M; Groh, M E; Wheeler, M T; Wollmann, R; McNally, E M

    2001-06-15

    The sarcoglycan complex is found normally at the plasma membrane of muscle. Disruption of the sarcoglycan complex, through primary gene mutations in dystrophin or sarcoglycan subunits, produces membrane instability and muscular dystrophy. Restoration of the sarcoglycan complex at the plasma membrane requires reintroduction of the mutant sarcoglycan subunit in a manner that will permit normal assembly of the entire sarcoglycan complex. To study sarcoglycan gene replacement, we introduced transgenes expressing murine gamma-sarcoglycan into muscle of normal mice. Mice expressing high levels of gamma-sarcoglycan, under the control of the muscle-specific creatine kinase promoter, developed a severe muscular dystrophy with greatly reduced muscle mass and early lethality. Marked gamma-sarcoglycan overexpression produced cytoplasmic aggregates that interfered with normal membrane targeting of gamma-sarcoglycan. Overexpression of gamma-sarcoglycan lead to the up-regulation of alpha- and beta-sarcoglycan. These data suggest that increased gamma-sarcoglycan and/or mislocalization of gamma-sarcoglycan to the cytoplasm is sufficient to induce muscle damage and provides a new model of muscular dystrophy that highlights the importance of this protein in the assembly, function, and downstream signaling of the sarcoglycan complex. Most importantly, gene dosage and promoter strength should be given serious consideration in replacement gene therapy to ensure safety in human clinical trials.

  12. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene confers drought tolerance in maize (Zea mays L.).

    PubMed

    Lu, Yao; Li, Yajun; Zhang, Jiachang; Xiao, Yitao; Yue, Yuesen; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2013-01-01

    Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H(2)O(2) content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.

  13. Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize (Zea mays L.)

    PubMed Central

    Zhang, Jiachang; Xiao, Yitao; Yue, Yuesen; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2013-01-01

    Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H2O2 content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses. PMID:23326325

  14. Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension

    PubMed Central

    Geraci, Mark W.; Gao, Bifeng; Shepherd, David C.; Moore, Mark D.; Westcott, Jay Y.; Fagan, Karen A.; Alger, Lori A.; Tuder, Rubin M.; Voelkel, Norbert F.

    1999-01-01

    Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway leading to prostacyclin (PGI2) production. Patients with severe pulmonary hypertension have a PGIS deficiency of their precapillary vessels, but the importance of this deficiency for lung vascular remodeling remains unclear. We hypothesized that selective pulmonary overexpression of PGIS may prevent the development of pulmonary hypertension. To study this hypothesis, transgenic mice were created with selective pulmonary PGIS overexpression using a construct of the 3.7-kb human surfactant protein-C (SP-C) promoter and the rat PGIS cDNA. Transgenic mice (Tg+) and nontransgenic littermates (Tg–) were subjected to a simulated altitude of 17,000 ft for 5 weeks, and right ventricular systolic pressure (RVSP) was measured. Histology was performed on the lungs. The Tg+ mice produced 2-fold more pulmonary 6-keto prostaglandin F1α (PGF1α) levels than did Tg– mice. After exposure to chronic hypobaric hypoxia, Tg+ mice have lower RVSP than do Tg– mice. Histologic examination of the lungs revealed nearly normal arteriolar vessels in the Tg+ mice in comparison with vessel wall hypertrophy in the Tg– mice. These studies demonstrate that Tg+ mice were protected from the development of pulmonary hypertension after exposure to chronic hypobaric hypoxia. We conclude that PGIS plays a major role in modifying the pulmonary vascular response to chronic hypoxia. This has important implications for the pathogenesis and treatment of severe pulmonary hypertension. PMID:10359560

  15. Steroidogenic acute regulatory protein (StAR) overexpression reduces inflammation and insulin resistance in obese mice.

    PubMed

    Qiu, Yanyan; Sui, Xianxian; Cao, Shengxuan; Li, Xiaobo; Ning, Yanxia; Wang, Songmei; Yin, Lianhua; Zhi, Xiuling

    2017-04-12

    Steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol delivery protein, plays a beneficial role in hyperlipidemia, NAFLD and endothelial inflammation. Elevated circulating fatty acids and low grade inflammation are known as key risk factors of insulin resistance and type 2 diabetes. In the present study, C57BL/6J mice were fed with a HFD and infected with recombinant adenovirus expressing StAR by tail-vein injection. Intraperitoneal glucose/insulin tolerance test was performed to assess the insulin sensitivity. Morphological analysis and intramuscular lipid determination were used to illustrate the adipose hypertrophy and ectopic fat accumulation in skeletal muscle. The levels of inflammatory factor and nitric oxide were determined by ELISA and classic Griess reagent methods respectively. The fatty acids composition was analysis using gas chromatography -mass spectrometry (GC-MS). The expression of genes associated with inflammation and insulin resistance were determined by Western blotting and qPCR to elucidate the underlying mechanism.We demonstrated that StAR overexpression ameliorated insulin resistance and systemic inflammatory response with the reduction of adipose hypertrophy and intramuscular lipid in HFD fed mice. In addition, StAR overexpression increased serum unsaturated fatty acids and PPARγ expression in muscle and adipose tissue of obese mice. In conclusion, StAR may activate PPARγ by increasing unsaturated fatty acids, which leads to a protective role in systemic inflammation and insulin resistance in obese mice. This article is protected by copyright. All rights reserved.

  16. Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3

    PubMed Central

    Snijder, Pauline M; Baratashvili, Madina; Grzeschik, Nicola A; Leuvenink, Henri G D; Kuijpers, Lucas; Huitema, Sippie; Schaap, Onno; Giepmans, Ben N G; Kuipers, Jeroen; Miljkovic, Jan Lj; Mitrovic, Aleksandra; Bos, Eelke M; Szabó, Csaba; Kampinga, Harm H; Dijkers, Pascale F; den Dunnen, Wilfred F A; Filipovic, Milos R; van Goor, Harry; Sibon, Ody C M

    2015-01-01

    Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies. PMID:26467707

  17. The Angiogenic Secretome in VEGF overexpressing Breast Cancer Xenografts

    PubMed Central

    Dore-Savard, Louis; Lee, Esak; Kakkad, Samata; Popel, Aleksander S.; Bhujwalla, Zaver M.

    2016-01-01

    The plasticity of cancer cells and the fluidity of the tumor microenvironment continue to present major challenges in the comprehensive understanding of cancer that is essential to design effective treatments. The tumor interstitial fluid (TIF) encompasses the secretome and holds the key to several of the phenotypic characteristics of cancer. Difficulties in sampling this fluid have resulted in limited characterization of its components. Here we have sampled TIF from triple negative and estrogen receptor (ER)-positive human breast tumor xenografts with or without VEGF overexpression. Angiogenesis-related factors were characterized in the TIF and plasma, to understand the relationship between the TIF and plasma secretomes. Clear differences were observed between the TIF and plasma angiogenic secretomes in triple negative MDA-MB-231 breast cancer xenografts compared to ER-positive MCF-7 xenografts with or without VEGF overexpression that provide new insights into TIF components and the role of VEGF in modifying the angiogenic secretome. PMID:27995973

  18. Overexpression of esterase D in kidney from trisomy 13 fetuses.

    PubMed Central

    Loughna, S; Bennett, P; Gau, G; Nicolaides, K; Blunt, S; Moore, G

    1993-01-01

    Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D was found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses. Images Figure 1 Figure 2 Figure 3 PMID:8213811

  19. Overexpression of ankyrin1 promotes pancreatic cancer cell growth

    PubMed Central

    Omura, Noriyuki; Mizuma, Masamichi; MacGregor, Anne; Hong, Seung-Mo; Ayars, Michael; Almario, Jose Alejandro; Borges, Michael; Kanda, Mitsuro; Li, Ang; Vincent, Audrey; Maitra, Anirban; Goggins, Michael

    2016-01-01

    The methylation status of a promoter influences gene expression and aberrant methylation during tumor development has important functional consequences for pancreatic and other cancers. Using methylated CpG island amplification and promoter microarrays, we identified ANK1 as hypomethylated in pancreatic cancers. Expression analysis determined ANK1 as commonly overexpressed in pancreatic cancers relative to normal pancreas. ANK1 was co-expressed with miR-486 in pancreatic cancer cells. Stable knockdown of ANK1 in the pancreatic cancer cell line AsPC1 led to changes in cell morphology, and decreases in colony formation. Stable knockdown of ANK1 also marked reduced the growth of tumors in athymic nude mice. Among patients undergoing pancreaticoduodenectomy, those with pancreatic cancers expressing ANK1 had a poorer prognosis than those without ANK1 expression. These findings indicate a role for ANK1 overexpression in mediating pancreatic cancer tumorigenicity. PMID:27144336

  20. Enhanced learning after genetic overexpression of a brain growth protein.

    PubMed

    Routtenberg, A; Cantallops, I; Zaffuto, S; Serrano, P; Namgung, U

    2000-06-20

    Ramón y Cajal proposed 100 years ago that memory formation requires the growth of nerve cell processes. One-half century later, Hebb suggested that growth of presynaptic axons and postsynaptic dendrites consequent to coactivity in these synaptic elements was essential for such information storage. In the past 25 years, candidate growth genes have been implicated in learning processes, but it has not been demonstrated that they in fact enhance them. Here, we show that genetic overexpression of the growth-associated protein GAP-43, the axonal protein kinase C substrate, dramatically enhanced learning and long-term potentiation in transgenic mice. If the overexpressed GAP-43 was mutated by a Ser --> Ala substitution to preclude its phosphorylation by protein kinase C, then no learning enhancement was found. These findings provide evidence that a growth-related gene regulates learning and memory and suggest an unheralded target, the GAP-43 phosphorylation site, for enhancing cognitive ability.

  1. Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse

    PubMed Central

    Coelho, Paula A.; Bury, Leah; Shahbazi, Marta N.; Liakath-Ali, Kifayathullah; Tate, Peri H.; Wormald, Sam; Hindley, Christopher J.; Huch, Meritxell; Archer, Joy; Skarnes, William C.; Zernicka-Goetz, Magdalena; Glover, David M.

    2015-01-01

    To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development. PMID:26701933

  2. Overexpression of the tomato pollen receptor kinase LePRK1 rewires pollen tube growth to a blebbing mode.

    PubMed

    Gui, Cai-Ping; Dong, Xin; Liu, Hai-Kuan; Huang, Wei-Jie; Zhang, Dong; Wang, Shu-Jie; Barberini, María Laura; Gao, Xiao-Yan; Muschietti, Jorge; McCormick, Sheila; Tang, Wei-Hua

    2014-09-01

    The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane-localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP), a Rop guanine nucleotide exchange factor. Here, we show that pollen tubes overexpressing LePRK1 or a truncated LePRK1 lacking its extracellular domain (LePRK1ΔECD) have enlarged tips but also extend their leading edges by producing "blebs." Coexpression of LePRK1 and tomato PLIM2a, an actin bundling protein that interacts with KPP in a Ca(2+)-responsive manner, suppressed these LePRK1 overexpression phenotypes, whereas pollen tubes coexpressing KPP, LePRK1, and PLIM2a resumed the blebbing growth mode. We conclude that overexpression of LePRK1 or LePRK1ΔECD rewires pollen tube growth to a blebbing mode, through KPP- and PLIM2a-mediated bundling of actin filaments from tip plasma membranes. Arabidopsis thaliana pollen tubes expressing LePRK1ΔECD also grew by blebbing. Our results exposed a hidden capability of the pollen tube cell: upon overexpression of a single membrane-localized molecule, LePRK1 or LePRK1ΔECD, it can switch to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila melanogaster stem cells. © 2014 American Society of Plant Biologists. All rights reserved.

