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Sample records for downstream effector mdm2

  1. P53 Mdm2 Inhibitors

    NARCIS (Netherlands)

    Khoury, Kareem; Doemling, Alex

    2012-01-01

    The protein-protein interaction (PPI) between p53 and its negative regulator MDM2 comprises one of the most important and intensely studied PPI's involved in preventing the initiation of cancer. The interaction between p53 and MDM2 is conformation-based and is tightly regulated on multiple levels. D

  2. Ribosomal protein S7 is both a regulator and a substrate of MDM2.

    Science.gov (United States)

    Zhu, Yan; Poyurovsky, Masha V; Li, Yingchun; Biderman, Lynn; Stahl, Joachim; Jacq, Xavier; Prives, Carol

    2009-08-14

    MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2's E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals.

  3. Serdemetan antagonizes the Mdm2-HIF1α axis leading to decreased levels of glycolytic enzymes.

    Directory of Open Access Journals (Sweden)

    Jason A Lehman

    Full Text Available Serdemetan (JNJ-26854165, an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1, were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises the possibility of side effects associated with therapeutically targeting Mdm2.

  4. Novel small molecule inhibitors of MDM2/4-p53 interaction, YH264 and its ethyl ester YH263: Preclinical evaluation

    NARCIS (Netherlands)

    Christner, Susan M.; Clausen, Dana M.; Beumer, Jan H.; Parise, Robert A.; Huang, Yi; Dömling, Alexander S.; Eiseman, Julie L.

    2012-01-01

    Introduction: In p53+/+ cells, expression of MDM2/4 leads to turnover of p53 and inhibition of downstream gene transcription decreasing cell cycle arrest or apoptosis. Prevention of MDM2/4-p53 interaction is a promising therapeutic strategy. Two in-house developed small molecule inhibitors of MDM2/4

  5. Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

    Science.gov (United States)

    D'Orsi, Beatrice; Bonner, Helena; Tuffy, Liam P; Düssmann, Heiko; Woods, Ina; Courtney, Michael J; Ward, Manus W; Prehn, Jochen H M

    2012-02-01

    Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

  6. Chemosensitization by antisense oligonucleotides targeting MDM2.

    Science.gov (United States)

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  7. Oncogene Mdm2 takes part in hepatocarcinogenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective:To investigate the role of Mdm2 expression in hepatocellular carcinoma.Methods: Streptavidin-peroxidase conjugation method (SP)was used to observe the expression of Mdm2 and p53 proteins in 61 cases of hepatocellular carcinoma(HCC)and 59 cases of corresponding paracancerous tissues,among which p53 mutations in exons 5~8 were detected in 21 cases by PCR-SSCP.Results:Positive nuclear P53 and Mdm2 immunostains were demonstrated in 57.38% and 26.23% of HCC,1.69% and 3.39% of corresponding paracancerous tissues respectively.The expression of p53 and Mdm2 proteins in hepatocellular carcinoma was significantly higher than that in paracancerous tissues(P<0.01).The expression of P53 and Mdm2 was not significantly correlated.42.86% of hepatocellualr carcinomas showed mutations in exon 7 of p53 gene,and no mutation was found in exons 5,6,8 and paracancerous tissues. 66.67% of mutational cases had P53 overexpression and 11.11%(1/9)showed overexpression of both P53 and Mdm2,Mdm2 overexpression also appeared in 25% of cases without mutations.Conclusions:Mdm2-induced p53 inactivation and p53 gene mutation play an important role in carcinogenesis of hepatocellular carcinoma.Tumorigenic property of Mdm2 itself,together with p53 mutation,may take part in hepatocarcinogenesis.

  8. The p53 inhibitor MDM2 facilitates Sonic Hedgehog-mediated tumorigenesis and influences cerebellar foliation.

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    Reem Malek

    Full Text Available Disruption of cerebellar granular neuronal precursor (GNP maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2(puro, which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1(+/- mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis.

  9. Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

    Science.gov (United States)

    Stoletov, Konstantin V; Terman, Bruce I

    2004-07-16

    We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

  10. Mdm2 ligase dead mutants did not act in a dominant negative manner to re-activate p53, but promoted tumor cell growth.

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    Swaroop, Manju; Sun, Yi

    2003-01-01

    Mdm2 (murine double minute 2) is an oncogene, first identified in BALB/c 3T3 cells. Over-expression and gene amplification of Mdm2 were found in a variety of human cancers. Recently, Mdm2 was found to be an E3 ubiquitin ligase that promotes degradation of p53, which contributes significantly to its oncogenic activity. In this study, we test a hypothesis that Mdm2 ligase dead mutants, which retained p53 binding activity but lost degradation activity, would act in a dominant negative manner to re-activate p53, especially upon stressed conditions. Five Mdm2 constructs expressing wild-type and E3 ligase-dead Mdm2 proteins were generated in a Tet-Off system and transfected into MCF-7 breast cancer cells (p53+/+ with Mdm2 overexpression) as well as MCF10A immortalized breast cells (p53+/+ without Mdm2 overexpression) as a normal control. We found that expression of Mdm2 mutants were tightly regulated by doxycycline. Withdrawal of doxycycline in culture medium triggered overexpression of Mdm2 mutants. However, expression of ligase dead mutants in MCF7 and MCF10A cells did not reactivate p53 as shown by a luciferase-reporter transcription assay and Western blot of p53 and its downstream target p21 under either unstressed condition or after exposure to DNA damaging agents. Biologically, over-expression of Mdm2 mutants had no effect on p53-induced apoptosis following DNA damage. Interestingly, over-expression of Mdm2 mutants promoted growth of MCF7 tumor cells probably via a p53-independent mechanism. Over-expression of Mdm2 mutants, however, had no effect on the growth of normal MCF10A cells and did not cause their transformation. Thus, ligase dead mutants of Mdm2 did not act in a dominant negative manner to reactivate p53 and they are not oncogenes in MCF10A cells.

  11. Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

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    Aishwarya G Jacob

    Full Text Available MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

  12. In vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of pyrazole-based small molecule inhibitors of Mdm2/4-p53 interaction

    NARCIS (Netherlands)

    Christner, Susan M; Clausen, Dana M; Beumer, Jan H; Parise, Robert A; Guo, Jianxia; Huang, Yijun; Dömling, Alexander S; Eiseman, Julie L

    2015-01-01

    PURPOSE: The interaction of p53 with its negative regulators Mdm2/4 has been widely studied (Khoury and Domling in Curr Pharm Des 18(30):4668-4678, 2012). In p53(+/+) cells, expression of Mdm2/4 leads to p53 turnover, inhibition of downstream transcription, decreasing cell cycle arrest, or apoptosis

  13. Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy.

    Science.gov (United States)

    Tovar, Christian; Rosinski, James; Filipovic, Zoran; Higgins, Brian; Kolinsky, Kenneth; Hilton, Holly; Zhao, Xiaolan; Vu, Binh T; Qing, Weiguo; Packman, Kathryn; Myklebost, Ola; Heimbrook, David C; Vassilev, Lyubomir T

    2006-02-07

    The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53-MDM2 interaction.

  14. Specific inhibition of Mdm2-mediated neddylation by Tip60

    DEFF Research Database (Denmark)

    Dohmesen, Christoph; Koeppel, Max; Dobbelstein, Matthias

    2007-01-01

    Tip60 is a histone acetyl transferase (HAT) and a cofactor of transcription, but also an interaction partner of the Mdm2 oncoprotein. The functional consequences of this interaction are only partially understood and were further explored in this study. We found that Tip60 is capable of selectively...... inhibiting the Mdm2- mediated conjugation of Nedd8 to p53, whereas it did not affect p53 ubiquitination. In contrast, the known Mdm2 antagonist p14arf preferentially blocked Ubiquitin conjugation by Mdm2. To identify underlying mechanisms, we studied the intracellular localization of Tip60 and Mdm2. Both...... proteins relocalized each other to the PML nuclear bodies, but a similar localization pattern was observed even in the absence of PML. Analysis of Tip60 deletion mutants revealed that some mutants, while still interacting with Mdm2, failed to relocalize it and to inhibit Mdm2-mediated neddylation...

  15. Proteomics analysis of cellular imatinib targets and their candidate downstream effectors.

    Science.gov (United States)

    Breitkopf, Susanne B; Oppermann, Felix S; Keri, Gyorgy; Grammel, Markus; Daub, Henrik

    2010-11-05

    Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Knowledge about direct cellular targets of kinase-selective drugs and the identification of druggable downstream mediators of oncogenic signaling are relevant for both initial therapy selection and the nomination of alternative targets in case molecular resistance emerges. To address these issues, we performed a proof-of-concept proteomics study designed to monitor drug effects on the pharmacologically tractable subproteome isolated by affinity purification with immobilized, nonselective kinase inhibitors. We applied this strategy to chronic myeloid leukemia cells that express the transforming Bcr-Abl fusion kinase. We used SILAC to measure how cellular treatment with the Bcr-Abl inhibitor imatinib affects protein binding to a generic kinase inhibitor resin and further quantified site-specific phosphorylations on resin-retained proteins. Our integrated approach indicated additional imatinib target candidates, such as flavine adenine dinucleotide synthetase, as well as repressed phosphorylation events on downstream effectors not yet implicated in imatinib-regulated signaling. These included activity-regulating phosphorylations on the kinases Btk, Fer, and focal adhesion kinase, which may qualify them as alternative target candidates in Bcr-Abl-driven oncogenesis. Our approach is rather generic and may have various applications in kinase drug discovery.

  16. Novel Insights into the Molecular Mechanisms Governing Mdm2 Ubiquitination and Destruction

    OpenAIRE

    Fukushima, Hidefumi; Inuzuka, Hiroyuki; Shaik, Shavali; Wei, Wenyi

    2010-01-01

    The Mdm2/p53 pathway is compromised in more than 50% of all human cancers, therefore it is an intensive area of research to understand the upstream regulatory pathways governing Mdm2/p53 activity. Mdm2 is frequently overexpressed in human cancers while the molecular mechanisms underlying the timely destruction of Mdm2 remain unclear. We recently reported that Casein Kinase I phosphorylates Mdm2 at multiple sites to trigger Mdm2 interaction with, and subsequent ubiquitination and destruction b...

  17. Potential Landscape and Flux of p53-Mdm2 Oscillator Mediated by Mdm2 Degradation Rate

    Science.gov (United States)

    Bi, Yuanhong; Yang, Zhuoqin

    The dynamics of the tumor suppressor p53 can play a crucial role in deciding cell fate after DNA damage. In this paper, we explore the dynamics and stability of p53 mediated by Mdm2 degradation rate in p53-Mdm2 oscillator through bifurcation, the potential landscape and flux. Based on the investigation of the bifurcation, we find that p53 can exhibit rich dynamics including monostability, bistability of two stable steady states and oscillation behaviors as well as bistability between a stable steady state and an oscillatory state. The stability of these states are further validated by the potential landscape. In addition, oscillatory behaviors of p53 are explored by means of the negative gradient of the potential landscape and the probability flux. It is shown that the negative gradient of the potential landscape can attract the system towards the oscillatory path and the flux can drive oscillation along the path. Moreover, the quicker the flux runs, the smaller the period is. Besides, stability and sensitivity of the system are explored by the barrier height and the entropy production rate in a single cell level, and we further compare the potential landscapes at single and population cell levels. Our results may be useful for understanding the regulation of p53 signaling pathways in response to DNA damage.

  18. The p53-MDM2 network: from oscillations to apoptosis

    Indian Academy of Sciences (India)

    Indrani Bose; Bhaswar Ghosh

    2007-08-01

    The p53 protein is well-known for its tumour suppressor function. The p53-MDM2 negative feedback loop constitutes the core module of a network of regulatory interactions activated under cellular stress. In normal cells, the level of p53 proteins is kept low by MDM2, i.e. MDM2 negatively regulates the activity of p53. In the case of DNA damage, the p53-mediated pathways are activated leading to cell cycle arrest and repair of the DNA. If repair is not possible due to excessive damage, the p53-mediated apoptotic pathway is activated bringing about cell death. In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective. We discuss a number of key predictions, related to some specific aspects of cell cycle arrest and cell death, which could be tested in experiments.

  19. Mdm2 Splice isoforms regulate the p53/Mdm2/Mdm4 regulatory circuit via RING domain-mediated ubiquitination of p53 and Mdm4.

    Science.gov (United States)

    Fan, Chuandong; Wang, Xinjiang

    2017-02-06

    p53 is regulated by heterodimer E3 ligase Mdm2-Mdm4 via RING domain interaction. Mdm2 transcripts undergo alternative splicing, and Mdm2 splice isoforms are increased in cancer and induced by DNA damage. Although two major Mdm2 splice isoforms that do not bind to p53 were reported to impact the p53 pathway, the underlying biochemical mechanisms were not understood. Here, we show that these Mdm2 splice isoforms ubiquitinate Mdm2 and Mdm4 in vitro and regulate the activity of Mdm2-Mdm4 E3 complex in cells. The Mdm2 isoforms are capable of promoting p53 ubiquitination in the absence of Mdm2 or Mdm4. The two isoforms stimulate Mdm2 or Mdm4 activity for p53 ubiquitination in vitro and promote degradation of p53 and Mdm4 in cells. However, the Mdm2 isoforms have opposing effects on the steady-state p53 levels depending on the stoichiometric ratios of Mdm2, Mdm4 and the isoforms, causing either decreased or increased p53 levels in cells. Our data indicate that the Mdm2 splice isoforms can act as independent E3 ligases for p53 when Mdm2 and Mdm4 are absent, form potent heterodimer E3 ligases with either Mdm2 or Mdm4 for targeting p53 degradation, or act as inhibitory regulators of Mdm2-Mdm4 E3 ligase activity by downregulating Mdm4. These findings suggest that Mdm2 splice isoforms may play critical roles in the regulatory loop of p53/Mdm2-Mdm4 via a RING domain-mediated biochemical mechanism.

  20. mRNA display selection of an optimized MDM2-binding peptide that potently inhibits MDM2-p53 interaction.

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    Hirokazu Shiheido

    Full Text Available p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins.

  1. p53 and MDM2 protein expression in actinic cheilitis.

    Science.gov (United States)

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  2. p53 and MDM2 protein expression in actinic cheilitis

    Directory of Open Access Journals (Sweden)

    Maria da Conceição Andrade de Freitas

    2008-12-01

    Full Text Available Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976 parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  3. A dynamic P53-MDM2 model with time delay

    Energy Technology Data Exchange (ETDEWEB)

    Mihalas, Gh.I. [Department of Biophysics and Medical Informatics, University of Medicine and Pharmacy, Piata Eftimie Murgu, nr. 3, 300041 Timisoara (Romania)]. E-mail: mihalas@medinfo.umft.ro; Neamtu, M. [Department of Forecasting, Economic Analysis, Mathematics and Statistics, West University of Timisoara, Str. Pestalozzi, nr. 14A, 300115 Timisoara (Romania)]. E-mail: mihaela.neamtu@fse.uvt.ro; Opris, D. [Department of Applied Mathematics, West University of Timisoara, Bd. V. Parvan, nr. 4, 300223 Timisoara (Romania)]. E-mail: opris@math.uvt.ro; Horhat, R.F. [Department of Biophysics and Medical Informatics, University of Medicine and Pharmacy, Piata Eftimie Murgu, nr. 3, 300041 Timisoara (Romania)]. E-mail: rhorhat@yahoo.com

    2006-11-15

    Specific activator and repressor transcription factors which bind to specific regulator DNA sequences, play an important role in gene activity control. Interactions between genes coding such transcription factors should explain the different stable or sometimes oscillatory gene activities characteristic for different tissues. Starting with the model P53-MDM2 described into [Mihalas GI, Simon Z, Balea G, Popa E. Possible oscillatory behaviour in P53-MDM2 interaction computer simulation. J Biol Syst 2000;8(1):21-9] and the process described into [Kohn KW, Pommier Y. Molecular interaction map of P53 and MDM2 logic elements, which control the off-on switch of P53 in response to DNA damage. Biochem Biophys Res Commun 2005;331:816-27] we enveloped a new model of this interaction. Choosing the delay as a bifurcation parameter we study the direction and stability of the bifurcating periodic solutions. Some numerical examples are finally given for justifying the theoretical results.

  4. Oncoprotein MDM2 Overexpression is Associated with Poor Prognosis in Distinct Non-Hodgkin's Lymphoma Entities

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

    1999-01-01

    MDM2 is an oncoprotein involved in the regulation of p53. MDM2 exerts its tumorigenic potential through p53-dependent and -independent mechanisms. It is frequently overexpressed in various malignancies. Little is known about the prognostic value of MDM2 expression in non-Hodgkin's lymphomas (NHL......). We analyzed MDM2 expression immunohistochemically in 188 NHL cases from a prospective population-based NHL registry. The aim was to identify MDM2 expression profiles in various histological NHL subtypes and analyze whether MDM2 expression correlated with clinical variables and p53 status. MDM2...... overexpression was present in 42 (22%) of 188 cases. The frequency was highest in aggressive/very aggressive NHL (P MDM2 overexpression was associated with higher-grade disease (P = .008). MDM2 overexpression was not related to a phenotype indicating...

  5. The p53-MDM2/MDMX axis - A chemotype perspective

    NARCIS (Netherlands)

    Khoury, Kareem; Popowicz, Grzegorz M.; Holak, Tad A.; Doemling, Alexander

    The protein-protein interaction (PPI) of the tumor suppressor p53 and its negative regulator MDM2 consists of the most intense studied PPI with a group of small molecular weight antagonists described and many more disclosed in patent literature. Due to the A-level structural insight into p53

  6. Immunohistochemical detection of P53 and Mdm2 in vitiligo

    Science.gov (United States)

    Bakry, Ola A.; Hammam, Mostafa A.; Wahed, Moshira M. Abdel

    2012-01-01

    Background: Vitiligo is a common depigmented skin disorder that is caused by selective destruction of melanocytes. It is generally accepted that the main function of melanin resides in the protection of skin cells against the deleterious effect of ultraviolet rays (UVRs). Association of vitiligo and skin cancer has been a subject of controversy. Occurrence of skin cancer in long-lasting vitiligo is rare despite multiple evidences of DNA damage in vitiliginous skin. Aim: To detect the expression of P53 and Mdm2 proteins in both depigmented and normally pigmented skin of vitiligo patients and to compare it to control subjects suffering from nonmelanoma skin cancer (NMSC). Materials and Methods: Thirty-four patients with vitiligo and 30 age and sex-matched patients with nodulo-ulcerative basal cell carcinoma (BCC) as a control group were selected. Both patients and control subjects had outdoor occupations. Skin biopsies were taken from each case and control subjects. Histopathological examination of Hematoxylin and eosin-stained sections was done. Expression of P53 and Mdm2 proteins were examined immunohistochemically. Results: Both P53 and Mdm2 were strongly expressed in depigmented as well as normally pigmented skin of vitiligo patients. This expression involved the epidermis, skin adnexa and blood vessels with significant differences between cases and controls. Conclusions: The overexpression of P53 and Mdm2 proteins in both normally pigmented and depigmented skin of patients with vitiligo could contribute to the decreased occurrence of actinic damage and NMSC in these patients. PMID:23189248

  7. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.

    Science.gov (United States)

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-04-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

  8. Therapeutic considerations for Mdm2: not just a one trick pony

    Science.gov (United States)

    Lehman, Jason A.; Eitel, Jacob A.; Batuello, Christopher N.; Mayo, Lindsey D.

    2008-01-01

    Background The mdm2 proto-oncogene is elevated in numerous late stage cancers. The Mdm2 protein manifests its oncogenic properties in part through inactivation of the tumor suppressor protein p53. Recent efforts in anti-cancer drug design have focused on the identification of small molecules that disrupt the Mdm2-p53 interaction, in hopes of re-engaging the p53 pathway. Objective In addition to binding p53, Mdm2 complexes with numerous proteins involved in DNA repair, translation, metabolic activities, tumor growth and apoptosis. Additional biochemical analysis is required to understand how Mdm2 integrates into all of these cellular processes. Post-translational modifications to Mdm2 can alter its ability to associate with numerous proteins. Changes in protein structure may also affect the ability of small molecule inhibitors to effectively antagonize Mdm2. Conclusion The complexity of Mdm2 modification has been largely neglected during the development of previous Mdm2 inhibitors. Future high-throughput or in silico screening efforts will need to recognize the importance of post-translational modifications to Mdm2. Furthermore, the identification of molecules that target other domains in Mdm2 may provide a tool to prevent other pivotal p53-independent functions of Mdm2. These aims provide a useful roadmap for the discovery of new Mdm2 binding compounds with therapeutic potency that may exceed its predecessors. PMID:19738896

  9. Amotl1 mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict heart growth

    Science.gov (United States)

    Ragni, Chiara V.; Diguet, Nicolas; Le Garrec, Jean-François; Novotova, Marta; Resende, Tatiana P.; Pop, Sorin; Charon, Nicolas; Guillemot, Laurent; Kitasato, Lisa; Badouel, Caroline; Dufour, Alexandre; Olivo-Marin, Jean-Christophe; Trouvé, Alain; McNeill, Helen; Meilhac, Sigolène M

    2017-01-01

    Although in flies the atypical cadherin Fat is an upstream regulator of Hippo signalling, the closest mammalian homologue, Fat4, has been shown to regulate tissue polarity rather than growth. Here we show in the mouse heart that Fat4 modulates Hippo signalling to restrict growth. Fat4 mutant myocardium is thicker, with increased cardiomyocyte size and proliferation, and this is mediated by an upregulation of the transcriptional activity of Yap1, an effector of the Hippo pathway. Fat4 is not required for the canonical activation of Hippo kinases but it sequesters a partner of Yap1, Amotl1, out of the nucleus. The nuclear translocation of Amotl1 is accompanied by Yap1 to promote cardiomyocyte proliferation. We, therefore, identify Amotl1, which is not present in flies, as a mammalian intermediate for non-canonical Hippo signalling, downstream of Fat4. This work uncovers a mechanism for the restriction of heart growth at birth, a process which impedes the regenerative potential of the mammalian heart. PMID:28239148

  10. Mdm2’s Dilemma: To Degrade or To Translate p53?

    OpenAIRE

    2012-01-01

    In this issue of Cancer Cell, Gajjar et al. provide insight into how Mdm2 can both inhibit and enhance p53 activity. In the basal setting, Mdm2 binds p53 and promotes p53 degradation. Under stress conditions, ATM-dependent phosphorylation of Mdm2 results in its recruitment to p53 mRNA, thereby stimulating p53 translation.

  11. Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP

    Directory of Open Access Journals (Sweden)

    Hsueh Chung-Tsen

    2011-04-01

    Full Text Available Abstract We reviewed preclinical data and clinical development of MDM2 (murine double minute 2, ALK (anaplastic lymphoma kinase and PARP (poly [ADP-ribose] polymerase inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC. Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.

  12. Stress-specific response of the p53-Mdm2 feedback loop

    Directory of Open Access Journals (Sweden)

    Jensen Mogens H

    2010-07-01

    Full Text Available Abstract Background The p53 signalling pathway has hundreds of inputs and outputs. It can trigger cellular senescence, cell-cycle arrest and apoptosis in response to diverse stress conditions, including DNA damage, hypoxia and nutrient deprivation. Signals from all these inputs are channeled through a single node, the transcription factor p53. Yet, the pathway is flexible enough to produce different downstream gene expression patterns in response to different stresses. Results We construct a mathematical model of the negative feedback loop involving p53 and its inhibitor, Mdm2, at the core of this pathway, and use it to examine the effect of different stresses that trigger p53. In response to DNA damage, hypoxia, etc., the model exhibits a wide variety of specific output behaviour - steady states with low or high levels of p53 and Mdm2, as well as spiky oscillations with low or high average p53 levels. Conclusions We show that even a simple negative feedback loop is capable of exhibiting the kind of flexible stress-specific response observed in the p53 system. Further, our model provides a framework for predicting the differences in p53 response to different stresses and single nucleotide polymorphisms.

  13. Structure of a stapled peptide antagonist bound to nutlin-resistant Mdm2.

    Directory of Open Access Journals (Sweden)

    Sharon Min Qi Chee

    Full Text Available As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A, which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 Å crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.

  14. mdm-2 gene amplification in 3T3-L1 preadipocytes.

    Science.gov (United States)

    Berberich, S J; Litteral, V; Mayo, L D; Tabesh, D; Morris, D

    1999-05-01

    In this study the regulation of the murine double minute-2 (mdm-2) gene was examined in NIH 3T3-L1 preadipocytes. The 3T3-L1 cell line, under proper conditions, has the capacity to differentiate from fibroblasts into adipocytes [15]. A recent report demonstrated that mdm-2 overexpression could block myogenesis [12]. While examining the regulation of the mdm-2 gene during adipogenesis, it was discovered that 3T3-L1 cells possess a 36-fold elevation of mdm-2 mRNA relative to A31 cells, another immortalized Balb/c 3T3 fibroblast cell line that lacks the capacity to differentiate. Based on Southern blot analysis, the increase in mdm-2 mRNA was the result of a mdm-2 gene amplification. The level of Mdm-2 protein in undifferentiated 3T3-L1 cells was elevated relative to A31 fibroblasts and resulted from translation of mRNA transcripts initiating from the p53-independent P1 promoter. We also examined how mdm-2 and p53 levels changed as undifferentiated fibroblasts converted to adipocytes. While mdm-2 mRNA levels remained elevated, p53 mRNA, protein, and DNA-binding activity decreased. These results suggest that adipogenesis is unaffected by elevated Mdm-2 levels and that the overexpression of mdm-2 mRNA is predominantly p53 independent.

  15. Structural Basis of Competitive Recognition of p53 and MDM2 by HAUSP/USP7: Implications for the Regulation of the p53-MDM2 Pathway.

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7, a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53-MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor-receptor associated factor (TRAF-like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP-MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-A and 1.7-A resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53-MDM2 pathway by HAUSP.

  16. 睾丸肿瘤组织中mdm-2基因表达及临床意义%Mdm-2 Gene Expression in Human Testicular cancer

    Institute of Scientific and Technical Information of China (English)

    吴先旺; 徐元虎

    2003-01-01

    探讨mdm-2基因与睾丸肿瘤发生及预后的关系.应用免疫组化方法检测27 例睾丸肿瘤中mdm-2蛋白表达水平.发现有12 例标本mdm-2蛋白阳性表达,并且与睾丸肿瘤的病理分级和临床分期有关,提示睾丸肿瘤恶性程度与mdm-2蛋白表达具有明显相关性.mdm-2蛋白表达可能成为睾丸肿瘤预后的指标.

  17. A dynamic p53-mdm2 model with distributed delay

    Science.gov (United States)

    Horhat, Raluca; Horhat, Raul Florin

    2014-12-01

    Specific activator and repressor transcription factors which bind to specific regulator DNA sequences, play an important role in gene activity control. Interactions between genes coding such transcripion factors should explain the different stable or sometimes oscillatory gene activities characteristic for different tissues. In this paper, the dynamic P53-Mdm2 interaction model with distributed delays is investigated. Both weak and Dirac kernels are taken into consideration. For Dirac case, the Hopf bifurcation is investigated. Some numerical examples are finally given for justifying the theoretical results.

  18. Escape, or Vanish: Control the Fate of p53 through MDM2-Mediated Ubiquitination.

    Science.gov (United States)

    Wei, Jinlian; Yang, Yingrui; Lu, Mengchen; Xu, Lili; Liu, Fang; Yuan, Zhenwei; Bao, Qichao; Jiang, Zhengyu; Xu, Xiaoli; Guo, Xiaoke; Zhang, Xiaojin; You, Qidong; Sun, Haopeng

    2015-01-01

    p53 protein is a prominent tumor suppressor to induce cell cycle arrest, apoptosis and senescence, which attracts significant interest to cancer treatment. Therefore, it would be particularly important to restore the wild-type p53 that retains latent functions in the approximately 50% of tumors. MDM2 (murine double minute 2), the principal cellular antagonist of p53, has long been believed to suppress p53 activity through two main mechanisms: promoting degradation via its E3 ligase activity and masking p53 transcriptional activation by direct binding. Targeting MDM2 E3 ligase activity is becoming a potential antitumor strategy resulting from MDM2's decisive role in controlling the fate of p53: p53 is going to degradation when entrapped into MDM2-mediated ubiquitination, where p53 can escape by abrogating MDM2 E3 ligase activity using regulators. The intensive focus on regulating MDM2 ubiquitin E3 ligase activity has led to the rapid progress of its inhibitors, which may be possible to help p53 escape from degradation and restore its function to control tumor growth. This review summarizes the current inhibitors of MDM2 E3 ligase in cancer therapy based on the understanding the regulation of MDM2 E3 ubiquitin ligase activity, including post-translational modification, interactions between MDM2 and its cofactors, and regulation of MDM2 stability.

  19. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Kasim, Vivi, E-mail: vivikasim78@gmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Yang, Li [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Miyagishi, Makoto [Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566 (Japan); Wu, Shourong, E-mail: shourongwu@hotmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  20. Mdm2 RING mutation enhances p53 transcriptional activity and p53-p300 interaction.

    Directory of Open Access Journals (Sweden)

    Hilary V Clegg

    Full Text Available The p53 transcription factor and tumor suppressor is regulated primarily by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 to target it for proteasomal degradation. Aside from its ubiquitin ligase function, Mdm2 has been believed to be capable of suppressing p53's transcriptional activity by binding with and masking the transactivation domain of p53. The ability of Mdm2 to restrain p53 activity by binding alone, without ubiquitination, was challenged by a 2007 study using a knockin mouse harboring a single cysteine-to-alanine point mutation (C462A in Mdm2's RING domain. Mouse embryonic fibroblasts with this mutation, which abrogates Mdm2's E3 ubiquitin ligase activity without affecting its ability to bind with p53, were unable to suppress p53 activity. In this study, we utilized the Mdm2(C462A mouse model to characterize in further detail the role of Mdm2's RING domain in the control of p53. Here, we show in vivo that the Mdm2(C462A protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2(C462A actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2(C462A facilitates the interaction between p53 and the acetyltransferase CBP/p300, and it fails to heterodimerize with its homolog and sister regulator of p53, Mdmx, suggesting that a fully intact RING domain is required for Mdm2's inhibition of the p300-p53 interaction and for its interaction with Mdmx. These findings help us to better understand the complex regulation of the Mdm2-p53 pathway and have important implications for chemotherapeutic agents targeting Mdm2, as they suggest that inhibition of Mdm2's E3 ubiquitin ligase activity may be sufficient for increasing p53 activity in vivo, without the need to block Mdm2-p53 binding.

  1. Clinicopathological significance of p53 and mdm2 protein expression in human pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Ming Dong; Gang Ma; Wei Tu; Ke-Jian Guo; Yu-Lin Tian; Yu-Ting Dong

    2005-01-01

    AIM: To study the clinicopathological significance of p53 and mdm2 protein expression in human pancreatic cancer. METHODS: To investigate the expression of p53 and mdm2 in pancreatic cancer by immunohistochemistry, and the relationships between the p53 and mdm2 protein expression and clinicopathological parameters in pancreatic cancer.RESULTS: The positive expression of p53 protein was found in 40 of 59 patients (67.8%) and that of mdm2 protein in 17 of 59 patients (28.8%). No obvious relationships were found between p53 as well as mdm2 expression and sex, tumor site, TNM staging and histological differentiation. p53 expression was increased in patients younger than 65 years old, while mdm2 had no relationship with age. The survival time of the patients with the positive expression of p53 and mdm2 proteins was obviously shorter than the other groups. CONCLUSION: Both p53 and mdm2 presented relatively high expression in human pancreatic cancer. The overexpression of p53 and mdm2 might reflect the malignant proliferation of pancreatic cancer and their co-expression might be helpful to evaluate the prognosis of the patients with pancreatic cancer.

  2. Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Feddersen, Søren; Francoz, S.

    2012-01-01

    The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel r...... in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination. Cell Death and Differentiation (2012) 19, 1381-1389; doi:10.1038/cdd.2012.15; published online 2 March 2012...

  3. Mouse models of Mdm2 and Mdm4 and their clinical implications

    Institute of Scientific and Technical Information of China (English)

    Shunbin Xiong

    2013-01-01

    Mdm2 and Mdm4 are two key negative regulators of the tumor suppressor p53.Deletion of either Mdm2 or Mdm4 induces p53-dependent early embryonic lethality in knockout mouse models.The tissuespecific deletion of Mdm2 induces p53-dependent apoptosis,whereas the deletion of Mdm4 induces both p53-dependent apoptosis and cell cycle arrest.Compared to Mdm4 deletion,Mdm2 deletion causes more severe phenotypic defects.Disrupting the Mdm2 and Mdm4 interaction using knockin mice models causes embryonic lethality that can be completely rescued by the concomitant loss of p53,suggesting that Mdm2 and Mdm4 heterodimerization is critical to inhibit p53 activity during embryogenesis.Overexpression of Mdm2 and Mdm4 in mice induces spontaneous tumorigenesis,which clearly indicates that Mdm2 and Mdm4 are bona fide oncogenes.Studies from these mouse models strongly suggest that blocking Mdm2-and Mdm4-mediated p53 inhibition is an appealing therapeutic strategy for cancer patients with wild-type p53 alleles.

  4. Primary extraskeletal osteosarcoma: a clinicopathological study of 18 cases focusing on MDM2 amplification status.

    Science.gov (United States)

    Yamashita, Kyoko; Kohashi, Kenichi; Yamada, Yuichi; Nishida, Yoshihiro; Urakawa, Hiroshi; Oda, Yoshinao; Toyokuni, Shinya

    2017-05-01

    Extraskeletal osteosarcoma (ESOS) is an uncommon malignant neoplasm. Most ESOSs are high grade, although some low-grade cases have been reported. A few cases of ESOS with MDM2 amplification have also been reported, suggesting some similarity to skeletal low-grade osteosarcoma such as parosteal osteosarcoma, where MDM2 is often amplified. However, the frequency of low-grade cases and cases with MDM2 amplification among ESOSs remains unknown, and their relationship is unclear. To clarify this, we examined 18 primary ESOS cases clinically, pathologically, and genetically, focusing on their MDM2 amplification status. Our cases comprised 10 men and 8 women whose mean age was 58.6 years; the most common site of the lesion was the thigh and buttock. There were one histologically low-grade case evaluated by biopsy specimen with an aggressive course and 2 relatively low-grade cases whose lesions were of low grade for the most part. MDM2 amplification status was revealed by fluorescence in situ hybridization in all 18 cases; 2 patients-histologically intermediate- and high-grade cases-were found to have MDM2 amplification. In conclusion, this study indicates that histologically low-grade and relatively low-grade cases of ESOS are not always associated with MDM2 amplification. The ESOS case with MDM2 amplification could be high grade, although MDM2-amplified dedifferentiated liposarcoma with osteogenic differentiation should be ruled out in making the diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3

    DEFF Research Database (Denmark)

    Hallenborg, P.; Siersbæk, M.; Barrio-Hernandez, I.

    2016-01-01

    The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies...... on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each...

  6. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function.

    Science.gov (United States)

    Chen, D; Zhang, Z; Li, M; Wang, W; Li, Y; Rayburn, E R; Hill, D L; Wang, H; Zhang, R

    2007-08-01

    As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2-p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2-interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2-p53 interaction and has implications in cancer prevention and therapy.

  7. Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis

    Science.gov (United States)

    Kamio, Takuya; Gu, Bai-wei; Olson, Timothy S.; Zhang, Yanping; Mason, Philip J.; Bessler, Monica

    2016-01-01

    MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed

  8. Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis.

    Directory of Open Access Journals (Sweden)

    Takuya Kamio

    Full Text Available MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA and in 5q- myelodysplastic syndrome (MDS. DBA and 5q- MDS are associated with inherited (DBA or acquired (5q- MDS haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F, retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM, these mice showed a significant decrease in Ter119hi cells compared to wild type (WT littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01. This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko. Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK cells, accompanied by significant decreases in multipotent progenitor (MPP cells (p < 0.01. Competitive BM repopulation experiments

  9. Association between MDM2-SNP309 and hepatocellular carcinoma in Taiwanese population

    Institute of Scientific and Technical Information of China (English)

    Jyh-Der Leu; I-Feng Lin; Ying-Fang Sun; Su-Mei Chen; Chih-Chao Liu; Yi-Jang Lee

    2009-01-01

    AIM: To investigate the risk association and compare the onset age of hepatocellular carcinoma (HCC)patients in Taiwan with different genotypes of MDM2-SNP309.METHODS: We analyzed MDM2-SNP309 genotypes from 58 patients with HCC and 138 cancer-free healthy controls consecutively. Genotyping of MDM2-SNP309 was conducted by restriction fragment length polymorphism assay.RESULTS: The proportion of homozygous MDM2-SNP309 genotype (G/G) in cases and cancer-free healthy controls was similar (17.2% vs 16.7%). Multivariate analysis showed that the risk of G/G genotype of MDM2-SNP309 vs wild-type T/T genotype in patients with HCC was not significant (OR = 1.265, 95%CI = 0.074-21.77) after adjustment for sex, hepatitis B or C virus infection, age, and cardiovascular disease/diabetes. Nevertheless, there was a trend that GG genotype of MDM2-SNP309 might increase the risk in HCC patients infected with hepatitis virus (OR = 2.568,95% CI = 0.054-121.69). Besides, the homozygous MDM2-SNP309 genotype did not exhibit a significantly earlier age of onset for HCC.CONCLUSION: Current data suggest that the association between MDM2-SNP309 GG genotype and HCC is not significant, while the risk may be enhanced in patients infected by hepatitis virus in Taiwan.

  10. MDM2 expression during mouse embryogenesis and the requirement of p53.

    Science.gov (United States)

    Léveillard, T; Gorry, P; Niederreither, K; Wasylyk, B

    1998-06-01

    We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206).

  11. Scaffold hopping via ANCHOR.QUERY : beta-lactams as potent p53-MDM2 antagonists

    NARCIS (Netherlands)

    Shaabani, S.; Neochoritis, C. G.; Twarda-Clapa, Aleksandra; Musielak, Bogdan; Holak, Tad A.; Domling, A.

    2017-01-01

    Using the pharmacophore-based virtual screening platform ANCHOR.QUERY, we morphed our recently described Ugi-4CR scaffold towards a beta-lactam scaffold with potent p53-MDM2 antagonizing activities. 2D-HSQC and FP measurements confirm potent MDM2 binding. Molecular modeling studies were used to unde

  12. MDM2 antagonist Nutlin-3a potentiates antitumour activity of cytotoxic drugs in sarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Lothe Ragnhild A

    2011-05-01

    Full Text Available Abstract Background Frequent failure and severe side effects of current sarcoma therapy warrants new therapeutic approaches. The small-molecule MDM2 antagonist Nutlin-3a activates the p53 pathway and efficiently induces apoptosis in tumours with amplified MDM2 gene and overexpression of MDM2 protein. However, the majority of human sarcomas have normal level of MDM2 and the therapeutic potential of MDM2 antagonists in this group is still unclear. We have investigated if Nutlin-3a could be employed to augment the response to traditional therapy and/or reduce the genotoxic burden of chemotherapy. Methods A panel of sarcoma cell lines with different TP53 and MDM2 status were treated with Nutlin-3a combined with Doxorubicin, Methotrexate or Cisplatin, and their combination index determined. Results Clear synergism was observed when Doxorubicin and Nutlin-3a were combined in cell lines with wild-type TP53 and amplified MDM2, or with Methotrexate in both MDM2 normal and amplified sarcoma cell lines, allowing for up to tenfold reduction of cytotoxic drug dose. Interestingly, Nutlin-3a seemed to potentiate the effect of classical drugs as Doxorubicin and Cisplatin in cell lines with mutated TP53, but inhibited the effect of Methotrexate. Conclusion The use of Nutlin in combination with classical sarcoma chemotherapy shows promising preclinical potential, but since clear biomarkers are still lacking, clinical trials should be followed up with detailed tumour profiling.

  13. Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

    Science.gov (United States)

    Ranaweera, Ruchira S.; Yang, Xiaolu

    2013-01-01

    The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

  14. Bitter, sweet and umami taste receptors and downstream signaling effectors: Expression in embryonic and growing chicken gastrointestinal tract.

    Science.gov (United States)

    Cheled-Shoval, Shira L; Druyan, Shelly; Uni, Zehava

    2015-08-01

    Taste perception is a crucial biological mechanism affecting food and water choices and consumption in the animal kingdom. Bitter taste perception is mediated by a G-protein-coupled receptor (GPCR) family-the taste 2 receptors (T2R)-and their downstream proteins, whereas sweet and umami tastes are mediated by the GPCR family -taste 1 receptors (T1R) and their downstream proteins. Taste receptors and their downstream proteins have been identified in extra-gustatory tissues in mammals, such as the lungs and gastrointestinal tract (GIT), and their GIT activation has been linked with different metabolic and endocrinic pathways in the GIT. The chicken genome contains three bitter taste receptors termed ggTas2r1, ggTas2r2, and ggTas2r7, and the sweet/umami receptors ggTas1r1 and ggTas1r3, but it lacks the sweet receptor ggTas1r2. The aim of this study was to identify and determine the expression of genes related to taste perception in the chicken GIT, both at the embryonic stage and in growing chickens. The results of this study demonstrate for the first time, using real-time PCR, expression of the chicken taste receptor genes ggTas2r1, ggTas2r2, ggTas2r7, ggTas1r1, and ggTas1r3 and of their downstream protein-encoding genes TRPM5, α-gustducin, and PLCβ2 in both gustatory tissues-the palate and tongue, and extra-gustatory tissues-the proventriculus, duodenum, jejunum, ileum, cecum, and colon of embryonic day 19 (E19) and growing (21 d old) chickens. Expression of these genes suggests the involvement of taste pathways for sensing carbohydrates, amino acids and bitter compounds in the chicken GIT. © 2015 Poultry Science Association Inc.

  15. Mouse modeling of the MDM2/MDMX-p53 signaling axis.

    Science.gov (United States)

    Tackmann, Nicole R; Zhang, Yanping

    2017-01-17

    It is evident that p53 activity is critical for tumor prevention and stress response through its transcriptional activation of genes affecting cellular senescence, apoptosis, cellular metabolism, and DNA repair. The regulation of p53 is highly complex, and MDM2 and MDMX are thought to be critical for deciding the fate of p53, both through inhibitory binding and posttranslational modification. Many mouse models have been generated to study the regulation of p53 in vivo, and they have altered our interpretations of how p53 is regulated by MDM2 and MDMX. Although MDM2 is absolutely required for p53 regulation, certain functions are dispensable under unstressed conditions, including the ability of MDM2 to degrade p53. MDMX, on the other hand, may only be required in select situations, like embryogenesis. These models have also clarified how cellular stress signals modify the p53-inhibiting activities of MDM2 and MDMX in vivo It is clear that more work will need to be performed to further understand the contexts for each of these signals and the requirements of various MDM2 and MDMX functions. Here, we will discuss what we have learned from mouse modeling of MDM2 and MDMX and underscore the ways in which these models could inform future therapies.

  16. Systems biology analysis reveals role of MDM2 in diabetic nephropathy

    Science.gov (United States)

    Saito, Rintaro; Rocanin-Arjo, Anaïs; You, Young-Hyun; Darshi, Manjula; Van Espen, Benjamin; Miyamoto, Satoshi; Pham, Jessica; Pu, Minya; Romoli, Simone; Natarajan, Loki; Ju, Wenjun; Kretzler, Matthias; Nelson, Robert; Ono, Keiichiro; Thomasova, Dana; Mulay, Shrikant R.; Ideker, Trey; D’Agati, Vivette; Beyret, Ergin; Belmonte, Juan Carlos Izpisua; Anders, Hans Joachim

    2016-01-01

    To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers. PMID:27777973

  17. Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1 and Caspase-9/-3 activation

    DEFF Research Database (Denmark)

    Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna;

    2016-01-01

    ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21(Waf1/Cip1), Bax, Noxa, MDM2, and activation of caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter...