  3. Law: toxic lead aftermath

    SciTech Connect

    Goldstein, E.A.

    1983-03-01

    The paper describes the events which began with an EPA proposal to weaken the lead-in-gas regulations. Because of the outcry from environmentalists and expert testimony from the medical community, the EPA reversed its policy and issued new standards which would reduce lead emissions between 1983 and 1990 by 34 percent (128,000 tons). Scientific evidence presented showed a clear reduction in blood lead levels from 1976-1980 which paralleled decreases of lead in gasoline. Results from lead poisoning clinics which linked chronic low lead exposures to decreased classroom performance and other learning disabilities were presented. Lawyers from several environmental groups took the agency to court on the related issue of attaining national ambient air quality standards for lead. (JMT)

  4. Lead in the environment

    USGS Publications Warehouse

    Pattee, O.H.; Pain, D.J.; Hoffman, David J.; Rattner, Barnett A.; Burton, G. Allen; Cairns, John=

    2003-01-01

    Anthropogenic uses of lead have probably altered its availability and environmental distribution more than any other toxic element. Consequently, lead concentrations in many living organisms may be approaching thresholds of toxicity for the adverse effects of lead. Such thresholds are difficult to define, as they vary with the chemical and physical form of lead, exposure regime, other elements present and also vary both within and between species. The technological capability to accurately quantify low lead concentrations has increased over the last decade, and physiological and behavioral effects have been measured in wildlife with tissue lead concentrations below those previously considered safe for humans.s.236 Consequently. lead criteria for the protection of wildlife and human health are frequently under review, and 'thresholds' of lead toxicity are being reconsidered. Proposed lead criteria for the protection of natural resources have been reviewed by Eisler. Uptake of lead by plants is limited by its generally low availability in soils and sediments, and toxicity may be limited by storage mechanisms and its apparently limited translocation within most plants. Lead does not generally accumulate within the foliar parts of plants, which limits its transfer to higher trophic levels. Although lead may concentrate in plant and animal tissues, no evidence of biomagnification exists. Acid deposition onto surface waters and soils with low buffering capacity may influence the availability of lead for uptake by plants and animals, and this may merit investigation at susceptible sites. The biological significance of chronic low-level lead exposure to wildlife is sometimes difficult to quantify. Animals living in urban environments or near point sources of lead emission are inevitably subject to greater exposure to lead and enhanced risk of lead poisoning. Increasingly strict controls on lead emissions in many countries have reduced exposure to lead from some sources

  5. Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

    SciTech Connect

    Yu, Suyeun; Jang, Yeogil; Paik, Donggi; Lee, Eunil; Park, Joong-Jean

    2015-10-02

    NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan. - Highlights: • Ubiquitous Nmdmc overexpression enhanced lifespan and stress tolerance. • Nmdmc overexpression in the fat body extended longevity. • Fat body-specific Nmdmc overexpression increased oxidative stress resistance. • Nmdmc overexpression decreased Hsp22 transcript levels and ROS. • Nmdmc overexpression increased mitochondrial DNA copy number.

  6. Overexpression of Lymphotoxin in T Cells Induces Fulminant Thymic Involution

    PubMed Central

    Heikenwalder, Mathias; Prinz, Marco; Zeller, Nicolas; Lang, Karl S.; Junt, Tobias; Rossi, Simona; Tumanov, Alexei; Schmidt, Hauke; Priller, Josef; Rülicke, Thomas; Macpherson, Andrew J.; Holländer, Georg A.; Nedospasov, Sergei A.; Aguzzi, Adriano

    2008-01-01

    Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTβR), representing two non-redundant pathways. Multiple lines of transgenic Ltαβ and Ltα mice show such a phenotype, which was not observed on overexpression of LTβ alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTβR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTβR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1−/− mice. These mice displayed elevated TNFα in both thymus and plasma, as well as increased LTs on both CD8+ and CD4−CD8− thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTβR signaling in pathological conditions and possibly also in normal aging. PMID:18483211

  7. Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation.

    PubMed

    Sola Carvajal, Agustín; McKenna, Tomás; Wallén Arzt, Emelie; Eriksson, Maria

    2015-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental progeroid disorder commonly caused by a point mutation in the LMNA gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS LMNA mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR) expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype.

  8. Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation

    PubMed Central

    Sola Carvajal, Agustín; McKenna, Tomás; Wallén Arzt, Emelie; Eriksson, Maria

    2015-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental progeroid disorder commonly caused by a point mutation in the LMNA gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS LMNA mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR) expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype. PMID:26053873

  9. Role of overexpressed CFA/I fimbriae in bacterial swimming

    NASA Astrophysics Data System (ADS)

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, SangMu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

  10. Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

    PubMed Central

    Nguyen, Hao G.; Yu, Guangyao; Makitalo, Maria; Yang, Dan; Xie, Hou-Xiang; Jones, Matthew R.; Ravid, Katya

    2005-01-01

    Megakaryocyte (MK)–specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)–off system to conditionally over-express genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)–tTA responsive promoter driving prokaryotic β-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA–viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by β-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16, that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo. PMID:15890684

  11. Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

    PubMed Central

    Schnell, Eric; Bensen, AeSoon L.; Washburn, Eric K.; Westbrook, Gary L.

    2012-01-01

    Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons. PMID:23110172

  12. Overexpression of calreticulin sensitizes SERCA2a to oxidative stress.

    PubMed

    Ihara, Yoshito; Kageyama, Kan; Kondo, Takahito

    2005-04-22

    Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.

  13. Subcellular protein overexpression to develop abiotic stress tolerant plants

    PubMed Central

    Nouri, Mohammad-Zaman; Komatsu, Setsuko

    2012-01-01

    Environmental stresses are major factors limiting growth and development of crops. Plants respond to the stresses through a wide range of reactions from morphological changes to alterations in the patterns of protein expression. Understanding the mechanisms involved in the stress response is the first step to develop abiotic stress tolerant crops. Proteomics is a powerful tool in evaluating regulated proteins in the cell under stress and it is an efficient technique in studying stress tolerant plants. Because of the nature of abiotic stress, intracellular compartments play a main role in the stress response. Subcellular proteins such as ion and water transporters, reactive oxygen species (ROS) scavengers, and the proteins related to signaling and transcriptional regulation are frequently reported as being involved in stress tolerance. Overexpression of stress-responsive protein through generation of transgenic plants is one the main practical approaches in production of tolerant plants. In this article, recent studies on transgenic plants overexpressing subcellular proteins are reviewed and the role of organelles and over-expressed proteins is classified. PMID:23346093

  14. Overexpression of follistatin in trout stimulates increased muscling.

    PubMed

    Medeiros, Erika F; Phelps, Michael P; Fuentes, Fernando D; Bradley, Terence M

    2009-07-01

    Deletion or inhibition of myostatin in mammals has been demonstrated to markedly increase muscle mass by hyperplasia, hypertrophy, or a combination of both. Despite a remarkably high degree of conservation with the mammalian protein, the function of myostatin remains unknown in fish, many species of which continue muscle growth throughout the lifecycle by hyperplasia. Transgenic rainbow trout (Oncorhynchus mykiss) overexpressing follistatin, one of the more efficacious antagonists of myostatin, were produced to investigate the effect of this protein on muscle development and growth. P(1) transgenics overexpressing follistatin in muscle tissue exhibited increased epaxial and hypaxial muscling similar to that observed in double-muscled cattle and myostatin null mice. The hypaxial muscling generated a phenotype reminiscent of well-developed rectus abdominus and intercostal muscles in humans and was dubbed "six pack." Body conformation of the transgenic animals was markedly altered, as measured by condition factor, and total muscle surface area increased. The increased muscling was due almost exclusively to hyperplasia as evidenced by a higher number of fibers per unit area and increases in the percentage of smaller fibers and the number of total fibers. In several individuals, asymmetrical muscling was observed, but no changes in mobility or behavior of follistatin fish were observed. The findings indicate that overexpression of follistatin in trout, a species with indeterminate growth rate, enhances muscle growth. It remains to be determined whether the double muscling in trout is due to inhibition of myostatin, other growth factors, or both.

  15. Role of overexpressed CFA/I fimbriae in bacterial swimming.

    PubMed

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, Sangmu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

  16. PACSIN3 Overexpression Increases Adipocyte Glucose Transport through GLUT1

    PubMed Central

    Roach, William; Plomann, Markus

    2007-01-01

    PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT exofacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUT1, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUT1 trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUT1, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes. PMID:17320047

  17. Lead in candle emissions.

    PubMed

    Wasson, Shirley J; Guo, Zhishi; McBrian, Jenia A; Beach, Laura O

    2002-09-16

    The candle-using public should be made aware that the core of candle wicks may contain lead. Used as a stiffening agent to keep the wick out of the molten wax, lead can be emitted as particulates to the air and then deposited on indoor surfaces. To define the problem, 100 sets of candles (two or more identical candles) were purchased locally. The criterion for purchase was that the candles must appear to contain a metal-cored wick or be covered by a metallic pigment. Of the candles purchased, 8% contained lead wicks. The wicks were 39-74% lead (the remainder was fabric or paper) and the lead cores (approx. 100% lead) had linear densities of 13-27 mg/cm. Candles were burned to completion in a closed chamber to capture the air emissions, and the candle residue was extracted to assess the lead mass balance. It was found that individual candles emitted lead to the air at average rates that ranged from 100 to 1700 microg/h. Assuming realistic indoor conditions, these emission rates were modeled to project room air concentration, child exposure by inhalation, and indoor deposition. Results showed that burning single candles can easily raise the source room concentration above the ambient air lead concentration limit of 1.5 microg/m3 set by EPA. Burning multiple candles can elevate it above OSHA permissible exposure limits of 50 microg/m3. Although blood lead levels have dropped precipitously in the United States since lead was phased out of gasoline in 1986, nearly 900,000 children still had levels above 10 microg/dl during NHANES III. Considering that candle sales in the US are estimated at $1-2 billion per year, and that children may spend as much as 88% of their time indoors, it is reasonable to suspect that some blood lead elevation in children arises from indoor micro-environments where lead-wick candles are burned.

  18. Neuroprotective potential of pleiotrophin overexpression in the striatonigral pathway compared with overexpression in both the striatonigral and nigrostriatal pathways

    PubMed Central

    Gombash, SE; Manfredsson, FP; Mandel, RJ; Collier, TJ; Fischer, DL; Kemp, CJ; Kuhn, NM; Wohlgenant, SL; Fleming, SM; Sortwell, CE

    2015-01-01

    Intrastriatal injection of recombinant adeno-associated viral vector serotype 2/1 (rAAV2/1) to overexpress the neurotrophic factor pleiotrophin (PTN) provides neuroprotection for tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc), increases THir neurite density in the striatum (ST) and reverses functional deficits in forepaw use following 6-hydroxydopamine (6-OHDA) toxic insult. Glial cell line-derived neurotrophic factor (GDNF) gene transfer studies suggest that optimal neuroprotection is dependent on the site of nigrostriatal overexpression. The present study was conducted to determine whether enhanced neuroprotection could be accomplished via simultaneous rAAV2/1 PTN injections into the ST and SN compared with ST injections alone. Rats were unilaterally injected in the ST alone or injected in both the ST and SN with rAAV2/1 expressing either PTN or control vector. Four weeks later, all rats received intrastriatal injections of 6-OHDA. Rats were euthanized 6 or 16 weeks relative to 6-OHDA injection. A novel selective total enumeration method to estimate nigral THir neuron survival was validated to maintain the accuracy of stereological assessment. Long-term nigrostriatal neuroprotection and functional benefits were only observed in rats in which rAAV2/1 PTN was injected into the ST alone. Results suggest that superior preservation of the nigrostriatal system is provided by PTN overexpression delivered to the ST and restricted to the ST and SN pars reticulata and is not improved with overexpression of PTN within SNpc neurons. PMID:24807806

  19. Neuroprotective potential of pleiotrophin overexpression in the striatonigral pathway compared with overexpression in both the striatonigral and nigrostriatal pathways.

    PubMed

    Gombash, S E; Manfredsson, F P; Mandel, R J; Collier, T J; Fischer, D L; Kemp, C J; Kuhn, N M; Wohlgenant, S L; Fleming, S M; Sortwell, C E

    2014-07-01

    Intrastriatal injection of recombinant adeno-associated viral vector serotype 2/1 (rAAV2/1) to overexpress the neurotrophic factor pleiotrophin (PTN) provides neuroprotection for tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc), increases THir neurite density in the striatum (ST) and reverses functional deficits in forepaw use following 6-hydroxydopamine (6-OHDA) toxic insult. Glial cell line-derived neurotrophic factor (GDNF) gene transfer studies suggest that optimal neuroprotection is dependent on the site of nigrostriatal overexpression. The present study was conducted to determine whether enhanced neuroprotection could be accomplished via simultaneous rAAV2/1 PTN injections into the ST and SN compared with ST injections alone. Rats were unilaterally injected in the ST alone or injected in both the ST and SN with rAAV2/1 expressing either PTN or control vector. Four weeks later, all rats received intrastriatal injections of 6-OHDA. Rats were euthanized 6 or 16 weeks relative to 6-OHDA injection. A novel selective total enumeration method to estimate nigral THir neuron survival was validated to maintain the accuracy of stereological assessment. Long-term nigrostriatal neuroprotection and functional benefits were only observed in rats in which rAAV2/1 PTN was injected into the ST alone. Results suggest that superior preservation of the nigrostriatal system is provided by PTN overexpression delivered to the ST and restricted to the ST and SN pars reticulata and is not improved with overexpression of PTN within SNpc neurons.

  20. Alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 can support immune responses toward tumors overexpressing ganglioside D3 in mice.

    PubMed

    Eby, Jonathan M; Barse, Levi; Henning, Steven W; Rabelink, Martijn J W E; Klarquist, Jared; Gilbert, Emily R; Hammer, Adam M; Fernandez, Manuel F; Yung, Nathan; Khan, Safia; Miller, Hannah G; Kessler, Edward R; Garrett-Mayer, Elizabeth; Dilling, Daniel F; Hoeben, Rob C; Le Poole, I Caroline

    2017-01-01

    An immunotherapeutic strategy is discussed supporting anti-tumor activity toward malignancies overexpressing ganglioside D3. GD3 can be targeted by NKT cells when derived moieties are presented in the context of CD1d. NKT cells can support anti-tumor responses by secreting inflammatory cytokines and through cytotoxicity toward CD1d(+)GD3(+) tumors. To overexpress GD3, we generated expression vector DNA and an adenoviral vector encoding the enzyme responsible for generating GD3 from its ubiquitous precursor GM3. We show that DNA encoding α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (SIAT8) introduced by gene gun vaccination in vivo leads to overexpression of GD3 and delays tumor growth. Delayed tumor growth is dependent on CD1d expression by host immune cells, as shown in experiments engaging CD1d knockout mice. A trend toward greater NKT cell populations among tumor-infiltrating lymphocytes is associated with SIAT8 vaccination. A single adenoviral vaccination introduces anti-tumor activity similarly to repeated vaccination with naked DNA. Here, greater NKT tumor infiltrates were accompanied by marked overexpression of IL-17 in the tumor, later switching to IL-4. Our results suggest that a single intramuscular adenoviral vaccination introduces overexpression of GD3 by antigen-presenting cells at the injection site, recruiting NKT cells that provide an inflammatory anti-tumor environment. We propose adenoviral SIAT8 (AdV-SIAT8) can slow the growth of GD3 expressing tumors in patients.

  1. Polymer-DNA Nanoparticle-Induced CXCR4 Overexpression Improves Stem Cell Engraftment and Tissue Regeneration in a Mouse Hindlimb Ischemia Model

    PubMed Central

    Deveza, Lorenzo; Choi, Jeffrey; Lee, Jerry; Huang, Ngan; Cooke, John; Yang, Fan

    2016-01-01

    Peripheral arterial disease affects nearly 202 million individuals worldwide, sometimes leading to non-healing ulcers or limb amputations in severe cases. Genetically modified stem cells offer potential advantages for therapeutically inducing angiogenesis via augmented paracrine release mechanisms and tuned dynamic responses to environmental stimuli at disease sites. Here, we report the application of nanoparticle-induced CXCR4-overexpressing stem cells in a mouse hindlimb ischemia model. We found that CXCR4 overexpression improved stem cell survival, modulated inflammation in situ, and accelerated blood reperfusion. These effects, unexpectedly, led to complete limb salvage and skeletal muscle repair, markedly outperforming the efficacy of the conventional angiogenic factor control, VEGF. Importantly, assessment of CXCR4-overexpressing stem cells in vitro revealed that CXCR4 overexpression induced changes in paracrine signaling of stem cells, promoting a therapeutically desirable pro-angiogenic and anti-inflammatory phenotype. These results suggest that nanoparticle-induced CXCR4 overexpression may promote favorable phenotypic changes and therapeutic efficacy of stem cells in response to the ischemic environment. PMID:27279910

  2. Overexpression and mutation as a genetic mechanism of fluconazole resistance in Candida albicans isolated from human immunodeficiency virus patients in Indonesia.

    PubMed

    Rosana, Yeva; Yasmon, Andi; Lestari, Delly Chipta

    2015-09-01

    Fluconazole is the standard treatment for oropharyngeal candidiasis, which is the third most common opportunistic infection in human immunodeficiency virus (HIV)/AIDS patients in Indonesia. Overuse of this drug could lead to the emergence of resistance. The objective of this study was to analyse the role of ERG11, CDR1, CDR2 and MDR1 gene overexpression and mutations in the ERG11 gene as a genetic mechanism of fluconazole resistance in Candida albicans isolated from HIV patients in Indonesia. Overexpression of ERG11, CDR1, CDR2 and MDR1 was analysed by real-time reverse transcription PCR, while ERG11 gene mutation analysis was performed using sequencing methods. Seventeen isolates out of 92 strains of C. albicans isolated from 108 HIV patients were found to be resistant to azole antifungals. The highest gene overexpression of ERG11 was found in C. albicans resistant to single fluconazole, while the highest gene overexpression of CDR2 was detected in all isolates of C. albicans resistant to multiple azoles. Amino acid substitutions were observed at six positions, i.e. D116E, D153E, I261V, E266D, V437I and V488I. The amino acid substitution I261V was identified in this study and was probably associated with fluconazole resistance. The combination of overexpression of CDR2 and ERG11 and mutation in the ERG11 gene was found to be a genetic mechanism of fluconazole resistance in C. albicans isolated from HIV patients in Indonesia.

  3. Lead Poison Detection

    NASA Technical Reports Server (NTRS)

    1976-01-01

    With NASA contracts, Whittaker Corporations Space Science division has developed an electro-optical instrument to mass screen for lead poisoning. Device is portable and detects protoporphyrin in whole blood. Free corpuscular porphyrins occur as an early effect of lead ingestion. Also detects lead in urine used to confirm blood tests. Test is inexpensive and can be applied by relatively unskilled personnel. Similar Whittaker fluorometry device called "drug screen" can measure morphine and quinine in urine much faster and cheaper than other methods.

  4. [Familial lead poisoning].

    PubMed

    Ríos, E; Dal Borgo, P; Riveros, A; Díaz, S M

    1989-06-01

    A 1 year and 9 month old patient was admitted with ataxia. CBC showed a microcytic, hypocromic anemia with intense basophilic sttipling of erythrocytes. Lead poisoning was suspected and confirmed with a blood lead level of 167 micrograms/dl. The patient was treated with EDTA and BAL. It was discovered that family burned old car batteries for food cooking. Four members were intoxicated, with blood lead levels at or above 50 micrograms/dl.

  5. Immunosuppressive effects of lead

    USGS Publications Warehouse

    Franson, J. Christian; Feierabend, J.Scott; Russell, A.Brooke

    1986-01-01

    Immunosuppressive effects of lead were reported as early as 1966, when it was noted that lead increased the sensitivity of rats to bacterial endotoxins (Selye et al. 1966). Since then a substantial body of literature has demonstrated adverse effects of lead on the immune system in a variety of laboratory animals, but very little has been done in this area with avian species. Such immunosuppressive effects could be of significance to waterfowl populations, considering the potential for lead ingestion by waterfowl and subsequent exposure of these birds to disease agents.

  6. Lead poisoning: case studies.

    PubMed

    Gordon, J N; Taylor, A; Bennett, P N

    2002-05-01

    Early clinical features of lead toxicity are non-specific and an occupational history is particularly valuable. Lead in the body comprises 2% in the blood (t1/2 35 days) and 95% in bone and dentine (t1/2 20-30 years). Blood lead may remain elevated for years after cessation from long exposure, due to redistribution from bone. Blood lead concentration is the most widely used marker for inorganic lead exposure. Zinc protoporphyrin (ZPP) concentration in blood usefully reflects lead exposure over the prior 3 months. Symptomatic patients with blood lead concentration >2.4 micromol l-1 (50 microg dl-1) or in any event >3.8 micromol l-1 (80 microg dl-1) should receive sodium calciumedetate i.v., followed by succimer by mouth for 19 days. Asymptomatic patients with blood lead concentration >2.4 micromol l-1 (50 microg dl-1) may be treated with succimer alone. Sodium calciumedetate should be given with dimercaprol to treat lead encephalopathy.

  7. Immunosuppressive effects of lead

    USGS Publications Warehouse

    Franson, J. Christian; Feierabend, J.Scott; Russell, A.Brooke

    1986-01-01

    Immunosuppressive effects of lead were reported as early as 1966, when it was noted that lead increased the sensitivity of rats to bacterial endotoxins (Selye et al. 1966). Since then a substantial body of literature has demonstrated adverse effects of lead on the immune system in a variety of laboratory animals, but very little has been done in this area with avian species. Such immunosuppressive effects could be of significance to waterfowl populations, considering the potential for lead ingestion by waterfowl and subsequent exposure of these birds to disease agents.

  8. Acute lead arsenate poisoning.

    PubMed

    Tallis, G A

    1989-12-01

    Three cases of acute lead arsenate poisoning which occurred in South Australia during a 12 month interval are described. The case reports demonstrate a number of features of the characteristic clinical syndrome which may follow ingestion of lead arsenate. The recommended management is immediate gastric lavage and subsequent chelation therapy with calcium EDTA and dimercaprol. Early gastric lavage may prevent significant lead absorption. However, arsenic acid (produced in the stomach when lead arsenate reacts with hydrochloric acid) is relatively water soluble and prompt gastric lavage is unlikely to prevent extensive arsenic absorption. It remains controversial as to whether chelation with dimercaprol prevents arsenical neuropathy.