  18. Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1 and Caspase-9/-3 activation

    DEFF Research Database (Denmark)

    Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna

    2016-01-01

    ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21(Waf1/Cip1), Bax, Noxa, MDM2, and activation of caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter...

  19. Novel small molecule inhibitors of the p53-MDM2 /MDM4 interaction for induction of apoptosis in AML

    NARCIS (Netherlands)

    Köhler, L.M.; Beck, B.; Huang, H.; Holak, T.; Dömling, A.; Subklewe, M.

    2012-01-01

    A promising new approach in cancer treatment is the inhibition of murine double minute 2 and 4 (MDM2/MDM4) which is a negative regulator of p53. In neoplasia with unmutated p53 the inactivation of MDM2/4 promotes apoptosis and growth arrest. A derivative of the first described p53/MDM2 antagonist Nu

  20. MdmX Protects p53 from Mdm2-Mediated Degradation

    OpenAIRE

    2000-01-01

    The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be exami...

  1. Regulation of transcription functions of the p53 tumor suppressor by the mdm-2 oncogene.

    OpenAIRE

    1995-01-01

    BACKGROUND: Mdm-2, a zinc finger protein, negatively regulates the p53 tumor suppressor gene product by binding to it and preventing transcriptional activation (16). MATERIALS AND METHODS: Assays for p53 mediated transcription, repression and activation by mutant and wild-type p53 proteins were used to measure the ability of mdm-2 to block each activity. RESULTS: Mdm-2 was able to inhibit all three functions of the wild-type and mutant p53 activities; transcriptional activation by the wild-ty...

  2. Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer

    Directory of Open Access Journals (Sweden)

    Clifford John

    2009-01-01

    Full Text Available Abstract Background Eukaryotic initiation factor 4E (eIF4E is elevated in many cancers and is a prognostic indicator in breast cancer. Many pro-tumorigenic proteins are selectively translated via eIF4E, including c-Myc, cyclin D1, ornithine decarboxylase (ODC, vascular endothelial growth factor (VEGF and Tousled-like kinase 1B (TLK1B. However, western blot analysis of these factors in human breast cancer has been limited by the availability of fresh frozen tissue and the labor-intensive nature of the multiple assays required. Our goal was to validate whether formalin-fixed, paraffin-embedded tissues arranged in a tissue microarray (TMA format would be more efficient than the use of fresh-frozen tissue and western blot to test multiple downstream gene products. Results Breast tumor TMAs were stained immunohistochemically and quantitated using the ARIOL imaging system. In the TMAs, eIF4E levels correlated strongly with c-Myc, cyclin D1, TLK1B, VEGF, and ODC. Western blot comparisons of eIF4E vs. TLK1B were consistent with the immunohistochemical results. Consistent with our previous western blot results, eIF4E did not correlate with node status, ER, PR, or HER-2/neu. Conclusion We conclude that the TMA technique yields similar results as the western blot technique and can be more efficient and thorough in the evaluation of several products downstream of eIF4E.

  3. An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence

    Directory of Open Access Journals (Sweden)

    Ashworth Alan

    2011-07-01

    Full Text Available Abstract Background Cellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism. Results To identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells. Conclusion The senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action.

  4. Polymorphism of MDM2 promoter 309 (rs 2279744) and the risk of PCOS.

    Science.gov (United States)

    Chan, Ying; Jiang, Hongguo; Yang, Xiaoling; Li, Dongya; Ma, Lan; Luo, Ying; Tang, Wenru

    2016-01-01

    This study aimed at evaluating possible association between MDM2 SNP309 polymorphism (rs 2279744) and polycystic ovary syndrome (PCOS). One hundred and twenty-five women with PCOS and two hundred and fifty women without PCOS were collected from the department of reproductive medicine of college hospital in this case-control study. Peripheral blood samples were collected from all participants and DNA was extracted, MDM2 SNP309 polymorphism (rs 2279744) was determined from the 125 cases and 250 controls. Women were grouped into PCOS (n = 125) group and control group (n = 250). Odds ratios (OR) and 95% confidence intervals (CI) were used to evaluate the association between MDM2 SNP309 polymorphism (rs 2279744) and PCOS. The distribution of T allele was significant higher in PCOS cases than controls. MDM2 SNP 309 T allele is associated with PCOS.

  5. Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Michael I. Carr

    2016-09-01

    Full Text Available ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2S394A knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eμ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2S394A mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2S394A mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies.

  6. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

    Directory of Open Access Journals (Sweden)

    Elena Elizabeth Bagley

    2014-06-01

    effector.

  7. Identification of functional DNA variants in the constitutive promoter region of MDM2.

    Science.gov (United States)

    Lalonde, Marie-Eve; Ouimet, Manon; Larivière, Mathieu; Kritikou, Ekaterini A; Sinnett, Daniel

    2012-09-01

    Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (-1494 G > A; indel 40 bp; and -182 C > G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40 bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.

  8. Prospective virtual screening for novel p53-MDM2 inhibitors using ultrafast shape recognition

    Science.gov (United States)

    Patil, Sachin P.; Ballester, Pedro J.; Kerezsi, Cassidy R.

    2014-02-01

    The p53 protein, known as the guardian of genome, is mutated or deleted in approximately 50 % of human tumors. In the rest of the cancers, p53 is expressed in its wild-type form, but its function is inhibited by direct binding with the murine double minute 2 (MDM2) protein. Therefore, inhibition of the p53-MDM2 interaction, leading to the activation of tumor suppressor p53 protein presents a fundamentally novel therapeutic strategy against several types of cancers. The present study utilized ultrafast shape recognition (USR), a virtual screening technique based on ligand-receptor 3D shape complementarity, to screen DrugBank database for novel p53-MDM2 inhibitors. Specifically, using 3D shape of one of the most potent crystal ligands of MDM2, MI-63, as the query molecule, six compounds were identified as potential p53-MDM2 inhibitors. These six USR hits were then subjected to molecular modeling investigations through flexible receptor docking followed by comparative binding energy analysis. These studies suggested a potential role of the USR-selected molecules as p53-MDM2 inhibitors. This was further supported by experimental tests showing that the treatment of human colon tumor cells with the top USR hit, telmisartan, led to a dose-dependent cell growth inhibition in a p53-dependent manner. It is noteworthy that telmisartan has a long history of safe human use as an approved anti-hypertension drug and thus may present an immediate clinical potential as a cancer therapeutic. Furthermore, it could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against variety of cancers. Importantly, the present study demonstrates that the adopted USR-based virtual screening protocol is a useful tool for hit identification in the domain of small molecule p53-MDM2 inhibitors.

  9. Low Prevalence of TP53 Mutations and MDM2 Amplifications in Pediatric Rhabdomyosarcoma

    Directory of Open Access Journals (Sweden)

    Simona Ognjanovic

    2012-01-01

    Full Text Available The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. The reported prevalence of mutations in rhabdomyosarcoma (RMS varies widely, with recent larger studies suggesting that TP53 mutations in pediatric RMS may be extremely rare. Overexpression of MDM2 also attenuates p53 function. We have performed TP53 mutation/MDM2 amplification analyses in the largest series analyzed thus far, including DNA isolated from 37 alveolar and 38 embryonal RMS tumor samples obtained from the Cooperative Human Tissue Network (CHTN. Available samples were frozen tumor tissues (N=48 and histopathology slides. TP53 mutations in exons 4–9 were analyzed by direct sequencing in all samples, and MDM2 amplification analysis was performed by differential PCR on a subset of 22 samples. We found only one sample (1/75, 1.3% carrying a TP53 mutation at codon 259 (p.D259Y and no MDM2 amplification. Two SNPs in the TP53 pathway, associated with accelerated tumor onset in germline TP53 mutation carriers, (TP53 SNP72 (rs no. 1042522 and MDM2 SNP309 (rs no. 2279744, were not found to confer earlier tumor onset. In conclusion, we confirm the extremely low prevalence of TP53 mutations/MDM2 amplifications in pediatric RMS (1.33% and 0%, respectively. The possible inactivation of p53 function by other mechanisms thus remains to be elucidated.

  10. MDM2 is a novel E3 ligase for HIV-1 Vif

    Directory of Open Access Journals (Sweden)

    Tomonaga Mitsunori

    2009-01-01

    Full Text Available Abstract The human immunodeficiency virus type 1 (HIV-1 Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G. Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3 complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.

  11. MDM2-MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance.

    Science.gov (United States)

    Moscetti, Ilaria; Teveroni, Emanuela; Moretti, Fabiola; Bizzarri, Anna Rita; Cannistraro, Salvatore

    Murine double minute 2 (MDM2) and 4 (MDM4) are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2-MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2-MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (KD ) in the micromolar range for the MDM2-MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2-MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2-MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation.

  12. Recombinant human MDM2 oncoprotein shows sequence composition selectivity for binding to both RNA and DNA.

    Science.gov (United States)

    Challen, Christine; Anderson, John J; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Lunec, John

    2012-03-01

    MDM2 is a 90 kDa nucleo-phosphoprotein that binds p53 and other proteins contributing to its oncogenic properties. Its structure includes an amino proximal p53 binding site, a central acidic domain and a carboxy region which incorporates Zinc and Ring Finger domains suggestive of nucleic acid binding or transcription factor function. It has previously been reported that a bacculovirus expressed MDM2 protein binds RNA in a sequence-specific manner through the Ring Finger domain, however, its ability to bind DNA has yet to be examined. We report here that a bacterially expressed human MDM2 protein binds both DNA as well as the previously defined RNA consensus sequence. DNA binding appears selective and involves the carboxy-terminal domain of the molecule. RNA binding is inhibited by an MDM2 specific antibody, which recognises an epitope within the carboxy region of the protein. Selection cloning and sequence analysis of MDM2 DNA binding sequences, unlike RNA binding sequences, revealed no obvious DNA binding consensus sequence, but preferential binding to oligopurine:pyrimidine-rich stretches. Our results suggest that the observed preferential DNA binding may occur through the Zinc Finger or in a charge-charge interaction through the Ring Finger, thereby implying potentially different mechanisms for DNA and RNA MDM2 binding.

  13. Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes

    Directory of Open Access Journals (Sweden)

    Natasha Chaudhary

    2016-12-01

    Full Text Available Insulin activation of phosphatidylinositol 3-kinase (PI3K regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin’s effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types.

  14. Expression and significance of P53 protein and MDM-2 protein in human gliomas

    Institute of Scientific and Technical Information of China (English)

    WANG An-liu; LIU Zhao-xia; LI Guang; ZHANG Li-wei

    2011-01-01

    Background P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed.Methods The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade Ⅰ-Ⅱ group: 5 cases of grade Ⅰ, 119 cases of grade Ⅱ; and grade Ⅲ-Ⅳ group: 53 cases of grade Ⅲ, and 35 cases of grade Ⅳ).Results The P53 positive rate was significantly higher in the glioma groups than in the control group (P <0.0001). The P53 positive rate was significantly higher in glioma tissues of grade Ⅲ-V than in glioma tissues of grade Ⅰ-Ⅱ group (P=0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P <0.0001).There was no significant difference in the MDM-2 positive rate between the two glioma groups (P=0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P=0.069)Conclusions Overexpression of P53 protein might be related to the occurrence and progression of glioma.Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.

  15. The oncoprotein HBXIP modulates the feedback loop of MDM2/p53 to enhance the growth of breast cancer.

    Science.gov (United States)

    Li, Hang; Liu, Qian; Wang, Zhen; Fang, Runping; Shen, Yu; Cai, Xiaoli; Gao, Yuen; Li, Yinghui; Zhang, Xiaodong; Ye, Lihong

    2015-09-11

    MDM2 and p53 form a negative feedback loop, in which p53 as a transcription factor positively regulates MDM2 and MDM2 negatively regulates tumor suppressor p53 through promoting its degradation. However, the mechanism of the feedback loop is poorly understood in cancers. We had reported previously that the oncoprotein hepatitis B X-interacting protein (HBXIP) is a key oncoprotein in the development of cancer. Thus, we supposed that HBXIP might be involved in the event. Here, we observed that the expression levels of HBXIP were positively correlated to those of MDM2 in clinical breast cancer tissues. Interestingly, HBXIP was able to up-regulate MDM2 at the levels of mRNA and protein in MCF-7 breast cancer cells. Mechanically, HBXIP increased the promoter activities of MDM2 through directly binding to p53 in the P2 promoter of MDM2. Strikingly, we identified that the acetyltransferase p300 was recruited by HBXIP to p53 in the promoter of MDM2. Moreover, we validated that HBXIP enhanced the p53 degradation mediated by MDM2. Functionally, the knockdown of HBXIP or/and p300 inhibited the proliferation of breast cancer cells in vitro, and the depletion of MDM2 or overexpression of p53 significantly blocked the HBXIP-promoted growth of breast cancer in vitro and in vivo. Thus, we concluded that highly expressed HBXIP accelerates the MDM2-mediated degradation of p53 in breast cancer through modulating the feedback loop of MDM2/p53, resulting in the fast growth of breast cancer cells. Our findings provide new insights into the mechanism of the acceleration of the MDM2/p53 feedback loop in the development of cancer.

  16. p53 expression and relationship with MDM2 amplification in breast carcinomas.

    Science.gov (United States)

    Buyukpinarbasili, Nur; Gucin, Zuhal; Ersoy, Yeliz Emine; İlbak, Ayca; Kadioglu, Huseyin; Muslumanoglu, Mahmut

    2016-04-01

    Carcinoma of the breast, like other malignancies, is a genetic disease with multiple genetic events leading to the malignant phenotype. p53 mutations are the most common genetic events in human cancer. Inactivation of p53 can be a result of mutation in gene sequence. One of the main structures that regulate p53 stabilization is MDM2. It suppresses p53 transcriptional activation by recognizing transactivation domain of p53. The loss of MDM2 function on p53 regulation results in deprivation of p53 tumor suppressor ability. Single nucleotide polymorphisms (SNP309 T->G exchange) or MDM2 amplification has been proposed to play a role in this issue. In the present study, our aim is to analyze p53 and MDM2 status and investigate their interactions in human sporadic breast carcinoma. The study groups were separated according to their molecular classifications. In each group, histologic type of the tumor, conventional prognostic parameters, p53, and MDM2 interactions were compared statistically. Tumors are divided into 4 subtypes due to estrogen and progesterone receptor status, HER-2, and Ki-67 proliferation index results. According to this classification, 23 cases are in the luminal A, 32 cases are in the luminal B, 15 cases are in the HER-2 positive, and 22 cases are in the triple-negative group, with a total of 92 cases. p53 expression is low in luminal breast carcinomas than HER-2 and triple-negative subtypes. MDM2 amplification frequency was found to be 5.4% in total. MDM2 gene amplification does not have a significant role in breast carcinogenesis, but other possible mechanisms may play a role in its inactivation.

  17. A new subtype of high-grade mandibular osteosarcoma with RASAL1/MDM2 amplification.

    Science.gov (United States)

    Guérin, Maxime; Thariat, Juliette; Ouali, Mounia; Bouvier, Corinne; Decouvelaere, Anne-Valérie; Cassagnau, Elisabeth; Aubert, Sébastien; Lepreux, Sébastien; Coindre, Jean-Michel; Valmary-Degano, Séverine; Larousserie, Frédérique; Meilleroux, Julie; Projetti, Fabrice; Stock, Nathalie; Galant, Christine; Marie, Béatrice; Peyrottes, Isabelle; de Pinieux, Gonzague; Gomez-Brouchet, Anne

    2016-04-01

    In contrast to long bone osteosarcoma, mandibular osteosarcoma is highly heterogeneous and morphologically overlaps with benign tumors, obscuring diagnosis and treatment selection. Molecular characterization is difficult due to the paucity of available specimens of this rare disease. We aimed to characterize the spectrum of mandibular osteosarcoma using immunohistochemistry and molecular techniques (quantitative polymerase chain reaction and sequencing) and compare them with benign fibro-osseous lesions. Forty-nine paraffin-embedded mandible osteosarcoma tissue samples were collected retrospectively and compared with 10 fibrous dysplasia and 15 ossifying fibroma cases. These were analyzed for molecular markers thought to differ between the different diseases and subtypes: MDM2 (murine double-minute type 2) overexpression, GNAS (guanine nucleotide-binding protein/α subunit) mutations, and amplification of MDM2 and/or RASAL1 (RAS protein activator like 1). Five fibroblastic high-grade osteosarcoma subtypes showed MDM2 amplification, including 2 with a microscopic appearance of high-grade osteosarcoma with part low-grade osteosarcoma (differentiated/dedifferentiated osteosarcoma) and MDM2 overexpression. The other 3 contained a coamplification of MDM2 and RASAL1, a signature also described for juvenile ossifying fibroma, with no overexpression of MDM2. These were of the giant cell-rich high-grade osteosarcoma, with areas mimicking juvenile ossifying fibroma (ossifying fibroma-like osteosarcoma). Our results show that some diagnosed high-grade osteosarcomas are differentiated/dedifferentiated osteosarcomas and harbor an overexpression and amplification of MDM2. In addition, juvenile ossifying fibromas can potentially evolve into giant cell-rich high-grade osteosarcomas and are characterized by a RASAL1 amplification (osteosarcoma with juvenile ossifying fibroma-like genotype). Thus, the presence of a RASAL1 amplification in ossifying fibroma may indicate a requirement

  18. Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors.

    Science.gov (United States)

    Kienitz, Marie-Cécile; Vladimirova, Dilyana; Müller, Christian; Pott, Lutz; Rinne, Andreas

    2016-12-16

    Activation of Gq protein-coupled receptors (GqPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the Gq signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of Gq We quantified signaling events downstream of GqPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (IKs) were measured as a functional readout of receptor-specific activation. Activation of muscarinic M1 receptors (M1-Rs) caused robust and reversible inhibition of IKs. In contrast, activation of α1B-adrenergic receptors (α1B-ARs) induced transient inhibition of IKs, which turned into delayed facilitation after agonist withdrawal. As a novel finding, we demonstrate that GqPCR-specific kinetics of IKs modulation are determined by receptor-specific desensitization, evident at the level of Gαq activation, phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, and diacylglycerol production. Sustained IKs inhibition during M1-R stimulation is attributed to robust membrane PIP2 depletion, whereas the rapid desensitization of α1B-AR delimits PIP2 reduction and augments current activation by protein kinase C (PKC). Overexpression of Ca(2+)-independent PKCδ did not affect the time course of α1B-AR-induced diacylglycerol formation, excluding a contribution of PKCδ to α1B-AR desensitization. Pharmacological inhibition of Ca(2+)-dependent PKC isoforms abolished fast α1B receptor desensitization and augmented IKs reduction, but did not affect IKs facilitation. These data indicate a contribution of Ca(2+)-dependent PKCs to α1B-AR desensitization, whereas IKs facilitation is induced by Ca(2+)-independent PKC isoforms. In contrast, neither inhibition of Ca(2+)-dependent/Ca(2+)-independent isoforms nor overexpression of PKCδ induced M1 receptor desensitization, excluding a contribution of

  19. Immunohistochemical detection of p53 and MDM2 expressions in liposarcoma with World health organization classification

    Directory of Open Access Journals (Sweden)

    A Arici

    2013-01-01

    Full Text Available Background: Liposarcomas are among the most common soft tissue sarcomas in adulthood. Aim: The purpose of the study is to perform a histopathologic typing according to World Health Organization (WHO classification of cases diagnosed with liposarcoma and to examine the difference of p53 and MDM2 expressions. Materials and Methods: The haematoxylin-eosin stained sections of 48 subjects enrolled in the study have been evaluated on the basis of the WHO classification for liposarcoma and sections stained using p53 and MDM2. Statistical Analysis Used: Chi-Square test was applied. Results: 20 subjects were diagnosed with well-differentiated liposarcoma (WLS, 16 myxoid liposarcoma (ML, 7 pleomorphic liposarcoma (PL, and 5 de-differentiated liposarcoma (DLS. The number of cases stained positive with MDM2 and p53 were positive correlated in all subjects (P = 0.02. p53 and MDM2 positivity increased in high grade tumors (P = 0.01. Conclusion: p53 and MDM2 immuno-reactivity was found to be potentially useful in liposarcoma diagnosis but a definitive implication would be rather unhealthy due to the small number of cases in our study.

  20. MDM2 inhibitor Nutlin-3a suppresses proliferation and promotes apoptosis in osteosarcoma cells

    Institute of Scientific and Technical Information of China (English)

    Bo Wang; Liming Fang; Hui Zhao; Tong Xiang; Dechun Wang

    2012-01-01

    Restoring p53 activity by inhibiting the interaction between p53 and the mouse double minutes clone 2 (MDM2) offers an attractive approach to cancer therapy.Nutlin-3a is a small-molecule inhibitor that inhibits MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling.In this study,we determined the efficacy of Nutlin-3a in inducing p53-mediated cell death in osteosarcoma (OS) cell lines both in vivo and in vitro.Targeted disruption of the p53-MDM2 interaction by Nutlin-3a stabilizes p53 and selectively activates the p53 pathway only in OS cells with wild-type p53,resulting ina pronounced anti-proliferative and cytotoxic effect due to G1 cell cycle arrest and apoptosis both in vitro and in vivo.p53 dependence of these alternative outcomes of Nutlin-3a treatment was shown by the abrogation of these effects when p53 was knocked-down by small interfering RNA.These data suggest that the disruption of p53-MDM2 interaction by Nutlin-3a might be beneficial for OS patients with MDM2 amplification and wt p53 status.

  1. MDM2 E3 ubiquitin ligase mediates UT-A1 urea transporter ubiquitination and degradation.

    Science.gov (United States)

    Chen, Guangping; Huang, Haidong; Fröhlich, Otto; Yang, Yuan; Klein, Janet D; Price, S Russ; Sands, Jeff M

    2008-11-01

    UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UT-A1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH2-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation.

  2. EGFR, p53, IDH-1 and MDM2 immunohistochemical analysis in glioblastoma: therapeutic and prognostic correlation

    Directory of Open Access Journals (Sweden)

    Richard Murdoch Montgomery

    2015-07-01

    Full Text Available We studied 36 glioblastoma cases at HC-UNICAMP from 2008 to 2012 and classified the immunohistochemical distribution of the wild-type epidermal growth factor receptor (EGFR, mutated forms of p53 protein and isocitrate dehydrogenase-1 (IDH-1 and murine double protein 2 (MDM2. Immunostaining findings were correlated with clinical data and response to treatment (surgery, chemotherapy and radiotherapy. About 97% of the tumors were primary, most of them localized in the frontal lobe. Mean time free of clinical or symptomatic disease and free time of radiological disease were 7.56 and 7.14 months, respectively. We observed a significant positive correlation between expressions of p53 and MDM2, EGFR and MDM2. Clinical, radiological and overall survivals also showed a significant positive correlation. p53 staining and clinical survival showed a significant negative correlation. The current series provides clinical and histopathological data that contribute to knowledge on glioblastoma in Brazilians.

  3. Expression and significance of p53 and mdm2 in patients with leukoplakia cancer

    Institute of Scientific and Technical Information of China (English)

    Juan-Juan Cui; Xiao-Lan Han; Wei-Min Wang

    2013-01-01

    Objective:To study the relationship of the expressions of p53 and mdm2 in leukoplakia cancer. Methods:RT-PCR was used to detect the mRNA of p53, mdm2 in patients with leukoplakia cancer.The frequencies of p53, mdm2 in peripheral blood were detected by flow cytometric analysis.Results:The expression of p53mRNA in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were7.7%,27.3%,33.3%,56.8%, respectively. The frequencies of p53 in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were(0.3±0.1)%,(1.6±0.9)%,(1.9±1.1)%,(3.4±1.8)%.The expression of mdm2 mRNA in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were0.0%,6.8%,11.1%,37.8%, respectively.The frequencies ofmdm2 in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were(0.1±0.1)%,(0.8±0.6)%,(1.2±0.8)%,(1.2±0.8)%.There was a positively correlation between p53 mRNA and mdm2 mRNA.Conclusions:The positive rate of p53 and mdm2 cells in the peripheral blood increases in patients with leukoplakia cancer tissue and has positive correlation with the severity of leukoplakia cancer.

  4. MDM2/MDMX: Master negative regulators for p53 and RB.

    Science.gov (United States)

    Hu, Linshan; Zhang, Haibo; Bergholz, Johann; Sun, Shengnan; Xiao, Zhi-Xiong Jim

    2016-03-01

    MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB.

  5. Quantitative assessment of the p53-Mdm2 feedback loop using protein lysate microarrays.

    Science.gov (United States)

    Ramalingam, Sundhar; Honkanen, Peter; Young, Lynn; Shimura, Tsutomu; Austin, John; Steeg, Patricia S; Nishizuka, Satoshi

    2007-07-01

    Mathematical simulations of the p53-Mdm2 feedback loop suggest that both proteins will exhibit impulsive expression characteristics in response to high cellular stress levels. However, little quantitative experimental evaluation has been done, particularly of the phosphorylated forms. To evaluate the mathematical models experimentally, we used lysate microarrays from an isogenic pair of gamma-ray-irradiated cell lysates from HCT116 (p53(+/+) and p53(-/-)). Both p53 and Mdm2 proteins showed expected pulses in the wild type, whereas no pulses were seen in the knockout. Based on experimental observations, we determined model parameters and generated an in silico "knockout," reflecting the experimental data, including phosphorylated proteins.

  6. Core Binding Factor β Protects HIV, Type 1 Accessory Protein Viral Infectivity Factor from MDM2-mediated Degradation.

    Science.gov (United States)

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Yoshinaga, Noriyoshi; Shirakawa, Kotaro; Kobayashi, Masayuki; Takaori-Kondo, Akifumi

    2016-11-25

    HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ.

  7. Activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in pituitary adenomas and their effects on downstream effectors.

    Science.gov (United States)

    Dworakowska, D; Wlodek, E; Leontiou, C A; Igreja, S; Cakir, M; Teng, M; Prodromou, N; Góth, M I; Grozinsky-Glasberg, S; Gueorguiev, M; Kola, B; Korbonits, M; Grossman, A B

    2009-12-01

    Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.

  8. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells*

    Science.gov (United States)

    Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A.; Zhang, Feng; Lei, Hetian

    2016-01-01

    The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. PMID:27246850

  9. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

    Science.gov (United States)

    Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A; Zhang, Feng; Lei, Hetian

    2016-07-29

    The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. MDM2/p53通路与心血管疾病的研究进展%The Research Progress of Mdm2/p53 Pathway and Cardiovascular Disease

    Institute of Scientific and Technical Information of China (English)

    羊暑艳

    2011-01-01

    鼠双微基因2(MDM2)是一原癌基因,因mRNA的不同剪切可形成多种不同相对分子质量的变异体,如p57、p64、p85、p90等.在正常细胞中,MDM2和野生型p53有着精细的平衡,其相互调节形成负反馈回路:p53诱导MDM2表达,MDM2与p53结合形成p53-MDM2复合物,使p53泛素化而被蛋白酶降解.MDM2-p53反馈体系与其他信号转导途径一起形成调控网络,参与细胞生长抑制、凋亡、细胞周期调控等各种生物进程.现对MDM2/p53途径在心血管疾病中的研究进展进行综述.%Murine double minute 2( Mdm2 )gene is an oncogene,due to the different cuttings of mRNA, a variety of variants with different molecular weight can be formed, such as p57, p64, p85, p90 and so on. In normal cells, Mdm2 and wild - type p53 has a delicate balance. The Interaction between them form a negative feedback loop:p53 induces the expression of Mdm2,the combination of Mdm2 and p53 forms a compound named p53-Mdm2. It makes p53 to be ubiquitinationed and then degraded by protease. Regulatory networks formed by Mdm2-p53 feedback system and other signaling pathways involved in cell growth inhibition,apop-tosis, cell cycle regulation and other biological processes. Here is to review the progress of the Mdm2/p53 pathways in cardiovascular disease research.

  11. mdm-2扩-增与胃癌及淋巴转移的关系%The relationship between mdm-2 amplification and gastric cancer,its lymph node metastasis

    Institute of Scientific and Technical Information of China (English)

    时军; 杨书文; 陈道达; 吕有勇

    2000-01-01

    目的探索mdm-2扩增与胃癌及其淋巴转移的关系.方法用DC-PCR技术定量分析32例胃癌及转移灶中mdm-2扩增.结果 mdm-2在转移淋巴结中的扩增频率(57.1%)高于胃原发癌中的扩增频率(37.5%),3例淋巴管内有瘤栓、淋巴结转移阴性的原发癌灶中发现mdm-2扩增.结论 mdm-2扩增与胃癌的淋巴转移关系较密切,它可能成为监测胃癌淋巴转移的潜在性分子标志物.

  12. MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3.

    Science.gov (United States)

    Hallenborg, P; Siersbæk, M; Barrio-Hernandez, I; Nielsen, R; Kristiansen, K; Mandrup, S; Grøntved, L; Blagoev, B

    2016-06-30

    The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last but not least, the binding of CRTC2 to a transcriptional enhancer that interacts with the Cebpd promoter was dramatically decreased upon JAK inhibition. Our data reveal the existence of an unusual functional interplay between STATs and CREB at the onset of adipogenesis through shared CRTC cofactors.

  13. Regulation of kidney development by the Mdm2/Mdm4-p53 axis.

    Science.gov (United States)

    El-Dahr, Samir; Hilliard, Sylvia; Saifudeen, Zubaida

    2017-01-17

    While p53 activity is required for tumor suppression, unconstrained p53 activity on the other hand is detrimental to the organism, resulting in inappropriate cellular death or proliferation defects. Unimpeded p53 activity is lethal in the developing embryo, underlining the need for maintaining a tight control on p53 activity during this period. The critical role of the negative regulators of p53, Mdm2 and Mdm4, in vertebrate development came to light by fatal disruption of embryogenesis that was observed with Mdm2 and Mdm4 gene deletions in mice. Embryonic lethality was rescued only by superimposing p53 removal. Here we summarize the contribution of the Mdm2/Mdm4-p53 axis that occurs at multiple steps of kidney development. Conditional, cell type-specific deletions reveal distinct functions of these proteins in renal morphogenesis. The severe impact on the renal phenotype from targeted gene deletions underscores the critical role played by the Mdm2/Mdm4-p53 nexus on nephrogenesis, and emphasizes the need to monitor patients with aberrations in this pathway for kidney function defects and associated cardiovascular dysfunction.

  14. Outside the p53 RING: Transcription Regulation by Chromatin-Bound MDM2.

    Science.gov (United States)

    Jain, Abhinav K; Barton, Michelle C

    2016-06-16

    Evidence mounts, via two studies published in Molecular Cell (Riscal et al., 2016; Wienken et al., 2016), that chromatin-bound MDM2 impacts pluripotency and metabolism to promote survival and proliferation of cancer cells, independently of p53 degradation.

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

    Science.gov (United States)

    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.

  16. Influence of MDM2 polymorphisms on squamous cell carcinoma susceptibility: a meta-analysis

    Science.gov (United States)

    Yu, Huanxin; Li, Haiyan; Zhang, Jinling; Liu, Gang

    2016-01-01

    Purpose Controversial associations between single-nucleotide polymorphisms (rs2279744, rs937283, rs3730485) of the MDM2 gene and the etiology of squamous cell carcinomas (SCCs) have been reported. This merits further comprehensive assessment. Materials and methods We systematically reviewed the available data and conducted an updated meta-analysis to evaluate the genetic effect of MDM2 polymorphisms in SCC susceptibility, using Stata/SE 12.0 software. Results After screening, 7,987 SCC cases and 12,954 controls from 26 eligible case–control studies were enrolled. Overall, compared with the control group, a significantly increased SCC risk was observed for the MDM2 rs2279744 polymorphism in the Asian population (test of association: odds ratio [OR] 1.12, P=0.027 for G vs T; OR 1.26, P=0.016 for GG vs TT; OR 1.25, P0.05). Conclusion Our results highlight a positive association between the GG genotype of MDM2 rs2279744 polymorphism and an increased risk of esophageal SCC in the Asian population, which needs to be clarified by more large-scale studies.

  17. TP53 and MDM2 gene polymorphisms and risk of hepatocellular carcinoma among Italian patients

    Directory of Open Access Journals (Sweden)

    Buonaguro Franco M

    2011-08-01

    Full Text Available Abstract Background Single-nucleotide polymorphisms within TP53 gene (codon 72 exon 4, rs1042522, encoding either arginine or proline and MDM2 promoter (SNP309; rs2279744, have been independently associated with increased risk of several cancer types. Few studies have analysed the role of these polymorphisms in the development of hepatocellular carcinoma. Methods Genotype distribution of TP53 codon 72 and MDM2 SNP309 in 61 viral hepatitis-related hepatocellular carcinoma cases and 122 blood samples (healthy controls from Italian subjects were determined by PCR and restriction fragment length polymorphism (RFLP. Results Frequencies of TP53 codon 72 alleles were not significantly different between cases and controls. A significant increase of MDM2 SNP309 G/G and T/G genotypes were observed among hepatocellular carcinoma cases (Odds Ratio, OR = 3.56, 95% Confidence Limits, 95% CI = 1.3-9.7; and OR = 2.82, 95% CI = 1.3-6.4, respectively. Conclusions These results highlight a significant role of MDM2 SNP309 G allele as a susceptibility gene for the development of viral hepatitis-related hepatocellular carcinoma among Italian subjects.

  18. A fluorescent probe for imaging p53-MDM2 protein-protein interaction.

    Science.gov (United States)

    Liu, Zhenzhen; Miao, Zhenyuan; Li, Jin; Fang, Kun; Zhuang, Chunlin; Du, Lupei; Sheng, Chunquan; Li, Minyong

    2015-04-01

    In this article, we describe a no-wash small-molecule fluorescent probe for detecting and imaging p53-MDM2 protein-protein interaction based on an environment-sensitive fluorescent turn-on mechanism. After extensive biological examination, this probe L1 exhibited practical activity and selectivity in vitro and in cellulo.

  19. Combined targeting of MDM2 and CDK4 is synergistic in dedifferentiated liposarcomas.

    Science.gov (United States)

    Laroche-Clary, Audrey; Chaire, Vanessa; Algeo, Marie-Paule; Derieppe, Marie-Alix; Loarer, François L; Italiano, Antoine

    2017-06-19

    MDM2 and CDK4 are frequently co-amplified in well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). We aimed to determine whether combined MDM2/CDK4 targeting is associated with higher antitumour activity than a single agent in preclinical models of DDLPS. DDLPS cells were exposed to RG7388 (MDM2 antagonist) and palbociclib (CDK4 inhibitor), and apoptosis and signalling/survival pathway perturbations were monitored by flow cytometry and Western blotting. Xenograft mouse models were used to assess tumour growth and survival. Treatment efficacy was assessed by Western blotting, histopathology and tumour volume. RG7388 and palbociclib together exerted a greater antitumour effect than either drug alone, with significant differences in cell viability after a 72-h treatment with RG7388 and/or palbociclib. The combination treatment significantly increased apoptosis compared to the single agents. We then analysed the in vivo antitumour activity of RG7388 and palbociclib in a xenograft model of DDLPS. The combination regimen reduced the tumour growth rate compared with a single agent alone and significantly increased the median progression-free survival. Our results provide a strong rationale for evaluating the therapeutic potential of CDK4 inhibitors as potentiators of MDM2 antagonists in DDLPS and justify clinical trials in this setting.

  20. Study of MDM2 and SUMO-1 expression in actinic cheilitis and lip cancer.

    Science.gov (United States)

    Oliveira Alves, Mônica Ghislaine; da Mota Delgado, Adriana; Balducci, Ivan; Carvalho, Yasmin Rodarte; Cavalcante, Ana Sueli Rodrigues; Almeida, Janete Dias

    2014-11-01

    Actinic cheilitis exhibits a potential of malignant transformation in 10-20 % of cases. The objective of this study was to compare the expression of MDM2 and SUMO-1 proteins between actinic cheilitis (AC) and squamous cell carcinoma (SCC) of the lip. The sample consisted of lower lip mucosa specimens obtained from cases with a clinical and histopathological diagnosis of AC (n = 26) and SCC (n = 25) and specimens of labial semi-mucosa (n = 15) without clinical alterations or inflammation. The tissue samples were stained with hematoxylin-eosin and anti-MDM2 and anti-SUMO-1 antibodies. Data were analyzed by the Kruskal-Wallis and Dunn's tests (5 %). The median expression of MDM2 (kW = 36.8565; df = 3-1 = 2; p = 0.0001) and SUMO-1 (kW = 32.7080; df = 3-1 = 2; p = 0.0001) was similar in cases of AC and SCC of the lip, but differed significantly from that observed for normal labial semi-mucosa. Despite the limitations of the present study, immunohistochemistry demonstrated the overexpression of important proteins (MDM2 and SUMO-1) related to regulatory mechanisms of apoptosis in AC and SCC of the lip, but further studies are needed.

  1. Chlamydia infection depends on a functional MDM2-p53 axis.

    Science.gov (United States)

    González, Erik; Rother, Marion; Kerr, Markus C; Al-Zeer, Munir A; Abu-Lubad, Mohammad; Kessler, Mirjana; Brinkmann, Volker; Loewer, Alexander; Meyer, Thomas F

    2014-11-13

    Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection.

  2. The p53 Tumor Suppressor Protein Does Not Regulate Expression of Its Own Inhibitor, MDM2, Except under Conditions of Stress

    OpenAIRE

    2000-01-01

    MDM2 is an important regulator of the p53 tumor suppressor protein. MDM2 inhibits p53 by binding to it, physically blocking its ability to transactivate gene expression, and stimulating its degradation. In cultured cells, mdm2 expression can be regulated by p53. Hence, mdm2 and p53 can interact to form an autoregulatory loop in which p53 activates expression of its own inhibitor. The p53/MDM2 autoregulatory loop has been elucidated within cultured cells; however, regulation of mdm2 expression...

  3. Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of the MDM2-p53 interaction

    NARCIS (Netherlands)

    Surmiak, Ewa; Neochoritis, Constantinos G; Musielak, Bogdan; Twarda-Clapa, Aleksandra; Kurpiewska, Katarzyna; Dubin, Grzegorz; Camacho, Carlos; Holak, Tad A; Dömling, Alexander

    2016-01-01

    Using the computational pharmacophore-based ANCHOR.QUERY platform a new scaffold was discovered. Potent compounds evolved inhibiting the protein-protein interaction p53-MDM2. An extensive SAR study was performed based on our four-point pharmacophore model, yielding derivatives with affinity to MDM2

  4. Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of the MDM2-p53 interaction

    NARCIS (Netherlands)

    Surmiak, Ewa; Neochoritis, Constantinos G; Musielak, Bogdan; Twarda-Clapa, Aleksandra; Kurpiewska, Katarzyna; Dubin, Grzegorz; Camacho, Carlos; Holak, Tad A; Dömling, Alexander

    2016-01-01

    Using the computational pharmacophore-based ANCHOR.QUERY platform a new scaffold was discovered. Potent compounds evolved inhibiting the protein-protein interaction p53-MDM2. An extensive SAR study was performed based on our four-point pharmacophore model, yielding derivatives with affinity to MDM2

  5. Overexpression of MDM2 protein in ameloblastomas as compared to adenomatoid odontogenic tumor

    Directory of Open Access Journals (Sweden)

    A Krishna

    2012-01-01

    Full Text Available Background: Recent studies on odontogenic tumors have identified various molecular alterations responsible for their development, and determination of epithelial proliferation is a useful means of investigating the differences in biologic behavior of these tumors. One such specific marker to identify proliferative activity and tumor aggressiveness by immunohistochemistry (IHC is MDM2, 90-95kDa protein. Objective: This immunohistochemical study using MDM2 expression was undertaken to understand better the diverse biological activity of two groups of odontogenic tumors namely ameloblastoma and adenomatoid odontogenic tumor (AOT based on their cell proliferation activity. Materials and Methods: A total of 50 cases, comprising of 36 ameloblastoma samples and 14 AOT samples, were subjected to heat-induced antigen retrieval method using citrate buffer in a pressure cooker. Consequently, the sections were stained with MDM2 monoclonal antibody and visualized using an LSAB+ kit. Results: In ameloblastomas, statistically significant association was seen between plexiform ameloblastomas, follicular ameloblastomas with granular cell changes, desmoplastic and unicystic variants. The predominant nuclear staining by MDM2 revealed overexpression in ameloblastomas as compared to AOT. Conclusion: The MDM2 overexpression noticed in plexiform ameloblastoma, follicular ameloblastoma with granular cell changes and acanthomatous ameloblastoma when compared to simple unicystic and desmoplastic ameloblastoma suggest a relatively enhanced proliferative phenotype of these solid multicystic variants of ameloblastomas. On overall comparison, higher expression was noted in ameloblastomas when compared to AOT. This indicates differences in the aggressive nature between these two groups of odontogenic tumors favoring the perception of a greater aggressive nature of ameloblastomas.

  6. MDM2 gene amplification: a new independent factor of adverse prognosis in non-small cell lung cancer (NSCLC).

    Science.gov (United States)

    Dworakowska, Dorota; Jassem, Ewa; Jassem, Jacek; Peters, Brigitte; Dziadziuszko, Rafał; Zylicz, Maciej; Jakóbkiewicz-Banecka, Joanna; Kobierska-Gulida, Grazyna; Szymanowska, Amelia; Skokowski, Jan; Roessner, Albert; Schneider-Stock, Regine

    2004-03-01

    The prognostic impact of MDM2 amplification in non-small cell lung cancer (NSCLC) remains unknown. In this study, we investigated the occurrence of MDM2 amplification in surgically treated NSCLC patients. Molecular data were correlated with clinicopathological factors and evaluated for their prognostic value. The study group included 116 NSCLC patients who underwent pulmonary resection between 1996 and 1999. MDM2 amplification was assessed by real-time PCR using hybridization probe format on a LightCycler (Roche). The calculated ratio was a MDM2 value normalized to the amplification of the housekeeping gene phenylalaninhydroxylase (PAH). Survival curves were drawn according to the Kaplan-Meier method and compared with the use of the log-rank test. Multivariate analysis was based on Cox regression analysis. MDM2 amplification was found in 24 patients (21%). There was no relationship between MDM2 amplification and clinicopathological factors, such as sex, age and stage of disease, pT, pN, histology and tumor differentiation. Median disease-free survival (DFS) in patients with and without MDM2 amplification was 3 and 31 months, and 5-year DFS 24 and 33%, respectively (log-rank, P = 0.02). Likewise, median overall survival (OS) in patients with and without MDM2 amplification was 9 and 33 months, respectively, and 5-year OS 24 and 39%, respectively (log-rank, P = 0.01). The strong prognostic relevance of MDM2 amplification for both DFS and OS was confirmed in multivariate analysis (P < 0.01 for both comparisons). Our results suggest that MDM2 gene amplification analysis provides additional prognostic information in surgically treated NSCLC patients.

  7. MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4.

    Directory of Open Access Journals (Sweden)

    Lynnette Marcar

    Full Text Available Melanoma antigen A (MAGE-A proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i uncouple the ubiquitin ligase and degradation functions of MDM2; (ii act as potent inhibitors of E3 ligase function; and (iii regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically.

  8. MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4

    Science.gov (United States)

    Marcar, Lynnette; Ihrig, Bianca; Hourihan, John; Bray, Susan E.; Quinlan, Philip R.; Jordan, Lee B.; Thompson, Alastair M.; Hupp, Ted R.; Meek, David W.