  9. Childhood lead poisoning.

    PubMed

    Linakis, J G

    1995-01-01

    Lead poisoning has been referred to as the most important environmental health hazard for children in New England. Medical professionals are in a unique position to perform a number of interventions that could make a lasting impact. First, physicians and nurses, particularly in the areas of pediatrics and family medicine, can provide anticipatory guidance to all families with young children. Lead poisoning, in contrast to long held beliefs, is an affliction that affects all socioeconomic groups. Parents should thus be informed regarding sources of lead, including occupational and hobby sources, and basic nutritional and abatement information should be provided. Second, health care workers should encourage lead screening in appropriately aged children at recommended intervals based on known risk factors. Once a blood lead concentration greater than 20[symbol: see text]g/dl has been obtained in a child, treatment or referral to an established lead clinic should be undertaken in a timely fashion. For children with low or moderate lead levels, many pediatricians or family physicians prefer to supervise their patients' treatment, including chelation therapy. For children with higher levels or in instances when the health care professional elects to refer, there are several lead clinics throughout New England whose clinicians are experienced in the treatment of childhood lead poisoning. Finally the medical profession needs to publicly recognize, as child advocates, that lead poisoning is one of the most common pediatric health problems in the United States and that it is entirely preventable. Fortunately, after many years and much hard work, Rhode Island finally has laws that start to deal with the lead problem in an appropriately aggressive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. LEAD IN CANDLE EMISSIONS

    EPA Science Inventory

    The candle-using public should be made aware that the core of candle wicks may contain lead. Used as a stiffening agent to keep the wick out of the molten wax, lead can be emitted as particulate to the air and then deposited on indoor surfaces. To define the problem, 100 sets of ...

  11. Lead carbonate scintillator materials

    DOEpatents

    Derenzo, Stephen E.; Moses, William W.

    1991-01-01

    Improved radiation detectors containing lead carbonate or basic lead carbonate as the scintillator element are disclosed. Both of these scintillators have been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to other known scintillator materials. The radiation detectors disclosed are favorably suited for use in general purpose detection and in medical uses.

  12. Supersonic Leading Edge Receptivity

    NASA Technical Reports Server (NTRS)

    Maslov, Anatoly A.

    1998-01-01

    This paper describes experimental studies of leading edge boundary layer receptivity for imposed stream disturbances. Studies were conducted in the supersonic T-325 facility at ITAM and include data for both sharp and blunt leading edges. The data are in agreement with existing theory and should provide guidance for the development of more complete theories and numerical computations of this phenomena.

  13. Rapid Lead Screening Test

    MedlinePlus

    ... and treated earlier before the damaging effects of lead poisoning occur. U.S. Department of Health and Human Services ... exceed 10μg/dL, the threshold used to indicate lead poisoning. The American Academy of Pediatrics (AAP) estimates one ...

  14. Lead Poisoning in Children.

    ERIC Educational Resources Information Center

    Lin-Fu, Jane S.

    This publication is a guide to help social and health workers plan a preventive campaign against lead poisoning, a cause of mental retardation other neurological handicaps, and death among children. The main victims are 1- to 6-year-olds living in areas where deteriorating housing prevails. Among the causes of lead poisoning are: ingestion of…

  15. Lead toxicity: a review

    PubMed Central

    Ara, Anjum; Usmani, Jawed Ahmad

    2015-01-01

    Lead toxicity is an important environmental disease and its effects on the human body are devastating. There is almost no function in the human body which is not affected by lead toxicity. Though in countries like US and Canada the use of lead has been controlled up to a certain extent, it is still used vehemently in the developing countries. This is primarily because lead bears unique physical and chemical properties that make it suitable for a large number of applications for which humans have exploited its benefits from historical times and thus it has become a common environmental pollutant. Lead is highly persistent in the environment and because of its continuous use its levels rise in almost every country, posing serious threats. This article reviews the works listed in the literature with recent updates regarding the toxicity of lead. Focus is also on toxic effects of lead on the renal, reproductive and nervous system. Finally the techniques available for treating lead toxicity are presented with some recent updates. PMID:27486361

  16. Lead Poisoning (For Parents)

    MedlinePlus

    ... blood cells and limit their ability to carry oxygen to the organs and tissues that need it, thus causing anemia. Most lead ends up in the bone, where it causes even more problems. Lead can interfere with the production of blood cells and the absorption of calcium ...

  17. Lead toxicity: a review.

    PubMed

    Wani, Ab Latif; Ara, Anjum; Usmani, Jawed Ahmad

    2015-06-01

    Lead toxicity is an important environmental disease and its effects on the human body are devastating. There is almost no function in the human body which is not affected by lead toxicity. Though in countries like US and Canada the use of lead has been controlled up to a certain extent, it is still used vehemently in the developing countries. This is primarily because lead bears unique physical and chemical properties that make it suitable for a large number of applications for which humans have exploited its benefits from historical times and thus it has become a common environmental pollutant. Lead is highly persistent in the environment and because of its continuous use its levels rise in almost every country, posing serious threats. This article reviews the works listed in the literature with recent updates regarding the toxicity of lead. Focus is also on toxic effects of lead on the renal, reproductive and nervous system. Finally the techniques available for treating lead toxicity are presented with some recent updates.

  18. LEAD IN CANDLE EMISSIONS

    EPA Science Inventory

    The candle-using public should be made aware that the core of candle wicks may contain lead. Used as a stiffening agent to keep the wick out of the molten wax, lead can be emitted as particulate to the air and then deposited on indoor surfaces. To define the problem, 100 sets of ...

  19. Bonding aluminum beam leads

    NASA Technical Reports Server (NTRS)

    Burkett, F. S.

    1978-01-01

    Report makes it relatively easy for hybrid-circuit manufacturers to convert integrated circuit chips with aluminum bead leads. Report covers: techniques for handling tiny chips; proper geometries for ultrasonic bonding tips; best combinations of pressure, pulse time, and ultrasonic energy for bonding; and best thickness for metal films to which beam leads are bonded.

  20. Lead polluters get punished

    SciTech Connect

    Not Available

    1991-08-09

    The Environmental Protection Agency (EPA) and the Department of Justice last week cracked down on 36 US companies for polluting the environment with lead. EPA slapped fines totaling more than $10 million on 12 of the offending companies, and Justice filed 24 civil complaints. Hank Habicht, deputy administrator of the EPA, said that his agency's initiative comes after 8 months of intense - and presumably successful - efforts at locating and documenting lead pollution in the soil, air, and water supply. Most feared has been lead's ability to damage the intellectual development of children. This caused the agency, Habicht said, to look beyond the usual suspect - lead in the water supply - to lead-laced dirt in residential areas. Meanwhile, the Department of Justice is using the EPA contamination data as well. Twenty US attorneys have been assigned to pore over the federal environmental statutes, including the Clean Water Act and the Superfund Law, in order to file civil complaints.

  1. Lead contamination in Uruguay.

    PubMed

    Mañay, N; Pereira, L; Cousillas, Z

    1999-01-01

    Uruguay is a developing country of South America with about 3 million people, half of whom live in its principal city, Montevideo. This city has several lead pollution sources as emitting industries, most of them surrounded by residential neighborhoods, some still using lead pipes in drinking water systems of old buildings, and has areas of heavy traffic with cars that are still fueled with leaded gasoline. The toxic effects of this heavy metal are well known. Children are a very sensitive population and their early symptoms of intoxication are not always taken into account. Blood lead is a good indicator of recent exposure to lead influenced by inhalation and ingestion. The systematic data assessment of lead pollution and people exposure in Uruguay was not well known when the Department of Toxicology and Environmental Hygiene of the Faculty of Chemistry began to analyze lead in biological samples, first from exposed workers and next from children and the general population, including sensitive animal species like dogs. Several described studies were carried out analyzing for blood lead to assess lead uptake and to obtain reference values for Uruguayan populations. Since 1986, that Department is the only laboratory where blood lead analyses are done, and the analytical method has been controlled by an interlaboratory quality control program of the Ministry of Labour of Spain and confirmed by experts from the Laboratory of Occupational and Environmental Medicine of Lund, Sweden. Financial and technical support was obtained from Sweden (SAREC) and also from the University of the Republic of Uruguay. Uruguayan lead workers have always been the principally studied population because their lead exposure assessment as well as their health protection education is not always done properly. Uruguay has adopted ACGIH reference values (150 micrograms/m3 in total lead dust, 50 micrograms/m3 respirable lead dust, 300 micrograms/L blood), and the high blood lead levels indicate

  2. Glyoxalase 1 overexpression does not affect atherosclerotic lesion size and severity in ApoE-/- mice with or without diabetes.

    PubMed

    Hanssen, Nordin M J; Brouwers, Olaf; Gijbels, Marion J; Wouters, Kristiaan; Wijnands, Erwin; Cleutjens, Jack P M; De Mey, Jo G; Miyata, Toshio; Biessen, Erik A; Stehouwer, Coen D A; Schalkwijk, Casper G

    2014-10-01

    Advanced glycation end-products (AGEs) and their precursors have been associated with the development of atherosclerosis. We recently discovered that glyoxalase 1 (GLO1), the major detoxifying enzyme for AGE precursors, is decreased in ruptured human plaques, and that levels of AGEs are higher in rupture-prone plaques. We here investigated whether overexpression of human GLO1 in ApoE(-/-) mice could reduce the development of atherosclerosis. We crossed C57BL/6 ApoE(-/-) mice with C57BL/6 GLO1 overexpressing mice (huGLO1(+/-)) to generate ApoE(-/-) (n = 16) and ApoE(-/-) huGLO1(+/-) (n = 20) mice. To induce diabetes, we injected a subset with streptozotocin (STZ) to generate diabetic ApoE(-/-) (n = 8) and ApoE(-/-) huGLO1(+/-) (n = 13) mice. All mice were fed chow and sacrificed at 25 weeks of age. The GLO1 activity was three-fold increased in huGLO1(+/-) aorta, but aortic root lesion size and phenotype did not differ between mice with and without huGLO1(+/-) overexpression. We detected no differences in gene expression in aortic arches, in AGE levels and cytokines, in circulating cells, and endothelial function between ApoE(-/-) mice with and without huGLO1(+/-) overexpression. Although diabetic mice showed decreased GLO1 expression (P < 0.05) and increased lesion size (P < 0.05) in comparison with non-diabetic mice, GLO1 overexpression also did not affect the aortic root lesion size or inflammation in diabetic mice. In ApoE(-/-) mice with or without diabetes, GLO1 overexpression did not lead to decreased atherosclerotic lesion size or systemic inflammation. Increasing GLO1 levels does not seem to be an effective strategy to reduce glycation in atherosclerotic lesions, likely due to increased AGE formation through GLO1-independent mechanisms. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  3. Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps.