    2015-01-01

    Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically. PMID:26001071

  9. p53蛋白和MDM 2蛋白在食管鳞状细胞癌中的表达及意义%Expression of p53 and MDM 2 Protein and their Significance in Esophageal Carcinomas

    Institute of Scientific and Technical Information of China (English)

    赵仲生; 张梅; 徐文娟

    1998-01-01

    为了探讨肿瘤抑制基因p53和癌基因MDM 2在食管鳞状细胞癌中的表达及其意义,采用免疫组织化学链菌素亲生物素蛋白过氧化物酶连接法(SP法),检测了68例食管鳞状细胞癌中p53蛋白和MDM 2蛋白的表达,结果显示p53蛋白和MDM 2蛋白阳性率分别为60.3%(41/68)和42.6%(29/68),p53蛋白阳性率与食管鳞状细胞癌分级呈显著正相关(P<0.01),MDM 2蛋白阳性率与食管鳞状细胞癌分级呈显著负相关(P<0.01),并与食管鳞状细胞癌临床病理分期呈显著负相关(P<0.05).在68例食管鳞状细胞癌中,p53蛋白阳性表达者41例,其中p53蛋白和MDM 2蛋白表达均阳性者12例,MDM 2蛋白表达阴性者29例;在68例食管癌中,MDM 2蛋白阳性表达者29例,其中MDM2与p53蛋白均阳性者12例,p53蛋白表达阴性者17例,两者呈显著负相关(P<0.01),p53蛋白和MDM 2蛋白表达均阴性者10例.可以认为p53蛋白和MDM 2蛋白表达可作为食管鳞状细胞癌病理分级参考指标之一,MDM 2蛋白表达还可作为食管鳞状细胞癌临床病理分期的参考指标之一,并间接证明MDM 2蛋白对p53蛋白表达具有负性调节作用.

  10. LC-MS/MS-based targeted proteomics quantitatively detects the interaction between p53 and MDM2 in breast cancer.

    Science.gov (United States)

    Zhang, Wen; Zhong, Ting; Chen, Yun

    2017-01-30

    In breast cancer, p53 could be functionally compromised by interaction with several proteins. Among those proteins, MDM2 serves as a pivotal negative regulator and counteracts p53 activation. Thus, the ability to quantitatively and accurately monitor the changes in level of p53-MDM2 interaction with disease state can enable an improved understanding of this protein-protein interaction (PPI), provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI including p53-MDM2 interaction so far. In this study, a LC-MS/MS-based targeted proteomics assay was developed and coupled with co-immunoprecipitation (Co-IP) for the quantification of p53-MDM2 complex. A p53 antibody with the epitope residing at 156-214 residues achieved the greatest IP efficiency. 321KPLDGEYFTLQIR333 (p53) and 327ENWLPEDK334 (MDM2) were selected as surrogate peptides in the targeted analysis. Stable isotope-labeled synthetic peptides were used as internal standards. An LOQ (limit of quantification) of 2ng/mL was obtained. Then, the assay was applied to quantitatively detect total p53, total MDM2 and p53-MDM2 in breast cells and tissue samples. Western blotting was performed for a comparison. Finally, a quantitative time-course analysis in MCF-7 cells with the treatment of nutlin-3 as a PPI inhibitor was also monitored.

  11. Protein-peptide molecular docking with large-scale conformational changes: the p53-MDM2 interaction

    Science.gov (United States)

    Ciemny, Maciej Pawel; Debinski, Aleksander; Paczkowska, Marta; Kolinski, Andrzej; Kurcinski, Mateusz; Kmiecik, Sebastian

    2016-12-01

    Protein-peptide interactions are often associated with large-scale conformational changes that are difficult to study either by classical molecular modeling or by experiment. Recently, we have developed the CABS-dock method for flexible protein-peptide docking that enables large-scale rearrangements of the protein chain. In this study, we use CABS-dock to investigate the binding of the p53-MDM2 complex, an element of the cell cycle regulation system crucial for anti-cancer drug design. Experimental data suggest that p53-MDM2 binding is affected by significant rearrangements of a lid region - the N-terminal highly flexible MDM2 fragment; however, the details are not clear. The large size of the highly flexible MDM2 fragments makes p53-MDM2 intractable for exhaustive binding dynamics studies using atomistic models. We performed extensive dynamics simulations using the CABS-dock method, including large-scale structural rearrangements of MDM2 flexible regions. Without a priori knowledge of the p53 peptide structure or its binding site, we obtained near-native models of the p53-MDM2 complex. The simulation results match well the experimental data and provide new insights into the possible role of the lid fragment in p53 binding. The presented case study demonstrates that CABS-dock methodology opens up new opportunities for protein-peptide docking with large-scale changes of the protein receptor structure.

  12. TBP-like Protein (TLP) Disrupts the p53-MDM2 Interaction and Induces Long-lasting p53 Activation.

    Science.gov (United States)

    Maeda, Ryo; Tamashiro, Hiroyuki; Takano, Kazunori; Takahashi, Hiro; Suzuki, Hidefumi; Saito, Shinta; Kojima, Waka; Adachi, Noritaka; Ura, Kiyoe; Endo, Takeshi; Tamura, Taka-Aki

    2017-02-24

    Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions.

  13. The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation

    DEFF Research Database (Denmark)

    Guerra, B; Götz, C; Wagner, P

    1997-01-01

    The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro 0.5-1 mol of phosphate were incorporated per mol MDM2 protein. The catalytic subunit of protein kinase CK2 (alpha-subunit) catalyzed the incorporation of twice as much phosphate into the MDM2 protein as it was obtained...... with the holoenzyme. Polylysine stimulated MDM2 phosphorylation by CK2 holoenzyme threefold in contrast to the alpha-subunit-catalyzed MDM2 phosphorylation which was reduced by about 66% when polylysine was added. Full length p53, but also a peptide representing a C-terminal fragment of the tumor suppressor gene......(264-393) was on the average close to twofold and inhibition in the case of the alpha-subunit-catalyzed MDM2 phosphorylation was about 40%. Phosphorylation of MDM2 by CK2 holoenzyme in the presence of the p21(WAF1/CIP1), known to be a potent inhibitor of cyclin-dependent protein kinases, also led to a significant reduction...

  14. A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2.

    Science.gov (United States)

    Nicholson, Judith; Scherl, Alex; Way, Luke; Blackburn, Elizabeth A; Walkinshaw, Malcolm D; Ball, Kathryn L; Hupp, Ted R

    2014-06-01

    Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors.

  15. Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

    Science.gov (United States)

    Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic st...

  16. MDM2 SNP309, gene-gene interaction, and tumor susceptibility: an updated meta-analysis

    Directory of Open Access Journals (Sweden)

    Wu Wei

    2011-05-01

    Full Text Available Abstract Background The tumor suppressor gene p53 is involved in multiple cellular pathways including apoptosis, transcriptional control, and cell cycle regulation. In the last decade it has been demonstrated that the single nucleotide polymorphism (SNP at codon 72 of the p53 gene is associated with the risk for development of various neoplasms. MDM2 SNP309 is a single nucleotide T to G polymorphism located in the MDM2 gene promoter. From the time that this well-characterized functional polymorphism was identified, a variety of case-control studies have been published that investigate the possible association between MDM2 SNP309 and cancer risk. However, the results of the published studies, as well as the subsequent meta-analyses, remain contradictory. Methods To investigate whether currently published epidemiological studies can clarify the potential interaction between MDM2 SNP309 and the functional genetic variant in p53 codon72 (Arg72Pro and p53 mutation status, we performed a meta-analysis of the risk estimate on 27,813 cases with various tumor types and 30,295 controls. Results The data we reviewed indicated that variant homozygote 309GG and heterozygote 309TG were associated with a significant increased risk of all tumor types (homozygote comparison: odds ratio (OR = 1.25, 95% confidence interval (CI = 1.13-1.37; heterozygote comparison: OR = 1.10, 95% CI = 1.03-1.17. We also found that the combination of GG and Pro/Pro, TG and Pro/Pro, GG and Arg/Arg significantly increased the risk of cancer (OR = 3.38, 95% CI = 1.77-6.47; OR = 1.88, 95% CI = 1.26-2.81; OR = 1.96, 95% CI = 1.01-3.78, respectively. In a stratified analysis by tumor location, we also found a significant increased risk in brain, liver, stomach and uterus cancer (OR = 1.47, 95% CI = 1.06-2.03; OR = 2.24, 95%CI = 1.57-3.18; OR = 1.54, 95%CI = 1.04-2.29; OR = 1.34, 95%CI = 1.07-1.29, respectively. However, no association was seen between MDM2 SNP309 and tumor susceptibility

  17. Rare Aggressive Behavior of MDM2-Amplified Retroperitoneal Dedifferentiated Liposarcoma, with Brain, Lung and Subcutaneous Metastases

    Science.gov (United States)

    Ben Salha, Imen; Zaidi, Shane; Noujaim, Jonathan; Miah, Aisha B.; Fisher, Cyril; Jones, Robin L.; Thway, Khin

    2016-01-01

    Dedifferentiated liposarcoma (DDL) is a histologically pleomorphic sarcoma, traditionally defined as well-differentiated liposarcoma with abrupt transition to high grade, non-lipogenic sarcoma. It can occur as part of recurrent well-differentiated liposarcoma, or may arise de novo. DDL most frequently occurs within the retroperitoneum, and while it is prone to local recurrence, it usually has a lower rate of metastasis than other pleomorphic sarcomas. We describe a case of retroperitoneal dedifferentiated liposarcoma in a 63-year-old male, who showed MDM2 amplification with fluorescence in situ hybridization, which displayed unusually aggressive behavior, with brain, lung and subcutaneous soft tissue metastases. As previous reports of metastatic liposarcoma have largely grouped DDL in with other (genetically and clinically distinct) liposarcoma subtypes, we highlight and discuss the rare occurrence of brain metastasis in MDM2-amplified retroperitoneal liposarcoma.

  18. Heterotrimeric G-protein, Gα16, is a critical downstream effector of non-canonical Wnt signaling and a potent inhibitor of transformed cell growth in non small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Sreedevi Avasarala

    Full Text Available G-protein-coupled receptors (GPCR are the largest family of cell surface molecules that play important role/s in a number of biological and pathological processes including cancers. Earlier studies have highlighted the importance of Wnt7a signaling via its cognate receptor Frizzled9, a GPCR, in inhibition of cell proliferation, anchorage-independent growth, and reversal of transformed phenotype in non small cell lung cancer primarily through activation of the tumor suppressor, PPARγ. However, the G-protein effectors that couple to this important tumor suppressor pathway have not been identified, and are of potential therapeutic interest. In this study, by using two independent Wnt7a/Frizzled9-specific read-outs, we identify Gα16 as a novel downstream effector of Wnt7a/Frizzled9 signaling. Interestingly, Gα16 expression is severely down-regulated, both at the messenger RNA levels and protein levels, in many non small cell lung cancer cell lines. Additionally, through gene-specific knock-downs and expression of GTPase-deficient forms (Q212L of Gα16, we also establish Gα16 as a novel regulator of non small cell lung cancer cell proliferation and anchorage-independent cell growth. Taken together, our data not only establish the importance of Gα16 as a critical downstream effector of the non-canonical Wnt signaling pathway but also as a potential therapeutic target for the treatment of non small cell lung cancer.

  19. MDM2 SNP309 contributes to tumor susceptibility: A meta-analysis

    Institute of Scientific and Technical Information of China (English)

    Xiaoman Wo; Dong Han; Haiming Sun; Yang Liu; Xiangning Meng; Jing Bai; Feng Chen

    2011-01-01

    The potentially functional polymorphism,SNP309,in the promoter region of MDM2 gene has been implicated in cancer risk,but individual published studies showed inconclusive results.To obtain a more precise estimate of the association between MDM2 SNP309 and risk of cancer,we performed a meta-analysis of 70 individual studies in 59 publications that included 26,160 cases with different types of tumors and 33,046 controls.Summary odds ratios (OR) and corresponding 95% confidence intervals (CIs) were estimated using fixed- and random-effects models when appropriate.Overall,the variant genotypes were associated with a significantly increased cancer risk for all cancer types in different genetic models (GG vs.TT:OR,1.123; 95% CI,1.056-1.193; GG/GT vs.TT:OR,1.028; 95% CI,1.006-1.050).In the stratified analyses,the increased risk remained for the studies of most types of cancers,Asian populations,and hospital-/population-based studies in different genetic models,whereas significantly decreased risk was found in prostate cancer (GG vs.TT:OR,0.606; 95% CI,0.407-0.903; GG/GT vs.TT:OR,0.748; 95% CI,0.579-0.968).In conclusion,the data of meta-analysis suggests that MDM2 SNP309 is a potential biomarker for cancer risk.

  20. Genetic variants in TP53 and MDM2 associated with male infertility in Chinese population

    Institute of Scientific and Technical Information of China (English)

    Cong Huang; Wei Liu; Gui-Xiang Ji; Ai-Hua Gu; Jian-Hua Qu; Ling Song; Xin-Ru Wang

    2012-01-01

    The TP53,a transcriptional regulator and tumor suppressor,is functionally important in spermatogenesis.MDM2 is a key regulator of the p53 pathway and modulates p53 activity.Both proteins have been functionally linked to germ cell apoptosis,which may affect human infertility,but very little is known on how common polymorphisms in these genes may influence germ cell apoptosis and the risk of male infertility.Thus,this study was designed to test whether three previously described polymorphisms 72Arg>Pro (rs1042522) and the Ex2+ 19C>T (rs2287498) in TP53,and the 5' untranslated region (5' UTR) 309T>G (rs937283) in MDM2,are associated with idiopathic male infertility in a Chinese population.The three polymorphisms were genotyped using OpenArray assay in a hospital-based case-control study,including 580 infertile patients and 580 fertile controls.Our analyses revealed that TP53 Ex2+ 19C>T and MDM2309T>G polymorphisms are associated with mate infertility.Furthermore,we detected a nearly statistically significant additive interaction between TP53 rs2287498 and MDM2 rs937283 for the development of male.infertility (Pinteraction=0.055).In summary,this study found preliminary evidence,demonstrating that genetic variants in genes of the TP53 pathway are risk factors for male infertility.

  1. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

    Directory of Open Access Journals (Sweden)

    Christopher J Brown

    Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

  2. Bcl-2/MDM2 Dual Inhibitors Based on Universal Pyramid-Like α-Helical Mimetics.

    Science.gov (United States)

    Wang, Ziqian; Song, Ting; Feng, Yingang; Guo, Zongwei; Fan, Yudan; Xu, Wenjie; Liu, Lu; Wang, Anhui; Zhang, Zhichao

    2016-04-14

    No α-helical mimetic that exhibits Bcl-2/MDM2 dual inhibition has been rationally designed due to the different helicities of the α-helixes at their binding interfaces. Herein, we extracted a one-turn α-helix-mimicking ortho-triarene unit from o-phenylene foldamers. Linking benzamide substrates with a rotatable C-N bond, we constructed a novel semirigid pyramid-like scaffold that could support its two-turn α-helix mimicry without aromatic stacking interactions and could adopt the different dihedral angles of the key residues of p53 and BH3-only peptides. On the basis of this universal scaffold, a series of substituent groups were installed to capture the key residues of both p53TAD and BimBH3 and balance the differences of the bulks between them. Identified by FP, ITC, and NMR spectroscopy, a compound 6e (zq-1) that directly binds to Mcl-1, Bcl-2, and MDM2 with balanced submicromolar affinities was obtained. Cell-based experiments demonstrated its antitumor ability through Bcl-2/MDM2 dual inhibition simultaneously.

  3. MDM2 Amplification and PI3KCA Mutation in a Case of Sclerosing Rhabdomyosarcoma.

    Science.gov (United States)

    Kikuchi, Ken; Wettach, George R; Ryan, Christopher W; Hung, Arthur; Hooper, Jody E; Beadling, Carol; Warrick, Andrea; Corless, Christopher L; Olson, Susan B; Keller, Charles; Mansoor, Atiya

    2013-01-01

    A rare sclerosing variant of rhabdomyosarcoma characterized by prominent hyalinization and pseudovascular pattern has recently been described as a subtype biologically distinct from embryonal, alveolar, and pleomorphic forms. We present cytogenetic and molecular findings as well as experimental studies of an unusual case of sclerosing rhabdomyosarcoma. The primary lesion arose within the plantar subcutaneous tissue of the left foot of an otherwise healthy 23-year-old male who eventually developed pulmonary nodules despite systemic chemotherapy. Two genetic abnormalities identified in surgical and/or autopsy samples of the tumor were introduced into 10T1/2 murine fibroblasts to determine whether these genetic changes cooperatively facilitated transformation and growth. Cytogenetic analysis revealed a complex abnormal hyperdiploid clone, and MDM2 gene amplification was confirmed by fluorescence in situ hybridization. Cancer gene mutation screening using a combination of multiplexed PCR and mass spectroscopy revealed a PIK3CA exon 20 H1047R mutation in the primary tumor, lung metastasis, and liver metastasis. However, this mutation was not cooperative with MDM2 overexpression in experimental assays for transformation or growth. Nevertheless, MDM2 and PIK3CA are genes worthy of further investigation in patients with sclerosing rhabdomyosarcoma and might be considered in the enrollment of these patients into clinical trials of targeted therapeutics.

  4. MDM2 Amplification and PI3KCA Mutation in a Case of Sclerosing Rhabdomyosarcoma

    Directory of Open Access Journals (Sweden)

    Ken Kikuchi

    2013-01-01

    Full Text Available A rare sclerosing variant of rhabdomyosarcoma characterized by prominent hyalinization and pseudovascular pattern has recently been described as a subtype biologically distinct from embryonal, alveolar, and pleomorphic forms. We present cytogenetic and molecular findings as well as experimental studies of an unusual case of sclerosing rhabdomyosarcoma. The primary lesion arose within the plantar subcutaneous tissue of the left foot of an otherwise healthy 23-year-old male who eventually developed pulmonary nodules despite systemic chemotherapy. Two genetic abnormalities identified in surgical and/or autopsy samples of the tumor were introduced into 10T1/2 murine fibroblasts to determine whether these genetic changes cooperatively facilitated transformation and growth. Cytogenetic analysis revealed a complex abnormal hyperdiploid clone, and MDM2 gene amplification was confirmed by fluorescence in situ hybridization. Cancer gene mutation screening using a combination of multiplexed PCR and mass spectroscopy revealed a PIK3CA exon 20 H1047R mutation in the primary tumor, lung metastasis, and liver metastasis. However, this mutation was not cooperative with MDM2 overexpression in experimental assays for transformation or growth. Nevertheless, MDM2 and PIK3CA are genes worthy of further investigation in patients with sclerosing rhabdomyosarcoma and might be considered in the enrollment of these patients into clinical trials of targeted therapeutics.

  5. The silence of p66(Shc) in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway.

    Science.gov (United States)

    Zhang, Ling; Zhu, Shengtao; Shi, Xuesen; Sha, Weihong

    2015-01-01

    Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66(Shc) protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66(Shc) in the progress of colon cancer still unknown. In this study, we found that p66(Shc) highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66(Shc) in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66(Shc) siRNA. Furthermore, after HCT8 cells treated with p66(Shc) siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66(Shc) in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell.

  6. The silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway

    Science.gov (United States)

    Zhang, Ling; Zhu, Shengtao; Shi, Xuesen; Sha, Weihong

    2015-01-01

    Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66Shc protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66Shc in the progress of colon cancer still unknown. In this study, we found that p66Shc highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66Shc in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66Shc siRNA. Furthermore, after HCT8 cells treated with p66Shc siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell. PMID:26464652

  7. 睾丸肿瘤组织中mdm-2基因表达及临床意义%Mdm-2 Gene Expression in Human Testicular Cancer

    Institute of Scientific and Technical Information of China (English)

    徐元虎; 张培华

    2001-01-01

    为了探讨mdm-2基因与睾丸肿瘤发生及预后的关系,应用免疫组化方法检测27例睾丸肿瘤中mdm-2蛋白表达水平,发现有12例为mdm-2蛋白阳性表达,并且与睾丸肿瘤的病理分级和临床分期有关.提示睾丸肿瘤恶性程度与mdm-2蛋白表达具有明显相关性.mdm-2蛋白表达可能成为睾丸肿瘤预后的指标.

  8. MDM-2、bcl-2 和Ki-67在子宫颈癌中的表达及其意义%Expression and Evaluation of MDM-2, bcl-2 and Ki-67 in Cervical Carcinoma

    Institute of Scientific and Technical Information of China (English)

    李宏艳; 张建中

    2008-01-01

    目的 探讨MDM-2、bcl-2和Ki-67在子宫颈癌的发生、发展过程中的作用.方法 采用免疫组化法检测MDM-2、bcl-2和Ki-67在31例子宫颈癌、13例子宫颈非典型性增生、15例正常子宫颈组织中的表达水平.结果 在31例子宫颈癌组织中MDM-2表达阳性11例,bcl-2阳性13例,Ki-67阳性15例.bcl-2、MDM-2和Ki-67三者共同阳性9例(29.03%).对31例子宫颈癌组织中MDM-2与bcl-2共同阳性者、MDM-2与Ki-67共同阳性者及Ki-67与bcl-2共同阳性者进行配对资料χ2检验,P<0.05.子宫颈癌组织中bcl-2、MDM-2和Ki-6阳性表达率彼此间存在着明显的正性相关关系.结论 联合检测MDM-2、bcl-2和Ki-67在子宫颈癌中的表达,显示三者具有明显相关性,与子宫颈癌的发生、发展有关.

  9. Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain.

    Science.gov (United States)

    Vasileiou, Zoe; Barlos, Kostas; Gatos, Dimitrios

    2009-12-01

    The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis.

  10. P53 and Murine Double Mimute 2 (MDM2) Expression Changes and Significance in Different Types of Endometrial Lesions

    Science.gov (United States)

    Jiang, Zhongyong; Xu, Wanqing; Dan, Gang; Liu, Yuan; Xiong, Jie

    2016-01-01

    Background Endometrial lesions are common in obstetrics and gynecology, including endometrial polyps, uterine adenomyosis, and malignant endometrial adenocarcinoma. Endometrial lesions seriously affect women’s health, fertility, quality of life, and life safety. As a pro-apoptosis gene, p53 is considered to be closely related with human tumors. Murine double mimute 2 (MDM2) is an oncogene that can promote tumor occurrence and development. P53 and MDM2 expression and significance in different types of endometrial lesions have not been fully elucidated. Material/Methods Normal endometrium, endometrial polyps, uterine adenomyosis, and endometrial adenocarcinoma tissue samples were collected. Real-time PCR was used to detect p53 and MDM2 mRNA expression. Immunohistochemical staining and Western blot analysis were applied to test p53 and MDM2 protein expression. Their correlation with clinical staging of endometrial adenocarcinoma was analyzed. Results P53 and MDM2 mRNA and protein expression were significantly elevated in the endometrial polyps group and the endometrial adenocarcinoma group compared with the normal control group (Pendometrial adenocarcinoma compared with endometrial polyps (P0.05). P53 and MDM2 mRNA and protein level showed a positive correlation. Significantly higher expression of p53 or MDM2 was observed in patients with stage III compared to those in patients with stage II. Higher expression was also observed in patients with stage II than in patients with stage I. Conclusions P53 and MDM2 mRNA and protein were elevated in endometrial polyps and endometrial adenocarcinoma and their expressions were correlated with clinical staging of endometrial adenocarcinoma. They can promote cancer occurrence and development, and can be treated to assist diagnosis and provide a reference for treatment. PMID:27924072

  11. MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition.

    Science.gov (United States)

    Kovatcheva, Marta; Liu, David D; Dickson, Mark A; Klein, Mary E; O'Connor, Rachael; Wilder, Fatima O; Socci, Nicholas D; Tap, William D; Schwartz, Gary K; Singer, Samuel; Crago, Aimee M; Koff, Andrew

    2015-04-10

    CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy.

  12. MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53

    DEFF Research Database (Denmark)

    Wienken, Magdalena; Dickmanns, Antje; Nemajerova, Alice

    2016-01-01

    The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion...... in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically...... associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell...

  13. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response.

    Science.gov (United States)

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-08-04

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage.

  14. Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice.

    Science.gov (United States)

    Carr, Michael I; Roderick, Justine E; Zhang, Hong; Woda, Bruce A; Kelliher, Michelle A; Jones, Stephen N

    2016-12-27

    The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2(S394A) mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2(Y393F)) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2(Y393F) mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2(Y393F/S394A) mice and Mdm2(S394A) mice display similar phenotypes.

  15. Expression and clinical significance of Pokemon and mdm2 proteins in breast cancer%Pokemon、mdm2蛋白在乳腺癌中的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    刘海峰; 曲宏岩

    2008-01-01

    目的 探讨Pokemon和mdm2蛋白与乳腺癌发生、发展的关系及其临床意义.方法 采用免疫组织化学S-P法检测54例乳腺癌及18例正常乳腺组织中Pokemon和mdm2蛋白的表达水平,分析其与乳腺癌临床病理学特征的关系,以及Pokemon与mdm2蛋白的相关性.结果 在乳腺癌和正常乳腺组织中,Pokemon蛋白的阳性表达率分别为74.1%(40/54)、16.7%(3/18);mdm2蛋白的阳性表达率分别为81.5%(44/54)、22.2%(4/18).两组中Pokemon与mdm2蛋白的阳性表达率均有统计学意义(P0.05 ).Pokemon蛋白表达与mdm2蛋白表达呈正相关(rs=0.588 ,P<0.05).结论 Pokemon与mdm2蛋白可能共同参与了乳腺癌的发生、发展和转移.Pokemon有望成为乳腺癌治疗的一个有效的靶基因,从而为乳腺癌基因治疗开辟新的途径.

  16. The Expression and Clinical Significance of P21WAF1 MDM-2 in Pancreatic Carcinoma%人胰腺癌中P21WAF1、MDM-2的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    刘振华; 冯一中; 李峰; 柴玉海

    2007-01-01

    目的 探讨P21WAF1、MDM-2在胰腺癌中的表达及其在胰腺癌发生发展中的作用.方法 应用免疫组织化学S-P法检测P21WAF1、MDM-2在10例正常胰腺组织、53例胰腺癌组织中的表达,并分析其与临床病理参数之间的关系.结果 胰腺癌组织中P21WAE1的表达显著低于正常胰腺组织(P<0.05),MDM-2的表达显著高于正常胰腺组织(P<0.05).53例胰腺癌组织中P21WAF1、MDM-2的表达随肿瘤的分化程度降低而减少,差异具有显著性(P<0.05).有淋巴结转移的胰腺癌P21WAF1的表达明显低于没有淋巴结转移者(P<0.05).P21WAF1、MDM-2的表达与患者的生存率有关.P21WAF1、MDM-2的表达与性别、年龄、肿瘤部位、肿瘤大小及临床分期无关.结论 P21WAF1、MDM-2在胰腺癌的发生、发展中起重要作用,可作为该肿瘤预后判断的理论依据.

  17. Aberrant splicing of the DMP1-ARF-MDM2-p53 pathway in cancer.

    Science.gov (United States)

    Inoue, Kazushi; Fry, Elizabeth A

    2016-07-01

    Alternative splicing (AS) of mRNA precursors is a ubiquitous mechanism for generating numerous transcripts with different activities from one genomic locus in mammalian cells. The gene products from a single locus can thus have similar, dominant-negative or even opposing functions. Aberrant AS has been found in cancer to express proteins that promote cell growth, local invasion and metastasis. This review will focus on the aberrant splicing of tumor suppressor/oncogenes that belong to the DMP1-ARF-MDM2-p53 pathway. Our recent study shows that the DMP1 locus generates both tumor-suppressive DMP1α (p53-dependent) and oncogenic DMP1β (p53-independent) splice variants, and the DMP1β/α ratio increases with neoplastic transformation of breast epithelial cells. This process is associated with high DMP1β protein expression and shorter survival of breast cancer (BC) patients. Accumulating pieces of evidence show that ARF is frequently inactivated by aberrant splicing in human cancers, demonstrating its involvement in human malignancies. Splice variants from the MDM2 locus promote cell growth in culture and accelerate tumorigenesis in vivo. Human cancers expressing these splice variants are associated with advanced stage/metastasis, and thus have negative clinical impacts. Although they lack most of the p53-binding domain, their activities are mostly dependent on p53 since they bind to wild-type MDM2. The p53 locus produces splice isoforms that have either favorable (β/γ at the C-terminus) or negative impact (Δ40, Δ133 at the N-terminus) on patients' survival. As the oncogenic AS products from these loci are expressed only in cancer cells, they may eventually become targets for molecular therapies.

  18. The regulation of the p53/MDM2 feedback loop by microRNAs

    OpenAIRE

    2015-01-01

    Tumor suppressor p53 and its signaling pathway play a central role in tumor prevention. The E3 ubiquitin ligase MDM2, which is a direct p53 transcriptional target and also the most critical negative regulator of p53, forms an autoregulatory negative feedback loop with p53 in the cell to tightly regulate the levels and activity of p53. MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that play a critical role in the post-translational regulation of gene expression. Recent st...

  19. MDM2 promoter del1518 polymorphism and cancer risk: evidence from 22,931 subjects

    Directory of Open Access Journals (Sweden)

    Hua WF

    2017-07-01

    Full Text Available Wenfeng Hua,1,* Anqi Zhang,2,* Ping Duan,2,* Jinhong Zhu,3 Yuan Zhao,2 Jing He,4 Zhi Zhang1 1Department of Laboratory Medicine and Central Laboratories, Guangdong Second Provincial General Hospital, Guangzhou, Guangdong, 2Department of Obstetrics and Gynecology, The Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 3Molecular Epidemiology Laboratory and Department of Laboratory Medicine, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 4Department of Pediatric Surgery, Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic of China *These authors contributed equally to this work Abstract: Studies have shown that single-nucleotide polymorphisms in MDM2 gene may play important roles in the development of malignant tumor. The association of del1518 polymorphism (rs3730485 in the MDM2 promoter with cancer susceptibility has been extensively studied; however, the results are contradictory. To quantify the association between this polymorphism and overall cancer risk, we conducted a meta-analysis with 12,905 cases and 10,026 controls from 16 eligible studies retrieved from PubMed, Embase, and Chinese Biomedical (CBM databases. We assessed the strength of the connection using odds ratios (ORs and 95% confidence intervals (CIs. In summary, no significant associations were discovered between the del1518 polymorphism and overall cancer risk (Del/Del vs Ins/Ins: OR =1.01, 95% CI =0.90–1.14; Ins/Del vs Ins/Ins: OR =1.03, 95% CI =0.96–1.12; recessive model: OR =0.98, 95% CI =0.90–1.07; dominant model: OR =1.03, 95% CI =0.94–1.12; and Del vs Ins: OR =1.01, 95% CI =0.94–1.07. In the stratified analysis by source of control, quality score, cancer type, and ethnicity, no significant associations were found. Despite some limitations, the current meta-analysis provides solid

  20. Homozygous mdm2 SNP309 cancer cells with compromised transcriptional elongation at p53 target genes are sensitive to induction of p53-independent cell death.

    Science.gov (United States)

    Rosso, Melissa; Polotskaia, Alla; Bargonetti, Jill

    2015-10-27

    A single nucleotide polymorphism (T to G) in the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression promoting chemotherapy resistant cancers. Two mdm2 G/G SNP309 cancer cell lines, MANCA and A875, have compromised wild-type p53 that co-localizes with MDM2 on chromatin. We hypothesized that MDM2 in these cells inhibited transcription initiation at the p53 target genes p21 and puma. Surprisingly, following etoposide treatment transcription initiation occurred at the compromised target genes in MANCA and A875 cells similar to the T/T ML-1 cell line. In all cell lines tested there was equally robust recruitment of total and initiated RNA polymerase II (Pol II). We found that knockdown of MDM2 in G/G cells moderately increased expression of subsets of p53 target genes without increasing p53 stability. Importantly, etoposide and actinomycin D treatments increased histone H3K36 trimethylation in T/T, but not G/G cells, suggesting a G/G correlated inhibition of transcription elongation. We therefore tested a chemotherapeutic agent (8-amino-adenosine) that induces p53-independent cell death for higher clinically relevant cytotoxicity. We demonstrated that T/T and G/G mdm2 SNP309 cells were equally sensitive to 8-amino-adenosine induced cell death. In conclusion for cancer cells overexpressing MDM2, targeting MDM2 may be less effective than inducing p53-independent cell death.

  1. MDM2–MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance

    Directory of Open Access Journals (Sweden)

    Moscetti I

    2016-08-01

    Full Text Available Ilaria Moscetti,1 Emanuela Teveroni,2,3 Fabiola Moretti,3 Anna Rita Bizzarri,1 Salvatore Cannistraro1 1Biophysics and Nanoscience Centre, Department DEB, Università della Tuscia, Viterbo, Italy; 2Department of Endocrinology and Metabolism, Università Cattolica di Roma, Roma, Italy; 3Institute of Cell Biology and Neurobiology, Consiglio Nazionale delle Ricerche (CNR, Roma, Italy Abstract: Murine double minute 2 (MDM2 and 4 (MDM4 are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2–MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2–MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (KD in the micromolar range for the MDM2–MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2–MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2–MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation. Keywords: MDM2, MDM4, atomic force spectroscopy, surface plasmon resonance

  2. Expression of p53 and MDM2 in the retinas of three RP mice during the development of disease%p53和MDM2在3种RP小鼠视网膜中的表达

    Institute of Scientific and Technical Information of China (English)

    申煌煊; 张清炯; 肖学珊; 黎仕强; 贾小云; 郭向明

    2002-01-01

    目的了解凋亡相关基因p53和MDM2与视网膜色素变性(retinitis pigmentosa, RP)发病的关系.方法以RP模式动物rds小鼠、rd小鼠、C3H小鼠及正常对照C3B小鼠为材料,提取不同发病时间(出生后7、12、17、23、29、37和50d)视网膜的总RNA,以β-actin和L19 核糖体蛋白基因为内对照,RT-PCR分析小鼠发病过程中视网膜p53和MDM2的表达.结果在C3B小鼠视网膜中,p53和MDM2的表达完全同步.出生后7d,高表达;7~12d期间,表达量迅速下降;12~50d期间,表达水平低,但基本稳定.p53和MDM2在rds小鼠的表达水平低,但基本上是同步、稳定表达.在rd小鼠中,p53的表达变化不大,但MDM2的表达则与p53不同步,类似于C3B小鼠MDM2的表达.在C3H小鼠的视网膜,p53和MDM2的表达也不同步.7~23d期间,p53的表达基本稳定;自23d后逐步下降,其中23~29d期间,p53的表达下降最快.而MDM2的表达总体变化不大.结论在这3种RP模式动物中,虽然凋亡相关基因p53和MDM2存在着表达差异,但其发病过程中视网膜细胞的变性死亡可能是通过p53非依赖型途径进行的.

  3. Interplay among p53, MDM2, and MDMX and Progress in Related Studies%p53与MDM2及MDMX 的相互作用和研究进展

    Institute of Scientific and Technical Information of China (English)

    李小龙

    2012-01-01

    p53是重要的抑癌基因,在细胞周期阻滞、DNA损伤修复及细胞凋亡等生物过程中发挥重要作用,并已成为潜在的肿瘤治疗靶点.MDM2(Mdm2 p53 binding protein homolog)及MDMX(Mdm4 p53 binding protein homolog)是p53的主要抑制因子,两者相互协同并通过不同的信号途径抑制p53的活性.MDM2是p53的E3连接酶,介导p53的泛素化从而降低p53的稳定性.MDMX则主要通过与p53的转录活性区结合,抑制p53对其下游基因的转录活性,但并不介导p53的降解.MDM2与MDMX通过不同机制协同对p53产生抑制作用,其具体分子过程及作用机制繁多且复杂.本文就p53、MDM2及MDMX的相互作用及各蛋白的功能进行综述.%P53 is one of the most important tumor suppressors. The resukts from functional analysis revealed that p53 is a potent stress responder that regulates cell-cycle arrest, DNA repair, cell senescence, and apoptosis. P53 is also recognized as a potential therapy target. MDM2 and MDMX are two major p53 suppressors, which can both inhibit p53 activity via different mechanisms. MDM2 is an E3 ligase of p53, which can promote p53 ubiquitination and can regulate p53 degradation and stabilization. MDMX does not have E3 ligase activity, but can directly bind to the N terminus of p53 and can inhibit p53 transactivation. Studies have shown that MDM2 and MDMX could work together to regulate p53 activity in a more complicated mechanism. The present work reviews the interplay among p53, MDM2, and MDMX.

  4. Expressions of P16, P53 and MDM2 protein in retinoblastoma%视网膜母细胞瘤中MDM2、P16和P53的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    雷晓华; 朱润庆; 胡学斌

    2006-01-01

    目的 研究P16、P53和癌基因MDM2在视网膜母细胞瘤的表达作用.方法 对已明确诊断的42例视网膜母细胞瘤标本采用免疫组织化学方法检测P16、P53和MDM2表达.结果 42例标本中P16、P53和MDM2阳性检出率分别是35.7%、69.1%和38.1%,P53和MDM2的共表达率为23.8%.结论 P16、P53和MDM2在视网膜母细胞瘤的产生和发展过程中起重要作用.

  5. The dynamics of stress p53-Mdm2 network regulated by p300 and HDAC1.

    Science.gov (United States)

    Arora, Akshit; Gera, Saurav; Maheshwari, Tanuj; Raghav, Dhwani; Alam, Md Jahoor; Singh, R K Brojen; Agarwal, Subhash M

    2013-01-01

    We construct a stress p53-Mdm2-p300-HDAC1 regulatory network that is activated and stabilised by two regulatory proteins, p300 and HDAC1. Different activation levels of [Formula: see text] observed due to these regulators during stress condition have been investigated using a deterministic as well as a stochastic approach to understand how the cell responds during stress conditions. We found that these regulators help in adjusting p53 to different conditions as identified by various oscillatory states, namely fixed point oscillations, damped oscillations and sustain oscillations. On assessing the impact of p300 on p53-Mdm2 network we identified three states: first stabilised or normal condition where the impact of p300 is negligible, second an interim region where p53 is activated due to interaction between p53 and p300, and finally the third regime where excess of p300 leads to cell stress condition. Similarly evaluation of HDAC1 on our model led to identification of the above three distinct states. Also we observe that noise in stochastic cellular system helps to reach each oscillatory state quicker than those in deterministic case. The constructed model validated different experimental findings qualitatively.

  6. The dynamics of stress p53-Mdm2 network regulated by p300 and HDAC1.

    Directory of Open Access Journals (Sweden)

    Akshit Arora

    Full Text Available We construct a stress p53-Mdm2-p300-HDAC1 regulatory network that is activated and stabilised by two regulatory proteins, p300 and HDAC1. Different activation levels of [Formula: see text] observed due to these regulators during stress condition have been investigated using a deterministic as well as a stochastic approach to understand how the cell responds during stress conditions. We found that these regulators help in adjusting p53 to different conditions as identified by various oscillatory states, namely fixed point oscillations, damped oscillations and sustain oscillations. On assessing the impact of p300 on p53-Mdm2 network we identified three states: first stabilised or normal condition where the impact of p300 is negligible, second an interim region where p53 is activated due to interaction between p53 and p300, and finally the third regime where excess of p300 leads to cell stress condition. Similarly evaluation of HDAC1 on our model led to identification of the above three distinct states. Also we observe that noise in stochastic cellular system helps to reach each oscillatory state quicker than those in deterministic case. The constructed model validated different experimental findings qualitatively.

  7. Characterizing the Free-Energy Landscape of MDM2 Protein-Ligand Interactions by Steered Molecular Dynamics Simulations.

    Science.gov (United States)

    Hu, Guodong; Xu, Shicai; Wang, Jihua

    2015-12-01

    Inhibition of p53-MDM2 interaction by small molecules is considered to be a promising approach to re-activate wild-type p53 for tumor suppression. Several inhibitors of the MDM2-p53 interaction were designed and studied by the experimental methods and the molecular dynamics simulation. However, the unbinding mechanism was still unclear. The steered molecular dynamics simulations combined with Brownian dynamics fluctuation-dissipation theorem were employed to obtain the free-energy landscape of unbinding between MDM2 and their four ligands. It was shown that compounds 4 and 8 dissociate faster than compounds 5 and 7. The absolute binding free energies for these four ligands are in close agreement with experimental results. The open movement of helix II and helix IV in the MDM2 protein-binding pocket upon unbinding is also consistent with experimental MDM2-unbound conformation. We further found that different binding mechanisms among different ligands are associated with H-bond with Lys51 and Glu25. These mechanistic results may be useful for improving ligand design.

  8. The cholesterol metabolite 27-hydroxycholesterol regulates p53 activity and increases cell proliferation via MDM2 in breast cancer cells.

    Science.gov (United States)

    Raza, Shaneabbas; Ohm, Joyce E; Dhasarathy, Archana; Schommer, Jared; Roche, Conor; Hammer, Kimberly D P; Ghribi, Othman

    2015-12-01

    Estrogen is synthesized from cholesterol and high cholesterol levels are suggested to be associated with increased risk of estrogen receptor(ER)-positive breast cancer. The cholesterol metabolite 27-hydroxycholesterol (27-OHC) was recently identified as a selective estrogen receptor modulator (SERM) and may therefore impact breast cancer progression. However, the mechanisms by which 27-OHC may contribute to breast cancer are not all known. We determined the extent to which 27-OHC regulates cell proliferation in MCF7 ER-positive breast cancer cell line involving the tumor suppressor protein p53. We found that treatment of MCF7 cells with 27-OHC resulted reduced p53 transcriptional activity. Conversely, treatment of the ER-negative MDA-MB 231 cells with 27-OHC induced no significant change in p53 activity. Exposure of MCF7 cells to 27-OHC was also associated with increased protein levels of the E3 ubiquitin protein ligase MDM2 and decreased levels of p53. Moreover, 27-OHC also enhanced physical interaction between p53 and MDM2. Furthermore, 27-OHC-induced proliferation was attenuated using either the p53 activator Tenovin-1 or the MDM2 inhibitor Nutlin-3 and Mdm2 siRNA. Taken together, our results indicate that 27-OHC may contribute to ER-positive breast cancer progression by disrupting constitutive p53 signaling in an MDM2-dependent manner.

  9. PTCH-1 and MDM2 expression in ameloblastoma from a West African sub-population: implication for chemotherapeutics.

    Science.gov (United States)

    Udeabor, Samuel Ebele; Adisa, Akinyele Olumuyiwa; Lawal, Ahmed Oluwatoyin; Barbeck, Mike; Booms, Patrick; Sader, Robert Alexander; Ghanaati, Shahram

    2015-01-01

    Ameloblastoma is a slow growing, painless odontogenic swelling which can attain sizes that result in severe deformities of the craniofacial complex. It is the most commonly encountered odontogenic tumor in Nigeria. Surgical intervention is currently the method of treatment; however identification of altered molecular pathways may inform chemotherapeutic potential. The Protein Patched homolog 1 (PTCH-1) is overexpressed in ameloblastoma. Also, mutation in the MDM2 gene can reduce the tumor suppressor function of p53 and promote ameloblastoma growth. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing chemotherapeutic alternatives. The objective was to characterize the PTCH-1 genetic profile of Ameloblastoma in Nigerian patients as a first step in investigating its potential for chemotherapeutic intervention. Twenty-eight FFPE blocks of ameloblastoma cases from Nigerian patients were prepared for antibody processing to PTCH-1 (Polyclonal Anti-PTCH antibody ab39266) and MDM2 (Monoclonal Anti-MDM2 antibody (2A10) ab16895). Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2. Moderate and strong expressions for PTCH in ameloblast and stellate reticulum were 78.6% and 60.7% respectively. Only 3 (10.7%) cases expressed MDM2. The importance of our study is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases and also infers the possible additional use of anti-p53 agents.

  10. 荧光定量PCR法检测MDM2T309G位点多态性%Novel real-time PCR methods for clinical detection of MDM2 T309G polymorphism

    Institute of Scientific and Technical Information of China (English)

    陈红; 谢丽; 胡文静; 禹立霞; 钱晓萍; 刘宝瑞

    2011-01-01

    Objective To establish a simple, convenient method for genotyping of MDM2 SNP309. Methods Using SYBR GREEN PCR with Tm-shift primers and genomic DNA purified with TIANamp DNA blood kit, we established a new method to detect genotyping of MDM2 SNP309. And we also genotyped MDM2 SNP3O9 by SYBR GREEN PCR with Tm-shift primers using whole blood DNA extracted by boiling method. Furthermore, genomic DNA were purified using boiling method from different blood samples kept at room temperature for different time or treated with multiple freezing and thawing as well as hematoiogic abnormal blood samples. Quantitative PCR were carried out to observe the amplification efficiency of the obtained DNA templates to assess the DNA extraction method. Results We established a novel, SYBR GREEN PCR method for MDM2 SNP309 genotyping. DNA purified by both methods mentioned before were suitable for MDM2 SNP309 SYBR GREEN PCR. DNA extracted using boiling method could be used for poly-merase chain reaction even when the blood sample were disposed in different ways. Conclusion In the present research, we established methods for MDM2 SNP309 genotype. Boiling method for DNA extraction is proved to be a simpler and quicker way of processing whole-blood samples compared with the DNA templates, and is effective for both PCR-RFLP and SYBR GREEN PCR.%目的 建立一种简便易行的MDM2基因单核苷酸多态性(SNP)309位点的检测方法.方法 用DNA提取试剂盒提取外周血DNA,建立MDM2 SNP309位点荧光定量PCR熔解曲线检测法.应用煮沸法处理血样取得粗制DNA,行荧光定量PCR检测,建立位点特异引物法检测MDM2 SNP309位点基因型.比较静置不同时间、反复冻融等处理以及存在血液学指标异常的血样经煮沸法提取DNA对荧光定量PCR扩增反应的影响,通过观察PCR扩增情况,评估不同处理方法对PCR扩增反应的影响.结果 荧光定量建立MDM2 SNP309位点PCR检测方法.煮沸法提取的DNA可用于普通PCR

  11. Expression of Brn-3a and MDM-2 in Cervical Neoplasia%Brn-3a和MDM-2在宫颈癌及癌前病变中的表达

    Institute of Scientific and Technical Information of China (English)

    黄雅; 冯玉昆; 李建军; 于璐

    2009-01-01

    目的:探讨Brn-3a和MDM-2在宫颈癌和癌前病变中的表达及其作为宫颈癌和癌前病变生物标志物的可行性和临床意义.方法:利用SP免疫组化法检测110例蜡块标本中Brn-3a和MDM-2的表达.结果:宫颈癌及癌前病变各组的Brn-3a表达阳性率均高于对照组,差异有统计学意义(P<0.05);SCC组及CINⅢ组的MDM-2表达阳性率均高于对照组,差异有统计学意义(P<0.05),SCC组与CIN Ⅰ组比较,差异有统计学意义(P<0.05).Brn-3a NtMDM-2在各临床分期及组织学分级中的阳性表达无统计学差异(P>0.05)Brn-3a与MDM-2蛋白阳性表达之间无相关关系(P>0.05).结论:Brn-3a和MDM-2可作为宫颈癌前病变的生物标志物.

  12. The Expression of MDM-2 and Rb in Pancreatic Carcinoma and Its Clinical Significance%MDM-2、Rb在胰腺癌中的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    刘振华; 冯一中; 柴玉海; 李峰; 任苏勤

    2007-01-01

    目的 探讨MDM-2、Rb在胰腺癌生长、转移过程中的作用,为患者预后判断提供理论依据.方法 应用免疫组织化学S-P法,检测MDM.2、Rb在53例胰腺癌、10非肿瘤性胰腺组织中的表达,并分析它们与胰腺癌临床病理参数及患者生存率的关系.结果 (1)MDM-2在胰腺癌中的阳性表达率明显高于非肿瘤性胰腺组织(P<0.05),与胰腺癌的病理分级成正相关(P<0.05).(2)Rb的阳性表达率在胰腺癌中显著低于非肿瘤性胰腺组织(P<0.01),与胰腺癌的病理分级成负相关(p<0.05).(3)MDM-2与Rb在胰腺癌中的阳性表达无显著相关.(4)MDM-2及Rb的阳性表达经生存单因素分析显示与患者的预后显著相关(P<0.01,P<0.05).结论 MDM-2、Rb在胰腺癌的发生发展中均发挥一定的作用,它们在胰腺癌的发生中可能起协同作用.

  13. Hispolon from Phellinus linteus has antiproliferative effects via MDM2-recruited ERK1/2 activity in breast and bladder cancer cells.

    Science.gov (United States)

    Lu, Te-Ling; Huang, Guan-Jhong; Lu, Te-Jung; Wu, Jin-Bin; Wu, Chieh-Hsi; Yang, Tung-Chuan; Iizuka, Akira; Chen, Yuh-Fung

    2009-08-01

    The MDM2 proto-oncogene is overexpressed in many human tumors. Although MDM2 inhibits tumor-suppressor function of p53, there exists a p53-independent role for MDM2 in tumorigenesis. Therefore, downregulation of MDM2 has been considered an attractive therapeutic strategy. Hispolon extracted from Phellinus species was found to induce epidermoid and gastric cancer cell apoptosis. However, the mechanisms are not fully understood. Herein, we report our findings that hispolon inhibited breast and bladder cancer cell growth, regardless of p53 status. Furthermore, p21(WAF1), a cyclin-dependent kinase inhibitor, was elevated in hispolon-treated cells. MDM2, a negative regulator of p21(WAF1), was ubiquitinated and degraded after hispolon treatment. We also found that activated ERK1/2 (extracellular signal-regulated kinase1/2) was recruited to MDM2 and involved in mediating MDM2 ubiquitination. Based on this finding, we investigated whether the sensitivity of cells to hispolon was related to ERK1/2 activity. The results indicated that cells with higher ERK1/2 activity were more sensitive to hispolon. In addition, hispolon-induced caspase-7 cleavage was inhibited by the ERK1/2 inhibitor, U0126. In conclusion, hispolon ubiquitinates and downregulates MDM2 via MDM2-recruited activated ERK1/2. Therefore, hispolon may be a potential anti-tumor agent in breast and bladder cancers.

  14. A re-examination of the MDM2/p53 interaction leads to revised design criteria for novel inhibitors.

    Science.gov (United States)

    Vasilevich, Natalya I; Afanasyev, Ilya I; Kovalskiy, Dmitry A; Genis, Dmitry V; Kochubey, Valery S

    2014-11-01

    The general model of epitope-type MDM2 inhibitor was developed based on the structural information on the complexes between MDM2 and various low molecular weight ligands found in the PDB database. Application of this model to our in-house library has led us to a new scaffold capable of interrupting protein-protein interactions. A synthetic library based on this and related scaffolds resulted in new classes of compounds that possess biochemical and cellular activity and good pharmacokinetic properties. We assume that such general approach to PPI inhibitors design may be useful for the development of inhibitors of various PPI types, including Bcl/XL.

  15. Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors.

    Science.gov (United States)

    Yu, Zhiliang; Zhuang, Chunlin; Wu, Yuelin; Guo, Zizhao; Li, Jin; Dong, Guoqiang; Yao, Jianzhong; Sheng, Chunquan; Miao, Zhenyuan; Zhang, Wannian

    2014-09-05

    A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2) inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2.

  16. Expression and clinical significance of Pokemon and mdm2 proteins in gallbladder cancer%Pokemon及mdm2蛋白在胆囊癌组织中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    李达; 张水军; 赵永福; 吴阳; 唐哲

    2012-01-01

    目的 探讨Pokemon及mdm2蛋白与胆囊癌的发生、发展的关系及其临床意义.方法 采用免疫组织化学S-P法检测40例胆囊癌、12例胆囊腺瘤、20例胆囊息肉及20例胆囊炎组织中Pokemon和mdm2蛋白的表达水平,分析其与胆囊癌临床病理学特征的关系,以及Pokemon和mdm2蛋白在组织中表达的相关性.结果 在检测的胆囊癌、胆囊腺瘤、胆囊息肉及胆囊炎组织中Pokemon蛋白的阳性表达率分别为60.0%、25.0%、30.0%、25.0%,mdm2 蛋白的阳性表达率分别为57.5%、16.7%、35.0%、30.0%,胆囊癌组织中Pokemon及mdm2蛋白的阳性表达率高于其他组,差异有统计学意义(P< 0.05);Pokemon及mdm2蛋白的表达与肿瘤的病理学分级及Nevin分期相关(P<0.05),与患者性别、年龄、肿瘤组织学类型及是否合并结石无关(P>0.05).Poketnon蛋白的表达与mdm2蛋白的表达呈正相关(r=0.434,p<0.05).结论 Pokemon蛋白和mdm2蛋白可能共同参与了胆囊癌的发生、发展和转移的过程,Pokemon基因有望成为胆囊癌治疗的一个有效的靶基因,从而为胆囊癌的治疗开辟新的途径.%Objective To investigate the relationship between POK erythroid myeloid ontogenic factor (Pokemon) and mdm2 proteins expressions and gallbladder cancer and its clinical significance.Methods The Pokemon and mdm2 proteins were analyzed in 40 gallbladder cancer patients,12 gallbladder adenoma patients,20 gallbladder polyps patients and 20 chronic cholecystitis patients.The relationship between the expressions of Pokemon and mdm2 and clinic-pathological features of gallbladder cancer were analyzed.The correlation of Pokemon expression with mdm2 expression in tissues was analyzed as well.Results In the tissues of gallbladder cancer,gallbladder adenoma,gallbladder polyps and chronic cholecystitis,the positive expression rates of Pokemon protein were 60.0%,25.0%,30.0%,25.0%,the positive expression rates of mdm2 were 57

  17. Vangl1 protein acts as a downstream effector of intestinal trefoil factor (ITF)/TFF3 signaling and regulates wound healing of intestinal epithelium.

    Science.gov (United States)

    Kalabis, Jiri; Rosenberg, Ian; Podolsky, Daniel K

    2006-03-10

    The intestinal trefoil factor (ITF/TFF3) protects intestinal epithelia from a range of insults and contributes to mucosal repair. However, the signaling events that mediate healing responses are only partially understood. To identify ITF signaling pathways, proteins that were Ser/Thr phosphorylated in response to ITF stimulation were immunoprecipitated from human colon carcinoma cell lines and identified by mass spectrometry. We demonstrated that Van Gogh-like protein 1 (also designated Vang-like 1 or Vangl1), a protein with four transmembrane domains, was Ser/Thr phosphorylated in response to ITF stimulation. Vangl1 was present in normal human colon and all intestinal epithelial cell lines (IEC) tested. In transfected IEC, FLAG-Vangl1 was mostly present in the Nonidet P-40 soluble fraction as detected by Western blotting, corresponding to the localization of endogenous protein in cytoplasmic vesicular structures by confocal microscopy with rabbit polyclonal anti-human Vangl1 antibody (alpha-Vangl1). Vangl1 cell membrane association increased with differentiation, as demonstrated by co-localization with E-cadherin in differentiated IEC. Increased Vangl1 phosphorylation after stimulation with ITF corresponded to decreased cell membrane association with E-cadherin. Functionally, Vangl1 overexpression enhanced ITF unstimulated and stimulated wound closure of IEC, whereas siRNA directed against Vangl1 inhibited the migratory response to ITF. Vangl1 protein may serve as an effector mediating the ITF healing response of the intestinal mucosa.

  18. The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Tamotsu, E-mail: nishida@gene.mie-u.ac.jp [Department of Human Functional Genomics, Life Science Research Center, Mie University, 1577 Kurima-machiya, Tsu 514-8507 (Japan); Yamada, Yoshiji [Department of Human Functional Genomics, Life Science Research Center, Mie University, 1577 Kurima-machiya, Tsu 514-8507 (Japan)

    2011-03-11

    Research highlights: {yields} SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. {yields} SMT3IP1 competes with p53 for binding to the central acidic domain of Mdm2. {yields} SMT3IP1 binding to Mdm2 inhibits Mdm2-mediated p53 ubiquitination and degradation. {yields} We postulate that SMT3IP1 acts as a new regulator of the p53-Mdm2 pathway. -- Abstract: SUMO (small ubiquitin-like modifier) modification plays multiple roles in several cellular processes. Sumoylation is reversibly regulated by SUMO-specific proteases. SUMO-specific proteases have recently been implicated in cell proliferation and early embryogenesis, but the underlying mechanisms remain unknown. Here, we show that a nucleolar SUMO-specific protease, SMT3IP1/SENP3, controls the p53-Mdm2 pathway. We found that SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. Overexpression of SMT3IP1 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein. In addition, SMT3IP1 bound to the acidic domain of Mdm2, which also mediates the p53 interaction, and competed with p53 for binding. Increasing expression of SMT3IP1 suppressed Mdm2-mediated p53 ubiquitination and subsequent proteasomal degradation. Interestingly, the desumoylation activity of SMT3IP1 was not necessary for p53 stabilization. These results suggest that SMT3IP1 is a new regulator of the p53-Mdm2 pathway.

  19. 核糖体蛋白家族成员与 MDM 2-p53通路调控的研究进展%Research progress of the relationship between ribosomal protein and MDM 2-p 5 3 pathway

    Institute of Scientific and Technical Information of China (English)

    刘弘扬; 孙冬琳; 孙海明; 金焰

    2016-01-01

    肿瘤是危害人类生命健康的最主要的疾病之一。 p53是目前为止研究最多的抑癌基因,p53的功能紊乱是肿瘤发生最主要的原因。核糖体蛋白发挥核糖体外功能抑制鼠双微体基因2( murine double minute 2, MDM 2)并激活 P 53是近十年的研究热点。本文就核糖体蛋白与 MDM 2-p 53通路调控关系及其与肿瘤治疗的研究进展进行综述。%Cancer is one of the most life threatening diseases .The dysfunction of the p 5 3 , as one of the most studied tumor-suppressor genes so far , is the main reason for tumorigenesis .Many studies over the past decades reveal that the ribosome-free ribosomal protein inhibits the MDM 2 and actives the P 5 3 .This review focuseon the progress in studying on the relationship between the ribosomal protein on the MDM 2-p 5 3 regulatory pathway and its roles incancer therapy .

  20. Association of N-cadherin levels and downstream effectors of Rho GTPases with dendritic spine loss induced by chronic stress in rat hippocampal neurons.

    Science.gov (United States)

    Castañeda, Patricia; Muñoz, Mauricio; García-Rojo, Gonzalo; Ulloa, José L; Bravo, Javier A; Márquez, Ruth; García-Pérez, M Alexandra; Arancibia, Damaris; Araneda, Karina; Rojas, Paulina S; Mondaca-Ruff, David; Díaz-Véliz, Gabriela; Mora, Sergio; Aliaga, Esteban; Fiedler, Jenny L

    2015-10-01

    Chronic stress promotes cognitive impairment and dendritic spine loss in hippocampal neurons. In this animal model of depression, spine loss probably involves a weakening of the interaction between pre- and postsynaptic cell adhesion molecules, such as N-cadherin, followed by disruption of the cytoskeleton. N-cadherin, in concert with catenin, stabilizes the cytoskeleton through Rho-family GTPases. Via their effector LIM kinase (LIMK), RhoA and ras-related C3 botulinum toxin substrate 1 (RAC) GTPases phosphorylate and inhibit cofilin, an actin-depolymerizing molecule, favoring spine growth. Additionally, RhoA, through Rho kinase (ROCK), inactivates myosin phosphatase through phosphorylation of the myosin-binding subunit (MYPT1), producing actomyosin contraction and probable spine loss. Some micro-RNAs negatively control the translation of specific mRNAs involved in Rho GTPase signaling. For example, miR-138 indirectly activates RhoA, and miR-134 reduces LIMK1 levels, resulting in spine shrinkage; in contrast, miR-132 activates RAC1, promoting spine formation. We evaluated whether N-cadherin/β-catenin and Rho signaling is sensitive to chronic restraint stress. Stressed rats exhibit anhedonia, impaired associative learning, and immobility in the forced swim test and reduction in N-cadherin levels but not β-catenin in the hippocampus. We observed a reduction in spine number in the apical dendrites of CA1 pyramidal neurons, with no effect on the levels of miR-132 or miR-134. Although the stress did not modify the RAC-LIMK-cofilin signaling pathway, we observed increased phospho-MYPT1 levels, probably mediated by RhoA-ROCK activation. Furthermore, chronic stress raises the levels of miR-138 in accordance with the observed activation of the RhoA-ROCK pathway. Our findings suggest that a dysregulation of RhoA-ROCK activity by chronic stress could potentially underlie spine loss in hippocampal neurons. © 2015 Wiley Periodicals, Inc.

  1. Association between polymorphism of MDM2 and susceptibility of primary hepatic carcinoma:A Meta analysis%MDM2基因多态与肝癌遗传易感性的Meta分析

    Institute of Scientific and Technical Information of China (English)

    万艳; 任仰武; 周宝森

    2011-01-01

    目的:根据已发表的相关文献,综合评估鼠双微体同源基因2(MDM2)多态与肝癌遗传易感性之间的关系.方法:按照统一的检索策略在相关中英文数据库中全面检索相关文献,对文献进行筛选、评价后获得相关研究的结果数据,然后应用Stata 10软件中Meta分析的方法,计算合并OR值及95%CI,并进行敏感性分析和发表偏倚的估计.结果:共有国内外5篇合格文献纳入本研究,累计病例和对照数分别为738和1 062例.合并结果显示,携带GG等位基因型者患肝癌的危险性是携带TT等位基因型的2.39倍(95%CI=1.81~3.15,P<0.001),携带TG等位基因型者患肝癌的危险性是携带TT等位基因型的1.65倍(95%CI=1.31~2.08,P<0.001).发表偏倚评估未发现明显的偏倚.结论:MDM2SNP309多态中GG、TG等位基因型可能与肝癌的易感性升高有关.%OBJECTIVE: To explore whether the polymorphism of murine double minute 2 (MDM2) contribute to the genetic susceptibility to primary hepatic carcinoma (PHC) based on the published studies. METHODS: All studies related to MDM2 and primary hepatic carcinoma were published in English and Chinese language and the allele frequencies of MDM2 and outcomes were abstracted after assessed under the same criteria. The pooled Ors with their 95%Cis were calculated using Meta methods in Stata 10 software. Publication bias and sensitivity were evaluated at the same time. RESULTS: Five studies with 738 cases and 1062 controls were included based on the selection criteria. The pooled results indicated MDM2 309GG genotype could increase the susceptibility to PHC with OR 2. 39(95%CI = 1. 81 - 3. 15, P<0. 001) compared to the wild allele MDM2 309TT, and MDM2 309TG genotype could increase the susceptibility to PHC with OR 1. 65 (95%CI=1.31-2. 08, P<0. 001) compared to the wild allele MDM2 309TT. The publication bias was not significant. CONCLUSION: MDM2 309GG, 309TG genotype seems to be involved in the risk of

  2. Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ)*

    Science.gov (United States)

    Gandhirajan, Rajesh Kumar; Jain, Manaswita; Walla, Benedikt; Johnsen, Marc; Bartram, Malte P.; Huynh Anh, Minh; Rinschen, Markus M.; Benzing, Thomas; Schermer, Bernhard

    2016-01-01

    Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo. This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway. PMID:27048650

  3. Molecular Features of the YAP Inhibitor Verteporfin: Synthesis of Hexasubstituted Dipyrrins as Potential Inhibitors of YAP/TAZ, the Downstream Effectors of the Hippo Pathway.

    Science.gov (United States)

    Gibault, Floriane; Bailly, Fabrice; Corvaisier, Matthieu; Coevoet, Mathilde; Huet, Guillemette; Melnyk, Patricia; Cotelle, Philippe

    2017-06-21

    Porphyrin derivatives, in particular verteporfin (VP), a photosensitizer initially designed for cancer therapy, have been identified as inhibitors of the YAP-TEAD interaction and transcriptional activity. Herein we report the efficient convergent synthesis of the dipyrrin half of protoporphyrin IX dimethyl ester (PPIX-DME), in which the sensitive vinyl group was created at the final stage by a dehydroiodination reaction. Two other dipyrrin derivatives were synthesized, including dipyrrin 19 [(Z)-2-((3,5-dimethyl-4-vinyl-2H-pyrrol-2-ylidene)methyl)-3,5-dimethyl-4-vinyl-1H-pyrrole], containing two vinyl groups. We found that VP and dipyrrin 19 showed significant inhibitory effects on TEAD transcriptional activity in MDA-MB-231 human breast cancer cells, whereas other compounds did not show significant changes. In addition, we observed a marked decrease in both YAP and TAZ levels following VP treatment, whereas dipyrrin 19 treatment primarily decreased the levels of YAP and receptor kinase AXL, a downstream target of YAP. Together, our data suggest that, due to their chemical structures, porphyrin- and dipyrrin-related derivatives can directly target YAP and/or TAZ proteins and inhibit TEAD transcriptional activity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Influence of zinc deficiency on AKT-MDM2-P53 signaling axes in normal and malignant human prostate cells

    Science.gov (United States)

    With prostate being the highest zinc-accumulating tissue before the onset of cancer, the effects of physiologic levels of zinc on Akt-Mdm2-p53 and Akt-p21 signaling axes in human normal prostate epithelial cells (PrEC) and malignant prostate LNCaP cells were examined. Cells were cultured for 6 d in...

  5. Positive effect of Mdm2 on p53 expression explains excitability of p53 in response to DNA damage.

    Science.gov (United States)

    Eliaš, Ján

    2017-04-07

    Most of the existing biological models consider Mdm2 as a dominant negative regulator of p53 appearing in several negative feedback loops. However, in addition to targeting p53 for degradation, Mdm2 in tight cooperation with MdmX can control expression levels of p53 through enhanced induction of p53 synthesis in response to DNA damage. Whilst ATM-dependent phosphorylation of p53 is not observed to be important in this enhanced synthesis, ATM-dependent phosphorylation of Mdm2 (as well as MdmX) is essential for its dual role, which is accompanied with widely oscillating p53. In the light of these new observations we formulate a novel molecular mechanism which, in silico, is capable of triggering p53 oscillations. The mechanism that is based on Mdm2's dual regulation of p53 can provide mechanistic insights into an excitability of the p53 network, thus it contributes to understanding of variability of p53 dynamics in response to single and double strand breaks.

  6. MDM2 inhibitor nutlin-3a induces apoptosis and senescence in cutaneous T-cell lymphoma: Role of p53

    DEFF Research Database (Denmark)

    Manfé, Valentina; Biskup, Edyta Urszula; Johansen, Peter

    2012-01-01

    P53 is rarely mutated in cutaneous T-cell lymphoma (CTCL) and is therefore a promising target for innovative therapeutic approaches. Nutlin-3a is an inhibitor of MDM2 (human homolog of murine double minute 2), which disrupts its interaction with p53, leading to the stabilization and activation of p...

  7. Next-generation repeat-free FISH probes for DNA amplification in glioblastoma in vivo: Improving patient selection to MDM2-targeted inhibitors.

    Science.gov (United States)

    Brunelli, Matteo; Eccher, Albino; Cima, Luca; Trippini, Tobia; Pedron, Serena; Chilosi, Marco; Barbareschi, Mattia; Scarpa, Aldo; Pinna, Giampietro; Cabrini, Giulio; Pilotto, Sara; Carbognin, Luisa; Bria, Emilio; Tortora, Giampaolo; Fioravanzo, Adele; Schiavo, Nicola; Meglio, Mario; Sava, Teodoro; Belli, Laura; Martignoni, Guido; Ghimenton, Claudio

    2017-01-01

    A next-generation FISH probe mapping to the MDM2 locus-specific region has recently been designed. The level of MDM2 gene amplification (high versus low) may allow selection of patients for cancer treatment with MDM2 inhibitors and may predict their responsiveness. We investigated the spectrum of MDM2 gene alterations using the new probes in vivo after visualizing single neoplastic cells in situ from a series of glioblastomas. Signals from next-generation repeat-free FISH interphase probes were identified in tissue microarrays that included 3 spots for each of the 48 cases. The murine double minutes (MDM2)-specific DNA probe and the satellite enumeration probe for chromosome 12 were used. Three cases (6%) showed more than 25 signals (high gene amplification), and 7 (15%) showed 3-10 signals (gains); among these, 4 cases (8%) had an equal number of MDM2 and centromeric signals on chromosome 12 (polyploidy). Genomic heterogeneity was observed only in 3 cases with low gene amplification. In our series, 6% of glioblastomas exhibited high MDM2 amplification (in vivo) with a pattern related to the known double minutes/chromothripsis phenomenon (in situ), and only cases with low amplification showed genomic heterogeneity. We concluded that the rate of MDM2 gene amplification can be a useful predictive biomarker to improve patient selection.

  8. MDM2 phenotypic and genotypic profiling, respective to TP53 genetic status, in diffuse large B-cell lymphoma patients treated with rituximab-CHOP immunochemotherapy

    DEFF Research Database (Denmark)

    Xu-Monette, Zijun Y; Møller, Michael; Tzankov, Alexander

    2013-01-01

    MDM2 is a key negative regulator of the tumor suppressor p53, however, the prognostic significance of MDM2 overexpression in diffuse large B-cell lymphoma (DLBCL) has not been defined convincingly. In a p53-genetically defined large cohort of de novo DLBCL patients treated with R-CHOP chemotherap...

  9. Structural Exploration and Conformational Transitions in MDM2 upon DHFR Interaction from Homo sapiens: A Computational Outlook for Malignancy via Epigenetic Disruption.

    Science.gov (United States)

    Banerjee, Arundhati; Ray, Sujay

    2016-01-01

    Structural basis for exploration into MDM2 and MDM2-DHFR interaction plays a vital role in analyzing the obstruction in folate metabolism, nonsynthesis of purines, and further epigenetic regulation in Homo sapiens. Therefore, it leads to suppression of normal cellular behavior and malignancy. This has been earlier documented via yeast two-hybrid assays. So, with a novel outlook, this study explores the molecular level demonstration of the best satisfactory MDM2 model selection after performing manifold modeling techniques. Z-scores and other stereochemical features were estimated for comparison. Further, protein-protein docking was executed with MDM2 and the experimentally validated X-ray crystallographic DHFR. Residual disclosure from the best suited simulated protein complex disclosed 18 side chain and 3 ionic interactions to strongly accommodate MDM2 protein into the pocket-like zone in DHFR due to the positive environment by charged residues. Lysine residues from MDM2 played a predominant role. Moreover, evaluation from varied energy calculations, folding rate, and net area for solvent accessibility implied the active participation of MDM2 with DHFR. Fascinatingly, conformational transitions from coils to helices and β-sheets after interaction with DHFR affirm the conformational strength and firmer interaction of human MDM2-DHFR. Therefore, this probe instigates near-future clinical research and interactive computational investigations with mutations.

  10. Structural Exploration and Conformational Transitions in MDM2 upon DHFR Interaction from Homo sapiens: A Computational Outlook for Malignancy via Epigenetic Disruption

    Science.gov (United States)

    Banerjee, Arundhati; Ray, Sujay

    2016-01-01

    Structural basis for exploration into MDM2 and MDM2-DHFR interaction plays a vital role in analyzing the obstruction in folate metabolism, nonsynthesis of purines, and further epigenetic regulation in Homo sapiens. Therefore, it leads to suppression of normal cellular behavior and malignancy. This has been earlier documented via yeast two-hybrid assays. So, with a novel outlook, this study explores the molecular level demonstration of the best satisfactory MDM2 model selection after performing manifold modeling techniques. Z-scores and other stereochemical features were estimated for comparison. Further, protein-protein docking was executed with MDM2 and the experimentally validated X-ray crystallographic DHFR. Residual disclosure from the best suited simulated protein complex disclosed 18 side chain and 3 ionic interactions to strongly accommodate MDM2 protein into the pocket-like zone in DHFR due to the positive environment by charged residues. Lysine residues from MDM2 played a predominant role. Moreover, evaluation from varied energy calculations, folding rate, and net area for solvent accessibility implied the active participation of MDM2 with DHFR. Fascinatingly, conformational transitions from coils to helices and β-sheets after interaction with DHFR affirm the conformational strength and firmer interaction of human MDM2-DHFR. Therefore, this probe instigates near-future clinical research and interactive computational investigations with mutations. PMID:27213086

  11. P53 Gene Mutation and Expression of MDM2, P53, P16 Protein and their Relationship in Human Glioma

    Institute of Scientific and Technical Information of China (English)

    CUI Wen; WU Renliang; CAO Huiling; GAO Jifa; WANG Xu; REN Qiwei

    2005-01-01

    To investigate the effect of P53 protein accumulation and p53 gene mutation in the pathogenesis of glioma and to study the role of MDM2, P53 and P16 protein in glioma formation and progression and their relationship with each other, LSAB immunohistochemical staining method and non-isotopic PCR-SSCP techniques were used to detect the expression of MDM2, P53 and P16 pro tein and p53 gene mutation in 48 cases of gliomas. The results showed that the positive expression rate of MDM2, P53 and the negative rate of P16 was 22.9 %, 41.7 % and 60.4 %, respectively.The latter two in high grade (grade Ⅲ , Ⅳ) gliomas had a significantly higher rate than in the low grade (grade Ⅱ ) gliomas. Moreover, the co-expression of MDM2 and P53 protein was confirmed in only 1 of 48 cases. No significant difference was found in the rate of the expression of MDM2 between high grade and low grade gliomas (P>0.1) . PCR SSCP results showed that mutation of 5-8 exons of p53 gene was detected in 17 out of 48 cases (35.42 %) . Mutation was detected in 16of 20 cases of positive p53 expression, and another one was detected in 28 cases of negative expression cases. The correlation between p53 mutation and p53 immunopositivity was observed in 89.6% of the cases. P53 gene mutation and the level of MDM2, P53 and P16 protein were not related to age, gender of the patients, tumor location and size. It is concluded that the mutation of p53 and deletion of p16 might play important roles in the tumorigenesis of gliomas and it was significantly associated with the grade of tumor differentiation. P53 protein accumulation can indirectly reflect p53 mutation. MDM2 amplification and overexpression might be an early event in the growth of human gliomas.

  12. Influence of the MDM2 single nucleotide polymorphism SNP309 on tumour development in BRCA1 mutation carriers

    Directory of Open Access Journals (Sweden)

    Johnson Peter W

    2006-03-01

    Full Text Available Abstract Background The MDM2 gene encodes a negative regulator of the p53 tumour suppressor protein. A single nucleotide polymorphism (SNP in the MDM2 promoter (a T to G exchange at nucleotide 309 has been reported to produce accelerated tumour formation in individuals with inherited p53 mutations. We have investigated the effect of the MDM2 SNP309 on clinical outcome in a cohort of patients with germline mutations of BRCA1. Methods Genomic DNA was obtained for 102 healthy controls and 116 patients with established pathogenic mutations of BRCA1 and Pyrosequencing technology™ was used to determine the genotype at the MDM2 SNP309 locus. Results The polymorphism was present in 52.9% of the controls (G/T in 37.3% and G/G in 15.6% and 58.6% of the BRCA1 mutation carriers (47.4% G/T and 11.2% G/G. Incidence of malignancy in female BRCA1 carriers was not significantly higher in SNP309 carriers than in wildtype (T/T individuals (72.7% vs. 75.6%, p = 1.00. Mean age of diagnosis of first breast cancer was 41.2 years in the SNP309 G/G genotype carriers, 38.6 years in those with the SNP309 G/T genotype and 39.0 years in wildtype subjects (p = 0.80. Conclusion We found no evidence that the MDM2 SNP309 accelerates tumour development in carriers of known pathogenic germline mutations of BRCA1.

  13. HIF-1α, MDM2, CDK4, and p16 expression in ischemic fasciitis, focusing on its ischemic condition.

    Science.gov (United States)

    Yamada, Yuichi; Kinoshita, Izumi; Kohashi, Kenichi; Yamamoto, Hidetaka; Kuma, Yuki; Ito, Takamichi; Koda, Kenji; Kisanuki, Atsushi; Kurosawa, Manabu; Yoshimura, Michiko; Furue, Masutaka; Oda, Yoshinao

    2017-07-01

    Ischemic fasciitis is a benign myofibroblastic lesion, occurring in the sacral region or proximal thigh of elderly or bedridden individuals. The pathogenesis of ischemic fasciitis is thought to be based on ischemic condition; however, it has never been demonstrated. In this study, we examined the expression of ischemia-associated proteins in ischemic fasciitis by immunohistochemical and genetic methods. Specifically, this study aimed to reveal the expression of HIF-1α, MDM2, CDK4, p16, and gene amplification of MDM2 gene. Seven cases of ischemic fasciitis from among the soft-tissue tumors registered at our institution were retrieved. Histopathological findings were as follows: poorly demarcated nodular masses, a proliferation of spindle-shaped fibroblastic or myofibroblastic cells with oval nuclei and eosinophilic or pale cytoplasm, zonal fibrinous deposition, pseudocystic degeneration, granulation-like proliferation of capillary vessels, ganglion-like cells, myxoid or hyalinized stroma, and chronic inflammatory infiltration. Immunohistochemically, the spindle cells were positive for HIF-1α (7/7 cases), MDM2 (4/7 cases), CDK4 (4/7 cases), p16 (7/7 cases), p53 (2/7 case), cyclin D1 (7/7 cases), and alpha-smooth muscle actin (6/7 cases). Neither MDM2 gene amplification nor USP6 gene split signal was detected in any case. Overexpression of the above proteins may be associated with the pathogenic mechanism of ischemic fasciitis. It is noted that the immunohistochemical positivity of MDM2, CDK4, and p16 do not necessarily indicate malignant neoplasm such as dedifferentiated liposarcoma.

  14. The role of NPM, p14arf and MDM2 in precursors of bronchial squamous cell carcinoma.

    Science.gov (United States)

    Mascaux, C; Bex, F; Martin, B; Burny, A; Haller, A; Paesmans, M; Willard-Gallo, K; Ninane, V; Sculier, J-P

    2008-09-01

    Murine double minute clone 2 (MDM2), p14 alternate reading frame (p14arf), and nucleophosmin (NPM) regulate p53 activity. A total of 200 biopsies, including normal bronchial, pre-invasive and invasive tissues, were examined for changes in NPM, p14arf, MDM2 and p53 expression patterns by immunohistochemistry and immunofluorescence with confocal microscopy. NPM and p14arf displayed a diffuse nuclear staining in most normal bronchial tissue. The fraction of biopsies displaying an increased MDM2 staining or a nucleolar relocalisation of NPM increased at mild and moderate dysplasia, respectively. Two different modifications occurred in p14arf expression, i.e. its loss or its nucleolar relocalisation, both increasing at severe dysplasia and both being associated with high MDM2 expression. In addition, the nucleolar relocalisation of p14arf was associated with that of NPM. Immunofluorescence staining indicated that NPM and p14arf either co-localised in the nucleoplasm or in the nucleoli, before and as a result of severe dysplasia, respectively. MDM2 was not detected in the nucleoli. Thus, changes occur in murine double minute clone 2, p14 alternate reading frame and nucleophosmin level of expression and/or cellular distribution during early steps of lung carcinogenesis. Their relative localisation as determined by immunofluorescence, supports the hypothesis that p14 alternate reading frame nucleolar relocalisation impairs p14 alternate reading frame-murine double minute clone 2 complex formation and that nucleophosmin might sequester p14 alternate reading frame. The demonstration of this hypothesis requires further functional studies.

  15. The MDM-2 Antagonist Nutlin-3 Promotes the Maturation of Acute Myeloid Leukemic Blasts

    Directory of Open Access Journals (Sweden)

    Paola Secchiero

    2007-10-01

    Full Text Available The small-molecule inhibitor of murine double minute (MDM-2, Nutlin-3, induced variable apoptosis in primary acute myeloid leukemia (AML blasts, promoted myeloid maturation of surviving cells, as demonstrated by analysis of CD11 b, CD14 surface antigens, by morphologic examination. Although the best-characterized activity of Nutlin-3 is activation of the p53 pathway, Nutlin-3 induced maturation also in one AML sample characterized by p53 deletion, as well as in the p53-/- human myeloblastic HL-60 cell line. At the molecular level, the maturational activity of Nutlin-3 in HL-60 cells was accompanied by the induction of E2F1 transcription factor, it was significantly counteracted by specific gene knockdown with small interfering RNA for E2F1. Moreover, Nutlin-3, as well as tumor necrosis factor (TNF α, potentiated the maturational activity of recombinant TNF-related apoptosis-inducing lig, (TRAIL in HL-60 cells. However, although TNF-α significantly counteracted the proapoptotic activity of TRAIL, Nutlin-3 did not interfere with the proapoptotic activity of TRAIL. Taken together, these data disclose a novel, potentially relevant therapeutic role for Nutlin-3 in the treatment of both p53 wild-type, p53-/- AML, possibly in association with recombinant TRAIL.

  16. MOXA推出支持MDM 2.0的Rcore平台

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Moxa在所有嵌入式计算机中都预装了操作系统,Moxa同时提供中间件、范例程序和开发工具,为编程人员带来了友善的应用软件开发环境,减少系统集成所需的开发时间和人力。Moxa Device Manager 2.0(MDM 2.0)作为简单易用的远程管理工具,可通过浏览器管理Moxa立即可用系列嵌八式计算机。Moxa的嵌入式计算机为现场数据采集和工业级控制应用提供了优越的前端控制主机;但这些前端主机通常位于远程场所。MDM就是为了让系统管理者更简单且快速地管理他们的远程嵌入式计算机而设计。

  17. 结肠腺癌患者癌组织MDM2、TBX2和Pokemon表达及相关性

    Institute of Scientific and Technical Information of China (English)

    崔婷; 魏强

    2014-01-01

    目的:分析结肠腺癌患者癌组织TBX2、MDM2和Pokemon表达及相关性。方法结肠腺癌患者119例为观察组,留取术后新鲜组织。收集距肿瘤边缘>5 cm的正常结肠黏膜新鲜组织78例为对照组。应用流式细胞术检测 TBX2、MDM2和 Pokemon表达。结果观察组 TBX2、MDM2和Pokemon表达量明显高于对照组(P<0.0001),观察组TBX2、MDM2和Pokemon表达与肿瘤分化程度、浸润深度、临床分期和Ki67表达密切相关。观察组TBX2和MDM2表达正相关,而TBX2和Pokemon、MDM2和Pokemon未见明显相关性。结论结肠腺癌患者术后组织TBX2、MDM2和Pokemon异常表达对肿瘤性病变的发生和进展有重要促进作用。

  18. Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

    Science.gov (United States)

    Jaako, P; Debnath, S; Olsson, K; Zhang, Y; Flygare, J; Lindström, M S; Bryder, D; Karlsson, S

    2015-11-01

    Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

  19. Ribosomal protein S7 regulates arsenite-induced GADD45α expression by attenuating MDM2-mediated GADD45α ubiquitination and degradation.

    Science.gov (United States)

    Gao, Ming; Li, Xiaoguang; Dong, Wen; Jin, Rui; Ma, Hanghang; Yang, Pingxun; Hu, Meiru; Li, Yi; Hao, Yi; Yuan, Shengtao; Huang, Junjian; Song, Lun

    2013-05-01

    The stress-responding protein, GADD45α, plays important roles in cell cycle checkpoint, DNA repair and apoptosis. In our recent study, we demonstrate that GADD45α undergoes a dynamic ubiquitination and degradation in vivo, which process can be blocked by the cytotoxic reagent, arsenite, resulting in GADD45α accumulation to activate JNKs cell death pathway, thereby revealing a novel mechanism for the cellular GADD45α functional regulation. But the factors involved in GADD45α stability modulations are unidentified. Here, we demonstrated that MDM2 was an E3 ubiquitin ligase for GADD45α. One of MDM2-binding partner, ribosomal protein S7, interacted with and stabilized GADD45α through preventing the ubiquitination and degradation of GADD45α mediated by MDM2. This novel function of S7 is unrelated to p53 but seems to depend on S7/MDM2 interaction, for the S7 mutant lacking MDM2-binding ability lost its function to stabilize GADD45α. Further investigations indicated that arsenite treatment enhanced S7-MDM2 interaction, resulting in attenuation of MDM2-dependent GADD45α ubiquitination and degradation, thereby leading to GADD45α-dependent cell death pathway activation. Silencing S7 expression suppressed GADD45α-dependent cytotoxicity induced by arsenite. Our findings thus identify a novel function of S7 in control of GADD45α stabilization under both basal and stress conditions and its significance in mediating arsenite-induced cellular stress.

  20. NF-kappaB inhibits T-cell activation-induced, p73-dependent cell death by induction of MDM2.

    Science.gov (United States)

    Busuttil, Valere; Droin, Nathalie; McCormick, Laura; Bernassola, Francesca; Candi, Eleonora; Melino, Gerry; Green, Douglas R

    2010-10-19

    NF-κB is a key transcription factor involved in the regulation of T-cell activation and proliferation upon engagement of the T-cell receptor (TCR). T cells that lack the IκB kinase (IKKβ) are unable to activate NF-κB, and rapidly undergo apoptosis upon activation. NF-κB activation following T-cell receptor engagement induces the expression of Mdm2 through interaction with NF-κB sites in its P1 promoter, and enforced expression of Mdm2 protected T cells deficient for NF-κB activation from activation-induced cell death. In T cells with intact NF-κB signaling, ablation or pharmacologic inhibition of Mdm2 resulted in activation-induced apoptosis. Mdm2 coprecipitates with p73 in activated T cells, and apoptosis induced by inhibition of Mdm2 was p73-dependent. Further, Bim was identified as a p73 target gene required for cell death induced by Mdm2 inhibition, and a p73-responsive element in intron 1 of Bim was characterized. Our results demonstrate a pathway for survival of activated T cells through NF-κB-induced Mdm2, which blocks Bim-dependent apoptosis through binding and inhibition of p73.

  1. Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway.

    Science.gov (United States)

    Pettersson, Susanne; Sczaniecka, Matylda; McLaren, Lorna; Russell, Fiona; Gladstone, Karen; Hupp, Ted; Wallace, Maura

    2013-03-15

    The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.

  2. Expression of MDM2 mRNA, MDM2, P53 and P16 Proteins in Urothelial Lesions in the View of the WHO 4th Edition Guidelines as A Molecular Insight towards Personalized Medicine

    Directory of Open Access Journals (Sweden)

    Olfat Hammam

    2017-08-01

    Full Text Available AIM: Here we imposed a multimarker molecular panel composed of P53, MDM2 protein & mRNA & P16 with the identification of sensitive and specific cut offs among the Egyptian urothelial carcinomas bilharzial or not emphasize the pathological and molecular classifications, pathways and prognosis as a privilege for adjuvant therapy. METHODS: Three hundred and ten urothelial lesions were pathologically evaluated and grouped as follows: 50 chronic cystitis as benign, 240 urothelial carcinomas and 20 normal bladder tissue as a control. Immunohistochemistry for MDM Protein, P16 & p53 and In Situ Hybridization for MDM2mRNA were done. RESULTS: MDM2mRNA overexpression correlated with low grade low stage non invasive tumors, while P53 > 40% & p16 40% & P16 10% from high grade, high stage invasive urothelial carcinomas (with p53 > 40, p16 40 & p16 < 10%, together with the histopathological features can distinguish in situ urothelial lesions from dysplastic and atypical lesions.

  3. Differential diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: utility of p16 in combination with MDM2 and CDK4 immunohistochemistry.

    Science.gov (United States)

    Kammerer-Jacquet, Solène-Florence; Thierry, Sixte; Cabillic, Florian; Lannes, Morgane; Burtin, Florence; Henno, Sébastien; Dugay, Frédéric; Bouzillé, Guillaume; Rioux-Leclercq, Nathalie; Belaud-Rotureau, Marc-Antoine; Stock, Nathalie

    2017-01-01

    The differential diagnosis between atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS) from their morphologic counterparts is challenging. Currently, the diagnosis is guided by MDM2 and CDK4 immunohistochemistry (IHC) and is confirmed by the amplification of the corresponding genes. Recently, p16 IHC has been proposed as a useful diagnostic biomarker. The objective was to assess the utility of p16 IHC in the differential diagnosis of ALT/WDLPS and DDLPS. Our series included 101 tumors that were previously analyzed using fluorescence in situ hybridization for MDM2 and CDK4 amplification. We compared sensitivity and specificity of p16 IHC to MDM2 and CDK4 IHC in the differential diagnosis of ALT-WDLPS (n=19) versus benign adipocytic tumors (n=44) and DDLPS (n=18) versus mimicking sarcomas (n=20). In the differential diagnosis of ALT-WDLPS, p16 had a sensitivity of 89.5% but a specificity of 68.2%, which was impaired by false-positive lipomas with secondary changes, especially in biopsies. Likewise, in the differential diagnosis of DDLPS, p16 had a sensitivity of 94.4% and a specificity of 70%, which hampered its use as a single marker. However, adding p16 to MDM2 and/or CDK4 increased diagnostic specificity. Indeed, MDM2+/p16+ tumors were all ALT-WDLPS, and MDM2-/p16- tumors were all benign adipocytic tumors. Moreover, all MDM2+/CDK4+/p16+ tumors were DDLPS, and the MDM2-/CDK4-/p16- tumor was an undifferentiated sarcoma. Although the use of p16 as a single immunohistochemical marker is limited by its specificity, its combination with MDM2 and CDK4 IHC may help discriminate ALT-WDLPS/DDLPS.