    PubMed

    Olivares Pacheco, Jorge; Alvarez-Ortega, Carolina; Alcalde Rico, Manuel; Martínez, José Luis

    2017-07-25

    It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H(+) accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations.IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been

  4. Lead toxicity: current concerns.

    PubMed Central

    Goyer, R A

    1993-01-01

    Over the 20-year period since the first issue of Environmental Health Perspectives was published, there has been considerable progress in the understanding of the potential toxicity of exposure to lead. Many of these advances have been reviewed in published symposia, conferences, and review papers in EHP. This brief review identifies major advances as well as a number of current concerns that present opportunities for prevention and intervention strategies. The major scientific advance has been the demonstration that blood lead (PbB) levels of 10-15 micrograms/dL in newborn and very young infants result in cognitive and behavioral deficits. Further support for this observation is being obtained by prospective or longitudinal studies presently in progress. The mechanism(s) for the central nervous system effects of lead is unclear but involve lead interactions within calcium-mediated intracellular messenger systems and neurotransmission. Effects of low-level lead exposure on blood pressure, particularly in adult men, may be related to the effect of lead on calcium-mediated control of vascular smooth muscle contraction and on the renin-angiotensin system. Reproductive effects of lead have long been suspected, but low-level effects have not been well studied. Whether lead is a carcinogen or its association with renal adenocarcinoma is a consequence of cystic nephropathy is uncertain. Major risk factors for lead toxicity in children in the United States include nutrition, particularly deficiencies of essential metals, calcium, iron, and zinc, and housing and socioeconomic status. A goal for the year 2000 is to reduce prevalence of blood lead levels exceeding 15 micrograms/dL. Images FIGURE 2. PMID:8354166

  5. Lead toxicity: Current concerns

    SciTech Connect

    Goyer, R.A. )

    1993-04-01

    Over the 20-year period since the first issue of Environmental Health Perspectives was published, there has been considerable progress in the understanding of the potential toxicity of exposure to lead. Many of these advances have been reviewed in published symposia, conferences, and review papers in EHP. This brief review identifies major advances as well as a number of current concerns that present opportunities for prevention and intervention strategies. The major scientific advance has been the demonstration that blood lead (PbB) levels of 10-15 micrograms/dL in newborn and very young infants result in cognitive and behavioral deficits. Further support for this observation is being obtained by prospective or longitudinal studies presently in progress. The mechanism(s) for the central nervous system effects of lead is unclear but involve lead interactions within calcium-mediated intracellular messenger systems and neurotransmission. Effects of low-level lead exposure on blood pressure, particularly in adult men, may be related to the effect of lead on calcium-mediated control of vascular smooth muscle contraction and on the renin-angiotensin system. Reproductive effects of lead have long been suspected, but low-level effects have not been well studied. Whether lead is a carcinogen or its association with renal adenocarcinoma is a consequence of cystic nephropathy is uncertain. Major risk factors for lead toxicity in children in the United States include nutrition, particularly deficiencies of essential metals, calcium, iron, and zinc, and housing and socioeconomic status. A goal for the year 2000 is to reduce prevalence of blood lead levels exceeding 15 micrograms/dL. 97 refs.

  6. Transgenic sugarcane overexpressing CaneCPI-1 negatively affects the growth and development of the sugarcane weevil Sphenophorus levis.

    PubMed

    Schneider, Vanessa Karine; Soares-Costa, Andrea; Chakravarthi, Mohan; Ribeiro, Carolina; Chabregas, Sabrina Moutinho; Falco, Maria Cristina; Henrique-Silva, Flavio

    2017-01-01

    Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

  7. Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases.

    PubMed

    Zhang, Yao; Luan, Xiao; Zhang, Huaiyuan; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

    2017-06-21

    γ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides. To enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain. These findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.

  8. NDRG2 overexpression suppresses hepatoma cells survival during metabolic stress through disturbing the activation of fatty acid oxidation.

    PubMed

    Pan, Tao; Zhang, Mei; Zhang, Fang; Yan, Guang; Ru, Yi; Wang, Qinhao; Zhang, Yao; Wei, Xuehui; Xu, Xinyuan; Shen, Lan; Zhang, Jian; Wu, Kaichun; Yao, Libo; Li, Xia

    2017-02-05

    Because of the high nutrient consumption and inadequate vascularization, solid tumor constantly undergoes metabolic stress during tumor development. Oncogenes and tumor suppressor genes participated in cancer cells' metabolic reprogramming. N-Myc downstream regulated gene 2 (NDRG2) is a recently identified tumor suppressor gene, but its function in cancer metabolism, particularly during metabolic stress, remains unclear. In this study, we found that NDRG2 overexpression significantly reduced hepatoma cell proliferation and enhanced cell apoptosis under glucose limitation. Moreover, NDRG2 overexpression aggravated energy imbalance and oxidative stress by decreasing the intracellular ATP and NADPH generation and increasing ROS levels. Strikingly, NDRG2 inhibited the activation of fatty acid oxidation (FAO), which preserves ATP and NADPH purveyance in the absence of glucose. Finally, mechanistic investigation showed that NDRG2 overexpression suppressed the glucose-deprivation induced AMPK/ACC pathway activation in hepatoma cells, whereas the expression of a constitutively active form of AMPK abrogated glucose-deprivation induced AMPK activation and cell apoptosis. Thus, as a negative regulator of AMPK, NDRG2 disturbs the induction of FAO genes by glucose limitation, leading to dysregulation of ATP and NADPH, and thus reduces the tolerance of hepatoma cells to glucose limitation.

  9. Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus.

    PubMed

    Moyano, Elisabet; Jouhikainen, Katja; Tammela, Päivi; Palazón, Javier; Cusidó, Rosa M; Piñol, M Teresa; Teeri, Teemu H; Oksman-Caldentey, Kirsi-Marja

    2003-01-01

    In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.

  10. Overexpression of serum response factor in astrocytes improves neuronal plasticity in a model of early alcohol exposure.

    PubMed

    Paul, A P; Medina, A E

    2012-09-27

    Neuronal plasticity deficits underlie many of the cognitive problems seen in fetal alcohol spectrum disorders (FASD). We have developed a ferret model showing that early alcohol exposure leads to a persistent disruption in ocular dominance (OD) plasticity. Recently, we showed that this deficit could be reversed by overexpression of serum response factor (SRF) in the primary visual cortex during the period of monocular deprivation (MD). Surprisingly, this restoration was observed throughout the extent of visual cortex and most of the cells transfected by the virus were positive for the astrocytic marker GFAP rather than the neuronal marker NeuN. Here we test whether overexpression of SRF exclusively in astrocytes is sufficient to restore OD plasticity in alcohol-exposed ferrets. To accomplish that, first we exposed cultured astrocytes to Sindbis viruses carrying either a constitutively active form of SRF (SRF+), a dominant negative (SRF-) or control Green Fluorescent Protein (GFP). After 24h, these astrocytes were implanted in the visual cortex of alcohol-exposed animals or saline controls one day before MD. Optical imaging of intrinsic signals showed that alcohol-exposed animals that were implanted with astrocytes expressing SRF, but not SRF- or GFP, showed robust restoration of OD plasticity in all visual cortex. These findings suggest that overexpression of SRF exclusively in astrocytes can improve neuronal plasticity in FASD. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. FGFR2 is overexpressed in myxoid liposarcoma and inhibition of FGFR signaling impairs tumor growth in vitro

    PubMed Central

    Künstlinger, Helen; Fassunke, Jana; Schildhaus, Hans-Ulrich; Brors, Benedikt; Heydt, Carina; Ihle, Michaela Angelika; Mechtersheimer, Gunhild; Wardelmann, Eva; Büttner, Reinhard; Merkelbach-Bruse, Sabine

    2015-01-01

    Myxoid liposarcomas account for more than one third of liposarcomas and about 10% of all adult soft tissue sarcomas. The tumors are characterized by specific chromosomal translocations leading to the chimeric oncogenes FUS-DDIT3 or EWS1R-DDIT3. The encoded fusion proteins act as aberrant transcription factors. Therefore, we implemented comparative expression analyses using whole-genome microarrays in tumor and fat tissue samples. We aimed at identifying differentially expressed genes which may serve as diagnostic or prognostic biomarkers or as therapeutic targets. Microarray analyses revealed overexpression of FGFR2 and other members of the FGF/FGFR family. Overexpression of FGFR2 was validated by qPCR, immunohistochemistry and western blot analysis in primary tumor samples. Treatment of the myxoid liposarcoma cell lines MLS 402 and MLS 1765 with the FGFR inhibitors PD173074, TKI258 (dovitinib) and BGJ398 as well as specific siRNAs reduced cell proliferation, induced apoptosis and delayed cell migration. Combination of FGFR inhibitors with trabectedin further increased the effect. Our study demonstrates overexpression of FGFR2 and a functional role of FGFR signaling in myxoid liposarcoma. As FGFR inhibition showed effects on proliferation and cell migration and induced apoptosis in vitro, our data indicate the potential use of FGFR inhibitors as a targeted therapy for these tumors. PMID:26036639

  12. Overexpression of circadian clock protein cryptochrome (CRY) 1 alleviates sleep deprivation-induced vascular inflammation in a mouse model.

    PubMed

    Qin, Bing; Deng, Yunlong

    2015-01-01

    Disturbance of the circadian clock by sleep deprivation has been proposed to be involved in the regulation of inflammation. However, the underlying mechanism of circadian oscillator components in regulating the pro-inflammatory process during sleep deprivation remains poorly understood. Using a sleep deprivation mouse model, we showed here that sleep deprivation increased the expression of pro-inflammatory cytokines expression and decreased the expression of cryptochrome 1 (CRY1) in vascular endothelial cells. Furthermore, the adhesion molecules including intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin were elevated in vascular endothelial cells and the monocytes binding to vascular endothelial cells were also increased by sleep deprivation. Interestingly, overexpression of CRY1 in a mouse model by adenovirus vector significantly inhibited the expression of inflammatory cytokines and adhesion molecules, and NF-κB signal pathway activation, as well as the binding of monocytes to vascular endothelial cells. Using a luciferase reporter assay, we found that CRY1 could repress the transcriptional activity of nuclear factor (NF)-κB in vitro. Subsequently, we demonstrated that overexpression of CRY1 inhibited the basal concentration of cyclic adenosine monophosphate (cAMP), leading to decreased protein kinase A activity, which resulted in decreased phosphorylation of p65. Taken together, these results suggested that the overexpression of CRY1 inhibited sleep deprivation-induced vascular inflammation that might be associated with NF-κB and cAMP/PKA pathways.

  13. FGFR2 is overexpressed in myxoid liposarcoma and inhibition of FGFR signaling impairs tumor growth in vitro.

    PubMed

    Künstlinger, Helen; Fassunke, Jana; Schildhaus, Hans-Ulrich; Brors, Benedikt; Heydt, Carina; Ihle, Michaela Angelika; Mechtersheimer, Gunhild; Wardelmann, Eva; Büttner, Reinhard; Merkelbach-Bruse, Sabine

    2015-08-21

    Myxoid liposarcomas account for more than one third of liposarcomas and about 10% of all adult soft tissue sarcomas. The tumors are characterized by specific chromosomal translocations leading to the chimeric oncogenes FUS-DDIT3 or EWS1R-DDIT3. The encoded fusion proteins act as aberrant transcription factors. Therefore, we implemented comparative expression analyses using whole-genome microarrays in tumor and fat tissue samples. We aimed at identifying differentially expressed genes which may serve as diagnostic or prognostic biomarkers or as therapeutic targets. Microarray analyses revealed overexpression of FGFR2 and other members of the FGF/FGFR family. Overexpression of FGFR2 was validated by qPCR, immunohistochemistry and western blot analysis in primary tumor samples. Treatment of the myxoid liposarcoma cell lines MLS 402 and MLS 1765 with the FGFR inhibitors PD173074, TKI258 (dovitinib) and BGJ398 as well as specific siRNAs reduced cell proliferation, induced apoptosis and delayed cell migration. Combination of FGFR inhibitors with trabectedin further increased the effect. Our study demonstrates overexpression of FGFR2 and a functional role of FGFR signaling in myxoid liposarcoma. As FGFR inhibition showed effects on proliferation and cell migration and induced apoptosis in vitro, our data indicate the potential use of FGFR inhibitors as a targeted therapy for these tumors.