  4. Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

    Science.gov (United States)

    Wu, W; Xu, C; Ling, X; Fan, C; Buckley, B P; Chernov, M V; Ellis, L; Li, F; Muñoz, I G; Wang, X

    2015-12-31

    Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

  5. The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.

    Science.gov (United States)

    Gibson, Monica Prasad; Zhu, Qinglin; Wang, Suzhen; Liu, Qilin; Liu, Ying; Wang, Xiaofang; Yuan, Baozhi; Ruest, L Bruno; Feng, Jian Q; D'Souza, Rena N; Qin, Chunlin; Lu, Yongbo

    2013-03-08

    Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

  6. MDM2 Inhibitor, Nutlin 3a, Induces p53 Dependent Autophagy in Acute Leukemia by AMP Kinase Activation.

    Directory of Open Access Journals (Sweden)

    Gautam Borthakur

    Full Text Available MDM2 (mouse double minute 2 inhibitors that activate p53 and induce apoptosis in a non-genotoxic manner are in clinical development for treatment of leukemias. P53 can modulate other programmed cell death pathways including autophagy both transcriptionally and non-transcriptionally. We investigated autophagy induction in acute leukemia by Nutlin 3a, a first-in-class MDM2 inhibitor. Nutlin 3a induced autophagy in a p53 dependent manner and transcriptional activation of AMP kinase (AMPK is critical, as this effect is abrogated in AMPK -/- mouse embryonic fibroblasts. Nutlin 3a induced autophagy appears to be pro-apoptotic as pharmacological (bafilomycin or genetic inhibition (BECLIN1 knockdown of autophagy impairs apoptosis induced by Nutlin 3a.

  7. Design, Synthesis and Biological Evaluation of Sulfamide and Triazole Benzodiazepines as Novel p53-MDM2 Inhibitors

    Directory of Open Access Journals (Sweden)

    Zhiliang Yu

    2014-09-01

    Full Text Available A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2 inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2.

  8. A synthetic form of frizzled 8-associated antiproliferative factor enhances p53 stability through USP2a and MDM2.

    Directory of Open Access Journals (Sweden)

    Jayoung Kim

    Full Text Available Frizzled 8-associated Antiproliferative Factor (APF is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS, a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1 were treated with asialo-APF (as-APF, a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2, and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.

  9. A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation

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    Kumar Sonia

    2011-02-01

    Full Text Available Abstract Background The mammalian DNA-damage response (DDR has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs, mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. Methods In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+ with the MDM2 antagonist, Nutlin-3. Results Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37. Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. Conclusion To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist.

  10. Polymorphisms at p53, p73, and MDM2 loci modulate the risk of tobacco associated leukoplakia and oral cancer.

    Science.gov (United States)

    Misra, Chaitali; Majumder, Mousumi; Bajaj, Swati; Ghosh, Saurabh; Roy, Bidyut; Roychoudhury, Susanta

    2009-09-01

    Polymorphisms at loci controlling cellular processes such as cell cycle, DNA repair, and apoptosis may modulate the risk of cancer. We examined the association of two linked polymorphisms (G4C14-A4T14) at p73 and one polymorphism (309G > T) at MDM2 promoter with the risk of leukoplakia and oral cancer. The p73 and MDM2 genotypes were determined in 197 leukoplakia patients, 310 oral cancer patients and in 348 healthy control subjects. The p73 GC/AT genotype increased the risk of leukoplakia (OR = 1.6, 95% CI = 1.1-2.3) and oral cancer (OR = 2.4, 95% CI = 1.7-3.3) but the 309G > T MDM2 polymorphism independently could not modify the risk of any of the diseases. Stratification of the study population into subgroups with different tobacco habits showed that the risk of the oral cancer is not modified further for the individuals carrying p73 risk genotype. However, leukoplakia patients with smokeless tobacco habit showed increased risk with combined GC/AT and AT/AT (OR = 3.0, 95% CI = 1.3-7.0) genotypes. A combined analysis was done with our previous published data on p53 codon 72 pro/arg polymorphism. Analysis of pair wise genotype combinations revealed increase in risk for specific p73-MDM2 and p73-p53 genotype combinations. Finally, the combined three loci analyses revealed that the presence of at least one risk allele at all three loci increases the risk of both leukoplakia and oral cancer.

  11. MDM2 antagonists synergize with PI3K/mTOR inhibition in well-differentiated/dedifferentiated liposarcomas.

    Science.gov (United States)

    Laroche, Audrey; Chaire, Vanessa; Algeo, Marie-Paule; Karanian, Marie; Fourneaux, Benjamin; Italiano, Antoine

    2017-08-15

    Well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS) are characterized by a consistent amplification of the MDM2 gene. The PI3K/AKT/mTOR pathway has been suggested to play also an important role in their tumorigenesis. Our goal was to determine whether combined MDM2 and PI3K/AKT/mTOR targeting is associated with higher anti-tumor activity than single agent alone in preclinical models of WDLPS/DDLPS. WDLPS/DDLPS cells were exposed to RG7388 (MDM2 antagonist) and BEZ235 (PI3K/mTOR dual inhibitor) after which apoptosis and signaling/survival pathway perturbations were monitored by flow cytometry and Western blot analysis. Xenograft mouse models were used to assess tumor growth and animal survival. Western blotting, histopathology, and tumor volume evolution were used for the assessment of treatment efficacy. The PI3K/AKT/mTOR was upregulated in up to 81% of the human WDLPS/DDLPS samples analysed. Treatment with RG7388 and BEZ235 resulted in a greater tumor activity than either drug alone with a significant difference in terms of cell viability after 72h of treatment with RG-73888 alone, BEZ235 alone and a combination of both agents. Consistent with these observations, we found a significant increase in apoptosis with the combination versus the single agent treatment alone. We then analysed the in vivo antitumor activity of RG7388 and BEZ235 in a xenograft model of DDLPS. The combination regimen significantly reduced tumor growth rate in comparison with single agent alone. Our results represent the first in vivo evidence of synergy between MDM2 and PI3K/AKT/mTOR antagonists and represent a strong rationale to evaluate the therapeutic potential of such a combination in WDLPS/DDLPS.

  12. E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis

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    Kuan-Ho Lin

    2017-04-01

    Full Text Available Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2, for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs. In addition, protein phosphatase such as protein phosphatase 1 (PP1 and calcineurin (PP2B are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.

  13. Frequent alteration of MDM2 and p53 in the molecular progression of recurring non-Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

    2002-01-01

    -Hodgkin's lymphoma. METHODS AND RESULTS: We have analysed sequential biopsies from 42 non-Hodgkin's lymphoma patients immunohistochemically for p53 alterations (based on p53 and p21Waf1 expression), as well as for expression of MDM2, p27Kip1 and cyclin D3. Relapse of follicle centre lymphoma was associated with p53......-Hodgkin's lymphoma, as 2/5 (40%) diffuse large B-cell lymphomas and 3/9 (33%) T-cell non-Hodgkin's lymphomas with normal p53 at diagnosis showed p53 alterations at relapse. No indolent non-Hodgkin's lymphoma case showed MDM2 over-expression at diagnosis, whereas 4/5 (80%) transformed diffuse large B-cell lymphomas...... developed MDM2 over-expression. CONCLUSION: Our data are consistent with the notion that p53 alterations are important for the histological transformation of follicle centre lymphoma. However, the data also suggest that relapsing follicle centre lymphomas without overt transformation often have p53...

  14. The pharmacodynamics of the p53-Mdm2 targeting drug Nutlin: the role of gene-switching noise.

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    Krzysztof Puszynski

    2014-12-01

    Full Text Available In this work we investigate, by means of a computational stochastic model, how tumor cells with wild-type p53 gene respond to the drug Nutlin, an agent that interferes with the Mdm2-mediated p53 regulation. In particular, we show how the stochastic gene-switching controlled by p53 can explain experimental dose-response curves, i.e., the observed inter-cell variability of the cell viability under Nutlin action. The proposed model describes in some detail the regulation network of p53, including the negative feedback loop mediated by Mdm2 and the positive loop mediated by PTEN, as well as the reversible inhibition of Mdm2 caused by Nutlin binding. The fate of the individual cell is assumed to be decided by the rising of nuclear-phosphorylated p53 over a certain threshold. We also performed in silico experiments to evaluate the dose-response curve after a single drug dose delivered in mice, or after its fractionated administration. Our results suggest that dose-splitting may be ineffective at low doses and effective at high doses. This complex behavior can be due to the interplay among the existence of a threshold on the p53 level for its cell activity, the nonlinearity of the relationship between the bolus dose and the peak of active p53, and the relatively fast elimination of the drug.

  15. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human ca...... method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.......ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human...

  16. Monitoring p53 by MDM2 and MDMX is required for endocrine pancreas development and function in a spatio-temporal manner.

    Science.gov (United States)

    Zhang, Yiwei; Zeng, Shelya X; Hao, Qian; Lu, Hua

    2017-03-01

    Although p53 is not essential for normal embryonic development, it plays a pivotal role in many biological and pathological processes, including cell fate determination-dependent and independent events and diseases. The expression and activity of p53 largely depend on its two biological inhibitors, MDM2 and MDMX, which have been shown to form a complex in order to tightly control p53 to an undetectable level during early stages of embryonic development. However, more delicate studies using conditional gene-modification mouse models show that MDM2 and MDMX may function separately or synergistically on p53 regulation during later stages of embryonic development and adulthood in a cell and tissue-specific manner. Here, we report the role of the MDM2/MDMX-p53 pathway in pancreatic islet morphogenesis and functional maintenance, using mouse lines with specific deletion of MDM2 or MDMX in pancreatic endocrine progenitor cells. Interestingly, deletion of MDM2 results in defects of embryonic endocrine pancreas development, followed by neonatal hyperglycemia and lethality, by inducing pancreatic progenitor cell apoptosis and inhibiting cell proliferation. However, unlike MDM2-knockout animals, mice lacking MDMX in endocrine progenitor cells develop normally. But, surprisingly, the survival rate of adult MDMX-knockout mice drastically declines compared to control mice, as blockage of neonatal development of endocrine pancreas by inhibition of cell proliferation and subsequent islet dysfunction and hyperglycemia eventually lead to type 1 diabetes-like disease with advanced diabetic nephropathy. As expected, both MDM2 and MDMX deletion-caused pancreatic defects are completely rescued by loss of p53, verifying the crucial role of the MDM2 and/or MDMX in regulating p53 in a spatio-temporal manner during the development, functional maintenance, and related disease progress of endocrine pancreas. Also, our study suggests a possible mouse model of advanced diabetic nephropathy

  17. Predictive and prognostic impact of TP53 mutations and MDM2 promoter genotype in primary breast cancer patients treated with epirubicin or paclitaxel.

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    Ranjan Chrisanthar

    Full Text Available BACKGROUND: TP53 mutations have been associated with resistance to anthracyclines but not to taxanes in breast cancer patients. The MDM2 promoter single nucleotide polymorphism (SNP T309G increases MDM2 activity and may reduce wild-type p53 protein activity. Here, we explored the predictive and prognostic value of TP53 and CHEK2 mutation status together with MDM2 SNP309 genotype in stage III breast cancer patients receiving paclitaxel or epirubicin monotherapy. EXPERIMENTAL DESIGN: Each patient was randomly assigned to treatment with epirubicin 90 mg/m(2 (n = 109 or paclitaxel 200 mg/m(2 (n = 114 every 3rd week as monotherapy for 4-6 cycles. Patients obtaining a suboptimal response on first-line treatment requiring further chemotherapy received the opposite regimen. Time from last patient inclusion to follow-up censoring was 69 months. Each patient had snap-frozen tumor tissue specimens collected prior to commencing chemotherapy. PRINCIPAL FINDINGS: While TP53 and CHEK2 mutations predicted resistance to epirubicin, MDM2 status did not. Neither TP53/CHEK2 mutations nor MDM2 status was associated with paclitaxel response. Remarkably, TP53 mutations (p = 0.007 but also MDM2 309TG/GG genotype status (p = 0.012 were associated with a poor disease-specific survival among patients having paclitaxel but not patients having epirubicin first-line. The effect of MDM2 status was observed among individuals harbouring wild-type TP53 (p = 0.039 but not among individuals with TP53 mutated tumors (p>0.5. CONCLUSION: TP53 and CHEK2 mutations were associated with lack of response to epirubicin monotherapy. In contrast, TP53 mutations and MDM2 309G allele status conferred poor disease-specific survival among patients treated with primary paclitaxel but not epirubicin monotherapy.

  18. Induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway delays the initiation but fails to eradicate established murine acute myeloid leukemia.

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    Jaako, P; Ugale, A; Wahlestedt, M; Velasco-Hernandez, T; Cammenga, J; Lindström, M S; Bryder, D

    2017-01-01

    Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

  19. MDM2 promoter polymorphism and p53 codon 72 polymorphism in chronic myeloid leukemia: the association between MDM2 promoter genotype and disease susceptibility, age of onset, and blast-free survival in chronic phase patients receiving imatinib.

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    Liu, Yi-Chang; Hsiao, Hui-Hua; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Yang, Ming-Yu; Lin, Pai-Mei; Hsu, Jui-Feng; Lee, Ching-Ping; Lin, Sheng-Fung

    2014-12-01

    The genetic or functional inactivation of the p53 pathway plays an important role with regards to disease progression from the chronic phase (CP) to blast phase (BP) and imatinib treatment response in chronic myeloid leukemia (CML). Two functional single nucleotide polymorphisms (SNPs), p53 R72P and MDM2 SNP309, are associated with alternation of p53 activity, however the association regarding CML susceptibility and BP transformation under imatinib treatment is unclear. The MDM2 SNP309 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism and confirmed by direct sequencing from 116 CML patients, including 104 in the CP at diagnosis, and 162 healthy Taiwanese controls. The p53 R72P polymorphism was examined in all CML patients. The SNP309 G/G genotype was associated with an increased risk of CML susceptibility (OR: 1.82, 95% CI: 1.03-3.22, P = 0.037), and an earlier age of disease onset (log-rank P = 0.005) compared with the T/T + T/G genotypes. Higher MDM2 mRNA expression was found in G/G genotype compared with T/T (P = 0.034) and T/T + T/G (P = 0.056) genotypes. No associations were found between the p53 R72P genotypes and clinical parameters and survival outcomes. Among 62 CP patients receiving imatinib as first-line therapy, the G/G genotype was associated with a shorter blast-free survival (log-rank P = 0.048) and more clonal evolution compared with the T/T + T/G genotypes. In patients with advanced diseases at diagnosis, the G/G genotype was associated with a poor overall survival (log-rank P = 0.006). Closely monitoring CML patients harboring the G/G genotype and further large-scale studies are warranted.

  20. Effects of the single nucleotide polymorphism at MDM2 309 on breast cancer patients with/without BRCA1/2 mutations

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    Sharon Nir

    2009-02-01

    Full Text Available Abstract Background A germ line single nucleotide polymorphism (SNP in the first intron of the gene encoding MDM2 at position 309, an important modulator of p53, has been described. BRCA1/2 mutation have been associated with increased rates of breast cancers with mutated P53. It was shown that the presence of MDM2 309 SNP correlated with younger cancer onset age in individuals with a p53 mutations. The differential effects of this SNP were also linked to estrogen receptor activation. Here we report on our study of 453 Ashkenazi breast cancer patients of whom 180 were positive for the known Ashkenazi BRCA1/2 mutations Methods DNA from breast cancer patients was obtained for analysis of one of the three common BRCA1/2 mutations and MDM2 SNP309. Data regarding cancer onset and death ages was obtained from our database and Statistical analysis was performed using the SPSS® statistical package (SPCC Inc., Chicago, IL, and JMP® software (SAS Institute, Cary, NC. Results The percentage of MDM2 SNP309 in control and BRCA 1/2 population which is similar to that reported for other Jewish Ashkenazi populations at 52.2% for the heterozygotes and 25.0% for MDM2SNP309G/G and 22.8% for MDM2SNP309T/T. There was not a statistical significant difference in median age of disease onset in the different MDM2 SNP309 subgroups of the BRCA1/2 carriers. When we further divided the group into under and above 51 years old ( presumed menopause age in the BRCA1 positive subset we found that there were less patients of the MDM2SNP309 G/G versus the MDM2SNP309 T/T in the over 51 patient group (p = 0.049. This result has been obtained in a relatively small subgroup and is of borderline statistical significance. Interestingly, in the BRCA1/2 mutation carriers, we found a survival advantage for patients harboring the SNP309 G/G genotype (p = 0.0086 but not for the 272 patients not harbouring this mutations. Conclusion MDM2SNP309G/G main effect on BRCA1/2 positive mutation

  1. HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage.

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    Pääjärvi, Gerd; Roudier, Emilie; Crisby, Milita; Högberg, Johan; Stenius, Ulla

    2005-03-01

    3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, statins, are widely used cholesterol-lowering drugs and have been shown to have anticancer effects in many models. We have investigated the effect of statins on Mdm2, a p53-specific ubiquitin ligase. It was found that pravastatin induced Mdm2 phosphorylation at Ser166 and at 2A10 antibody-specific epitopes in HepG2 cells, while mRNA levels were unchanged. Furthermore, pravastatin was found to induce phosphorylation of mTOR at Ser2448. Ser166 phosphorylation of Mdm2 was abrogated by an inhibitor of mTOR, rapamycin, but not by the PI3-kinase inhibitors LY294002 and wortmannin. Ser166 phosphorylation of Mdm2 has been associated to active Mdm2 and has been shown to increase its ubiquitin ligase activity and lead to increased p53 degradation. Our data show that statins attenuated the p53 response to DNA damage. Thus, in HepG2 cells pravastatin and simvastatin pretreatment attenuated the p53 response to DNA damage induced by 5-fluorouracil and benzo(a)pyrene. Similar attenuation was induced when p53 stabilization was induced by the inhibitor of nuclear export, leptomycin B. Furthermore, in the DNA-damaged cells, half-lives of Mdm2 and p53 were decreased by statins, indicating a more rapid formation of p53/Mdm2 complexes and facilitated p53 degradation. The induction of p53 responsive genes and apoptosis was attenuated. Mdm2 and p53 were also studied in vivo in rat liver employing immunohistochemistry, and it was found that constitutive Mdm2 expression was changed in livers of pravastatin-treated rats. We also show that the p53 response to a challenging dose of diethylnitrosamine was attenuated in hepatocytes in situ and in primary cultures of hepatocytes by pravastatin pretreatment. Taken together, these data indicate that statins induce an mTOR-dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the duration and intensity of the p53 response to DNA damage in hepatocytes.

  2. MDM2 SNP309 promoter polymorphism and p53 mutations in urinary bladder carcinoma stage T1

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    Olsson Hans

    2013-01-01

    Full Text Available Abstract Background Urinary bladder carcinoma stage T1 is an unpredictable disease that in some cases has a good prognosis with only local or no recurrence, but in others can appear as a more aggressive tumor with progression to more advanced stages. The aim here was to investigate stage T1 tumors regarding MDM2 promoter SNP309 polymorphism, mutations in the p53 gene, and expression of p53 and p16 measured by immunohistochemistry, and subsequently relate these changes to tumor recurrence and progression. We examined a cohort of patients with primary stage T1 urothelial carcinoma of the bladder and their tumors. Methods After re-evaluation of the original slides and exclusions, the study population comprised 141 patients, all with primary stage T1 urothelial carcinoma of the bladder. The hospital records were screened for clinical parameters and information concerning presence of histologically proven recurrence and progression. The paraffin-embedded tumor material was evaluated by immunohistochemistry. Any mutations found in the p53 gene were studied by single-strand conformation analysis and Sanger sequencing. The MDM2 SNP309 polymorphism was investigated by pyrosequencing. Multivariate analyses concerning association with prognosis were performed, and Kaplan-Meier analysis was conducted for a combination of changes and time to progression. Results Of the 141 patients, 82 had at least one MDM2 SNP309 G allele, and 53 had a mutation in the p53 gene, but neither of those anomalies was associated with a worse prognosis. A mutation in the p53 gene was associated with immunohistochemically visualized p53 protein expression at a cut-off value of 50%. In the group with p53 mutation Kaplan-Meier analysis showed higher rate of progression and shorter time to progression in patients with immunohistochemically abnormal p16 expression compared to them with normal p16 expression (p = 0.038. Conclusions MDM2 SNP309 promoter polymorphism and mutations in

  3. d-Amino acid mutation of PMI as potent dual peptide inhibitors of p53-MDM2/MDMX interactions.

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    Li, Xiang; Liu, Chao; Chen, Si; Hu, Honggang; Su, Jiacan; Zou, Yan

    2017-09-07

    According to the previously reported potent dual l-peptide PMI of p53-MDM2/MDMX interactions, a series of d-amino acid mutational PMI analogues, PMI-1-4, with enhanced proteolytic resistence and in vitro tumor cell inhibitory activities were reported, of which Liposome-PMI-1 showed a stronger inhibitory activity against the U87 cell lines than Nutlin-3. This d-amino acid mutation strategy may give a hand for enhancing the potential of peptide drugs. Copyright © 2017. Published by Elsevier Ltd.

  4. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular tran-scriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells’ entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  5. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    GUO HongXiong; CUN Wei; LIU LongDing; WANG LiChun; ZHAO HongLing; DONG ChengHong; LI QiHan

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular transcriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells' entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  6. Expressions of MDM2, Livin and Caspase-3 protein and mRNA in endometrial adenocarcinomas%学位论文摘要

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To investigate the relationship of the expression of MDM2,Livin and Caspase-3 protein and mRNA in the development of endometrioid adenocarcinoma (EA). Methods The expression levels of MDM2, Livin and Caspase-3 proteins and mRNA in EA tissues (n = 72), endometrial hyperplasia tissues (n = 60) and normal tissues ( n = 30) were examined by tissue microarray technique, immunohistochemistry( SP method) and in situ hybridization method. Results The positive expression rates of MDM2, Livin and Caspase-3 protein and mRNA in EA were respectively 80. 6% ( 58/72 ), 80. 6% ( 58/72 ), 33.3% ( 24/72 ) and 73.6% ( 53/72 ), 75.0% ( 54/72 ),27.8% (20/72). The positive rates of both MDM2 and Livin protein and mRNA in EA were higher than that in normal endometrium and endometrial hyperplasia( P < 0. 01 ). However, the positive rate of Caspase-3 in EA was lower than that in normal endometrium and endometrial hyperplasia( P < 0. 01 ). The positive expressions of MDM2 protein and mRNA were not related to the histological grade, FIGO stage, depth of invasion and lymph node metastasis. The positive expressions of Livin and Caspase-3 protein and mRNA were related to histological grade (P <0. 01 ,P <0.05 ), but they were not related to FIGO stage, depth of invasion and the lymph node metastasis. The expressions of MDM2, Livin and Caspase-3 protein were positively correlated with their mRNA. The expression of Livin was negatively correlated Caspase-3. Conclusion The expressions of MDM2, Livin and Caspase-3 protein and mRNA correlate with the dedvelopment and progression of EA, which may be valuable biomarkers to detect the early carcinogenesis and prognosis of EA.

  7. Effect of sodium arsenite on the gene and protein expression of p53, mdm2 and Kras in islet cells%亚砷酸钠对胰岛细胞中 p53、mdm2和Kras基因及蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    姚晓峰; 孙睿; 姜丽平; 耿成燕; 仲来福; 郑白璐; 杨光; 刘爽; 孙鲜策

    2014-01-01

    Objective To investigate the effect of sodium arsenite on p53, mdm2 and Kras expressions in islet βcells (INS-1) of rat.Methods The levels of wild type p53 (Tp53), mdm2 and Kras in sodium arsenite-treated rat islet βcells were detected by real-time PCR.The expression of mutant p53 and mdm2 protein were detected by western blot. Results After treatment with sodium arsenite, the level of Tp53 decreased, but those of mdm2、Kras increased.The protein expressions of mutant p53 and mdm2 increased.Conclusion Sodium arsenite could induce the transformation of Tp53 to mutant p53, and increase the level of mdm2 and Kras in INS-1 cells.%目的:探讨砷对大鼠胰岛β细胞(INS-1)p53、mdm2和Kras基因和蛋白表达的影响。方法荧光实时定量PCR法检测亚砷酸钠对大鼠胰岛β细胞中野生型p53(Tp53)、mdm2和Kras基因表达的影响,Western blot检测亚砷酸钠对大鼠胰岛β细胞突变型p53和mdm2蛋白表达的影响。结果亚砷酸钠作用于INS-1细胞后,Tp53基因水平降低,mdm2、Kras基因水平升高;突变型p53和mdm2蛋白表达增加。结论亚砷酸钠可使INS-1细胞中抑癌基因Tp53向突变型p53转变,癌基因mdm2和Kras的水平升高。

  8. Aciculatin induces p53-dependent apoptosis via MDM2 depletion in human cancer cells in vitro and in vivo.

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    Chin-Yu Lai

    Full Text Available Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (-/- (p53-KO HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+. The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity.

  9. Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

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    Mariell Pettersson

    Full Text Available The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2 via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

  10. A stapled peptide antagonist of MDM2 carried by polymeric micelles sensitizes glioblastoma to temozolomide treatment through p53 activation.

    Science.gov (United States)

    Chen, Xishan; Tai, Lingyu; Gao, Jie; Qian, Jianchang; Zhang, Mingfei; Li, Beibei; Xie, Cao; Lu, Linwei; Lu, Wuyuan; Lu, Weiyue

    2015-11-28

    Antagonizing MDM2 and MDMX to activate the tumor suppressor protein p53 is an attractive therapeutic paradigm for the treatment of glioblastoma multiforme (GBM). However, challenges remain with respect to the poor ability of p53 activators to efficiently cross the blood-brain barrier and/or blood-brain tumor barrier and to specifically target tumor cells. To circumvent these problems, we developed a cyclic RGD peptide-conjugated poly(ethylene glycol)-co-poly(lactic acid) polymeric micelle (RGD-M) that carried a stapled peptide antagonist of both MDM2 and MDMX (sPMI). The peptide-carrying micelle RGD-M/sPMI was prepared via film-hydration method with high encapsulation efficiency and loading capacity as well as ideal size distribution. Micelle encapsulation dramatically increased the solubility of sPMI, thus alleviating its serum sequestration. In vitro studies showed that RGD-M/sPMI efficiently inhibited the proliferation of glioma cells in the presence of serum by activating the p53 signaling pathway. Further, RGD-M/sPMI exerted potent tumor growth inhibitory activity against human glioblastoma in nude mouse xenograft models. Importantly, the combination of RGD-M/sPMI and temozolomide--a standard chemotherapy drug for GBM increased antitumor efficacy against glioblastoma in experimental animals. Our results validate a combination therapy using p53 activators with temozolomide as a more effective treatment for GBM.

  11. Natural product ginsenoside 25-OCH3-PPD inhibits breast cancer growth and metastasis through down-regulating MDM2.

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    Wei Wang

    Full Text Available Although ginseng and related herbs have a long history of utility for various health benefits, their application in cancer therapy and underlying mechanisms of action are not fully understood. Our recent work has shown that 20(S-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH(3-PPD, a newly identified ginsenoside from Panax notoginseng, exerts activities against a variety of cancer cells in vitro and in vivo. This study was designed to investigate its anti-breast cancer activity and the underlying mechanisms of action. We observed that 25-OCH(3-PPD decreased the survival of breast cancer cells by induction of apoptosis and G1 phase arrest and inhibited the growth of breast cancer xenografts in vivo. We further demonstrated that, in a dose- and time-dependent manner, 25-OCH(3-PPD inhibited MDM2 expression at both transcriptional and post-translational levels in human breast cancer cells with various p53 statuses (wild type and mutant. Moreover, 25-OCH(3-PPD inhibited in vitro cell migration, reduced the expression of epithelial-to-mesenchymal transition (EMT markers, and prevented in vivo metastasis of breast cancer. In summary, 25-OCH(3-PPD is a potential therapeutic and anti-metastatic agent for human breast cancer through down-regulating MDM2. Further preclinical and clinical development of this agent is warranted.

  12. MDM-2和bcl-2在子宫颈癌中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    张巍

    2009-01-01

    宫颈癌是全球妇女恶性肿瘤中仅次于乳腺癌的第2个最常见恶性肿瘤。每年全世界新增病例大约500000例。在发展中国家妇女中发病率居第1位。子宫颈癌的早期诊断是子宫颈癌防治的重要策略之一。子宫颈癌的发生涉及多因素、多基因、多环节。本研究采用免疫组化法,检测了MDM-2和bcl-2在宫颈癌中的表达,旨在探讨MDM-2和bcl-2在宫颈癌的发生、发展过程中的相互关系。

  13. Npas4 Regulates Mdm2 and thus Dcx in Experience-Dependent Dendritic Spine Development of Newborn Olfactory Bulb Interneurons

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    Sei-ichi Yoshihara

    2014-08-01

    Full Text Available Sensory experience regulates the development of various brain structures, including the cortex, hippocampus, and olfactory bulb (OB. Little is known about how sensory experience regulates the dendritic spine development of OB interneurons, such as granule cells (GCs, although it is well studied in mitral/tufted cells. Here, we identify a transcription factor, Npas4, which is expressed in OB GCs immediately after sensory input and is required for dendritic spine formation. Npas4 overexpression in OB GCs increases dendritic spine density, even under sensory deprivation, and rescues reduction of dendrite spine density in the Npas4 knockout OB. Furthermore, loss of Npas4 upregulates expression of the E3-ubiquitin ligase Mdm2, which ubiquitinates a microtubule-associated protein Dcx. This leads to reduction in the dendritic spine density of OB GCs. Together, these findings suggest that Npas4 regulates Mdm2 expression to ubiquitinate and degrade Dcx during dendritic spine development in newborn OB GCs after sensory experience.

  14. Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

    Science.gov (United States)

    Pettersson, Mariell; Quant, Maria; Min, Jaeki; Iconaru, Luigi; Kriwacki, Richard W; Waddell, M Brett; Guy, R Kiplin; Luthman, Kristina; Grøtli, Morten

    2015-01-01

    The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

  15. Induction of apoptosis in Ehrlich ascites tumour cells via p53 activation by a novel small-molecule MDM2 inhibitor - LQFM030.

    Science.gov (United States)

    da Mota, Mariana F; Cortez, Alane P; Benfica, Polyana L; Rodrigues, Bruna Dos S; Castro, Thalyta F; Macedo, Larissa M; Castro, Carlos H; Lião, Luciano M; de Carvalho, Flávio S; Romeiro, Luiz A S; Menegatti, Ricardo; Verli, Hugo; Villavicencio, Bianca; Valadares, Marize C

    2016-09-01

    The activation of the p53 pathway through the inhibition of MDM2 has been proposed as a novel therapeutic strategy against tumours. A series of cis-imidazoline analogues, termed nutlins, were reported to displace the recombinant p53 protein from its complex with MDM2 by binding to MDM2 in the p53 pocket, and exhibited an antitumour activity both in vitro and in vivo. Thus, the purpose of this study was to evaluate the antitumour properties of LQFM030 (2), a nutlin analogue created by employing the strategy of molecular simplification. LQFM030 (2) cytotoxicity was evaluated in Ehrlich ascites tumour (EAT) cells, p53 wild type, by the trypan blue exclusion test, and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry, real-time PCR and Western blotting. Our results demonstrate that LQFM030 has dose-dependent antiproliferative activity and cytotoxic activity on EAT cells, induces the accumulation of p53 protein and promotes cell cycle arrest and apoptosis. p53 gene transcription was unaffected by LQFM030 (2); however, MDM2 mRNA increased and MDM2 protein decreased. These results suggest that the small-molecule p53 activator LQFM030 (2) has the potential for further development as a novel cancer therapeutic agent. © 2016 Royal Pharmaceutical Society.

  16. Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

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    Yuxin Zhang

    2012-02-01

    Full Text Available Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors.

  17. 树舌多糖GF对HepA瘤细胞MDM-2基因表达的影响研究

    Institute of Scientific and Technical Information of China (English)

    于赫; 于英君

    2005-01-01

    应用免疫组化技术对HepA瘤细胞中MDM-2基因的表达量进行分析,以探讨树舌多糖抗肿瘤的分子机制.目的:研究树舌多糖GF对HepA瘤细胞MDM-2表达的影响.方法:应用SP免疫组化染色技术和多媒体彩色病理图文分析系统测定HepA瘤细胞中MDM-2的表达.结果:树舌多糖组中MDM-2的表达量均显著低于模型组(P<0.01),树舌多糖组与猪苓多糖组无显著性差异.结论:初步认为树舌多糖GF可通过降低MDM-2的表达而抑制HepA瘤细胞的增殖.

  18. 膀胱癌中mdm2、p53蛋白和粘着斑激酶表达的病理意义

    Institute of Scientific and Technical Information of China (English)

    桂津; 罗金芳; 李如昌; 林梅绥; 许祖德

    2001-01-01

    目的探讨mdm2、p53蛋白和粘着斑激酶(FAK)在膀胱移行细胞癌(TCC)中的表达与肿瘤生物行为的关系.方法采用免疫组织化学方法,测定mdm2、p53蛋白和FAK在81例TCC中的表达情况.结果mdm2、p53蛋白和FAK在TCC组织中的表达明显高于癌旁粘膜,mdm2在p53阳性TCC组中的表达明显高于阴性组.结论mdm2和p53在TCC发展过程中可能具有协同作用,联合检测mdm2、p53蛋白和FAK可作为TCC有价值的诊断指标.

  19. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations.

    Science.gov (United States)

    Verma, Sharad; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-01-01

    p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin) by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy) have major contribution in binding free energy.

  20. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations.

    Directory of Open Access Journals (Sweden)

    Sharad Verma

    Full Text Available p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy have major contribution in binding free energy.

  1. Influences of MDM2 on epithelial mesenchymal transition of human breast cancer cells and its molecular mechanism%MDM2对人乳腺癌细胞上皮间质转化的影响及其机制研究

    Institute of Scientific and Technical Information of China (English)

    闫彩云; 仇金荣; 卢建磊; 殷咏梅

    2014-01-01

    目的:探讨鼠双微粒体2( MDM2)在上皮间质转化( EMT )过程中的作用及其分子机制。方法采用Western blotting检测人乳腺癌 MCF-7、MDA-MB-231和 MDA-MB-435细胞株中 MDM2及 EMT相关标记分子( E-cadherin、N-cadherin及Vimentin)水平;分别于MCF-7细胞中瞬时转染MDM2空质粒pcmv、MDM2过表达质粒pcmv-MDM2,MDA-MB-231细胞中瞬时转染针对MDM2三个不同靶点的干扰质粒36h后观察细胞形态,并采用Western blotting和免疫荧光检测EMT相关标记分子及MDM2的表达情况;采用Western blotting及实时定量PCR检测在MCF-7、MDA-MB-231细胞过表达MDM2后对促EMT转录因子Snail1、Twist 蛋白和mRNA水平的影响。结果 MCF-7细胞过表达MDM2后形态由鹅卵石形变为纺锤形, E-cadherin蛋白随MDM2表达水平的上调表达降低;MDA-MB-231细胞敲低MDM2后形态由长梭形变为卵圆形,Vimentin、N-cadherin蛋白随MDM2表达水平的下调表达依次降低;MCF-7及MDA-MB-231细胞过表达MDM2后Snail1、Twist的蛋白及mRNA水平均升高。结论在乳腺癌细胞株中MDM2可促进 EMT发生,其可能通过上调转录因子 Snail1和 Twist表达来实现。%Objective To explore the influence of murine double minute 2 ( MDM2) on epithelial mesenchymal transition ( EMT) of human breast cancer cells and its molecular mechanism. Methods Western blotting method was performed to examine the protein expressions of MDM2 and EMT-related proteins ( E-cadherin, N-cadherin and Vimentin) in human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435. The optical microscope was used to observe the morphological changes at 36th hour after transiently transfection of MCF-7 cells with empty plasmid ( pcmv) or MDM2 overexpression plasmid ( pcmv-MDM2) and MDA-MB-231 cells with three different interference plasmids. Meanwhile, the expressions of MDM2 and EMT related molecular markers were measured by Western blotting and immunofluorescence assays

  2. p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in Esophageal Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Memar Bahram

    2010-04-01

    Full Text Available Abstract Background Tumor suppressor genes p53 and p16INK4a and the proto-oncogene MDM2 are considered to be essential G1 cell cycle regulatory genes whose loss of function is associated with ESCC carcinogenesis. We assessed the aberrant methylation of the p16 gene and its impact on p16INK4a protein expression and correlations with p53 and MDM2 protein expressions in patients with ESCC in the Golestan province of northeastern Iran in which ESCC has the highest incidence of cancer, well above the world average. Methods Cancerous tissues and the adjacent normal tissue obtained from 50 ESCC patients were assessed with Methylation-Specific-PCR to examine the methylation status of p16. The expression of p16, p53 and MDM2 proteins was detected by immunohistochemical staining. Results Abnormal expression of p16 and p53, but not MDM2, was significantly higher in the tumoral tissue. p53 was concomitantly accumulated in ESCC tumor along with MDM2 overexpression and p16 negative expression. Aberrant methylation of the p16INK4a gene was detected in 31/50 (62% of esophageal tumor samples, while two of the adjacent normal mucosa were methylated (P p16INK4a aberrant methylation was significantly associated with decreased p16 protein expression (P = 0.033, as well as the overexpression of p53 (P = 0.020. Conclusions p16 hypermethylation is the principal mechanism of p16 protein underexpression and plays an important role in ESCC development. It is associated with p53 protein overexpression and may influence the accumulation of abnormally expressed proteins in p53-MDM2 and p16-Rb pathways, suggesting a possible cross-talk of the involved pathways in ESCC development.

  3. p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Taghavi, Noushin; Biramijamal, Firouzeh; Sotoudeh, Masoud; Khademi, Hooman; Malekzadeh, Reza; Moaven, Omeed; Memar, Bahram; A'rabi, Azadeh; Abbaszadegan, Mohammad Reza

    2010-04-13

    Tumor suppressor genes p53 and p16INK4a and the proto-oncogene MDM2 are considered to be essential G1 cell cycle regulatory genes whose loss of function is associated with ESCC carcinogenesis. We assessed the aberrant methylation of the p16 gene and its impact on p16INK4a protein expression and correlations with p53 and MDM2 protein expressions in patients with ESCC in the Golestan province of northeastern Iran in which ESCC has the highest incidence of cancer, well above the world average. Cancerous tissues and the adjacent normal tissue obtained from 50 ESCC patients were assessed with Methylation-Specific-PCR to examine the methylation status of p16. The expression of p16, p53 and MDM2 proteins was detected by immunohistochemical staining. Abnormal expression of p16 and p53, but not MDM2, was significantly higher in the tumoral tissue. p53 was concomitantly accumulated in ESCC tumor along with MDM2 overexpression and p16 negative expression. Aberrant methylation of the p16INK4a gene was detected in 31/50 (62%) of esophageal tumor samples, while two of the adjacent normal mucosa were methylated (P p16 protein expression (P = 0.033), as well as the overexpression of p53 (P = 0.020). p16 hypermethylation is the principal mechanism of p16 protein underexpression and plays an important role in ESCC development. It is associated with p53 protein overexpression and may influence the accumulation of abnormally expressed proteins in p53-MDM2 and p16-Rb pathways, suggesting a possible cross-talk of the involved pathways in ESCC development.

  4. Fluorescence In Situ Hybridization for MDM2 Amplification as a Routine Ancillary Diagnostic Tool for Suspected Well-Differentiated and Dedifferentiated Liposarcomas: Experience at a Tertiary Center

    Directory of Open Access Journals (Sweden)

    Khin Thway

    2015-01-01

    Full Text Available Background. The assessment of MDM2 gene amplification by fluorescence in situ hybridization (FISH has become a routine ancillary tool for diagnosing atypical lipomatous tumor (ALT/well-differentiated liposarcoma and dedifferentiated liposarcoma (WDL/DDL in specialist sarcoma units. We describe our experience of its utility at our tertiary institute. Methods. All routine histology samples in which MDM2 amplification was assessed with FISH over a 2-year period were included, and FISH results were correlated with clinical and histologic findings. Results. 365 samples from 347 patients had FISH for MDM2 gene amplification. 170 were positive (i.e., showed MDM2 gene amplification, 192 were negative, and 3 were technically unsatisfactory. There were 122 histologically benign cases showing a histology:FISH concordance rate of 92.6%, 142 WDL/DDL (concordance 96.5%, and 34 cases histologically equivocal for WDL (concordance 50%. Of 64 spindle cell/pleomorphic neoplasms (in which DDL was a differential diagnosis, 21.9% showed MDM2 amplification. Of the cases with discrepant histology and FISH, all but 3 had diagnoses amended following FISH results. For discrepancies of benign histology but positive FISH, lesions were on average larger, more frequently in “classical” (intra-abdominal or inguinal sites for WDL/DDL and more frequently core biopsies. Discrepancies of malignant histology but negative FISH were smaller, less frequently in “classical” sites but again more frequently core biopsies. Conclusions. FISH has a high correlation rate with histology for cases with firm histologic diagnoses of lipoma or WDL/DDL. It is a useful ancillary diagnostic tool in histologically equivocal cases, particularly in WDL lacking significant histologic atypia or DDL without corresponding WDL component, especially in larger tumors, those from intra-abdominal or inguinal sites or core biopsies. There is a significant group of well-differentiated adipocytic neoplasms

  5. Single-nucleotide polymorphisms p53 G72C and Mdm2 T309G in patients with psoriasis, psoriatic arthritis, and SAPHO syndrome.

    Science.gov (United States)

    Assmann, Gunter; Wagner, Annette D; Monika, Mueller; Pfoehler, Claudia; Pfreundschuh, Michael; Tilgen, Wolfgang; Roemer, Klaus

    2010-08-01

    Psoriasis (Ps), psoriatic arthritis (PsA), and SAPHO syndrome are diseases of unknown etiology that share common clinical features; however, family studies support the hypothesis of a genetic background for each of these diseases. To study the two common single-nucleotide polymorphisms (SNP) in the murine-double-minute-2-(Mdm2) and p53 genes in patients with Ps, PsA, and SAPHO syndrome. Genomic DNA was obtained from 187 patients with Ps, 50 with PsA, and 36 with SAPHO as well as 478 healthy controls. Mdm2-gene SNP T309G and p53-gene SNP G72C genotypes were determined by the polymerase chain reaction. Genotype and allele frequencies were analyzed with chi(2)-tests. Among the patients with Ps and PsA, no differences in allele or genotype frequencies of the p53-gene SNP G72C and Mdm2-gene SNP T309G were detected. However, in the SAPHO patients group, the frequencies of the Mdm2 SNP309 G allele and the genotype SNP 309 GG were significantly increased compared with the controls (G allele: 51.4 vs. 38.7%, P = 0.034; genotype GG: 36.1 vs. 14.2%, P = 0.002). In addition, the frequencies of the p53 SNP72 C allele and the genotype SNP 72 CC were also increased in the SAPHO patients cohort (C allele: 36.1 vs. 25.6%, P = 0.05; genotype CC: 16.7 vs. 6.3%, P = 0.05). SAPHO syndrome may be linked to an imbalance between MDM2 and p53 regulation with a "weak" p53-response associated with the Mdm2 SNP 309 G allele. In contrast, the p53 network does not seem to play a major role in pathogenesis of Ps or PsA.

  6. Clinical significance of mdm2 and p53 expression in orbital rhabdomyosarcoma%原癌基因mdm2及p53基因与眼眶横纹肌肉瘤生物学特性研究

    Institute of Scientific and Technical Information of China (English)

    顼晓琳; 李彬; 孙宪丽; 李辽青

    2004-01-01

    目的探讨原癌基因mdm2、p53基因在眼眶横纹肌肉瘤(ORMS)发病机制中的可能作用,初步论证mdm2、p53作为ORMS生物学特性标记物和预后指标的可行性.方法应用免疫组化法检测31例ORMS标本中mdm2、p53、Ki-67蛋白的表达,Ki-67蛋白表达作为增殖指标反映肿瘤细胞的增殖状态.应用原位杂交方法检测31例ORMS标本中mdm2、p53基因在mRNA水平的表达.分析mdm2、p53蛋白表达与年龄、性别、病理类型、分化程度、增殖程度等临床组织病理学指标之间的关系.结果 31例ORMS中,mdm2、p53蛋白水平表达阳性率分别为77.4%(24/31)和71.0%(22/31),mdm2与p53蛋白共同表达阳性率是61.3%.mdm2、p53在蛋白水平表达与其各自的mRNA水平表达之间具有较好一致性.(1)按肿瘤细胞分化程度分为高、中、低3个组:中、低分化组mdm2蛋白阳性率和mdm2与p53蛋白共同表达阳性率均较高分化组高,差异有显著意义(P=0.007、0.009).(2)按Ki-67表达与否分为高、低增殖组:高增殖组p53蛋白表达阳性率较低增殖组高,差异有显著意义(P=0.044).mdm2、p53蛋白表达与其他临床病理学指标之间无显著相关性(P>0.05).结论 mdm2和p53与ORMS的分化和增殖密切相关,mdm2与p53共同表达提示mdm2可能与p53相结合而调节其活性.mdm2和p53的异常表达可能导致细胞异型性和过度增殖,在ORMS的发生和发展中起重要作用.mdm2和p53在一定程度上能够反映肿瘤的分化和增殖特性,有可能成为判断该肿瘤预后的指标之一.

  7. The MDM2-p53-pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    DEFF Research Database (Denmark)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic...... oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53...

  8. Nondenaturing polyacrylamide gel electrophoresis to study the dissociation of the p53·MDM2/X complex by potentially anticancer compounds.

    Science.gov (United States)

    Sgammato, Roberta; Desiderio, Doriana; Lamberti, Anna; Raimo, Gennaro; Novellino, Ettore; Carotenuto, Alfonso; Masullo, Mariorosario

    2015-12-01

    A new analytical method to study the dissociation of the complexes between the oncosuppressor p53 and its negative modulators murine double-minute protein 2 (MDM2) or MDMX, is proposed. This technique is reliable to determine the dissociative power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX in a native polyacrylamide gel, when inhibitors are added to the complex mixture. Therefore, we propose this new approach to easily screen library of compounds, with potential pharmacological anticancer activity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Justina McEvoy

    Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

  10. Expression and combined expression of p53, MDM-2 and p21 in 190 non-Hodgkin lymphomas%非霍奇金淋巴瘤中p53、MDM-2和p21表达的意义

    Institute of Scientific and Technical Information of China (English)

    马怡晖; 朱有凯; 叶子茵; 林汉良; 林素霞; 李海刚

    2012-01-01

    目的 研究非霍奇金淋巴瘤(NHL)中p53、MDM-2和p21表达及联合表达的情况,探讨它们在NHL发生发展中的作用和在临床病理诊断工作中的应用价值.方法 应用组织芯片、免疫组化及原位凋亡检测等技术对190例NHL中p53、MDM-2、p21和Ki-67的表达以及凋亡进行检测并统计分析.结果 (1)p53、MDM-2和p21在B-NHL中的阳性表达率分别为36.00%、8.00%和17.33%,在T/NK-NHL中的阳性表达率分别为40.00%、18.55%和24.35%;(2)MDM-2主要在侵袭性较高的NHL中表达,p53+/MDM-2+表型的NHL具有较高增殖指数,其中9例ALCL有5例为ALK阴性;(3)p53与p21阳性表达在T/NK-NHL中显著正相关(P=0.010).结论 (1)MDM-2表达可能参与恶性程度较高类型NHL的发生发展;(2)p53+/MDM-2+和p53+/p21+表型可能与侵袭性较高NHL的高度恶性、侵袭性及不良预后相关.

  11. Development of cell-penetrating peptide-based drug leads to inhibit MDMX:p53 and MDM2:p53 interactions.

    Science.gov (United States)

    Philippe, Grégoire; Huang, Yen-Hua; Cheneval, Olivier; Lawrence, Nicole; Zhang, Zhen; Fairlie, David P; Craik, David J; de Araujo, Aline Dantas; Henriques, Sónia Troeira

    2016-11-01

    The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.

  12. The MDM2–p53–pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    Science.gov (United States)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao; Yang, Jin-Kui; Wang, Baile; Jiang, Xue; Zhou, Yawen; Hallenborg, Philip; Hoo, Ruby L. C.; Lam, Karen S. L.; Ikeda, Yasuhiro; Gao, Xin; Xu, Aimin

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53 alleviates defective GSIS in diabetic islets by restoring PC expression. Thus, the MDM2–p53–PC signalling axis links mitochondrial metabolism to insulin secretion and glucose homeostasis, and could represent a therapeutic target in diabetes. PMID:27265727

  13. Non-linear feedback control of the p53 protein-mdm2 inhibitor system using the derivative-free non-linear Kalman filter.

    Science.gov (United States)

    Rigatos, Gerasimos G

    2016-06-01

    It is proven that the model of the p53-mdm2 protein synthesis loop is a differentially flat one and using a diffeomorphism (change of state variables) that is proposed by differential flatness theory it is shown that the protein synthesis model can be transformed into the canonical (Brunovsky) form. This enables the design of a feedback control law that maintains the concentration of the p53 protein at the desirable levels. To estimate the non-measurable elements of the state vector describing the p53-mdm2 system dynamics, the derivative-free non-linear Kalman filter is used. Moreover, to compensate for modelling uncertainties and external disturbances that affect the p53-mdm2 system, the derivative-free non-linear Kalman filter is re-designed as a disturbance observer. The derivative-free non-linear Kalman filter consists of the Kalman filter recursion applied on the linearised equivalent of the protein synthesis model together with an inverse transformation based on differential flatness theory that enables to retrieve estimates for the state variables of the initial non-linear model. The proposed non-linear feedback control and perturbations compensation method for the p53-mdm2 system can result in more efficient chemotherapy schemes where the infusion of medication will be better administered.

  14. In squamous cell carcinoma of the vulva, overexpression of p53 is a late event and neither p53 nor mdm2 expression is a useful marker to predict lymph node metastases

    NARCIS (Netherlands)

    Emanuels, AG; Koudstaal, J; Burger, MPM; Hollema, H

    1999-01-01

    To offer more tailored treatment to individual patients with squamous cell carcinoma of the vulval more accurate prediction of lymph node metastases is required. As p53 and mdm2 are genes known to be involved in the development of other tumours, we studied expression of p53 and mdm2 in carcinogenesi

  15. In squamous cell carcinoma of the vulva, overexpression of p53 is a late event and neither p53 nor mdm2 expression is a useful marker to predict lymph node metastases

    NARCIS (Netherlands)

    Emanuels, AG; Koudstaal, J; Burger, MPM; Hollema, H

    1999-01-01

    To offer more tailored treatment to individual patients with squamous cell carcinoma of the vulval more accurate prediction of lymph node metastases is required. As p53 and mdm2 are genes known to be involved in the development of other tumours, we studied expression of p53 and mdm2 in carcinogenesi

  16. Association between MDM2 SNP309 T>G polymorphism and the risk of bladder cancer: new data in a Chinese population and an updated meta-analysis

    Directory of Open Access Journals (Sweden)

    Xie LG

    2015-12-01

    Full Text Available Linguo Xie,1,2,* Yan Sun,2,* Tao Chen,1,2,* Dawei Tian,1,2 Yujuan Li,3 Yu Zhang,1,2 Na Ding,2 Zhonghua Shen,1,2 Hao Xu,1,2 Xuewu Nian,4 Nan Sha,1,2 Ruifa Han,1,2 Hailong Hu,1,2 Changli Wu1,2 Objective: Human murine double minute 2 protein (MDM2 is mainly a negative regulator of p53 tumor suppressor pathway. We aimed to investigate the association between MDM2 SNP309 polymorphism and bladder cancer risk. Methods: A total of 535 bladder cancer patients and 649 health controls were recruited for our study. MDM2 SNP309 T>G polymorphism was genotyped by polymerase chain reaction-ligase detection reaction method. Logistic regression was used to analyze the relationship between the genotype and susceptibility of bladder cancer. Kaplan–Meier estimates and log-rank test were obtained to analyze the association between the genotype and risk of recrudesce in nonmuscle-invasive bladder cancer patients. A multivariable Cox proportional hazards model was fitted to identify independent prognostic factors. To further investigate the association, we conducted a meta-analysis including six studies. Results: The frequency of the MDM2 SNP309 T>G polymorphism showed no significant difference between cases and controls (all P>0.05. In the stratification analysis, the results showed that G allele carriers were prone to have a significant decrease in risk of low-grade bladder cancer (adjusted odds ratio: 0.613, 95% confidence interval: 0.427–0.881, and G variant was associated with a significantly reduced risk of recurrence in nonmuscle-invasive bladder cancer patients with or without chemotherapy (P<0.05. The results of the meta-analysis showed that G allele and GG genotype of MDM2 SNP309 polymorphism were significantly associated with increased risk of bladder cancer in Caucasians (both P<0.05, and no association was observed in total populations and Asians (P>0.05. Conclusion: MDM2 SNP309 T>G polymorphism has no influence on bladder cancer risk in Asians, but

  17. Study on expressions of MDM2 and SPIN1mRNA in cancer of larynx%MDM2和SPIN1mRNA 在喉癌组织中的表达及其相关性研究

    Institute of Scientific and Technical Information of China (English)

    马伟军; 任晓勇; 许珉

    2014-01-01

    Objective:To observe the expressions of MDM 2 and SPIN1 mRNA in tissue of cancer of lar-ynx and investigate their correlations and explore their roles in tumor genesis .Method :The expressions of MDM2 and SPIN1mRNA in 32 cancer of larynx tissues and adjacent tissues detected by RT-PCR .Result :There were signif-icant differences in the expressions of MDM2 of 32 samples ,MDM2 mRNA presented higher level expression in cancer of larynx tissues (P< 0 .01) ,while SPIN1mRNA expressions of two tissues show no significant difference . MDM2 mRNA expression increased along with the increased tumor differentiation degree of cancer of larynx (P<0 . 01) .There were no correlation between the expressions of MDM 2 and SPIN1 in cancer of larynx .Conclusion :MDM2 may play an important role in tumor genesis of cancer of larynx ,while SPIN1 may play an unimportant role in tumor genesis of cancer of larynx .Their relationship will be researched in further study .%目的:研究喉癌组织中MDM2和SPIN1mRNA的表达,探讨其相关性及在肿瘤发生发展中的作用。方法:应用real-time PCR对32例喉癌及癌旁组织标本的MDM2、SPIN1在mR-NA水平进行检测。结果:MDM2在喉癌组织中的表达明显高于癌旁正常组织,差异具有显著性( P<0.01);SPIN1在喉癌组织和癌旁正常组织中表达无显著性差异( P>0.05)。MDM2在喉癌中的表达与肿瘤的病理分化呈负相关(P<0.01)。SPIN1的表达和肿瘤分化无关( P >0.05)。喉癌组织中MDM2和SPIN1表达无显著相关性( P >0.05)。结论:MDM2基因在喉癌的发生发展中可能发挥重要作用,而SPIN1基因在喉癌的发生发展过程中可能不发挥主要作用,两者之间的相互作用机制有待进一步研究。

  18. DIMP53-1: A novel small-molecule dual inhibitor of p53-MDM2/X interactions with multifunctional p53-dependent anticancer properties.

    Science.gov (United States)

    Soares, Joana; Espadinha, Margarida; Raimundo, Liliana; Ramos, Helena; Gomes, Ana Sara; Gomes, Sara; Loureiro, Joana B; Inga, Alberto; Reis, Flávio; Gomes, Célia; Santos, Maria M M; Saraiva, Lucília

    2017-03-10

    The transcription factor p53 plays a crucial role in cancer development and dissemination, and thus p53-targeted therapies are amongst the most encouraging anticancer strategies. In human cancers with wild-type (wt) p53, its inactivation by interaction with murine double minute (MDM)2 and MDMX is a common event. Simultaneous inhibition of the p53 interaction with both MDMs is crucial to restore the tumor suppressor activity of p53. Here we describe the synthesis of the new tryptophanol-derived oxazoloisoindolinone DIMP53-1 and identify its activity as a dual inhibitor of the p53-MDM2/X interactions using a yeast-based assay. DIMP53-1 caused growth inhibition, mediated by p53 stabilization and upregulation of p53 transcriptional targets involved in cell cycle arrest and apoptosis, in wt p53-expressing tumor cells, including MDM2- or MDMX-overexpressing cells. Importantly, DIMP53-1 abolishes the p53-MDM2/X interactions by binding to p53, in human colon adenocarcinoma HCT116 cells. DIMP53-1 also inhibited the migration and invasion of HCT116 cells, and the migration and tube formation of HMVEC-D endothelial cells. Notably, in human tumor xenograft mice models, DIMP53-1 showed a p53-dependent antitumor activity through induction of apoptosis and inhibition of proliferation and angiogenesis. Finally, no genotoxicity or undesirable toxic effects were observed with DIMP53-1. In conclusion, DIMP53-1 is a novel p53 activator, which potentially binds to p53 inhibiting its interaction with MDM2 and MDMX. Although target-directed, DIMP53-1 has a multifunctional activity, targeting major hallmarks of cancer through its anti-proliferative, pro-apoptotic, anti-angiogenic, anti-invasive and anti-migratory properties. DIMP53-1 is a promising anticancer drug candidate and an encouraging starting point to develop improved derivatives for clinical application.

  19. Effects of MDM2, MDM4 and TP53 codon 72 polymorphisms on cancer risk in a cohort study of carriers of TP53 germline mutations.

    Directory of Open Access Journals (Sweden)

    Shenying Fang

    Full Text Available BACKGROUND: Previous studies have shown that MDM2 SNP309 and p53 codon 72 have modifier effects on germline P53 mutations, but those studies relied on case-only studies with small sample sizes. The impact of MDM4 polymorphism on tumor onset in germline mutation carriers has not previously been studied. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed 213 p53 germline mutation carriers including 168(78.9% affected with cancer and 174 who had genotypic data. We analyzed time to first cancer using Kaplan-Meier and Cox proportional hazards methods, comparing risks according to polymorphism genotypes. For MDM2 SNP309, a significant difference of 9.0 years in the average age of cancer diagnosis was observed between GG/GT and TT carriers (18.6 versus 27.6 years, P = 0.0087. The hazards ratio was 1.58 (P = 0.03 comparing risks among individuals with GG/GT to risk among TT, but this effect was only significant in females (HR = 1.60, P = 0.02. Compared to other genotypes, P53 codon 72 PP homozygotes had a 2.24 times (P = 0.03 higher rate for time to develop cancer. We observed a multiplicative joint effect of MDM2 and p53 codon72 polymorphism on risk. The MDM4 polymorphism had no significant effects. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the MDM2 SNP309 G allele is associated with cancer risk in p53 germline mutation carriers and accelerates time to cancer onset with a pronounced effect in females. A multiplicative joint effect exists between the MDM2 SNP309 G allele and the p53 codon 72 G allele in the risk of cancer development. Our results further define cancer risk in carriers of germline p53 mutations.

  20. Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf(-/-) mouse model.

    Science.gov (United States)

    Chapeau, Emilie A; Gembarska, Agnieszka; Durand, Eric Y; Mandon, Emeline; Estadieu, Claire; Romanet, Vincent; Wiesmann, Marion; Tiedt, Ralph; Lehar, Joseph; de Weck, Antoine; Rad, Roland; Barys, Louise; Jeay, Sebastien; Ferretti, Stephane; Kauffmann, Audrey; Sutter, Esther; Grevot, Armelle; Moulin, Pierre; Murakami, Masato; Sellers, William R; Hofmann, Francesco; Jensen, Michael Rugaard

    2017-03-21

    Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf(-/-) mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

  1. Rapid detection of SNP (c.309T>G in the MDM2 gene by the Duplex SmartAmp method.

    Directory of Open Access Journals (Sweden)

    Yasuaki Enokida

    Full Text Available BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

  2. MDM2 and HIF1alpha expression levels in different histologic subtypes of malignant pleural mesothelioma: correlation with pathological and clinical data.

    Science.gov (United States)

    Pasello, Giulia; Urso, Loredana; Mencoboni, Manlio; Grosso, Federica; Ceresoli, Giovanni Luca; Lunardi, Francesca; Vuljan, Stefania Edith; Bertorelle, Roberta; Sacchetto, Valeria; Ciminale, Vincenzo; Rea, Federico; Favaretto, Adolfo; Conte, PierFranco; Calabrese, Fiorella

    2015-12-08

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features.

  3. Potential genotype-specific single nucleotide polymorphism interaction of common variation in p53 and its negative regulator mdm2 in cholangiocarcinoma susceptibility.

    Science.gov (United States)

    Zimmer, Vincent; Höblinger, Aksana; Mihalache, Florentina; Assmann, Gunter; Acalovschi, Monica; Lammert, Frank

    2012-07-01

    Aberrant cell cycle control and apoptosis deregulation are involved in biliary carcinogenesis. The tumor suppressor gene p53 and its key negative regulator murine double minute 2 (mdm2) cooperate in modulating these basic cell functions and germline p53 alteration promotes cholangiocarcinoma (CCA) formation in animal models. The potential association between common functional genetic variation in p53 (SNP72 G/C) and mdm2 (SNP309 T/G) and susceptibility to bile duct cancer, however, has not been studied. p53/SNP72 G/C (rs1042522) and mdm2/SNP309 T/G (rs2279744) were genotyped in 182 Caucasian CCA patients and 350 controls using TaqMan assays. Allelic and genotypic differences, including exploratory data analyses (according to gender, tumor localization, early onset and genotypic interactions) were compared in contingency tables using the χ(2) and Fisher's exact tests. The overall comparison of allele and genotype frequencies yielded no significant association between either SNP and CCA susceptibility. Similarly, gender- and localization-specific analyses did not reveal deviations in allelic or genotypic distributions. In carriers of the low-apoptotic p53 genotype CC, the mdm2 SNP309 T allele conferred borderline significant CCA risk [P=0.049; odds ratio (OR), 4.36; 95% CI, 0.92-20.77]. Power analysis confirmed adequate statistical power to exclude major SNP effects (each >97% for OR 1.7). Collectively, the results we obtained from the largest European CCA cohort do not support the hypothesis of a prominent role of common p53 and mdm2 variation in the genetic susceptibility to bile duct cancer. However, epistatic effects may modulate genetic CCA risk in individual subsets.

  4. MDM2 and HIF1alpha expression levels in different histologic subtypes of malignant pleural mesothelioma: correlation with pathological and clinical data

    Science.gov (United States)

    Mencoboni, Manlio; Grosso, Federica; Ceresoli, Giovanni Luca; Lunardi, Francesca; Vuljan, Stefania Edith; Bertorelle, Roberta; Sacchetto, Valeria; Ciminale, Vincenzo; Rea, Federico; Favaretto, Adolfo; Conte, PierFranco; Calabrese, Fiorella

    2015-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p < 0.001), HIF1alpha (p = 0.013), necrosis (p = 0.013) and proliferation index (p < 0.001) were seen mainly in sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features. PMID:26544728

  5. The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

    Science.gov (United States)

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

    2016-08-15

    Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. © 2016 The Author(s).

  6. miR-339-5p regulates the p53 tumor-suppressor pathway by targeting MDM2

    DEFF Research Database (Denmark)

    Jansson, M D; Djodji Damas, Nkerorema; Lees, M

    2014-01-01

    MicroRNAs (miRNAs) regulate many key cancer-relevant pathways and may themselves possess oncogenic or tumor-suppressor functions. Consequently, miRNA dysregulation has been shown to be a prominent feature in many human cancers. The p53 tumor suppressor acts as a negative regulator of cell...... proliferation in response to stress and represents the most commonly lost and mutated gene in human cancers. The function of p53 is inhibited by the MDM2 oncoprotein. Using a high-throughput screening approach, we identified miR-339-5p as a regulator of the p53 pathway. We demonstrate that this regulation...... inhibition of miR-339-5p function perturbs the p53 response in cancer cells, allowing an increased proliferation rate. In addition, miR-339-5p expression is downregulated in tumors harboring wild-type TP53, suggesting that reduction of miR-339-5p level helps to suppress the p53 response in p53-competent...

  7. Evidence that proteasome-dependent degradation of the retinoblastoma protein in cells lacking A-type lamins occurs independently of gankyrin and MDM2.

    Directory of Open Access Journals (Sweden)

    Ryan T Nitta

    Full Text Available BACKGROUND: A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB stabilization through unknown mechanism(s. Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna-/- fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna-/- fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16(ink4a-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna-/- cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna-/- cells with p14(arf. p14(arf expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna-/- cells. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that pRB degradation in Lmna-/- cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna-/- fibroblasts. Second, Lmna-/- cells are refractory to

  8. 胃黏膜病变中突变型p53、Mdm2和PCNA蛋白表达及与幽门螺杆菌感染的关系%Expressions of mutant p53, Mdm2 and PCNA protein in gastric mucosal lesions and their relationship with Helicobacter pylori infection

    Institute of Scientific and Technical Information of China (English)

    齐淑文; 舒徐; 陈江; 吕农华

    2009-01-01

    目的 探讨胃黏膜病变中突变型p53、Mdm2、增殖细胞核抗原(PCNA)蛋白的表达及其与幽门螺杆菌(Hp)感染的关系.方法 应用免疫组化PV-9000法染色及Giemsa染色检测胃黏膜病变中突变型p53、Mdm2、PCNA蛋白的表达和Hp感染情况.结果 在慢性浅表性胃炎(CSG)、慢性萎缩性胃炎伴肠上皮化生(CAG伴IM)、不典型增生(Dys)及胃癌(GC)中,突变型p53、Mdm2、PCNA阳性表达随病变发展而逐渐升高,突变型p53在CSG组高于GC组,GC组高于CAG伴IM组(P均<0.05).不同胃黏膜病变之间Mdm2比较均有统计学差异(P<0.05).在同一病变中Hp阳性者突变型p53、Mdm2、PCNA阳性表达一般高于Hp阴性者,突变型p53在CAG伴IM组Hp阳性与阴性者间有统计学差异(P<0.05),PCNA和Mdm2在Dys组Hp阳性与阴性者有统计学差异(P<0.05).GC组中突变型p53、Mdm2、PCNA蛋白表达无相关关系.结论 突变型p53、Mdm2和PCNA蛋白的表达及Hp感染共同参与胃黏膜病变的发生、发展.

  9. Expression signature based on TP53 target genes doesn't predict response to TP53-MDM2 inhibitor in wild type TP53 tumors.

    Science.gov (United States)

    Sonkin, Dmitriy

    2015-10-22

    A number of TP53-MDM2 inhibitors are currently under investigation as therapeutic agents in a variety of clinical trials in patients with TP53 wild type tumors. Not all wild type TP53 tumors are sensitive to such inhibitors. In an attempt to improve selection of patients with TP53 wild type tumors, an mRNA expression signature based on 13 TP53 transcriptional target genes was recently developed (Jeay et al. 2015). Careful reanalysis of TP53 status in the study validation data set of cancer cell lines considered to be TP53 wild type detected TP53 inactivating alterations in 23% of cell lines. The subsequent reanalysis of the remaining TP53 wild type cell lines clearly demonstrated that unfortunately the 13-gene signature cannot predict response to TP53-MDM2 inhibitor in TP53 wild type tumors.

  10. Markov models of the apo-MDM2 lid region reveal diffuse yet two-state binding dynamics and receptor poses for computational docking

    Science.gov (United States)

    Mukherjee, Sudipto; Pantelopulos, George A.; Voelz, Vincent A.

    2016-08-01

    MDM2 is a negative regulator of p53 activity and an important target for cancer therapeutics. The N-terminal lid region of MDM2 modulates interactions with p53 via competition for its binding cleft, exchanging slowly between docked and undocked conformations in the absence of p53. To better understand these dynamics, we constructed Markov State Models (MSMs) from large collections of unbiased simulation trajectories of apo-MDM2, and find strong evidence for diffuse, yet two-state folding and binding of the N-terminal region to the p53 receptor site. The MSM also identifies holo-like receptor conformations highly suitable for computational docking, despite initiating trajectories from closed-cleft receptor structures unsuitable for docking. Fixed-anchor docking studies using a test set of high-affinity small molecules and peptides show simulated receptor ensembles achieve docking successes comparable to cross-docking studies using crystal structures of receptors bound by alternative ligands. For p53, the best-scoring receptor structures have the N-terminal region lid region bound in a helical conformation mimicking the bound structure of p53, suggesting lid region association induces receptor conformations suitable for binding. These results suggest that MD + MSM approaches can sample binding-competent receptor conformations suitable for computational peptidomimetic design, and that inclusion of disordered regions may be essential to capturing the correct receptor dynamics.

  11. Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

    Science.gov (United States)

    Chang, Yong S; Graves, Bradford; Guerlavais, Vincent; Tovar, Christian; Packman, Kathryn; To, Kwong-Him; Olson, Karen A; Kesavan, Kamala; Gangurde, Pranoti; Mukherjee, Aditi; Baker, Theresa; Darlak, Krzysztof; Elkin, Carl; Filipovic, Zoran; Qureshi, Farooq Z; Cai, Hongliang; Berry, Pamela; Feyfant, Eric; Shi, Xiangguo E; Horstick, James; Annis, D Allen; Manning, Anthony M; Fotouhi, Nader; Nash, Huw; Vassilev, Lyubomir T; Sawyer, Tomi K

    2013-09-03

    Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

  12. p21-Activated kinase 6 (PAK6) inhibits prostate cancer growth via phosphorylation of androgen receptor and tumorigenic E3 ligase murine double minute-2 (Mdm2).

    Science.gov (United States)

    Liu, Tong; Li, Yang; Gu, Hui; Zhu, Ge; Li, Jiabin; Cao, Liu; Li, Feng

    2013-02-01

    The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.

  13. Common polymorphisms in TP53 and MDM2 and the relationship to TP53 mutations and clinical outcomes in women with ovarian and peritoneal carcinomas.

    Science.gov (United States)

    Galic, Vijaya; Willner, Julia; Wollan, Melissa; Garg, Ruchi; Garcia, Rochelle; Goff, Barbara A; Gray, Heidi J; Swisher, Elizabeth M

    2007-03-01

    The importance of somatic TP53 mutations and germline TP53 codon 72 genotype in the survival of women with epithelial ovarian cancer is controversial. Recent data suggest that a promoter polymorphism in the MDM2 gene may influence age of cancer onset in a gender-specific fashion. We sought to determine the relationship between somatic TP53 mutations, germline genotypes at TP53 codon 72 and MDM2 SNP309, and overall survival and response to chemotherapy in a large series of patients with ovarian and peritoneal carcinomas. Of the 188 cancers, 103 (54.8%) had a TP53 mutation, of which 71% were missense mutations and 29% were null mutations. TP53 mutation status and mutation type (null vs. missense) did not influence response to therapy or overall survival. Women with the codon 72 Pro/Pro had a decreased overall survival (median, 29 months) compared with women with one or two arginine alleles (median, 49 months; P=0.04). Somatic mutation or deletion was equally common for either codon 72 allele. Age of diagnosis was not influenced by codon 72 but showed a trend for younger age in women with somatic TP53 mutations and the MDM2 G/G genotype. Copyright (c) 2006 Wiley-Liss, Inc.

  14. Expression of p53, MDM2, p21, heat shock protein 70, and HPV 16/18 E6 proteins in oral verrucous carcinoma and oral verrucous hyperplasia.

    Science.gov (United States)

    Lin, Hung-Pin; Wang, Yi-Ping; Chiang, Chun-Pin

    2011-03-01

    Oral verrucous hyperplasia is a precancerous lesion of oral verrucous carcinoma. This study used immunohistochemistry to examine the expression of p53, murine double minute 2 (MDM2), p21, heat shock protein 70 (HSP 70), and human papillomavirus (HPV) 16/18 E6 proteins in 48 oral verrucous carcinoma and 30 oral verrucous hyperplasia samples. The mean labeling indices of p53, MDM2, p21, HSP 70, and HPV 16/18 E6 proteins in oral verrucous carcinoma samples were 21%, 31%, 7%, 17%, and 0.5%, respectively, and those in oral verrucous hyperplasia samples were 19%, 35%, 11%, 14%, and 0.3%, respectively. Immunohistochemistry with the above-cited 5 biomarkers could not help differentiate oral verrucous hyperplasia from oral verrucous carcinoma. The low expression of p21 may partially explain abnormal epithelial overgrowth in both verrucous lesions. The pathogenesis of both verrucous lesions may be at least partially attributed to the overexpression of MDM2 protein and moderate expression of HSP 70 protein in both lesions. Copyright © 2010 Wiley Periodicals, Inc.

  15. Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells

    DEFF Research Database (Denmark)

    Hjerrild, M; Milne, D; Dumaz, N

    2001-01-01

    -site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser(267), which lies within...... the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser(267) protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5......,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser(267)-->Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser(267) is a cellular target for phosphorylation. Ser(267) mutants are still able to direct the degradation of p53, but in a slightly...

  16. Expression of Bcl- 2 and MDM- 2, and HPV- 16 Infection Rate in Esophageal Carcinoma and in Its Pre - cancerous Lesion%食管癌及其癌前病变中Bcl-2和MDM-2表达以及HPV-16感染率的研究

    Institute of Scientific and Technical Information of China (English)

    吕怀盛; 张建中; 景丽; 秦璟

    2004-01-01

    目的:对食管癌及其癌前病变中Bcl-2、MDM-2和HPV-16进行分析,以探讨食管癌发生原因及其机制.方法:采用免疫组化方法,对正常食管上皮、食管癌及其癌前病变中Bcl-2、MDM-2蛋白的表达和HPV-16进行对比观察.结果:9例正常食管鳞状上皮中有1例Bcl-2染色阳性(11.11%),MDM-2和HPV-16全部阴性;39例食管癌中Bcl-2、MDM-2、HPV-16染色分别有33例(84.61%)、25例(64.10%)和13例(33.33%)染色阳性;31例非典型增生中Bcl-2、MDM-2、HPV-16染色分别有7例(22.58%)、11例(35.48%)和3例(9.68%)染色阳性.三项指标在三组间比较均具有显著性差异(P<0.05);食管癌HPV-16阳性(33.33%)与非典型增生(9.68%)有显著性差异(χ2=4.14,P<0.05);食管癌MDM-2与Bcl-2染色阳性率间具有明显的相关关系(χ2=7.689,P<0.01).结论:食管癌常伴有HPV-16感染,食管癌的发生与Bcl-2和MDM-2基因异常表达有密切关系.

  17. Study on expression changes of MDM2 gene in SD rats' blood after chronic 137Cs γ ray irradiation%137 Cs γ射线慢性照射诱导大鼠血MDM2基因表达改变研究

    Institute of Scientific and Technical Information of China (English)

    王超; 刘建功; 党旭红; 刘红艳; 左雅慧; 段志凯; 刘占旗

    2013-01-01

    目的 探讨长期慢性137Cs γ射线照射对大鼠血中MDM2基因表达改变的影响.方法 无特定病原体级雄性SD大鼠120只,随机分成1个对照组,11个剂量组,每组均为10只,各剂量组137Cs γ射线慢性累积照射分别为0.05、0.10、0.20、0.40、0.60、0.80、1.00、2.00、3.00、4.00、5.00 Gy,对照组不予照射.照射完成后采腹主动脉血,利用实时荧光定量聚合酶链反应法检测各组血中MDM2基因表达的剂量-效应关系.结果 各剂量组和对照组比较,血中MDM2基因的表达量均发生变化,从整体水平看随受照剂量的增加,基因表达量先上升后下降.其中,0.05、0.10Gy剂量组中的基因表达量升高,其后0.20~1.00 Gy剂量范围MDM2基因的相对表达量出现一定程度回落,以0.20 Gy剂量组中的基因表达量降至最低点;0.20~2.00 Gy剂量范围随着照射剂量增大,MDM2基因的相对表达量呈线性关系升高,2.00 Gy剂量组的MDM2基因表达量最高;其后随着剂量的增加,MDM2基因的相对表达量再次出现下降趋势.拟合建立了0.20~2.00 Gy剂量范围内MDM2基因表达量与照射剂量之间的回归方程为多=1.918 9x+0.386 0,决定系数=0.99,P<0.01.结论 慢性累积照射可诱导大鼠血中MDM2基因表达发生改变,其总体表达趋势是先上升后下降,其中0.20~2.00 Gy剂量内MDM2基因表达量与照射剂量有较好的剂量-效应关系.

  18. Association of MDM-2 SNP309 polymorphism and age at onset and risk of hepatocellular carcinoma%鼠MDM-2 SNP309位点与肝细胞癌发病年龄与风险关系的分析

    Institute of Scientific and Technical Information of China (English)

    张艳艳; 毛良勤; 曾小云; 谢志春; 仇小强; 余红平

    2012-01-01

    目的:探讨广西地区人群鼠双微粒体-2基因(MDM-2)启动子区309位点单核苷酸多态性(SNP)与肝细胞癌(hepatocellular carcinoma,HCC)发病年龄和发病风险的关系.方法:运用聚合酶链反应-限制性片段长度多态性方法,对985例HCC病例和992例非肿瘤对照者的MDM-2 SNP309位点(T>G,rs2279744)基因型进行检测,并分析该SNP 与HCC发病年龄和发病风险的关系.结果:经年龄、性别、民族、吸烟、饮酒、HBV及HCV感染等因素校正后,MDM-2SNP309位点与HCC发病风险之间无统计学关联(TG vs TT∶ OR=1.19,95%CI∶ 0.86~1.65; GG vs TT∶ OR=1.28,95%CI∶0.89~1.85;TG+GG vs TT∶ OR=1.22,95%CI:0.90~1.66).在女性HCC患者中,与携带MDM-2SNP309位点TG+GG基因型的女性HCC患者相比(44.8岁),携带TT基因型的女性患者HCC发病年龄提前4.6岁(49.4岁),Log-rank检验:x2=7.372,P=0.007.在男性患者中未发现此类似结果.结论:MDM-2 SNP309位点多态性可能对HCC的发病风险无单独效应作用,但其TT基因型可能与女性HCC的发病年龄提前有关联.本研究结果需要大样本量的研究进一步验证.%OBJECTIVE: To explore associations between MDM-2 single nucleotide polymorphism (SNP) 309 (T> G, rs2279744) and the age at onset and risk of hepatocellular carcinoma (HCC) in Guangxi population. METHODS: A hospital-based case-control study was conducted in 985 cases with HCC and 992 cancer-free controls. SNP309 genotypes were detected using a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay, and associations of MDM-2 SNP309 with risk and age of onset of HCC were assessed. RESULTS: The variant genotypes of MDM-2 SNP309 were not significantly associated with risk of HCC (TG vs TT: OR=1. 19, 95%CI: 0. 86-1. 65; GG vs TT: OR=1. 28, 95%CI: 0.89-1.85; TG+GG vsTT: OR=1. 22, 95%CI: 0. 90-1. 66). It was found that female patients carrying the TT genotype (44. 8 year-old) showed a 4. 6-year earlier age at

  19. Correlation of biological behaviors with mdm-2 and p53 gene changes in primary gastric cancer and metastasis%mdm-2和p53基因变异与胃癌及淋巴转移生物学行为的关系

    Institute of Scientific and Technical Information of China (English)

    时军; 陈道达; 崔建涛; 吕有勇

    2000-01-01

    目的:分析癌基因mdm-2和抑癌基因p53在胃癌及其转移灶中的变异及相互关系,探讨胃癌及其转移的分子机制.方法:采用DC-PCR、PCR-SSCP及DNA测序技术检测32例胃癌及其转移灶中的mdm-2扩增和p53突变.结果:mdm-2在转移淋巴结中的扩增频率(12/21,57.1%)高于胃原发癌灶(12/32,37.5%),3例淋巴微转移灶中出现mdm 2的扩增.在7例肠型胃癌的原发癌或转移癌灶中同时检测出mdm-2扩增和p53突变.肝转移癌灶中p53突变位点和突变方式与胃原发癌之间有差异.结论:mdm-2扩增可能与胃癌细胞的淋巴高转移潜能有关.p53突变和mdm-2扩增多并存于肠型胃癌中.在胃原发癌和肝转移癌中癌细胞的基因改变存在着异质性.

  20. 急性淋巴细胞性白血病患儿细胞中MDM-2蛋白表达与化疗反应的关系

    Institute of Scientific and Technical Information of China (English)

    邵秀梅; 杨献华

    2001-01-01

    @@ 1998年1 2月至2000年3月,我们对25例住院的急性淋巴细胞白血病(ALL)患儿细胞中MDM-2蛋白的表达水平及其对化疗效果的影响进行了研究,以探讨MDM-2蛋白异常表达与化疗反应的关系.

  1. A functional polymorphism T309G in MDM2 gene promoter, intensified by Helicobacter pylori lipopolysaccharide, is associated with both an increased susceptibility and poor prognosis of gastric carcinoma in Chinese patients.

    Science.gov (United States)

    Pan, Xiaolin; Li, Yuqin; Feng, Jin; Wang, Xiaoyong; Hao, Bo; Shi, Ruihua; Zhang, Guoxin

    2013-03-18

    Studies on the association between MDM2 SNP309 (T > G) and gastric cancer have reported conflicting results. Thus, the aim of this study was to investigate whether MDM2 SNP309 is associated with susceptibility and prognosis of gastric carcinoma in Chinese patients. Total of 574 gastric carcinoma cases and 574 age- and sex-matched healthy controls were included. MDM2 polymorphism was detected by PCR- RFLP and infection of Helicobacter pylori (H. pylori) by a validated serology test. The functionality of MDM2 SNP309, with or without H. pylori lipopolysaccharide (LPS), was examined by dual-luciferase assay. Kaplan-Meier survival curves were used to evaluate survival. Additional, a meta-analysis was conducted to verity the findings. MDM2 SNP309G/G genotype was associated with an increased risk of gastric carcinoma when compared with T/T genotype or T carriers (both P gastric carcinoma risk. SNP309G/G was identified as an independent marker of poor overall survival of carcinoma. In vitro, the luciferase assay further showed an increased transcriptional activity of SNP309G allele compared with SNP309T allele, and the function of polymorphism T309G in MDM2 gene promoter was intensified by H. pylori LPS. Pooled results from the meta-analysis confirmed that SNP309G/G genotype had a significantly increased risk of gastric carcinoma compared with T/T genotype or T carriers, consistent with the case-control findings. MDM2 SNP309G allele is associated with an increased risk and poor prognosis of gastric carcinoma in Chinese patients. Additional, there is a joint effect of MDM2 SNP309G/G allele and H. pylori infection on gastric carcinoma development, which may attribute to H. pylori LPS.

  2. Targeting p53-MDM2 negative feedback loops for treatment of cancer%针对p53-鼠双微体基因负反馈环的肿瘤治疗

    Institute of Scientific and Technical Information of China (English)

    吴学元; 马巍

    2009-01-01

    MDM2 controls p53 function through inhibiting p53-mediated transcriptional activity and promoting p53 degradation.Over-expression of MDM2 inactivates p53 and induces wide-type p53 cells malignant transformation.So inhibition of the p53-MDM2 interaction with synthetic molecules should therefore lead to both the nuclear accumulation and the activation of p53 followed by the death of the tumor cells from apoptosis.Inhibitors of the p53-MDM2 interaction might be attractive new anticancer agents that could be used to activate wild-type p53 in tumors.%鼠双微体基因(MDM2)通过抑制p53介导的转录活性和促进p53降解来控制p53的功能.MDM2过度表达引起p53失活,导致p53野生细胞恶性转化.人工合成抑制p53-MDM2蛋白复合体相互作用的小分子可促使p53核内积聚、活化p53,从而诱导肿瘤细胞的死亡.针对p53-MDM2相互作用的抑制剂可成为治疗p53野生型肿瘤系的新型抗癌药物.

  3. EGCG经AKT1-Mdm-2-p53途径抑制卵巢癌HO-891O细胞的增殖%Epigallocatechin-3-gallate Inhibits Proliferation of Ovarian Cancer HO-8910 Cells by AKT 1-Mdm-2-p53 Pathway

    Institute of Scientific and Technical Information of China (English)

    李维; 罗瞳; 罗招阳

    2011-01-01

    Objective: To investigate mechanism of Epigallocatechin-3-gallate (EGCG) inhibited the proliferation of ovarian cancer HO-8910 cells. Methods: EGCG inhibited HO-8910 cellular proliferation was observed by the drawing cellular growth curve, plate colony formation and soft agar colony formation. The expressions of AKT1, Mdm-2 and p53 proteins were detected by Western-blotting. Results: 1. The results of cellular growth curve, plate colony formation and soft agar colony formation showed that EGCG active depressed the proliferation of HO-8910 cells with treatment time extended (n=3, P<0.05). 2. The western-blotting results exhibited that the expressions of AKT1 and Mdm-2 proteins in HO-8910 cells obviously decreased, but the expression of p53 protein increased after EGCG dealing with it (P<0.05). Conclusion: EGCG depressed the expressions of AKT1, Mdm-2 proteins in HO-8910 cells and increased the expression of p53 protein to inhibit the cellular proliferation.%初步探讨EGCG对卵巢癌HO-8910细胞增殖的抑制作用及其机制.方法:通过绘制细胞生长曲线、平皿克隆和软琼脂集落形成实验观察EGCG对HO-8910细胞增殖的抑制作用;Western-blotting检测AKT1、Mdm-2与p53蛋白的表达.结果:(1)细胞生长曲线、平皿克隆和软琼脂集落形成实验结果显示,EGCG可有效抑制HO-8910细胞的增殖(n=3,P<0.05).(2)Westemblotting检测结果显示,EGCG处理后AKT1与Mdm-2蛋白表达均降低,而p53蛋白表达升高(P<0.05).结论:EGCG通过抑制HO-8910细胞中AKT1与Mdm-2蛋白表达,促使p53蛋白表达而发挥其对细胞增殖的抑制作用.

  4. An MDM2 antagonist (MI-319 restores p53 functions and increases the life span of orally treated follicular lymphoma bearing animals

    Directory of Open Access Journals (Sweden)

    Yang Dajun

    2009-12-01

    Full Text Available Abstract Background MI-319 is a synthetic small molecule designed to target the MDM2-P53 interaction. It is closely related to MDM2 antagonists MI-219 and Nutlin-3 in terms of the expected working mechanisms. The purpose of this study was to evaluate anti-lymphoma activity of MI-319 in WSU-FSCCL, a B-cell follicular lymphoma line. For comparison purpose, MI-319, MI-219 and Nutlin-3 were assessed side by side against FSCCL and three other B-cell hematological tumor cell lines in growth inhibition and gene expression profiling experiments. Results MI-319 was shown to bind to MDM2 protein with an affinity slightly higher than that of MI-219 and Nutlin-3. Nevertheless, cell growth inhibition and gene expression profiling experiments revealed that the three compounds have quite similar potency against the tumor cell lines tested in this study. In vitro, MI-319 exhibited the strongest anti-proliferation activity against FSCCL and four patient cells, which all have wild-type p53. Data obtained from Western blotting, cell cycle and apoptosis analysis experiments indicated that FSCCL exhibited strong cell cycle arrest and significant apoptotic cell death; cells with mutant p53 did not show significant apoptotic cell death with drug concentrations up to 10 μM, but displayed weaker and differential cell cycle responses. In our systemic mouse model for FSCCL, MI-319 was tolerated well by the animals, displayed effectiveness against FSCCL-lymphoma cells in blood, brain and bone marrow, and achieved significant therapeutic impact (p 28% (%ILS, 14.4 days increase in median survival days. Conclusion Overall, MI-319 probably has an anti-lymphoma potency equal to that of MI-219 and Nutlin-3. It is a potent agent against FSCCL in vitro and in vivo and holds the promises to be developed further for the treatment of follicular lymphoma that retains wild-type p53.