  14. Amphiregulin impairs apoptosis-stimulating protein 2 of p53 overexpression-induced apoptosis in hepatoma cells.

    PubMed

    Liu, Kai; Lin, Dongdong; Ouyang, Yabo; Pang, Lijun; Guo, Xianghua; Wang, Shanshan; Zang, Yunjin; Chen, Dexi

    2017-03-01

    Overexpression of apoptosis-stimulating protein 2 of p53 (ASPP2) induces apoptotic cell death in hepatoma cells (e.g. HepG2 cells) by enhancing the transactivation activity of p53, but long-term ASPP2 overexpression fails to induce more apoptosis since activation of the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway impairs the pro-apoptotic role of ASPP2. In this study, in recombinant adenovirus-ASPP2-infected HepG2 cells, ASPP2 overexpression induces amphiregulin expression in a p53-dependent manner. Although amphiregulin initially contributes to ASPP2-induced apoptosis, it eventually impairs the pro-apoptotic function of ASPP2 by activating the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway, leading to apoptosis resistance. Moreover, blocking soluble amphiregulin with a neutralizing antibody also significantly increased apoptotic cell death of HepG2 cells due to treatment with methyl methanesulfonate, cisplatin, or a recombinant p53 adenovirus, suggesting that the function of amphiregulin involved in inhibiting apoptosis may be a common mechanism by which hepatoma cells escape from stimulus-induced apoptosis. Thus, our data elucidate an apoptosis-evasion mechanism in hepatocellular carcinoma and have potential implications for hepatocellular carcinoma therapy.

  15. Overexpressing the Sedum alfredii Cu/Zn Superoxide Dismutase Increased Resistance to Oxidative Stress in Transgenic Arabidopsis

    PubMed Central

    Li, Zhen; Han, Xiaojiao; Song, Xixi; Zhang, Yunxing; Jiang, Jing; Han, Qiang; Liu, Mingying; Qiao, Guirong; Zhuo, Renying

    2017-01-01

    Superoxide dismutase (SOD) is a very important reactive oxygen species (ROS)-scavenging enzyme. In this study, the functions of a Cu/Zn SOD gene (SaCu/Zn SOD), from Sedum alfredii, a cadmium (Cd)/zinc/lead co-hyperaccumulator of the Crassulaceae, was characterized. The expression of SaCu/Zn SOD was induced by Cd stress. Compared with wild-type (WT) plants, overexpression of SaCu/Zn SOD gene in transgenic Arabidopsis plants enhanced the antioxidative defense capacity, including SOD and peroxidase activities. Additionally, it reduced the damage associated with the overproduction of hydrogen peroxide (H2O2) and superoxide radicals (O2•-). The influence of Cd stress on ion flux across the root surface showed that overexpressing SaCu/Zn SOD in transgenic Arabidopsis plants has greater Cd uptake capacity existed in roots. A co-expression network based on microarray data showed possible oxidative regulation in Arabidopsis after Cd-induced oxidative stress, suggesting that SaCu/Zn SOD may participate in this network and enhance ROS-scavenging capability under Cd stress. Taken together, these results suggest that overexpressing SaCu/Zn SOD increased oxidative stress resistance in transgenic Arabidopsis and provide useful information for understanding the role of SaCu/Zn SOD in response to abiotic stress. PMID:28659953

  16. Overexpression of a brassinosteroid biosynthetic gene Dwarf enhances photosynthetic capacity through activation of Calvin cycle enzymes in tomato.

    PubMed

    Li, Xiao-Jing; Guo, Xie; Zhou, Yan-Hong; Shi, Kai; Zhou, Jie; Yu, Jing-Quan; Xia, Xiao-Jian

    2016-01-28

    Genetic manipulation of brassinosteroid (BR) biosynthesis or signaling is a promising strategy to improve crop yield and quality. However, the relationships between the BR-promoted growth and photosynthesis and the exact mechanism of BR-regulated photosynthetic capacity are not clear. Here, we generated transgenic tomato plants by overexpressing Dwarf, a BR biosynthetic gene that encodes the CYP85A1, and compared the photosynthetic capacity with the BR biosynthetic mutant d (im) and wild type. Overexpression of Dwarf promoted net photosynthetic rate (P N), whereas BR deficiency in d (im) led to a significant inhibition in P N as compared with WT. The activation status of RuBisCO, and the protein content and activity of RuBisCO activase, but not the total content and transcripts of RuBisCO were closely related to the endogenous BR levels in different genotypes. However, endogenous BR positively regulated the expression and activity of fructose-1,6-bisphosphatase. Dwarf overexpression enhanced the activity of dehydroascorbate reductase and glutathione reductase, leading to a reduced redox status, whereas BR deficiency had the contrasting effects. In addition, BR induced a reduction of 2-cystein peroxiredoxin without altering the protein content. BR plays a role in the regulation of photosynthesis. BR can increase the photosynthetic capacity by inducing a reduced redox status that maintains the activation states of Calvin cycle enzymes.

  17. Lead-free piezoceramics

    NASA Astrophysics Data System (ADS)

    Saito, Yasuyoshi; Takao, Hisaaki; Tani, Toshihiko; Nonoyama, Tatsuhiko; Takatori, Kazumasa; Homma, Takahiko; Nagaya, Toshiatsu; Nakamura, Masaya

    2004-11-01

    Lead has recently been expelled from many commercial applications and materials (for example, from solder, glass and pottery glaze) owing to concerns regarding its toxicity. Lead zirconium titanate (PZT) ceramics are high-performance piezoelectric materials, which are widely used in sensors, actuators and other electronic devices; they contain more than 60 weight per cent lead. Although there has been a concerted effort to develop lead-free piezoelectric ceramics, no effective alternative to PZT has yet been found. Here we report a lead-free piezoelectric ceramic with an electric-field-induced strain comparable to typical actuator-grade PZT. We achieved this through the combination of the discovery of a morphotropic phase boundary in an alkaline niobate-based perovskite solid solution, and the development of a processing route leading to highly <001> textured polycrystals. The ceramic exhibits a piezoelectric constant d33 (the induced charge per unit force applied in the same direction) of above 300picocoulombs per newton (pCN-1), and texturing the material leads to a peak d33 of 416pCN-1. The textured material also exhibits temperature-independent field-induced strain characteristics.

  18. Lead-free piezoceramics.

    PubMed

    Saito, Yasuyoshi; Takao, Hisaaki; Tani, Toshihiko; Nonoyama, Tatsuhiko; Takatori, Kazumasa; Homma, Takahiko; Nagaya, Toshiatsu; Nakamura, Masaya

    2004-11-04

    Lead has recently been expelled from many commercial applications and materials (for example, from solder, glass and pottery glaze) owing to concerns regarding its toxicity. Lead zirconium titanate (PZT) ceramics are high-performance piezoelectric materials, which are widely used in sensors, actuators and other electronic devices; they contain more than 60 weight per cent lead. Although there has been a concerted effort to develop lead-free piezoelectric ceramics, no effective alternative to PZT has yet been found. Here we report a lead-free piezoelectric ceramic with an electric-field-induced strain comparable to typical actuator-grade PZT. We achieved this through the combination of the discovery of a morphotropic phase boundary in an alkaline niobate-based perovskite solid solution, and the development of a processing route leading to highly <001> textured polycrystals. The ceramic exhibits a piezoelectric constant d33 (the induced charge per unit force applied in the same direction) of above 300 picocoulombs per newton (pC N(-1)), and texturing the material leads to a peak d33 of 416 pC N(-1). The textured material also exhibits temperature-independent field-induced strain characteristics.

  19. Lead in potatoes

    PubMed Central

    Warren, Harry V.

    1975-01-01

    There are specific environments where potatoes contain much larger amounts of lead than is generally realised. Nevertheless, if we accept the hypothesis that human adults are only likely to be harmed if they absorb more than 100 micrograms of lead daily for extended periods10 then only in rare instances are they apt to be adversely affected by eating potatoes. Medical data suggest that where children are involved the acceptable amounts of lead are significantly less. However, where potatoes do show evidence of contamination by virtue of their high lead content, the possibility of more general contamination should be investigated. The intake of lead from potatoes, if supplemented by lead provided from other foodstuffs, from air, and possibly from water, can well reach unacceptable amounts. Patterson's12 claim that most people in industrialised countries are suffering from a chronic lead insult, does seem justified but, because of the remarkable ability of humans to adapt to some conditions, but how much this insult constitutes a menace to heath must be dealt with by medical men. PMID:1177205

  20. CREB Overexpression Ameliorates Age-related Behavioral and Biophysical Deficits

    NASA Astrophysics Data System (ADS)

    Yu, Xiao-Wen

    Age-related cognitive deficits are observed in both humans and animals. Yet, the molecular mechanisms underlying these deficits are not yet fully elucidated. In aged animals, a decrease in intrinsic excitability of pyramidal neurons from the CA1 sub-region of hippocampus is believed to contribute to age-related cognitive impairments, but the molecular mechanism(s) that modulate both these factors has yet to be identified. Increasing activity of the transcription factor cAMP response element-binding protein (CREB) in young adult rodents has been shown to facilitate cognition, and increase intrinsic excitability of their neurons. However, how CREB changes with age, and how that impacts cognition in aged animals, is not clear. Therefore, we first systematically characterized age- and training-related changes in CREB levels in dorsal hippocampus. At a remote time point after undergoing behavioral training, levels of total CREB and activated CREB (phosphorylated at S133, pCREB) were measured in both young and aged rats. We found that pCREB, but not total CREB was significantly reduced in dorsal CA1 of aged rats. Importantly, levels of pCREB were found to be positively correlated with short-term spatial memory in both young and aged rats i.e. higher pCREB in dorsal CA1 was associated with better spatial memory. These findings indicate that an age-related deficit in CREB activity may contribute to the development of age-related cognitive deficits. However, it was still unclear if increasing CREB activity would be sufficient to ameliorate age-related cognitive, and biophysical deficits. To address this question, we virally overexpressed CREB in CA1, where we found the age-related deficit. Young and aged rats received control or CREB virus, and underwent water maze training. While control aged animals exhibited deficits in long-term spatial memory, aged animals with CREB overexpression performed at levels comparable to young animals. Concurrently, aged neurons

  1. Prevalent overexpression of prolyl isomerase Pin1 in human cancers.

    PubMed

    Bao, Lere; Kimzey, Amy; Sauter, Guido; Sowadski, Janusz M; Lu, Kun Ping; Wang, Da-Gong

    2004-05-01

    Phosphorylation of proteins on serine or threonine residues preceding proline (pSer/Thr-Pro) is a major regulatory mechanism in cell proliferation and transformation. Interestingly, the pSer/Thr-Pro motifs in proteins exist in two distinct cis and trans conformations, whose conversion rate is normally reduced on phosphorylation, but is catalyzed specifically by the prolyl isomerase Pin1. Pin1 can catalytically induce conformational changes in proteins after phosphorylation, thereby having profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location, and/or turnover of certain phosphorylated proteins. Recently, it has been shown that Pin1 is overexpressed in human breast cancer cell lines and cancer tissues and plays a critical role in the transformation of mammary epithelial cells by activating multiple oncogenic pathways. Furthermore, Pin1 expression is an excellent independent prognostic marker in prostate cancer. However, little is known about Pin1 expression in other human normal and cancerous tissues. In the present study, we quantified Pin1 expression in 2041 human tumor samples and 609 normal tissue samples as well as normal and transformed human cell lines. We found that Pin1 was usually expressed at very low levels in most normal tissues and its expression was normally associated with cell proliferation, with high Pin1 levels being found only in a few cell types. However, Pin1 was strikingly overexpressed in many different human cancers. Most tumors (38 of 60 tumor types) have Pin1 overexpression in more than 10% of the cases, as compared with the corresponding normal controls, which included prostate, lung, ovary, cervical, brain tumors, and melanoma. Consistent with these findings, Pin1 expression in human cancer cell lines was also higher than that in the normal cell lines examined. These results indicate that Pin1 overexpression is a prevalent and specific event in human cancers. Given previous findings

  2. Expression of Wnt3 activates Wnt/β-catenin pathway and promotes EMT-like phenotype in trastuzumab-resistant HER2-overexpressing breast cancer cells.