  5. Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway.

    Science.gov (United States)

    Wu, Chien-Ting; Lin, Tung-Yi; Hsu, Hsien-Yeh; Sheu, Fuu; Ho, Chau-Mei; Chen, Edmund I-T

    2011-12-01

    Ling Zhi-8 (LZ-8), an immunomodulatory protein, is derived from and has been cloned from the medicinal mushroom Ganoderma lucidum (Reishi or Ling Zhi); this protein exhibits immunomodulating and antitumor properties. We investigated the effects of recombinant LZ-8 protein (rLZ-8) on the proliferation of A549 human lung cancer cells. Here, we showed that rLZ-8 inhibits cell growth and that this is correlated with increased G(1) arrest. The treatment of A549 cells with rLZ-8 activated p53 and p21 expression, and both the G(1) arrest and the antigrowth effect were found to be p53 dependent. It was further demonstrated that rLZ-8 inhibited tumor growth in mice transplanted with Lewis lung carcinoma cells. Interestingly, rLZ-8 treatment was found to lead to nucleolar stress (or ribosomal stress) as evidenced by inhibition of precursor ribosomal RNA synthesis and reduced polysome formation in A549 cells. These changes resulted in an increasing binding of ribosomal protein S7 to MDM2 and a decreased interaction between MDM2 and p53. Taking these results together, we have identified a novel rLZ-8 antitumor function that positively modulates p53 via ribosomal stress and inhibits lung cancer cell growth in vitro and in vivo. Our current results suggest that rLZ-8 may have potential as a therapeutic intervention for the treatment of cancers that contain wild-type p53 and high expression of MDM2.

  6. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-09-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  7. P53、MDM2在鼻咽癌组织中的表达及其与鼻咽癌生物学行为、预后关系的分析研究%Expression of p53 and MDM2 in Nasopharyngeal Carcinoma and their Correlation with the Biological Behavior and Prognosis of Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    张鹏; 吴松珂; 徐鹏; 冯梅; 范子煊; 李厨荣; 付彬玉; 王卫东; 郎锦义

    2012-01-01

    目的:探讨P53、MDM2在鼻咽癌组织中的表达及与鼻咽癌生物学行为和生存率之间的关系.方法:回顾分析我院2005年~2009年行根治性放化疗治疗的96例鼻咽癌患者,所有患者治疗前行鼻咽部活检病理证实,均采用6MV-X线调强放疗联合以顺铂为主的方案同步化疗,随访3年以上.通过免疫组化SP法检测96例鼻咽癌标本中P53、MDM2蛋白的表达水平,并分析其与临床参数、预后之间关系.结果:P53、MDM2在96例鼻咽癌中阳性表达率分别为65.6%(63/96)、79.17%(76/96).P53的阳性表达率与不同T分期有关(P=0.000);MDM2在不同N分期的鼻咽癌组织中的表达的差异性具有统计学意义(P=0.001);P53的阳性表达在不同临床分期中的差异性具有统计学意义(P=0.037).P53与MDM2的表达呈负相关(r=-3.24,P<0.05).3年无瘤生存率(DFS)及总生存率(OS)分别为73.9%、84.4%.结论:P53、MDMZ的表达与鼻咽癌的生物学行为有关;P53与MDM2的表达呈负相关;P53表达是影响总生存率的预后因素,可以作为评价预后的指标.%Objective; To determine the expression of p53 and MDM2 in nasopharyngeal carcinoma and to investigate the relationship between them and biological behavior , survival rate of nasopharyngeal carcinoma. Methods: Clinical data of 96 nasopharyngeal carcinoma patients accepted radical radiotherapy and chemotherapy in our hospital from 2005 - 2009 were retrospective analyzed. All patients were diagnosed by biopsy of the nasopharynx before treatment. Patients accepted the 6-MV X-ray intensity-modulated radiotherapy combined with cisplatin-based chemotherapy regimen and were followed up for more than 3 years. The expression levels of P53, MDM2 protein in 96 cases of nasopharyngeal specimens were detected by immunohistochemical SP method and their relationship with the clinical parameters, prognosis were analyzed. Results; The positive expression rate of P53 and MDM2 was 65. 6% (63/96) , 79. 17% (76

  8. Synthesis of spiro[isoindole-1,5’-isoxazolidin]-3(2H)-ones as potential inhibitors of the MDM2-p53 interaction

    Science.gov (United States)

    Cirmi, Santa; Mancuso, Raffaella; Nicolò, Francesco; Lanza, Giuseppe; Legnani, Laura; Campisi, Agata; Chiacchio, Maria A; Navarra, Michele; Gabriele, Bartolo

    2016-01-01

    A series of spiro[isoindole-1,5-isoxazolidin]-3(2H)-ones has been synthesized by 1,3-dipolar cycloaddition of N-benzylnitrone with isoindolin-3-methylene-1-ones. The regio- and stereoselectivity of the process have been rationalized by computational methods. The obtained compounds show cytotoxic properties and antiproliferative activity in the range of 9–22 μM. Biological tests suggest that the antitumor activity could be linked to the inhibition of the protein–protein p53-MDM2 interaction. Docking measurements support the biological data. PMID:28144352

  9. Synthesis of spiro[isoindole-1,5’-isoxazolidin]-3(2H-ones as potential inhibitors of the MDM2-p53 interaction

    Directory of Open Access Journals (Sweden)

    Salvatore V. Giofrè

    2016-12-01

    Full Text Available A series of spiro[isoindole-1,5-isoxazolidin]-3(2H-ones has been synthesized by 1,3-dipolar cycloaddition of N-benzylnitrone with isoindolin-3-methylene-1-ones. The regio- and stereoselectivity of the process have been rationalized by computational methods. The obtained compounds show cytotoxic properties and antiproliferative activity in the range of 9–22 μM. Biological tests suggest that the antitumor activity could be linked to the inhibition of the protein–protein p53-MDM2 interaction. Docking measurements support the biological data.

  10. The isolation, total synthesis and structure elucidation of chlorofusin, a natural product inhibitor of the p53-MDM2 protein-protein interaction

    Science.gov (United States)

    Clark, Ryan C.; Lee, Sang Yeul; Searcey, Mark; Boger, Dale L.

    2009-01-01

    Inhibitors of key protein-protein interactions are emerging as exciting therapeutic targets for the treatment of cancer. One such interaction between MDM2 (HDM2) and p53, that silences the tumour suppression activities of p53, was found to be inhibited by the recently isolated natural product chlorofusin. Synthetic studies on this complex natural product summarized herein have served to reassign its chromophore relative stereochemistry, assign its absolute stereochemistry, and provided access to a series of key analogues and partial structures for biological evaluation. PMID:19642417

  11. 肺小细胞癌p53基因分析与mdm-2基因蛋白表达的关系%Relationship of the analyse of p53 gene and the expression of mdm-2 gene protein in small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    杨廷桐; 张俊; 郑杰; 吴秉铨

    1999-01-01

    目的:探讨肺小细胞癌(SCLC)的p53基因改变与mdm-2基因蛋白表达的关系.方法:应用免疫组化LSAB法和聚合酶链反应-单链构象多态性(PCR-SSCP)分析的方法,对14例SCLC的石蜡切片组织进行研究.结果:免疫组化染色p53蛋白阳性9例,阳性率为64.3%(9/14);mdm-2蛋白阳性4例,阳性率为28.6%(4/14).p53基因第5、6、7、8外显子,突变率分别为21.4%(3/14);14.3(2/14);14.3%(2/14);7.1%(1/14),总突变率为57.1%(8/14).结论:SCLC中存在较高的p53基因突变率和mdm-2基因蛋白的明显表达.

  12. Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma.

    Science.gov (United States)

    Chen, Lindi; Rousseau, Raphaël F; Middleton, Steven A; Nichols, Gwen L; Newell, David R; Lunec, John; Tweddle, Deborah A

    2015-04-30

    Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.

  13. Elucidating the digital control mechanism for DNA damage repair with the p53-Mdm2 system: single cell data analysis and ensemble modelling.

    Science.gov (United States)

    Ogunnaike, Babatunde A

    2006-02-22

    Recent experimental evidence about DNA damage response using the p53-Mdm2 system has raised some fundamental questions about the control mechanism employed. In response to DNA damage, an ensemble of cells shows a damped oscillation in p53 expression whose amplitude increases with increased DNA damage--consistent with 'analogue' control. Recent experimental results, however, show that the single cell response is a series of discrete pulses in p53; and with increase in DNA damage, neither the height nor the duration of the pulses change, but the mean number of pulses increase--consistent with 'digital' control. Here we present a system engineering model that uses published data to elucidate this mechanism and resolve the dilemma of how digital behaviour at the single cell level can manifest as analogue ensemble behaviour. First, we develop a dynamic model of the p53-Mdm2 system that produces non-oscillatory responses to a stress signal. Second, we develop a probability model of the distribution of pulses in a cell population, and combine the two with the simplest digital control algorithm to show how oscillatory responses whose amplitudes grow with DNA damage can arise from single cell behaviour in which each single pulse response is independent of the extent of DNA damage. A stochastic simulation of the hypothesized control mechanism reproduces experimental observations remarkably well.

  14. Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Ino, Y; Gerdes, A M

    1999-01-01

    The two gene products of the CDKN2A gene, p16 and p19ARF, have recently been linked to each of two major tumour suppressor pathways in human carcinogenesis, the RB1 pathway and the p53 pathway. p16 inhibits the phosphorylation of the retinoblastoma gene product by cyclin D-dependent kinases...... of aberrations of CDKN2A, MDM2 and p53, since this has not been analysed previously. We investigated 37 DLCL for aberrations of p15, p16, p19ARF, MDM2, and p53 at the epigenetic, genetic and/or protein levels. Homozygous deletion of CDKN2A was detected in seven (19%) of 37 tumours, and another three cases were...... hypermethylated at the 5' CpG island of p16. No point mutations were found in CDKN2B or CDKN2A. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue for p16 confirmed these results, as all tumours with alterations of CDKN2A were p16 immunonegative. We found p53 mutations in eight (22%) cases...

  15. Long non-coding RNA ENST00462717 suppresses the proliferation, survival, and migration by inhibiting MDM2/MAPK pathway in glioma.

    Science.gov (United States)

    Wang, Aiqin; Meng, Mingzhu; Zhao, Xiuhe; Kong, Lina

    2017-04-01

    Gliomas are the most common and aggressive primary malignant tumor in the central nervous system, and requires new biomarkers and therapeutic methods. Long noncoding RNAs (lncRNAs) are important factors in numerous human diseases, including cancer. But studies on lncRNAs and gliomas are limited. In this study, we investigated the expression patterns of lncRNAs in 3 pairs of glioma samples and adjacent non-tumor tissues via microarray and selected the most down-regulated lnc00462717 to further verify its roles in glioma. We observed that decreased lnc00462717 expression was associated with the malignant status in glioma. In vitro experiment demonstrated that lnc00462717 overexpression suppressed glioma cell proliferation, survival and migration while knockdown of lnc00462717 had an opposite result. Moreover, we identified MDM2 as a direct target of lnc00462717 and lnc00462717 played a role by partially regulating the MDM2/MAPK pathway. In conclusion, lnc00462717 may function in suppressing glioma cell proliferation, survival, migration and may potentially serve as a novel biomarker and therapeutic target for glioma.

  16. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

  17. P21WAF1、MDM-2、Rb在胰腺癌中的表达及临床意义%Study on expression of P21WAF1, MDM-2 and Rb and its clinical significance in pancreatic carcinoma

    Institute of Scientific and Technical Information of China (English)

    刘振华; 冯一中; 李峰; 柴玉海; 任苏勤

    2007-01-01

    目的:探讨P21WAF1、MDM-2、Rb在胰腺癌发生过程中的作用,为预后判断提供理论依据.方法:应用免疫组织化学技术SP法检测53例胰腺癌手术患者的组织标本.结果:(1)P21WAF1在胰腺癌中的阳性表达率显著低于非肿瘤性胰腺组织(P<0.05),P21WAF1的阳性表达率与胰腺癌的病理分级、淋巴结转移呈负相关(P<0.05).(2)MDM-2在胰腺癌中的阳性表达率明显高于非肿瘤性胰腺组织(P<0.05),与胰腺癌的病理分级呈正相关(P<0.05).(3)Rb的阳性表达率在胰腺癌中明显低于非肿瘤性胰腺组织(P<0.01),与胰腺癌的病理分级呈负相关(P<0.05).(4)P21WAF1与MDM-2、P21WAF1与Rb在胰腺癌中的阳性表达呈显著正相关(P<0.01).(5)生存单因素分析MDM-2及Rb均与胰腺癌的预后显著相关(P<0.01,P<0.05).结论:P21WAF1、MDM-2、Rb在胰腺癌的发生发展中均发挥一定的作用.

  18. Expression of P14ARF, MDM2 and mutant type P53 in skin tissue of coal-burning-type of endemic arseniasis patients%燃煤污染型地方性砷中毒患者皮肤组织P14ARF和MDM2及突变型P53蛋白表达

    Institute of Scientific and Technical Information of China (English)

    夏玉洁; 张爱华; 韩雪; 黄晓欣

    2012-01-01

    目的 观察燃煤污染型地方性砷中毒患者皮肤组织突变型P53( P53mt)蛋白及其下游基因P14ARG、MDM2蛋白的表达水平,为揭示砷致皮肤损害的分子机制提供依据.方法 以自愿手术治疗的60例燃煤污染型地方性砷中毒患者作为观察对象,依据皮肤病理学诊断分为一般皮肤病变组(35例)、癌前病变组(19例)、癌变组(6例);以某医院经病理学诊断无异常的非肿瘤手术患者的9例皮肤组织为对照,采用免疫组织化学EnVision二步法检测皮肤组织中P14ARF、MDM2和P53mt蛋白的表达情况.结果 4组皮肤组织P14ARF蛋白表达阳性率比较,差异有统计学意义(x2=9.39,P<0.05);其中癌前病变组、癌变组[46.1%(6/19)、33.3%(2/6)]明显低于对照组[88.9%(8/9),P均<0.05];且随着皮肤病变程度的加重,P14ARF蛋白表达呈降低趋势(P<0.05).4组皮肤组织MDM2、P53mt蛋白表达阳性率比较,差异有统计学意义(x2值分别为6.21、20.64,P均< 0.05);其中一般病变组、癌前病变组和癌变组[54.2%( 19/35)、63.2%(10/19)、66.7%(4/6)和25.7%(9/35)、73.7%(14/19)、83.3%(5/6)]明显高于对照组(0、0,P均<0.05);随着皮肤病变程度的加重,MDM2和P53mt蛋白表达呈增强趋势(P均<0.05).结论 燃煤污染型地方性砷中毒患者皮肤组织P53mt蛋白表达阳性率升高,且P14ARF和MDM2蛋白表达异常,后者所致的细胞周期和凋亡调控失常可能是砷致皮肤病损发生发展的原因之一.%Objective To determine the protein expression of P14ARF,MDM2 and mutant type P53 (P53mt) in skin specimens of coal-burning-type of endemic arseniasis patients and to reveal the molecular mechanism of the disease.Methods Sixty skin specimens from 60 endemic arseniasis patients including 35 of skin lesions patients,19 of precancerous lesion and 6 of skin cancer and 9 normal skin specimens from non-cancer patients were studied.Expression of P14~,MDM2 and P53mt was evaluated

  19. EXPRESSION OF △Np63 AND MDM2 PROTEINS IN TRANSITIONAL CELL CARCINOMA OF URINARY BLADDER%膀胱移行细胞癌组织△Np63和MDM2蛋白表达及意义

    Institute of Scientific and Technical Information of China (English)

    张鲁伟; 周荣祥

    2007-01-01

    目的 探讨缺乏酸性N端反式激活区的截短型p63(△Np63)和MDM2蛋白在膀胱移行细胞癌(TCC)组织中的表达及其临床意义.方法 用免疫组织化学法,检测35例石蜡包埋TCC组织标本中△Np63和MDM2的表达,以38例正常膀胱黏膜作为对照.结果 膀胱癌组30例(83.3%)△Np63蛋白呈阳性表达,膀胱黏膜组7例(18.4%)呈阳性表达,两组比较差异有显著性(uc=5.75,P<0.01).膀胱癌组15例(42.9%)MDM2蛋白呈阳性表达,膀胱黏膜组3例(7.9%)呈阳性表达,两组差异有显著性(x2=11.99,P<0.01).在不同病理分级、临床分期TCC中,△Np63表达差异有显著性(H=21.09、14.99,P<0.01).随着TCC病理分级、临床分期升高,△Np63表达明显增强,呈显著正相关(r=0.64、0.51,P<0.01).在不同病理分级、临床分期TCC中,MDM2表达差异有显著性(x2=16.63,P<0.01;x2=6.72,P<0.05).30例△Np63蛋白阳性表达TCC组织中MDM2蛋白阳性表达10例(33.3%),两者呈显著相关性(x2=7.78,P<0.05).△Np63阳性表达主要集中在低分化、浸润性TCC中,而MDM2则主要表达在高分化、浅表TCC中.结论 △Np63和MDM2与膀胱肿瘤的发生发展密切相关,可作为膀胱肿瘤早期诊断和判断恶性程度的参考指标.

  20. DDX3 loss by p53 inactivation promotes tumor malignancy via the MDM2/Slug/E-cadherin pathway and poor patient outcome in non-small-cell lung cancer.

    Science.gov (United States)

    Wu, D-W; Lee, M-C; Wang, J; Chen, C-Y; Cheng, Y-W; Lee, H

    2014-03-20

    P53 inactivation by p53 mutation and E6 oncoprotein has a crucial role in human carcinogenesis. DDX3 has been shown to be a target of p53. In this study, we hypothesized that DDX3 loss by p53 inactivation may promote tumor malignancy and poor patients' outcome. Mechanically, DDX3 loss by p53 knockdown and E6 overexpression was observed in A549 lung cancer cells. Conversely, DDX3 expression was markedly elevated by wild-type (WT) p53 ectopic expression in p53-null H1299 cells, E6-knockdown TL-1 lung cancer and SiHa cervical cancer cells. Interestingly, DDX3 loss promotes soft-agar growth and invasive capability; however, both capabilities were suppressed by DDX3 overexpression. We next expected that DDX3 loss might result in Slug-suppressed E-cadherin expression via decreased MDM2-mediated Slug degradation. As expected, MDM2 transcription is suppressed by DDX3 loss via decreased SP1 binding activity to the MDM2 promoter. Consequently, Slug expression was elevated by the reduction of MDM2 because of DDX3 loss, and E-cadherin expression was suppressed by Slug. Consistent observations in the correlation of DDX3 loss with MDM2, Slug and E-cadherin were seen in lung tumors from lung cancer patients. In addition, patients with low-DDX3 tumors had poorer survival and relapse than patients with high-DDX3 tumors. In conclusion, we suggest that DDX3 loss by p53 inactivation via MDM2/Slug/E-cadherin pathway promotes tumor malignancy and poor patient outcome.

  1. Evaluation of TP53 Pro72Arg and MDM2 SNP285-SNP309 polymorphisms in an Italian cohort of LFS suggestive patients lacking identifiable TP53 germline mutations.

    Science.gov (United States)

    Ponti, Francesca; Corsini, Serena; Gnoli, Maria; Pedrini, Elena; Mordenti, Marina; Sangiorgi, Luca

    2016-10-01

    Li-Fraumeni syndrome (LFS) is a rare genetic cancer predisposition disease, partly determined by the presence of a TP53 germline mutation; lacking thereof, in presence of a typical LFS phenotype, defines a wide group of 'LFS Suggestive' patients. Alternative LFS susceptibility genes have been investigated without promising results, thus suggesting other genetic determinants involvement in cancer predisposition. Hence, this study explores the single and combined effects of cancer risk, age of onset and cancer type of three single nucleotide polymorphisms (SNPs)-TP53 Pro72Arg, MDM2 SNP285 and SNP309-already described as modifiers on TP53 mutation carriers but not properly investigated in LFS Suggestive patients. This case-control study examines 34 Italian LFS Suggestive lacking of germline TP53 mutations and 95 tumour-free subjects. A significant prevalence of homozygous MDM2 SNP309 G in the LFS Suggestive group (p TP53 positive families. Conversely its anticipating role on tumour onset has not been confirmed, as in our results it was associated with the SNP309 T allele. A strong combined outcome with a 'dosage' effect has also been reported for TP53 P72 and MDM2 SNP309 G allele on cancer susceptibility (p < 0.0005). Whereas the MDM2 SNP285 C allele neutralizing effect on MDM2 SNP309 G variant is not evident in our population. Although it needs further evaluations, obtained results strengthen the role of MDM2 SNP309 as a genetic factor in hereditary predisposition to cancer, so improving LFS Suggestive patients management.

  2. Leptin's effect on hyperactivity : potential downstream effector mechanisms

    NARCIS (Netherlands)

    Hillebrand, J J G; Kas, M J H; van Elburg, A A; Hoek, H W; Adan, R A H

    2008-01-01

    Up to 80% of patients with Anorexia Nervosa (AN) demonstrate hyperactivity. Hyperactivity counteracts weight gain during treatment and is associated with poor outcome of the disease. We hypothesized that hyperactivity in AN patients has a neurobiological basis and used an animal model-based translat

  3. Leptin's effect on hyperactivity : Potential downstream effector mechanisms

    NARCIS (Netherlands)

    Hillebrand, J. J. G.; Kas, M.J.H.; van Elburg, A. A.; Hoek, H. W.; Adan, R. A. H.

    2008-01-01

    Up to 80% of patients with Anorexia Nervosa (AN) demonstrate hyperactivity. Hyperactivity counteracts weight gain during treatment and is associated with poor outcome of the disease. We hypothesized that hyperactivity in AN patients has a neurobiological basis and used an animal model-based translat

  4. Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin

    DEFF Research Database (Denmark)

    Siemer, S; Ornskov, D; Guerra, B

    1999-01-01

    Protein kinase CK2 is a pleiotropic serine/threonine kinase which has been shown to phosphorylate numerous substrates. Evidence is accumulating that CK2 may exist complexed to a variety of cellular proteins, e.g. p53, MDM2, and A-Raf. Here, we explored the effects of the chemotherapeutic drugs...... cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after...

  5. Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene.

    Science.gov (United States)

    Kong, Ya; Lu, Zong-Liang; Wang, Jia-Jia; Zhou, Rui; Guo, Jing; Liu, Jie; Sun, Hai-Lan; Wang, He; Song, Wei; Yang, Jian; Xu, Hong-Xia

    2016-09-01

    The objective of the present study was to explore the in vitro and in vivo anticancer effects of Platycodin D (PD), derived from Platycodin grandiflorum, on highly metastatic MDA-MB-231 breast cancer cells. Using the MTT assay, we found that PD inhibited MDA-MB-231 cell growth in a concentration-dependent manner, with an IC50 value of 7.77±1.86 µM. Further studies showed that PD had anti-proliferative effects and induced cell cycle arrest in the G0/G1 phase. To explore the detailed mechanism(s) by which PD suppressed MDA-MB-231 cell growth, western blot analyses were used to detect the expression levels of proteins related to cell proliferation and survival. The data showed that PD decreased the expression of proteins related to the G0/G1 phases, downregulated the protein expression of MDM2, MDMX, and mutant p53, and increased the expression levels of p21 and p27 in vitro. We verified the effects of PD on the expression of MDM2, MDMX, mutant p53, p21 and p27 using a pcDNA3-Flag-MDM2 plasmid and MDM2 siRNA transfection, and found that PD inhibited MDA-MB-231 cell viability by targeting MDM2 and mutant p53. Compared with the corresponding parental cells, the cells with siRNA-MDM2 transfection had a greater decrease in cell viability and proliferation, while those with pcDNA3-MDM2 plasmid transfection did not show any increase in the effects of PD. We also established a MDA-MB-231 xenograft model in BALB/c nude mice, and found that PD significantly inhibited the growth of MDA-MB-231 xenograft tumors in these mice. The expression levels of various proteins in the tumor tissue exhibited changes similar to those observed in vitro. These findings indicate that PD exerted in vitro and in vivo anticancer effects against MDA-MB-231 breast cancer cells, that PD is a potential MDM2/MDMX inhibitor, and that the anticancer effects of PD were likely associated with its inhibition of these proteins. Our observations help to identify a mechanism by which PD functions as

  6. An Integrative Analysis Reveals a Central Role of P53 Activation via MDM2 in Zika Virus Infection Induced Cell Death

    Directory of Open Access Journals (Sweden)

    Yue Teng

    2017-07-01

    Full Text Available Zika virus (ZIKV infection is an emerging global threat that is suspected to be associated with fetal microcephaly. However, the molecular mechanisms underlying ZIKV disease pathogenesis in humans remain elusive. Here, we investigated the human protein interaction network associated with ZIKV infection using a systemic virology approach, and reconstructed the transcriptional regulatory network to analyze the mechanisms underlying ZIKV-elicited microcephaly pathogenesis. The bioinformatics findings in this study show that P53 is the hub of the genetic regulatory network for ZIKV-related and microcephaly-associated proteins. Importantly, these results imply that the ZIKV capsid protein interacts with mouse double-minute-2 homolog (MDM2, which is involved in the P53-mediated apoptosis pathway, activating the death of infected neural cells. We also found that synthetic mimics of the ZIKV capsid protein induced cell death in vitro and in vivo. This study provides important insight into the relationship between ZIKV infection and brain diseases.

  7. The suppression of prostate LNCaP cancer cells growth by Selenium nanoparticles through Akt/Mdm2/AR controlled apoptosis.

    Science.gov (United States)

    Kong, Ling; Yuan, Qing; Zhu, Huarui; Li, Ying; Guo, Quanyi; Wang, Qin; Bi, Xiaolin; Gao, Xueyun

    2011-09-01

    The trace element Selenium is suggested having cancer prevention activity and used as food supplement. Previous results had shown Selenium nanoparticles are safer compared with other Selenium compounds like selenomethionine, sodium selenite and monomethylated Selenium, however, its anticancer activity and intrinsic mechanisms are still elusive. Here, we prepared Selenium nanoparticles and investigated its inherent anticancer mechanisms. We found Selenium nanoparticles inhibit growth of prostate LNCaP cancer cells partially through caspases mediated apoptosis. Selenium nanoparticles suppress transcriptional activity of androgen receptor via down-regulating its mRNA and protein expression. Moreover, Selenium nanoparticles activate Akt kinase by increasing its phosphorylation, promote Akt-dependent androgen receptor phosphorylation and Mdm2 regulated degradation through proteasome pathway. We suggest Selenium nanoparticles suppress prostate cancer cells growth by disrupting androgen receptor, implicating a potential application in cancer treatment.

  8. Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction.

    Science.gov (United States)

    Fortunato, O; Boeri, M; Moro, M; Verri, C; Mensah, M; Conte, D; Caleca, L; Roz, L; Pastorino, U; Sozzi, G

    2014-12-11

    Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21(WAF1/CIP1) in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.

  9. Giant cell rich osteosarcoma revisited-diagnostic criteria and histopathologic patterns, Ki67, CDK4, and MDM2 expression, changes in response to bisphosphonate and denosumab treatment.

    Science.gov (United States)

    Chow, Louis Tsun Cheung

    2016-06-01

    Defining giant cell-rich osteosarcoma (GCRO) as "an osteosarcoma in which more than 50% of the tumor consists of numerous uniformly distributed osteoclastic giant cells amidst oval or spindle mononuclear cells embedded in a fibrovascular stroma," eight such cases identified among 265 cases of osteosarcoma were analysed. Their age ranges from 11 to 33 years, with peak incidence in the second decade and equal sex distribution. Seventy-five percent presented with pain, commonest in the knee, affecting the metaphysis. Most appeared radiologically as well-circumscribed expansile multiloculated osteolytic lesions, and many are displayed periosteal reaction. They showed several distinct histologic patterns: the stromal and giant cell, fibrohistiocytic, aneurysmal-cystic, osteoblastoma-like, and parosteal and fibrous dysplasia-like patterns. Focal subtle lacelike osteoid deposition, permeative infiltration into adjacent native bony trabeculae and over 30 % Ki67 proliferative index were characteristic. There was no CDK4 and MDM2 amplification. In those having bisphosphonate and denosumab treatment, there was limited focal necrosis with reduction in the number of giant cells and broad trabecular woven bone formation but no giant osteoclast was seen. Two patients with initial diagnosis of giant cell tumor treated by curettage and local resection pursued aggressive clinical courses, died after 14 and 21 months. The others survived 12 to 110 months. GCRO accounts for about 3 % of all osteosarcomas and apart from its more frequent diaphyseal location and associated normal bone-specific alkaline phosphate levels; it shares with conventional high-grade osteosarcoma the same patient demographics, sites of occurrence, absence of CDK4 and MDM2 amplification, and probably clinical course.

  10. NOXA、PUMA等基因在乳腺癌中的表达及其意义%The Expression Characteristics of MDM2, PCNA, NOXA and PUMA in Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    王尚文; 佟钧; 诸晨; 平妮娜; 喻箴; 崔明; 何越峰

    2016-01-01

    目的 比较癌组织与正常组织中MDM2、PCNA、NOXA、PUMA表达的差异,了解乳腺癌与表达的关系.方法 通过SYBR Green实时荧光定量PCR方法检测乳腺癌组织和正常乳腺组织中MDM2、PCNA、NOXA、PUMA 4个基因的含量.结果 (1) NOXA、和PUMA在乳腺癌组织中表达升高(P<0.05);(2) PCNA和NOXA的表达存在正相关关系(r=0.82,P<0.05));PCNA和MDM2的表达存在正相关关系(r=0.80,P<0.05);PUMA和MDM2的表达存在正相关关系(r=0.82,P<0.05).结论 NOXA、PUMA在乳腺癌组织中处于高表达状态,4个基因表达与乳腺癌有关.

  11. Markers aiding the diagnosis of chondroid tumors: an immunohistochemical study including osteonectin, bcl-2, cox-2, actin, calponin, D2-40 (podoplanin), mdm-2, CD117 (c-kit), and YKL-40

    DEFF Research Database (Denmark)

    Daugaard, Søren; Christensen, Lise H; Høgdall, Estrid

    2009-01-01

    (s) for the different subgroups. Archival material from three extraskeletal myxoid chondrosarcomas, five chordomas, five chondromyxoid fibromas, five chondroblastomas and 25 chondrosarcomas was stained with antibodies against osteonectin, bcl-2, cox-2, actin, calponin, D2-40 (podoplanin), mdm-2, CD117 (c-kit) and YKL......) and extraskeletal myxoid chondrosarcomas (n=3) were positive for bcl-2. In contrast to all other tumors, two of three extraskeletal myxoid chondrosarcomas were also positive for CD17 and negative for osteonectin, cox-2, mdm-2 and actin. All five chordomas were negative for D2-40 and positive for mdm-2 and YKL-40....... The diagnosis of chondrosarcoma may be aided by its positivity for D2-40 and YKL-40 and its lack of reactivity for actin and CD117. This should be seen in the light of no reaction for D2-40 in chordomas and a corresponding lack of reaction for osteonectin, cox-2, mdm-2 and actin in extraskeletal myxoid...

  12. Evaluation of MDM2 and CDK4 amplification by real-time PCR on paraffin wax-embedded material: a potential tool for the diagnosis of atypical lipomatous tumours/well-differentiated liposarcomas.

    Science.gov (United States)

    Hostein, I; Pelmus, M; Aurias, A; Pedeutour, F; Mathoulin-Pélissier, S; Coindre, J M

    2004-01-01

    Atypical lipomatous tumours/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by 12q13-15 region amplification. In contrast, this molecular event has not been reported in benign lipomas. Within the 12q13-15 chromosomal region, the MDM2, SAS, HMGA2, and CDK4 genes are the most frequent targets of amplification. A series of lipomas (36 cases) and liposarcomas (48 cases) was analysed for MDM2 and CDK4 gene amplification by real-time PCR. MDM2 and CDK4 gene amplification was detected in 2.8% and 5.6% of lipomas and 98.2% and 82.4% of liposarcomas, respectively. Moreover, co-amplification of the two genes as well as a higher-level amplification was observed more frequently in dedifferentiated liposarcomas than in atypical lipomatous tumours/well-differentiated liposarcomas. Real-time PCR proved to be a fast and reliable method to characterize lipomas and liposarcomas by quantification of MDM2 and CDK4 gene amplification. It is applicable to paraffin wax-embedded tissues and could be useful when histological diagnosis is difficult.

  13. Molecular mechanisms of MYCN-dependent apoptosis and the MDM2-p53 pathway: an Achille’s heel to be exploited for the therapy of MYCN amplified neuroblastoma

    Directory of Open Access Journals (Sweden)

    Marialaura ePetroni

    2012-10-01

    Full Text Available The p53 oncosuppressor is very seldom mutated in neuroblastoma, but several mechanisms cooperate to its functional inactivation in this tumor. Increased MDM2 levels, due to genetic amplification or constitutive inhibition of p14ARF, significantly contribute to this event highlighting p53 reactivation as an attractive perspective for neuroblastoma treatment.In addition to its role in tumorigenesis, MYCN sensitizes untransformed and cancer cells to apoptosis. This is associated to a fine modulation of the MDM2-p53 pathway. Indeed MYCN induces p53 and MDM2 transcription, and, by evoking a DNA damage response (DDR, it stabilizes p53 and its proapoptotic kinase HIPK2. Through the regulation of the HIPK2-p53 inhibitor HMGA1 and the homeobox proteins BMI-1 and TWIST-1, MYCN establishes a delicate balance between pro- and anti-apoptotic molecules that might be easily perturbed by a variety of insults, leading to cell death. MDM2-p53 antagonists, such as Nutlin-3, are strikingly prone to inducing death in MYCN-amplified neuroblastoma, by further pushing on HIPK2 accumulation. Here we discuss implications and caveats of exploiting this pathway and its connections to MYCN-induced DDR for a tailored therapy of MYCN-amplified neuroblastoma.

  14. The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway.

    Science.gov (United States)

    Bailly, A; Perrin, A; Bou Malhab, L J; Pion, E; Larance, M; Nagala, M; Smith, P; O'Donohue, M-F; Gleizes, P-E; Zomerdijk, J; Lamond, A I; Xirodimas, D P

    2016-01-28

    The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

  15. 鼠双微粒体-2基因沉默对人肝癌HepG2细胞移植瘤生长的抑制作用%shRNA-mediated silencing of MDM2 inhibits growth of HepG2 hepatocellular carcinoma cells xenografted in nude mice

    Institute of Scientific and Technical Information of China (English)

    赵燕颖; 李亚刚; 孙远杰; 刘海鹏; 杨泽成; 张舵舵; 赵春燕

    2013-01-01

    目的 构建鼠双微粒体-2基因(MDM2)靶向小分子干扰RNA (siRNA)质粒,研究其对人肝癌HepG2细胞裸鼠皮下移植瘤生长的抑制作用,探讨其在肝癌基因治疗中的可行性.方法 构建MDM2的siRNA真核表达载体pSilencer-siRNA-MDM2 (siMDM2),建立裸鼠荷瘤模型,分别给予阴性对照质粒和siMDM2质粒处理.监测肿瘤生长变化,RT-PCR、Western blot方法检测MDM2及p53 mRNA和蛋白的表达变化.正态分布数据的组间比较用单因素方差分析及LSD检验.结果 裸鼠体内实验结果显示,转染siMDM2-1和siMDM2-2组与对照组相比,肿瘤增长受到明显抑制,抑瘤率分别为60.6%和54.6%.RT-PCR和Westem blot结果示MDM2的mRNA和蛋白质表达下调,而p53的mRNA和蛋白质表达上调.与空白组比较,转染siMDM2-1和siMDM2-2组的MDM2mRNA表达分别下调62.8%和61.5%,MDM2蛋白表达分别下调60.7%和59.5%;p53 mRNA表达分别上调47.1%和45.6%,p53蛋白表达分别上调45.9%和44.3%,差异均具有统计学意义(F值分别为75.099、47.860、17.676和235.770,P值均<0.01).结论 siRNA沉默MDM2基因能抑制人肝癌HepG2细胞裸鼠皮下移植瘤的形成,可能与其在体内有效下调MDM2和上调p53的mRNA及蛋白质表达有关.%Objecive To construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo.Methods Small interfering (si)RNAs were designed targeting the MDM2 gene (siMDM2-1 and siMDM2-2) and unrelated sequences (negative control) and cloned into the expression plasmid pGCSilencer-U6-neo-GFP.A HCC mouse model was established by subcutaneous inoculation of HepG2 cells (2 x 106 in 0.2 ml) into 20 nude mice.The inoculated mice were divided into four equal groups for tumor-localized injections of saline,negative control siRNA plasmid,siMDM2-1 plasmid,and siMDM2

  16. Effects of PPP2R5C Downregulation on Expression Profile of GSK-3β, MDM2, pTEN and TP53 Signal Pathway Regulating Genes in Jurkat Cells%下调PPP2R5C表达对Jurkat细胞GsK-3β、MDM2、pTEN、TP53通路相关调控基因表达谱的影响

    Institute of Scientific and Technical Information of China (English)

    陈宇; 黄欣; 刘思初; 杨力建; 陈少华; 罗更新; 李扬秋

    2015-01-01

    目的 利用基因芯片技术研究RNA干扰下调PPP2R5C对Jurkat细胞中GSK-3β、MDM2、pTEN、TP53通路相关基因表达的影响.方法 利用核转染技术将PPP2R5C-siRNA799转导Jurkat细胞,培养48 h后,提取细胞总RNA,纯化mRNA后反转录制备目标序列,与Affymetrix基因表达谱芯片3' IVT杂交,以Affymetrix GeneChip Scanner 3000扫描仪扫描得到原始图像数据,并应用GeneSpring GX 11.0软件进行差异表达谱分析.结果 基因芯片分析GSK-3β、MDM2、pTEN、TP53信号通路相关的47个基因,发现全部基因发生了差异表达,其中上调基因有22个,下调基因有25个.明显上调的基因有 SNAI1、APC、CDKN1A、ATM、MDM2和pTEN,而明显下调的基因只有GSK-3β.结论 下调Jurkat细胞PPP2R5C基因时,在一定程度上选择性调节涉及细胞增殖和凋亡相关的GSK-3β、MDM2、pTEN和TP53信号通路基因的表达,从而抑制Jurkat细胞增殖.

  17. End-effector microprocessor

    Science.gov (United States)

    Doggett, William R.

    1992-01-01

    The topics are presented in viewgraph form and include: automated structures assembly facility current control hierarchy; automated structures assembly facility purposed control hierarchy; end-effector software state transition diagram; block diagram for ideal install composite; and conclusions.

  18. Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models

    Directory of Open Access Journals (Sweden)

    Christian Lehmann

    2016-06-01

    Full Text Available Abstract Background Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML. In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. Methods The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Results Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that

  19. Expressions of p63 mRNA and murine double minute 2 in esophageal cancer and molecular mechanisms involved%食管癌p63mRNA及MDM-2的表达及其分子机制

    Institute of Scientific and Technical Information of China (English)

    王克文; 张桂英

    2005-01-01

    目的探讨p63mRNA、MDM-2蛋白在食管鳞癌中的表达及其在食管鳞癌形成和预后中的意义.方法采用RT-PCR和免疫组化技术分别检测38例食管鳞癌组织及其对应的正常组织中p63mRNA和MDM-2蛋白的表达水平.结果ΔNp63mRNA在食管鳞癌组织和正常组织中阳性检出率分别为63.15%和52.63%,二者比较无显著性差异;其中低分化食管鳞癌组ΔNp63mRNA的阳性表达率显著高于高/中分化食管鳞癌组,而与年龄、性别、淋巴结转移和临床分期无关.38例食管鳞癌组织中仅有1例呈TAp63 mRNA阳性表达,正常组织样品中未检出有TAp63 mRNA阳性表达.MDM-2蛋白在食管鳞癌组织中的阳性表达率为65.79%,显著高于正常组织,低分化食管鳞癌组MDM-2蛋白的阳性表达率显著高于高/中分化组,而且随临床分期的程度增高而阳性表达率升高,而与年龄、性别、淋巴结转移无关.随着MDM-2蛋白表达率的增加,ΔNp63mRNA的表达率相应增加,二者的表达呈正相关.结论 p63基因在食管鳞癌和食管上皮中表达的亚型主要是ΔNp63,ΔNp63参与了食管鳞癌的发生、发展,并与食管鳞癌的预后相关;TAp63不是食管鳞癌和食管上皮中主要的表达亚型,与食管鳞癌的形成基本无关;MDM-2蛋白的表达异常在食管鳞癌形成中起重要作用,而且与鳞癌的预后相关;ΔNp63通过MDM-2发挥其对细胞周期及凋亡的调节作用,进而导致食管鳞癌的形成是其主要的分子机制.

  20. The age of effectors

    NARCIS (Netherlands)

    Gibriel, Hesham A.Y.; Thomma, Bart P.H.J.; Seidl, Michael F.

    2016-01-01

    Microbial pathogens cause devastating diseases on economically and ecologically important plant species, threatening global food security, and causing billions of dollars of losses annually. During the infection process, pathogens secrete so-called effectors that support host colonization, often

  1. Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin

    DEFF Research Database (Denmark)

    Siemer, S; Ornskov, D; Guerra, B

    1999-01-01

    Protein kinase CK2 is a pleiotropic serine/threonine kinase which has been shown to phosphorylate numerous substrates. Evidence is accumulating that CK2 may exist complexed to a variety of cellular proteins, e.g. p53, MDM2, and A-Raf. Here, we explored the effects of the chemotherapeutic drugs...... cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after...... cisplatin and carboplatin treatment, the RNA level of p53 dropped by 59% +/- 9% and 86% +/- 8% respectively, whereas the observed level of p53 protein rose to 7 and 10 times over the untreated control, respectively. Treatment with 33 microM cisplatin prompted apoptosis as early as 4 h after drug treatment...

  2. Digital expression profiling identifies RUNX2, CDC5L, MDM2, RECQL4, and CDK4 as potential predictive biomarkers for neo-adjuvant chemotherapy response in paediatric osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Martin

    Full Text Available Osteosarcoma is the most common malignancy of bone, and occurs most frequently in children and adolescents. Currently, the most reliable technique for determining a patients' prognosis is measurement of histopathologic tumor necrosis following pre-operative neo-adjuvant chemotherapy. Unfavourable prognosis is indicated by less than 90% estimated necrosis of the tumor. Neither genetic testing nor molecular biomarkers for diagnosis and prognosis have been described for osteosarcomas. We used the novel nanoString mRNA digital expression analysis system to analyse gene expression in 32 patients with sporadic paediatric osteosarcoma. This system used specific molecular barcodes to quantify expression of a set of 17 genes associated with osteosarcoma tumorigenesis. Five genes, from this panel, which encoded the bone differentiation regulator RUNX2, the cell cycle regulator CDC5L, the TP53 transcriptional inactivator MDM2, the DNA helicase RECQL4, and the cyclin-dependent kinase gene CDK4, were differentially expressed in tumors that responded poorly to neo-adjuvant chemotherapy. Analysis of the signalling relationships of these genes, as well as other expression markers of osteosarcoma, indicated that gene networks linked to RB1, TP53, PI3K, PTEN/Akt, myc and RECQL4 are associated with osteosarcoma. The discovery of these networks provides a basis for further experimental studies of role of the five genes (RUNX2, CDC5L, MDM2, RECQL4, and CDK4 in differential response to chemotherapy.

  3. Relationship of Ki67, TP53, MDM-2 and BCL-2 expressions with WHO 1973 and WHO/ISUP grades, tumor category and overall patient survival in urothelial tumors of the bladder.