    PubMed

    Wu, Yanyuan; Ginther, Charles; Kim, Juri; Mosher, Nicole; Chung, Seyung; Slamon, Dennis; Vadgama, Jaydutt V

    2012-12-01

    To understand the mechanisms leading to trastuzumab resistance in HER2-overexpressing breast tumors, we created trastuzumab-insensitive cell lines (SKBR3/100-8 and BT474/100-2). The cell lines maintain HER2 receptor overexpression and show increase in EGF receptor (EGFR). Upon trastuzumab treatment, SKBR3/100-8 and BT474/100-2 cell lines displayed increased growth rate and invasiveness. The trastuzumab resistance in SKBR3/100-8 and BT474/100-2 was accompanied with activation of the Wnt/β-catenin signaling pathway. Further investigation found that Wnt3 overexpression played a key role toward the development of trastuzumab resistance. The expression of Wnt3 in trastuzumab-resistant cells increased nuclear expression of β-catenin and transactivated expression of EGFR. The increased Wnt3 in the trastuzumab-resistant cells also promoted a partial EMT-like transition (epithelial-to-mesenchymal transition); increased N-cadherin, Twist, Slug; and decreased E-cadherin. Knockdown of Wnt3 by siRNA restored cytoplasmic expression of β-catenin and decreased EGFR expression in trastuzumab-resistant cells. Furthermore, the EMT markers were decreased, E-cadherin was increased, and the cell invasiveness was inhibited in response to the Wnt3 downregulation. Conversely, SKBR3 cells which had been stably transfected with full-length Wnt3 exhibited EMT-like transition. The Wnt3 transfectants, SKBR3/Wnt3-7 and SKBR3/Wnt3-9, showed a significant decrease in E-cadherin and increase in N-cadherin, Twist, and Slug. The cells were less sensitive to trastuzumab than parental SKBR3 and vector-transfected cells. In summary, our data suggest that Wnt3 overexpression activates Wnt/β-catenin signaling pathway that leads to transactivation of EGFR and promotes EMT-like transition. This could be an important mechanism leading to trastuzumab resistance in HER2-overexpressing breast cancer cells.

  3. Over-expression of SlJA2 decreased heat tolerance of transgenic tobacco plants via salicylic acid pathway.

    PubMed

    Liu, Zhong-Ming; Yue, Meng-Meng; Yang, Dong-Yue; Zhu, Shao-Bo; Ma, Na-Na; Meng, Qing-Wei

    2017-04-01

    Over-expression of SlJA2 decreased the accumulation of SA, which resulted in significant physiological and gene expression changes in transgenic tobacco plants, leading to the decreased heat tolerance of transgenic tobacco. NAC family, the largest transcription factors in plants, responses to different environmental stimuli. Here, we isolated a typical NAC transcription factor (SlJA2) from tomato and got transgenic tobacco with SlJA2 over-expression. Expression of SlJA2 was induced by heat stress (42 °C), chilling stress (4 °C), drought stress, osmotic stress, abscisic acid, and salicylic acid. Over-expression of SlJA2 decreased the accumulation of salicylic acid by regulating expression of salicylic acid degradation gene under heat stress. Compared to WT plants, stomatal apertures and water loss increased in transgenic plants, and the damage of photosynthetic apparatus and chlorophyll breakdown were more serious in transgenic plants under heat stress. Meanwhile, more H2O2 and O2(·-) were accumulated transgenic plants and proline synthesis was restricted, which resulted in more serious oxidative damage compared to WT. qRT-PCR analysis showed that over-expression of SlJA2 could down-regulate genes involved in reactive oxygen species scavenging, proline biosynthesis, and response to heat stress. All the above results indicated that SlJA2 may be a negative regulator responded to plant's heat tolerance. Thus, this study provides new insight into roles of NAC family member in plant response to abiotic stress.

  4. Constitutive overexpression of soybean plasma membrane intrinsic protein GmPIP1;6 confers salt tolerance

    PubMed Central

    2014-01-01

    Background Under saline conditions, plant growth is depressed via osmotic stress and salt can accumulate in leaves leading to further depression of growth due to reduced photosynthesis and gas exchange. Aquaporins are proposed to have a major role in growth of plants via their impact on root water uptake and leaf gas exchange. In this study, soybean plasma membrane intrinsic protein 1;6 (GmPIP1;6) was constitutively overexpressed to evaluate the function of GmPIP1;6 in growth regulation and salt tolerance in soybean. Results GmPIP1;6 is highly expressed in roots as well as reproductive tissues and the protein targeted to the plasma membrane in onion epidermis. Treatment with 100 mM NaCl resulted in reduced expression initially, then after 3 days the expression was increased in root and leaves. The effects of constitutive overexpression of GmPIP1;6 in soybean was examined under normal and salt stress conditions. Overexpression in 2 independent lines resulted in enhanced leaf gas exchange, but not growth under normal conditions compared to wild type (WT). With 100 mM NaCl, net assimilation was much higher in the GmPIP1;6-Oe and growth was enhanced relative to WT. GmPIP1;6-Oe plants did not have higher root hydraulic conductance (Lo) under normal conditions, but were able to maintain Lo under saline conditions compared to WT which decreased Lo. GmPIP1;6-Oe lines grown in the field had increased yield resulting mainly from increased seed size. Conclusions The general impact of overexpression of GmPIP1;6 suggests that it may be a multifunctional aquaporin involved in root water transport, photosynthesis and seed loading. GmPIP1;6 is a valuable gene for genetic engineering to improve soybean yield and salt tolerance. PMID:24998596

  5. Overexpression of CDKN1B inhibits fibroblast proliferation in a rabbit model of experimental glaucoma filtration surgery.

    PubMed

    Yang, Jian-gang; Deng, Ying; Zhou, Ling-xiao; Li, Xiao-yan; Sun, Peng-rui; Sun, Nai-xue

    2013-01-14

    To investigate the potential antiproliferative effect of cyclin-dependent kinase inhibitor 1B (CDKN1B) overexpression in a rabbit model of glaucoma filtration surgery (GFS). The recombinant adenovector expressing exogenous CDKN1B was delivered to Tenon's capsule by subconjunctival injection during unilateral filtration surgery. The time course of CDKN1B expression was monitored by immunohistochemistry and Western blot analysis. Evaluation of proliferating activity was performed by proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizing region (AgNOR) staining, and fibroblast-specific protein 1 (FSP-1). Cyclin-dependent kinase 2 (Cdk2) and Cdk4 expression were detected with immunohistochemical analysis. The overexpression of CDKN1B in Tenon's capsule was monitored throughout the experimental period. Immunoreactivity to CDKN1B was mainly observed in the nucleus of fibroblasts. The increased expression of CDKN1B in sclera was detected up to 21 days after viral infection, whereas the level of CDKN1B protein in corneal stroma was not significantly increased. The overexpression of CDKN1B induced a significant decrease in AgNOR number/nucleus and area/nucleus, PCNA staining, FSP-1 positive cells, and the decreased expressions of Cdk2 and Cdk4, as evidenced by nuclear and cytoplasmic immunoreactivity to Cdk2 and Cdk4 antibodies in positive fibroblasts. The persistent overexpression of CDKN1B mediated by the recombinant adenovector expressing exogenous CDKN1B in Tenon's fibroblasts after GFS may lead to the inhibition of fibroblast proliferation and the downregulation of Cdk2 and Cdk4 activity, thereby reducing the severity of scar formation and the surgical outcome.

  6. Kinase Activity of Overexpressed HipA Is Required for Growth Arrest and Multidrug Tolerance in Escherichia coli▿

    PubMed Central

    Correia, Frederick F.; D'Onofrio, Anthony; Rejtar, Tomas; Li, Lingyun; Karger, Barry L.; Makarova, Kira; Koonin, Eugene V.; Lewis, Kim

    2006-01-01

    Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in Escherichia coli. HipA is a “toxin” that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the hipBA operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg2+-binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation. PMID:17041039

  7. Overexpression of MN1 Confers Resistance to Chemotherapy, Accelerates Leukemia Onset, and Suppresses p53 and Bim Induction

    PubMed Central

    Pardee, Timothy S.

    2012-01-01

    Background The transcriptional co-activator MN1 confers a worse prognosis for patients with acute myeloid leukemia (AML) when highly expressed; however, the mechanisms involved are unknown. We sought to model the effects of high MN1 expression in AML models to explore the underlying mechanisms. Methodology/Principal Findings We used cell lines and a genetically defined mouse model of AML to examine the effects of MN1 overexpression on prognosis and response to cytarabine and doxorubicin in vitro and in vivo. Murine AML that was engineered to overexpress MN1 became more aggressive in vivo, leading to shortened survival in both treated and control groups. In vitro murine AML cells that overexpressed MN1 became resistant to treatment with cytarabine and highly resistant to doxorubicin. This resistant phenotype was also seen in vivo, where treatment with the combination of cytarabine and doxorubicin selected for cells expressing MN1. When therapy-induced DNA damage levels were assessed by γH2AX foci, no reduction was seen in MN1 expressing cells arguing against a drug efflux mechanism. Despite no reduction in DNA damage, MN1-expressing cells showed less apoptosis as assessed by annexin V and propidium iodide staining. Following treatment, p53 and BIM induction were markedly reduced in cells expressing MN1. Pharmacologic inhibition of the p53 E3 ligase MDM2 resulted in increased p53 levels and improved response to doxorubicin in vitro. Conclusions/Significance MN1 overexpression accelerates an already aggressive leukemia, confers resistance to chemotherapy, and suppresses p53 and BIM induction, resulting in decreased apoptosis. This provides a mechanistic explanation of the poor prognosis observed with high MN1 expression and suggests that therapies directed at increasing p53 function may be useful for these patients. PMID:22905229

  8. Overexpression of the transcription factor Sp1 activates the OAS-RNAse L-RIG-I pathway.

    PubMed

    Dupuis-Maurin, Valéryane; Brinza, Lilia; Baguet, Joël; Plantamura, Emilie; Schicklin, Stéphane; Chambion, Solène; Macari, Claire; Tomkowiak, Martine; Deniaud, Emmanuelle; Leverrier, Yann; Marvel, Jacqueline; Michallet, Marie-Cécile

    2015-01-01

    Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.

  9. Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

    PubMed Central

    Dupuis-Maurin, Valéryane; Brinza, Lilia; Baguet, Joël; Plantamura, Emilie; Schicklin, Stéphane; Chambion, Solène; Macari, Claire; Tomkowiak, Martine; Deniaud, Emmanuelle; Leverrier, Yann

    2015-01-01

    Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen. PMID:25738304

  10. Overexpression of GLUTAMINE DUMPER1 Leads to Hypersecretion of Glutamine from Hydathodes of Arabidopsis LeavesW⃞

    PubMed Central

    Pilot, Guillaume; Stransky, Harald; Bushey, Dean F.; Pratelli, Réjane; Ludewig, Uwe; Wingate, Vincent P.M.; Frommer, Wolf B.

    2004-01-01

    Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants. PMID:15208395

  11. Cardiac-specific overexpression of dominant-negative CREB leads to increased mortality and mitochondrial dysfunction in female mice.

    PubMed

    Watson, Peter A; Birdsey, Nicholas; Huggins, Gordon S; Svensson, Eric; Heppe, Daniel; Knaub, Leslie

    2010-12-01

    Cardiac failure is associated with diminished activation of the transcription factor cyclic nucleotide regulatory element binding-protein (CREB), and heart-specific expression of a phosphorylation-deficient CREB mutant in transgenic mice [dominant negative CREB (dnCREB) mice] recapitulates the contractile phenotypes of cardiac failure (Fentzke RC, Korcarz CE, Lang RM, Lin H, Leiden JM. Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart. J Clin Invest 101: 2415-2426, 1998). In the present study, we demonstrated significantly elevated mortality and contractile dysfunction in female compared with male dnCREB mice. Female dnCREB mice demonstrated a 21-wk survival of only 17% compared with 67% in males (P < 0.05) and exclusively manifest decreased cardiac peroxisome proliferator-activated receptor-γ coactivator-1α and estrogen-related receptor-α content, suggesting sex-related effects on cardiac mitochondrial function. Hearts from 4-wk-old dnCREB mice of both sexes demonstrated diminished mitochondrial respiratory capacity compared with nontransgenic controls. However, by 12 wk of age, there was a significant decrease in mitochondrial density (citrate synthase activity) and deterioration of mitochondrial structure, as demonstrated by transmission electron microscopy, in female dnCREB mice, which were not found in male transgenic littermates. Subsarcolemmal mitochondria isolated from hearts of female, but not male, dnCREB mice demonstrated increased ROS accompanied by decreases in the expression/activity of the mitochondrial antioxidants MnSOD and glutathione peroxidase. These results demonstrate that heart-specific dnCREB expression results in mitochondrial respiratory dysfunction in both sexes; however, increased oxidant burden, reduced antioxidant expression, and disrupted mitochondrial structure are exacerbated by the female sex, preceding and contributing to the greater contractile morbidity and mortality. These results provide further support for the role of the CREB transcription factor in regulating mitochondrial integrity and identify a critical pathway that may contribute to sex differences in heart failure.

  12. Cardiac-specific overexpression of dominant-negative CREB leads to increased mortality and mitochondrial dysfunction in female mice

    PubMed Central

    Birdsey, Nicholas; Huggins, Gordon S.; Svensson, Eric; Heppe, Daniel; Knaub, Leslie

    2010-01-01

    Cardiac failure is associated with diminished activation of the transcription factor cyclic nucleotide regulatory element binding-protein (CREB), and heart-specific expression of a phosphorylation-deficient CREB mutant in transgenic mice [dominant negative CREB (dnCREB) mice] recapitulates the contractile phenotypes of cardiac failure (Fentzke RC, Korcarz CE, Lang RM, Lin H, Leiden JM. Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart. J Clin Invest 101: 2415–2426, 1998). In the present study, we demonstrated significantly elevated mortality and contractile dysfunction in female compared with male dnCREB mice. Female dnCREB mice demonstrated a 21-wk survival of only 17% compared with 67% in males (P < 0.05) and exclusively manifest decreased cardiac peroxisome proliferator-activated receptor-γ coactivator-1α and estrogen-related receptor-α content, suggesting sex-related effects on cardiac mitochondrial function. Hearts from 4-wk-old dnCREB mice of both sexes demonstrated diminished mitochondrial respiratory capacity compared with nontransgenic controls. However, by 12 wk of age, there was a significant decrease in mitochondrial density (citrate synthase activity) and deterioration of mitochondrial structure, as demonstrated by transmission electron microscopy, in female dnCREB mice, which were not found in male transgenic littermates. Subsarcolemmal mitochondria isolated from hearts of female, but not male, dnCREB mice demonstrated increased ROS accompanied by decreases in the expression/activity of the mitochondrial antioxidants MnSOD and glutathione peroxidase. These results demonstrate that heart-specific dnCREB expression results in mitochondrial respiratory dysfunction in both sexes; however, increased oxidant burden, reduced antioxidant expression, and disrupted mitochondrial structure are exacerbated by the female sex, preceding and contributing to the greater contractile morbidity and mortality. These results provide further support for the role of the CREB transcription factor in regulating mitochondrial integrity and identify a critical pathway that may contribute to sex differences in heart failure. PMID:20935148

  13. Overexpression of Tyro3 receptor tyrosine kinase leads to the acquisition of taxol resistance in ovarian cancer cells.

    PubMed

    Lee, Chuhee

    2015-07-01

    The majority of patients with ovarian cancer are diagnosed at the advanced stages (III/IV) and their 5-year-survival rate is relatively low. One of the major causes of the poor prognosis of ovarian cancer is the development of resistance to first-line chemotherapy, including platinum and taxol. Therefore, improvements in current understanding of chemoresistance is required for the successful treatment of ovarian cancer. In the present study, taxol-resistant ovarian cancer cells, SKOV3/TR, were established by exposing parental SKOV3 cells to increasing concentrations of taxol. . Briefly, cells were treated with 1.5 nM (for 4 weeks), 3 nM (for 4 weeks), 6 nM (for 5 weeks), 12 nM (for 5 weeks) and 24 nM taxol (for 8 weeks) over 6 months. The SKOV3/TR cells were found to be smaller in size and rounder in shape compared with their parental cells. Cell viability and colony formation assays demonstrated an increase in the population doubling time of the SKOV3/TR cells, indicating a reduction in the proliferative capacity of these cells. Reverse transcription-polymerase chain reaction and western blot analysis revealed that, among the TAM receptor tyrosine kinases (RTKs), the mRNA and protein expression levels of Tyro3 RTK were increased, while those of Axl and Mer RTK were decreased in the SKOV3/TR cells. In addition, restoration of the level of Tyro3 by transfecting Tyro3-specific small interfering RNA into the SKOV3/TR cells reduced the proliferative capacity of the cells, indicating that upregulation of the expression of Tyro3 in SKOV3/TR cells may promote survival in the presence of taxol, which eventually resulted in the acquisition of resistance upon taxol treatment. The present study subsequently found that, in the SKOV3/TR cells, the level of intracellular reactive oxygen species (ROS) was elevated, and antioxidant treatment with N-acetyl cysteine (NAC) exerted more profound antiproliferative effects compared with the parental cells. The western blot analysis demonstrated that treatment of the SKOV3/TR cells with NAC reduced the protein expression of Tyro3, and the inhibitory effect of NAC on the phosphorylation of Akt was increased, which may have had a positive effect on the proliferation of the SKOV3/TR cells. The levels of phosphorylation and protein expression of signal transducers and activators of transcription 3 (STAT3) were not affected by NAC treatment, indicating that the phosphorylation of Akt, but not expression or phosphorylation of STAT3, was associated with the increased intracellular ROS level in the SKOV3/TR cells. Taken together, the results of the present study demonstrated that the acquired taxol resistance of ovarian cancer cells was associated with ROS-dependent upregulation in the expression of Tyro3 RTK and the subsequent activation of Akt.

  14. Genomic Rearrangements Leading to Overexpression of Aldo-Keto Reductase YafB of Escherichia coli Confer Resistance to Glyoxal

    PubMed Central

    Kwon, Minsuk; Lee, Junghoon; Lee, Changhan

    2012-01-01

    Glyoxal is toxic and mutagenic α-oxoaldehyde generated in vivo as an oxidation by-product of sugar metabolism. We selected glyoxal-resistant mutants from an Escherichia coli strain lacking major glyoxal-detoxifying genes, gloA and yqhD, by growing cells in medium containing a lethal concentration of glyoxal. The mutants carried diverse genomic rearrangements, such as multibase deletions and recombination, in the upstream region of the yafB gene, encoding an aldo-keto reductase. Since these genomic lesions create transcriptional fusions of the yafB gene to the upstream rrn regulon or eliminate a negative regulatory site, the mutants generally enhanced an expression of the yafB gene. Glyoxal resistances of the mutants are correlated with the levels of yafB transcripts as well as the activities of aldo-keto reductase. An overproduction of YafB in the glyoxal-resistant mutant lacking the putative NsrR-binding site provides evidence that the yafB gene is negatively regulated by this protein. We also observed that the expression of yafB is enhanced with an increased concentration of glyoxal as well as a mutation in the fnr gene, encoding a putative regulator. The bindings of NsrR and Fnr to the yafB promoter were also demonstrated by gel mobility shift assays. PMID:22328670

  15. Ferritin overexpression in Drosophila glia leads to iron deposition in the optic lobes and late-onset behavioral defects.

    PubMed

    Kosmidis, Stylianos; Botella, Jose A; Mandilaras, Konstantinos; Schneuwly, Stephan; Skoulakis, Efthimios M C; Rouault, Tracey A; Missirlis, Fanis

    2011-07-01

    Cellular and organismal iron storage depends on the function of the ferritin protein complex in insects and mammals alike. In the central nervous system of insects, the distribution and relevance of ferritin remain unclear, though ferritin has been implicated in Drosophila models of Alzheimers' and Parkinsons' disease and in Aluminum-induced neurodegeneration. Here we show that transgene-derived expression of ferritin subunits in glial cells of Drosophila melanogaster causes a late-onset behavioral decline, characterized by loss of circadian rhythms in constant darkness and impairment of elicited locomotor responses. Anatomical analysis of the affected brains revealed crystalline inclusions of iron-loaded ferritin in a subpopulation of glial cells but not significant neurodegeneration. Although transgene-induced glial ferritin expression was well tolerated throughout development and in young flies, it turned disadvantageous at older age. The flies we characterize in this report contribute to the study of ferritin in the Drosophila brain and can be used to assess the contribution of glial iron metabolism in neurodegenerative models of disease. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Leading Causes of Blindness

    MedlinePlus

    ... Cataract. Photo courtesy of National Eye Institute, NIH Cataracts Cataracts are a clouding of the lenses in your ... older people. More than 22 million Americans have cataracts. They are the leading cause of blindness in ...

  17. Lead Poisoning Prevention Tips

    MedlinePlus

    ... North Dakota Ohio Oklahoma Oregon Pennsylvania Philadelphia Rhode Island South Carolina South Dakota Tennessee Texas Utah Vermont ... up paint debris after work is completed. Create barriers between living/play areas and lead sources. Until ...

  18. Lead and Your Baby

    MedlinePlus

    ... lead, talk to your boss about changing job responsibilities to help keep you and your baby safe ... Participate & Support Make a donation Giving opportunities Our corporate partners In Your Area Tools & Resources Careers Events ...

  19. Surface superconductivity in lead

    SciTech Connect

    Khlyustikov, I. N.

    2016-02-15

    A transition to the surface superconducting state is detected in lead single crystals at a temperature approximately 0.25 mK higher than the bulk superconducting transition temperature. The (H, T) phase diagram of this state is analyzed.

  20. American Lead Action Memorandum

    EPA Pesticide Factsheets

    ACTION MEMORANDUM— Request for a Time-Critical Removal Action andExemption from the $2 Million and 12-Month Statutory Limits at the AmericanLead Site, Indianapolis, Marion County, Indiana (Site ID #B56J)