    Science.gov (United States)

    Gönül, Ipek Işik; Akyürek, Nalan; Dursun, Ayşe; Küpeli, Bora

    2008-01-01

    Using the 1998 World Health Organization/International Society of Urological Pathology (WHO/ISUP) (2004 WHO), 1999 WHO/ISUP, and 1973 WHO classifications, we examined Ki67, BCL-2, TP53, and MDM-2 expressions in invasive and noninvasive urothelial neoplasias of the bladder of 72 patients, and compared the results regarding tumor category and grade with clinical outcome to determine the clinicopathological relevance of these classifications. Ki67 and TP53 expressions were correlated with tumor grades of the 1973 WHO classification, and they also distinguished "papillary urothelial neoplasm with low malignant potential" from other WHO/ISUP grades (p ISUP grades (p > 0.05). Neither tumor grade nor tumor category correlated with MDM-2 or BCL-2 expressions (p > 0.05). WHO/ISUP classifications are obviously not superior to the 1973 WHO classification for grading urothelial neoplasia of the bladder. However, if the "papillary urothelial neoplasm with low malignant potential" is distinguished from grade 1 tumors of the 1973 WHO classification, more precise prognostic information may be obtained.

  4. Hypermethylation of the 5′ CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

    Directory of Open Access Journals (Sweden)

    Nyiraneza Christine

    2012-06-01

    Full Text Available Abstract Background It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI, low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs. In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. Results Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%, whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%. Only seven of ninety-eight tumors (7% had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13 versus 0 to 17 (95% CI 0 to 2 in adjacent colon mucosa (P = 0.004. Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03, and MDM2 overexpression (P = 0.02, independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational

  5. Effector glycosyltransferases in Legionella

    Directory of Open Access Journals (Sweden)

    Yury eBelyi

    2011-04-01

    Full Text Available Legionella causes severe pneumonia in humans. The pathogen produces an array of effectors, which interfere with host cell functions. Among them are the glucosyltransferases Lgt1, Lgt2 and Lgt3 from L. pneumophila. Lgt1 and Lgt2 are produced predominately in the post-exponential phase of bacterial growth, while synthesis of Lgt3 is induced mainly in the lag-phase before intracellular replication of bacteria starts. Lgt glucosyltransferases are structurally similar to clostridial glucosylating toxins. The enzymes use UDP-glucose as a donor substrate and modify eukaryotic elongation factor eEF1A at serine-53. This modification results in inhibition of protein synthesis and death of target cells. In addition to Lgts, Legionella genomes disclose several genes, coding for effector proteins likely to possess glycosyltransferase activities, including SetA, which influences vesicular trafficking in the yeast model system and displays tropism for late endosomal/lysosomal compartments of mammalian cells. This review mainly discusses recent results on the structure-function relationship of Lgt glucosyltransferases.

  6. Two-axis angular effector

    Energy Technology Data Exchange (ETDEWEB)

    Vaughn, Mark R. (Albuquerque, NM); Robinett, III, Rush D. (Tijeras, NM); Phelan, John R. (Albuquerque, NM); Van Zuiden, Don M. (Albuquerque, NM)

    1997-01-21

    A new class of coplanar two-axis angular effectors. These effectors combine a two-axis rotational joint analogous to a Cardan joint with linear actuators in a manner to produce a wider range of rotational motion about both axes defined by the joint. This new class of effectors also allows design of robotic manipulators having very high strength and efficiency. These effectors are particularly suited for remote operation in unknown surroundings, because of their extraordinary versatility. An immediate application is to the problems which arise in nuclear waste remediation.

  7. Two-axis angular effector

    Energy Technology Data Exchange (ETDEWEB)

    Vaughn, M.R.; Robinett, R.D. III; Phelan, J.R.; Zuiden, D.M. Van

    1997-01-21

    A new class of coplanar two-axis angular effectors is described. These effectors combine a two-axis rotational joint analogous to a Cardan joint with linear actuators in a manner to produce a wider range of rotational motion about both axes defined by the joint. This new class of effectors also allows design of robotic manipulators having very high strength and efficiency. These effectors are particularly suited for remote operation in unknown surroundings, because of their extraordinary versatility. An immediate application is to the problems which arise in nuclear waste remediation. 11 figs.

  8. Long-term administration of the fungus toxin, sterigmatocystin, induces intestinal metaplasia and increases the proliferative activity of PCNA, p53, and MDM2 in the gastric mucosa of aged Mongolian gerbils.

    Science.gov (United States)

    Kusunoki, Masahiro; Misumi, Junichi; Shimada, Tatsuo; Aoki, Kazuo; Matsuo, Noritaka; Sumiyoshi, Hideaki; Yamaguchi, Takeshi; Yoshioka, Hidekatsu

    2011-07-01

    The causal agents of gastric cancer could include fungus toxins. Sterigmatocystin (ST), a fungus toxin, is a risk factor of gastric cancer. We investigated the effects of ST on the stomach tissues of Mongolian gerbils. Seventy-five-week-old male Mongolian gerbils received ST ad libitum at a concentration of 0 ppb (non-treated, n = 11), 100 ppb (n = 7), or 1000 ppb (n = 13) dissolved in drinking water for a period of 24 weeks. After administration, we tested the histopathological changes and immunostaining for proliferating cell nuclear antigen (PCNA), p53, and MDM2 expression. We investigated the histopathological changes and determined the incidence of histopathological changes in animals with various gastric diseases after ST administration at a dose of 0 ppb (non-treated control), 100, or 1,000 ppb as follows: firstly, indices for gastritis were 18.2, 100, and 100%, those for erosion events were 9.1, 100, and 92.3%, and those for polyps were 0, 71.4, and 61.5%, respectively. These incidences in the ST-administered groups (100 or 1000 ppb) showed significant increases compared with those in the non-treated control group. And, lastly, indices for intestinal metaplasia were 0, 100, and 15.4%, respectively. Furthermore, immunostaining for PCNA, p53, and MDM2 expression showed significantly greater rates in the ST-administered groups (100 or 1000 ppb) than in the non-treated control group. The histopathological and immunohistopathological findings of this study indicate that ST exerts a marked influence on gastric mucus and gland cells, showing dominant gastritis, erosion events, polyps, and intestinal metaplasia in these animals.

  9. An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation.

    Science.gov (United States)

    Shukla, Sanket Kumar; Jha, Hem Chandra; El-Naccache, Darine W; Robertson, Erle S

    2016-04-05

    Epstein-Barr virus (EBV), a gamma herpes virus is associated with B-cell malignancies. EBNA-3C is critical for in vitro primary B-cell transformation. Interestingly, the N terminal domain of EBNA3C which contains residues 130-159, interacts with various cellular proteins, such as p53, Mdm2, CyclinD1/Cdk6 complex, and E2F1. In the current reverse genetics study, we deleted the residues 130-159 aa within EBNA3C open reading frame (ORF) by BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the 130-159 aa showed a reduction in cell proliferation. Also, this recombinant virus showed with higher infectivity of human peripheral blood mononuclear cells (PBMCs) compared to wild type EBV. PBMCs- infected with recombinant EBV deleted for 130-159 residues have differential expression patterns for the p53/Mdm2, CyclinD1/Cdk6 and pRb/E2F1 pathways compared to wild type EBV-infected PBMCs. PBMCs infected with recombinant virus showed increased apoptotic cell death which further resulted in activation of polymerase 1 (PARP1), an important contributor to apoptotic signaling. Interestingly, cells infected with this recombinant virus showed a dramatic decrease in chromosomal instability, indicated by the presence of increased multinucleation and micronucleation. In addition infection with recombinant virus have increased cells in G0/G1 phase and decreased cells in S-G2M phase when compared to wild type infected cells. Thus, these differences in signaling activities due to 29 amino acid residues of EBNA3C is of particular significance in deregulation of cell proliferation in EBV-infected cells.

  10. 一组癌基因扩增与胃癌生物学行为的相关分析%Analysis of the correlation between the amplification of HER2, mdm-2 and C-myc in the gastric cancer and metastases

    Institute of Scientific and Technical Information of China (English)

    时军; 崔建涛; 吕有勇

    2000-01-01

    目的:探讨癌基因HER2、mdm-2和C-myc扩增与胃癌恶性程度、转移及预后的关系.方法:用差异竞争性多聚酶链反应检测胃癌原发灶、癌旁、转移淋巴结及远处脏器转移癌中HER2、mdm-2和C-myc的基因变异.结果:HER2扩增频率在近端胃癌组中明显高于远端胃癌组(分别是11/13和5/19, P0.05),在3例淋巴结微转移灶中发现mdm-2扩增.C-myc在胃癌原发灶及转移淋巴结中的扩增频率分别是6/32和5/21, 2例远处脏器转移癌中有扩增.结论:HER2 、mdm-2和C-myc基因扩增与胃癌的快速生长有关,三者联合检测可能为判断胃癌恶性程度、转移及预后提供有价值的信息.

  11. The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition.

    Science.gov (United States)

    Unterweger, Daniel; Miyata, Sarah T; Bachmann, Verena; Brooks, Teresa M; Mullins, Travis; Kostiuk, Benjamin; Provenzano, Daniele; Pukatzki, Stefan

    2014-04-01

    Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria producing corresponding effectors. As a consequence, strains with matching effector-immunity gene sets can coexist, while strains with different sets compete against each other. Thus, the V. cholerae T6SS contributes to the competitive behaviour of this species.

  12. Oomycetes, effectors, and all that jazz.

    Science.gov (United States)

    Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

    2012-08-01

    Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Cell volume homeostatic mechanisms: effectors and signalling pathways

    DEFF Research Database (Denmark)

    Hoffmann, E K; Pedersen, Stine Helene Falsig

    2011-01-01

    . Later work addressed the mechanisms through which cellular signalling pathways regulate the volume regulatory effectors or flux pathways. These studies were facilitated by the molecular identification of most of the relevant channels and transporters, and more recently also by the increased...... understanding of their structures. Finally, much current research in the field focuses on the most up- and downstream components of these paths: how cells sense changes in cell volume, and how cell volume changes in turn regulate cell function under physiological and pathophysiological conditions....

  14. Ascl1 is a required downstream effector of Gsx gene function in the embryonic mouse telencephalon

    Directory of Open Access Journals (Sweden)

    Allen Zegary J

    2009-02-01

    Full Text Available Abstract Background The homeobox gene Gsx2 (formerly Gsh2 is known to regulate patterning in the lateral ganglionic eminence (LGE of the embryonic telencephalon. In its absence, the closely related gene Gsx1 (previously known as Gsh1 can partially compensate in the patterning and differentiation of ventral telencephalic structures, such as the striatum. However, the cellular and molecular mechanisms underlying this compensation remain unclear. Results We show here that in the Gsx2 mutants Gsx1 is expressed in only a subset of the ventral telencephalic progenitors that normally express Gsx2. Based on the similarities in the expression of Gsx1 and Ascl1 (Mash1 within the Gsx2 mutant LGE, we examined whether Ascl1 plays an integral part in the Gsx1-based recovery. Ascl1 mutants show only modest alterations in striatal development; however, in Gsx2;Ascl1 double mutants, striatal development is severely affected, similar to that seen in the Gsx1;Gsx2 double mutants. This is despite the fact that Gsx1 is expressed, and even expands, in the Gsx2;Ascl1 mutant LGE, comparable to that seen in the Gsx2 mutant. Finally, Notch signaling has recently been suggested to be required for normal striatal development. In spite of the fact that Notch signaling is severely disrupted in Ascl1 mutants, it actually appears to be improved in the Gsx2;Ascl1 double mutants. Conclusion These results, therefore, reveal a non-proneural requirement of Ascl1 that together with Gsx1 compensates for the loss of Gsx2 in a subset of LGE progenitors.

  15. Mechanism of p53 downstream effectors p21 and Gadd45 in DNA damage surveillance

    Institute of Scientific and Technical Information of China (English)

    孟祥兵; 董燕; 孙志贤

    1999-01-01

    Both p21 (WAF1/CIP1) and Gadd45 were activated in a p53-dependent manner in MCF-7 cells after being exposed to ionizing radiation. In order to investigate their roles in DNA damage surveillance, p21as/MCF-7 cells stably transfected by p21 antisense expression plasmid pC-WAF1-AS and Gadd45as/MCF-7 stably transfected by Gadd45 antisense expression plasmid pCMVas45 were established. It was observed that G1 arrest induced by radiation was significantly reduced in Gadd45as/MCF-7 cells as well as in p21as/MCF-7 cells. Repair of radiation damaged report gene greatly reduced in Gadd45as/MCF-7 and p21as/MCF-7 cells. Apoptosis significantly increased in p21as/MCF-7 after exposure to radiation. These results suggest that both p21 and Gadd45 support cellular survival by taking roles in G1 arrest and DNA repair, furthermore, p21 protects cells from death by inhibiting apoptosis after exposure to ionizing radiation.

  16. Regulation of MDM2 Activity by Nucleolin

    Science.gov (United States)

    2005-06-01

    equtal1ly "actiye it nialn a it art 401 INA repl catirlt teit: kinase. J. [BitI. C’hem. 275:7803-781(10 imin i cleotide excisiotn repaiir, Prtie. Naitl . Acad...L.. L. F. Erdile, 1. U. Gilbert, D. Non Winkler, T. J. Kelly, and E. after DNA dainae’c. Pnite. Naitl . Acod, Sri, UISA 96’:1i9 21-02 Fa nning. 1992

  17. Regulation of MDM2 Activity by Nucleolin

    Science.gov (United States)

    2007-06-01

    assistance with FACS analysis, Eric Rubin (UMDNJ) for providing the GST-nucleolin expression vectors, Cris- tina Cardoso for the pENeGFP RPA34 plasmid, and...formation, and functional char- acterization. J. Biol. Chem. 269:11121–11132. 25. Huang, W., and R. L. Erikson . 1994. Constitutive activation of Mek1

  18. Continuous downstream processing of biopharmaceuticals.

    Science.gov (United States)

    Jungbauer, Alois

    2013-08-01

    Continuous manufacturing has been applied in many different industries but has been pursued reluctantly in biotechnology where the batchwise process is still the standard. A shift to continuous operation can improve productivity of a process and substantially reduce the footprint. Continuous operation also allows robust purification of labile biomolecules. A full set of unit operations is available to design continuous downstream processing of biopharmaceuticals. Chromatography, the central unit operation, is most advanced in respect to continuous operation. Here, the problem of 'batch' definition has been solved. This has also paved the way for implementation of continuous downstream processing from a regulatory viewpoint. Economic pressure, flexibility, and parametric release considerations will be the driving force to implement continuous manufacturing strategies in future.

  19. Sequential displacement of Type VI Secretion System effector genes leads to evolution of diverse immunity gene arrays in Vibrio cholerae

    Science.gov (United States)

    Kirchberger, Paul C.; Unterweger, Daniel; Provenzano, Daniele; Pukatzki, Stefan; Boucher, Yan

    2017-01-01

    Type VI secretion systems (T6SS) enable bacteria to engage neighboring cells in contact-dependent competition. In Vibrio cholerae, three chromosomal clusters each encode a pair of effector and immunity genes downstream of those encoding the T6SS structural machinery for effector delivery. Different combinations of effector-immunity proteins lead to competition between strains of V. cholerae, which are thought to be protected only from the toxicity of their own effectors. Screening of all publically available V. cholerae genomes showed that numerous strains possess long arrays of orphan immunity genes encoded in the 3′ region of their T6SS clusters. Phylogenetic analysis reveals that these genes are highly similar to those found in the effector-immunity pairs of other strains, indicating acquisition by horizontal gene transfer. Extensive genomic comparisons also suggest that successive addition of effector-immunity gene pairs replaces ancestral effectors, yet retains the cognate immunity genes. The retention of old immunity genes perhaps provides protection against nearby kin bacteria in which the old effector was not replaced. This mechanism, combined with frequent homologous recombination, is likely responsible for the high diversity of T6SS effector-immunity gene profiles observed for V. cholerae and closely related species. PMID:28327641

  20. Operational optimization in the downstream; Otimizacao operacional no downstream

    Energy Technology Data Exchange (ETDEWEB)

    Silberman, Luis; Cunha, Filipe Silveira Ramos da [Petroleo Ipiranga, Porto Alegre, RS (Brazil)

    2004-07-01

    On the present competitive down stream's market, there is a great necessity of optimization aiming to guarantee the best price and quality of our clients. Our goal is to attend these expectations while we guarantee an efficient operation. The greatest question is how far we are from the ideal model. This way, a lot of projects have been executed during the last years aiming the operational optimization of all our activities. We divide the projects in 4 areas: Logistic (new modals distribution), Transport (transport optimization - quality and more deliveries with less trucks), Client Support (Internet Ipiranga and Support Center), Distribution Terminals Productivity (automation and environment). This work intend to present our ideal, perfect and complete Downstream Operation model. We will talk about how close we are of this ideal model and we will present the projects that we had already developed and implanted on the automation of the terminals and the logistics area. (author)

  1. Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators.

    Science.gov (United States)

    Bhattacharjee, Saikat; Halane, Morgan K; Kim, Sang Hee; Gassmann, Walter

    2011-12-01

    Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll-interleukin-1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR-mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.

  2. Deciphering interplay between Salmonella invasion effectors.

    Directory of Open Access Journals (Sweden)

    Robert J Cain

    2008-04-01

    Full Text Available Bacterial pathogens have evolved a specialized type III secretion system (T3SS to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP can individually manipulate actin dynamics at the plasma membrane, which acts as a 'signaling hub' during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cisbinary entry effector interplay (BENEFIT screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen-host interaction.

  3. Molecular pathways: targeting the kinase effectors of RHO-family GTPases.

    Science.gov (United States)

    Prudnikova, Tatiana Y; Rawat, Sonali J; Chernoff, Jonathan

    2015-01-01

    RHO GTPases, members of the RAS superfamily of small GTPases, are adhesion and growth factor-activated molecular switches that play important roles in tumor development and progression. When activated, RHO-family GTPases such as RAC1, CDC42, and RHOA, transmit signals by recruiting a variety of effector proteins, including the protein kinases PAK, ACK, MLK, MRCK, and ROCK. Genetically induced loss of RHO function impedes transformation by a number of oncogenic stimuli, leading to an interest in developing small-molecule inhibitors that either target RHO GTPases directly, or that target their downstream protein kinase effectors. Although inhibitors of RHO GTPases and their downstream signaling kinases have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to facilitate pharmaceutical research and development and is a promising therapeutic strategy.

  4. Rapid parallel flow cytometry assays of active GTPases using effector beads.

    Science.gov (United States)

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-11-15

    We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

  5. SPRYSEC effector proteins in Globodera rostochiensis

    NARCIS (Netherlands)

    Rehman, S.

    2008-01-01

    Plant pathogens inject so-called effector molecules into the cells of a host plant to promote their growth and reproduction in these hosts. In plant parasitic nematodes, these effector molecules are produced in the salivary glands. The objective of this thesis was to identify and characterize effect

  6. Characterization of effectors from Fusarium graminearum

    Science.gov (United States)

    Fusarium graminearum is the causal agent of Fusarium head blight (FHB), which reduces crop yield and quality by producing various mycotoxins. Effectors play an important role in the pathogenesis of many bacterial and fungal pathogens. In this study, 26 effector candidates were selected for investiga...

  7. NOD-like receptor cooperativity in effector-triggered immunity.

    Science.gov (United States)

    Griebel, Thomas; Maekawa, Takaki; Parker, Jane E

    2014-11-01

    Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are basic elements of innate immunity in plants and animals. Whereas animal NLRs react to conserved microbe- or damage-associated molecular patterns, plant NLRs intercept the actions of diverse pathogen virulence factors (effectors). In this review, we discuss recent genetic and molecular evidence for functional NLR pairs, and discuss the significance of NLR self-association and heteromeric NLR assemblies in the triggering of downstream signaling pathways. We highlight the versatility and impact of cooperating NLR pairs that combine pathogen sensing with the initiation of defense signaling in both plant and animal immunity. We propose that different NLR receptor molecular configurations provide opportunities for fine-tuning resistance pathways and enhancing the host's pathogen recognition spectrum to keep pace with rapidly evolving microbial populations. Copyright © 2014. Published by Elsevier Ltd.

  8. GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

    Science.gov (United States)

    Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

    2015-01-01

    Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

  9. ROBOTIC TANK INSPECTION END EFFECTOR

    Energy Technology Data Exchange (ETDEWEB)

    Rachel Landry

    1999-10-01

    The objective of this contract between Oceaneering Space Systems (OSS) and the Department of Energy (DOE) was to provide a tool for the DOE to inspect the inside tank walls of underground radioactive waste storage tanks in their tank farms. Some of these tanks are suspected to have leaks, but the harsh nature of the environment within the tanks precludes human inspection of tank walls. As a result of these conditions only a few inspection methods can fulfill this task. Of the methods available, OSS chose to pursue Alternating Current Field Measurement (ACFM), because it does not require clean surfaces for inspection, nor any contact with the Surface being inspected, and introduces no extra by-products in the inspection process (no coupling fluids or residues are left behind). The tool produced by OSS is the Robotic Tank Inspection End Effector (RTIEE), which is initially deployed on the tip of the Light Duty Utility Arm (LDUA). The RTEE combines ACFM with a color video camera for both electromagnetic and visual inspection The complete package consists of an end effector, its corresponding electronics and software, and a user's manual to guide the operator through an inspection. The system has both coarse and fine inspection modes and allows the user to catalog defects and suspected areas of leakage in a database for further examination, which may lead to emptying the tank for repair, decommissioning, etc.. The following is an updated report to OSS document OSS-21100-7002, which was submitted in 1995. During the course of the contract, two related subtasks arose, the Wall and Coating Thickness Sensor and the Vacuum Scarifying and Sampling Tool Assembly. The first of these subtasks was intended to evaluate the corrosion and wall thinning of 55-gallon steel drums. The second was retrieved and characterized the waste material trapped inside the annulus region of the underground tanks on the DOE's tank farms. While these subtasks were derived from the original

  10. Epigenetic control of effectors in plant pathogens

    Directory of Open Access Journals (Sweden)

    Mark eGijzen

    2014-11-01

    Full Text Available Plant pathogens display impressive versatility in adapting to host immune systems. Pathogen effector proteins facilitate disease but can become avirulence (Avr factors when the host acquires discrete recognition capabilities that trigger immunity. The mechanisms that lead to changes to pathogen Avr factors that enable escape from host immunity are diverse, and include epigenetic switches that allow for reuse or recycling of effectors. This perspective outlines possibilities of how epigenetic control of Avr effector gene expression may have arisen and persisted in plant pathogens, and how it presents special problems for diagnosis and detection of specific pathogen strains or pathotypes.

  11. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    Science.gov (United States)

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  12. CNOOC Advancing into Downstream of Petroleum Industry

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

    @@ "China National Offshore Oil Corporation is starting to expand its business into the downstream sector after making achievements continuously in exploration and development," Wang Yan, president of the corporation,told reporter in an interview, adding that the downstream sector is the third development stage for China National Offshore Oil Corporation (CNOOC).

  13. Avian Interferons and Their Antiviral Effectors

    OpenAIRE

    Santhakumar, Diwakar; Rubbenstroth, Dennis; Martinez-Sobrido, Luis; Munir, Muhammad

    2017-01-01

    Interferon (IFN) responses, mediated by a myriad of IFN-stimulated genes (ISGs), are the most profound innate immune responses against viruses. Cumulatively, these IFN effectors establish a multilayered antiviral state to safeguard the host against invading viral pathogens. Considerable genetic and functional characterizations of mammalian IFNs and their effectors have been made, and our understanding on the avian IFNs has started to expand. Similar to mammalian counterparts, three types of I...

  14. Jet Engine Exhaust Nozzle Flow Effector

    Science.gov (United States)

    Turner, Travis L. (Inventor); Cano, Roberto J. (Inventor); Silox, Richard J. (Inventor); Buehrle, Ralph D. (Inventor); Cagle, Christopher M. (Inventor); Cabell, Randolph H. (Inventor); Hilton, George C. (Inventor)

    2014-01-01

    A jet engine exhaust nozzle flow effector is a chevron formed with a radius of curvature with surfaces of the flow effector being defined and opposing one another. At least one shape memory alloy (SMA) member is embedded in the chevron closer to one of the chevron's opposing surfaces and substantially spanning from at least a portion of the chevron's root to the chevron's tip.

  15. Combover/CG10732, a novel PCP effector for Drosophila wing hair formation.

    Science.gov (United States)

    Fagan, Jeremy K; Dollar, Gretchen; Lu, Qiuheng; Barnett, Austen; Pechuan Jorge, Joaquin; Schlosser, Andreas; Pfleger, Cathie; Adler, Paul; Jenny, Andreas

    2014-01-01

    The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh.

  16. Combover/CG10732, a novel PCP effector for Drosophila wing hair formation.

    Directory of Open Access Journals (Sweden)

    Jeremy K Fagan

    Full Text Available The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP, the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh and Rho Kinase (Rok are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC, similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh.

  17. Cellular effector mechanisms against Plasmodium liver stages.

    Science.gov (United States)

    Frevert, Ute; Nardin, Elizabeth

    2008-10-01

    Advances in our understanding of the molecular and cell biology of the malaria parasite have led to new vaccine development efforts resulting in a pipeline of over 40 candidates undergoing clinical phase I-III trials. Vaccine-induced CD4+ and CD8+ T cells specific for pre-erythrocytic stage antigens have been found to express cytolytic and multi-cytokine effector functions that support a key role for these T cells within the hepatic environment. However, little is known of the cellular interactions that occur during the effector phase in which the intracellular hepatic stage of the parasite is targeted and destroyed. This review focuses on cell biological aspects of the interaction between malaria-specific effector cells and the various antigen-presenting cells that are known to exist within the liver, including hepatocytes, dendritic cells, Kupffer cells, stellate cells and sinusoidal endothelia. Considering the unique immune properties of the liver, it is conceivable that these different hepatic antigen-presenting cells fulfil distinct but complementary roles during the effector phase against Plasmodium liver stages.

  18. MARTX toxins as effector delivery platforms.

    Science.gov (United States)

    Gavin, Hannah E; Satchell, Karla J F

    2015-12-01

    Bacteria frequently manipulate their host environment via delivery of microbial 'effector' proteins to the cytosol of eukaryotic cells. In the case of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin, this phenomenon is accomplished by a single, >3500 amino acid polypeptide that carries information for secretion, translocation, autoprocessing and effector activity. MARTX toxins are secreted from bacteria by dedicated Type I secretion systems. The released MARTX toxins form pores in target eukaryotic cell membranes for the delivery of up to five cytopathic effectors, each of which disrupts a key cellular process. Targeted cellular processes include modulation or modification of small GTPases, manipulation of host cell signaling and disruption of cytoskeletal integrity. More recently, MARTX toxins have been shown to be capable of heterologous protein translocation. Found across multiple bacterial species and genera--frequently in pathogens lacking Type 3 or Type 4 secretion systems--MARTX toxins in multiple cases function as virulence factors. Innovative research at the intersection of toxin biology and bacterial genetics continues to elucidate the intricacies of the toxin as well as the cytotoxic mechanisms of its diverse effector collection.

  19. Minimal Mimicry: Mere Effector Matching Induces Preference

    Science.gov (United States)

    Sparenberg, Peggy; Topolinski, Sascha; Springer, Anne; Prinz, Wolfgang

    2012-01-01

    Both mimicking and being mimicked induces preference for a target. The present experiments investigate the minimal sufficient conditions for this mimicry-preference link to occur. We argue that mere effector matching between one's own and the other person's movement is sufficient to induce preference, independent of which movement is actually…

  20. PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase.

    Science.gov (United States)

    Leenders, Frauke; Möpert, Kristin; Schmiedeknecht, Anett; Santel, Ansgar; Czauderna, Frank; Aleku, Manuela; Penschuck, Silke; Dames, Sibylle; Sternberger, Maria; Röhl, Thomas; Wellmann, Axel; Arnold, Wolfgang; Giese, Klaus; Kaufmann, Jörg; Klippel, Anke

    2004-08-18

    Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.

  1. Scleroglucan: Fermentative Production, Downstream Processing and Applications

    Directory of Open Access Journals (Sweden)

    Shrikant A. Survase

    2007-01-01

    Full Text Available Exopolysaccharides produced by a variety of microorganisms find multifarious industrial applications in foods, pharmaceutical and other industries as emulsifiers, stabilizers, binders, gelling agents, lubricants, and thickening agents. One such exopolysaccharide is scleroglucan, produced by pure culture fermentation from filamentous fungi of genus Sclerotium. The review discusses the properties, fermentative production, downstream processing and applications of scleroglucan.

  2. Downstream bioprocess characterisation within microfluidic devices

    DEFF Research Database (Denmark)

    Marques, Marco; Krühne, Ulrich; Szita, Nicolas

    2016-01-01

    Miniaturising bioprocess unit operation steps is a well-established approach to find novel routes for process intensification and improved process economics. While a number of microbioreactors have been presented over the last 15 years, miniaturised downstream unit operations (mDUO) are less deve...

  3. MITIGATION OF SEDIMENTATION HAZARDS DOWNSTREAM FROM RESERVOIRS

    Institute of Scientific and Technical Information of China (English)

    Ellen WOHL; Sara RATHBURN

    2003-01-01

    Many reservoirs currently in operation trap most or all of the sediment entering the reservoir,creating sediment-depleted conditions downstream. This may cause channel adjustment in the form of bank erosion, bed erosion, substrate coarsening, and channel planform change. Channel adjustment may also result from episodic sediment releases during reservoir operation, or from sediment evacuation following dam removal. Channel adjustment to increased sediment influx depends on the magnitude, frequency, duration and grain-size distribution of the sediment releases, and on the downstream channel characteristics. Channel adjustment may occur as a change in substrate sizedistribution, filling of pools, general bed aggradation, lateral instability, change in channel planform,and/or floodplain aggradation. The increased sediment availability may alter aquatic and riparian habitat, reduce water quality, distribute adsorbed contaminants along the river corridor, and provide germination sites for exotic vegetation. Mitigation of these sedimentation hazards requires: (1)mapping grain-size distribution within the reservoir and estimating the grain-size distributions of sediment that will be mobilized through time; (2) mapping shear stress and sediment transport capacity as a function of discharge on the basis of channel units for the length of the river likely to be affected; (3) mapping potential depositional zones, and aquatic habitat and "acceptable losses," along the downstream channel, and comparing these volumes to the total sediment volume stored in the reservoir as a means of estimating total transport capacity required to mobilize reservoir sediment delivered to the channel; (4) designing discharge and sediment release regime (magnitude, frequency,duration) to minimize adverse downstream impacts; and (5) developing plans to remove, treat, contain,or track contaminants, and to restrict establishment of exotic vegetation. The North Fork Poudre River in Colorado is used to

  4. Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava.

    Science.gov (United States)

    Cohn, Megan; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J

    2016-08-01

    Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator-like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells, where they are directed to the nucleus, bind DNA in plant promoters and activate the expression of downstream genes. The DNA-binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study, we used RNA sequencing to identify the full target repertoire of TAL14Xam668 in cassava, which includes over 50 genes. A subset of highly up-regulated genes was tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668 -mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to the narrowing down of biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site, and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems.

  5. India's Downstream Petroleum Sector

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2010-07-01

    This study provides a holistic examination of pricing and investment dynamics in India's downstream petroleum sector. It analyses the current pricing practices, highlights the tremendous fiscal cost of current pricing and regulatory arrangements, and examines the sectoral investment dynamics. It also looks at potential paths towards market-based reform along which the Indian government may move, while at the same time protecting energy market access for India's large poor population.

  6. Avian Interferons and Their Antiviral Effectors.

    Science.gov (United States)

    Santhakumar, Diwakar; Rubbenstroth, Dennis; Martinez-Sobrido, Luis; Munir, Muhammad

    2017-01-01

    Interferon (IFN) responses, mediated by a myriad of IFN-stimulated genes (ISGs), are the most profound innate immune responses against viruses. Cumulatively, these IFN effectors establish a multilayered antiviral state to safeguard the host against invading viral pathogens. Considerable genetic and functional characterizations of mammalian IFNs and their effectors have been made, and our understanding on the avian IFNs has started to expand. Similar to mammalian counterparts, three types of IFNs have been genetically characterized in most avian species with available annotated genomes. Intriguingly, chickens are capable of mounting potent innate immune responses upon various stimuli in the absence of essential components of IFN pathways including retinoic acid-inducible gene I, IFN regulatory factor 3 (IRF3), and possibility IRF9. Understanding these unique properties of the chicken IFN system would propose valuable targets for the development of potential therapeutics for a broader range of viruses of both veterinary and zoonotic importance. This review outlines recent developments in the roles of avian IFNs and ISGs against viruses and highlights important areas of research toward our understanding of the antiviral functions of IFN effectors against viral infections in birds.

  7. Avian Interferons and Their Antiviral Effectors

    Science.gov (United States)

    Santhakumar, Diwakar; Rubbenstroth, Dennis; Martinez-Sobrido, Luis; Munir, Muhammad

    2017-01-01

    Interferon (IFN) responses, mediated by a myriad of IFN-stimulated genes (ISGs), are the most profound innate immune responses against viruses. Cumulatively, these IFN effectors establish a multilayered antiviral state to safeguard the host against invading viral pathogens. Considerable genetic and functional characterizations of mammalian IFNs and their effectors have been made, and our understanding on the avian IFNs has started to expand. Similar to mammalian counterparts, three types of IFNs have been genetically characterized in most avian species with available annotated genomes. Intriguingly, chickens are capable of mounting potent innate immune responses upon various stimuli in the absence of essential components of IFN pathways including retinoic acid-inducible gene I, IFN regulatory factor 3 (IRF3), and possibility IRF9. Understanding these unique properties of the chicken IFN system would propose valuable targets for the development of potential therapeutics for a broader range of viruses of both veterinary and zoonotic importance. This review outlines recent developments in the roles of avian IFNs and ISGs against viruses and highlights important areas of research toward our understanding of the antiviral functions of IFN effectors against viral infections in birds. PMID:28197148

  8. Fish reproductive guilds downstream of dams.

    Science.gov (United States)

    Vasconcelos, L P; Alves, D C; Gomes, L C

    2014-11-01

    Fish reproductive guilds were used to evaluate the responses of species with different reproductive strategies during two different periods of post-dam construction. The data used for the comparisons were collected in the upper Paraná River floodplain (Brazil), downstream of the Porto Primavera dam, 2 and 10 years after impoundment. The abundance (catch per unit effort, CPUE), species richness, evenness and structure of communities, all within reproductive guilds, were used to test the hypothesis that these metrics vary spatially and temporally. The influence of damming on species structure and the diversity of fish reproductive guilds varied spatiotemporally, and species with opportunistic reproductive strategies tended to be less affected. Conversely, long-distance migratory species responded more markedly to spatiotemporal variations, indicating that the ecosystem dynamics exert greater effects on populations of these species. Thus, the effects of a dam, even if attenuated, may extend over several years, especially downstream. This finding emphasizes the importance of maintaining large undammed tributaries downstream of reservoirs.

  9. Impact of end effector technology on telemanipulation performance

    Science.gov (United States)

    Bejczy, A. K.; Szakaly, Z.; Ohm, T.

    1990-01-01

    Generic requirements for end effector design are briefly summarized as derived from generic functional and operational requirements. Included is a brief summary of terms and definitions related to end effector technology. The second part contains a brief overview of end effector technology work as JPL during the past ten years, with emphasis on the evolution of new mechanical, sensing and control capabilities of end effectors. The third and major part is devoted to the description of current end effector technology. The ongoing work addresses mechanical, sensing and control details with emphasis on mechanical ruggedness, increased resolution in sensing, and close electronic and control integration with overall telemanipulator control system.

  10. Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

    Science.gov (United States)

    Heidrich, Katharina; Wirthmueller, Lennart; Tasset, Céline; Pouzet, Cécile; Deslandes, Laurent; Parker, Jane E

    2011-12-01

    Pathogen effectors are intercepted by plant intracellular nucleotide binding-leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll-interleukin-1 receptor)-NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment-specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

  11. Expanded functions for a family of plant intracellular immune receptors beyond specific recognition of pathogen effectors.

    Science.gov (United States)

    Bonardi, Vera; Tang, Saijun; Stallmann, Anna; Roberts, Melinda; Cherkis, Karen; Dangl, Jeffery L

    2011-09-27

    Plants and animals deploy intracellular immune receptors that perceive specific pathogen effector proteins and microbial products delivered into the host cell. We demonstrate that the ADR1 family of Arabidopsis nucleotide-binding leucine-rich repeat (NB-LRR) receptors regulates accumulation of the defense hormone salicylic acid during three different types of immune response: (i) ADRs are required as "helper NB-LRRs" to transduce signals downstream of specific NB-LRR receptor activation during effector-triggered immunity; (ii) ADRs are required for basal defense against virulent pathogens; and (iii) ADRs regulate microbial-associated molecular pattern-dependent salicylic acid accumulation induced by infection with a disarmed pathogen. Remarkably, these functions do not require an intact P-loop motif for at least one ADR1 family member. Our results suggest that some NB-LRR proteins can serve additional functions beyond canonical, P-loop-dependent activation by specific virulence effectors, extending analogies between intracellular innate immune receptor function from plants and animals.

  12. Legionella metaeffector exploits host proteasome to temporally regulate cognate effector.

    Directory of Open Access Journals (Sweden)

    Tomoko Kubori

    Full Text Available Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of "metaeffector," a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.

  13. Innovation in Downstream Fashion Retail Networks

    DEFF Research Database (Denmark)

    Tambo, Torben

    2012-01-01

    , technology and service providers, ownership structures and local level supply chain facilities. This paper analyses theoretical and empirical views of innovation in international retail networks using the fashion industry as a case because this industry better than other industries maintain branded stores......-depth studies of the biggest Danish fashion brand owners and their respective retail networks. The study shows how brand owners can emphasise change by shifting from a passive and narrow observation of its downstream retail network to an active and broad observation, or at least develop a conscious approach...

  14. Functionally redundant RXLR effectors from Phytophthora infestans act at different steps to suppress early flg22-triggered immunity.

    Science.gov (United States)

    Zheng, Xiangzi; McLellan, Hazel; Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C; Gilroy, Eleanor M; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R J; Brunner, Frédéric

    2014-04-01

    Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular

  15. Functionally redundant RXLR effectors from Phytophthora infestans act at different steps to suppress early flg22-triggered immunity.

    Directory of Open Access Journals (Sweden)

    Xiangzi Zheng

    2014-04-01

    Full Text Available Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs, such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs, the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI, significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the

  16. Upstream and Downstream Influence in STBLI Instability

    Science.gov (United States)

    Martin, Pino; Priebe, Stephan; Helm, Clara

    2016-11-01

    Priebe and Martín (JFM, 2012) show that the low-frequency unsteadiness in shockwave and turbulent boundary layer interactions (STBLI) is governed by an inviscid instability. Priebe, Tu, Martín and Rowley (JFM, 2016) show that the instability is an inviscid centrifugal one, i.e Görtlerlike vortices. Previous works had given differing conclusions as to whether the low-frequency unsteadiness in STBLI is caused by an upstream or downstream mechanism. In this paper, we reconcile these opposite views and show that upstream and downstream correlations co-exist in the context of the nature of Görtler vortices. We find that the instability is similar to that in separated subsonic and laminar flows. Since the turbulence is modulated but passive to the global mode, the turbulent separated flows are amenable to linear global analysis. As such, the characteristic length and time scales, and the receptivity of the global mode might be determined, and low-order models that represent the low-frequency dynamics in STBLI might be developed. The centrifugal instability persists even under hypersonic conditions. This work is funded by the AFOSR Grant Number AF9550-15-1-0284 with Dr. Ivett Leyva.

  17. G Proteins and Regulation of Effector Function

    Directory of Open Access Journals (Sweden)

    A.R. Dehpour

    1991-07-01

    Full Text Available Cell surface receptors use a variety of membrane signalling mechanisms to translate information encoded in neurotransmitters, hormones, and growth factors into cellular responses.Collectively these mechanisms are refered to as transmembrane signalling or signal transduction. In the simplest example,the process involves a receptor protein-encompassed ion channel whose conductance is regulated by receptor activation.A second type of transmembrane signalling system involves the coupling of at least three separate components, a receptor protein, a guanine nucleotide binding protein (G protein , and an effector mechanism. In some receptor" effector systems the signal transduction pathways is entirely confined to the membrane, in which no intracellular messenger is involved.Alternatively, the activity of an enzyme may be changed to generate a specific intracellular signal molecule or second messenger. Receptors in this latter category may regulate the activity of adenylyl cyclase in a positive manner through a stimulatory G protein( G or in a negative manner through an inhibitory G protein( G. thereby controlling the intracellular level of cAMP. Another membrane- associated enzyme, similar to adenylate cyclase, is phospholipase C which catalizes the hydrolysis of PIP2into IP3and DAG. Phospholipase C coupled receptors are physiologically very important because both products of the reaction act as a second messenger; diacylglycerol activates protein kinase C and IP3 stimulates calcium release from Intracellular stores.

  18. Metabolic priming by a secreted fungal effector.

    Science.gov (United States)

    Djamei, Armin; Schipper, Kerstin; Rabe, Franziska; Ghosh, Anupama; Vincon, Volker; Kahnt, Jörg; Osorio, Sonia; Tohge, Takayuki; Fernie, Alisdair R; Feussner, Ivo; Feussner, Kirstin; Meinicke, Peter; Stierhof, York-Dieter; Schwarz, Heinz; Macek, Boris; Mann, Matthias; Kahmann, Regine

    2011-10-05

    Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

  19. Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

    Science.gov (United States)

    Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

    2015-10-13

    Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

  20. AND STEP-SHAPED DOWNSTREAM SECTION

    Directory of Open Access Journals (Sweden)

    Bal'zannikov Mihail Ivanovich

    2012-10-01

    The article also presents the results of the hydraulic research of the pilot model of the proposed structure. The research has proven that the suppression of any excessive kinetic energy of the discharged water flows falling onto the step-shaped and curving section or the step-shaped section the shape of which is similar to the one made of geosynthetic shells is more efficient that the suppression provided by the smooth section by 50 % to 60 %, and the above suppression is almost equal to the one provided by the well-known step-shaped section. This conclusion can serve as the proof of the high rate of energy suppression demonstrated by the earth-filled dam designed at Samara University of Architecture and Civil Engineering. The dam in question has geosynthetic shells that connect the downstream section to the body of the dam.

  1. Downstream Thermal Evolution of Vortex Cores

    Science.gov (United States)

    Gómez-Barea, A.; Herrada, M. A.; Pérez-Saborid, M.; Barrero, A.

    1999-11-01

    The downstream evolution of the total temperature field in a quasi-incompressible axisymmetric vortex core has been computed. Starting at an initial station (z=0) with velocity profiles of the Burgers type and given temperature distributions, the numerical results of the evolution show that, according to experimental results, the total temperature in the near-axis region decreases substantially due to the work done by pressure and viscous forces together with the effect of both convection and conduction of heat. Depending on the values of the parameters characterizing the initial profiles and on the value of the Prandtl number, the vortex either breaks down or eventually reaches a self-similar regime. The results obtained shed light on the basic physics involved in the thermal separation phenomenon which appears inside Ranque-Hilsch vortex tubes.

  2. Channel changes downstream from a dam

    Science.gov (United States)

    Hadley, R.F.; Emmett, W.W.

    1998-01-01

    A flood-control dam was completed during 1979 on Bear Creek, a small tributary stream to the South Platte River in the Denver, Colorado, area. Before and after dam closure, repetitive surveys between 1977 and 1992 at five cross sections downstream of the dam documented changes in channel morphology. During this 15-year period, channel width increased slightly, but channel depth increased by more than 40 percent. Within the study reach, stream gradient decreased and median bed material sizes coarsened from sand in the pools and fine gravel on the riffle to a median coarse gravel throughout the reach. The most striking visual change was from a sparse growth of streamside grasses to a dense growth of riparian woody vegetation.

  3. Ammonia downstream from HH 80 North

    Science.gov (United States)

    Girart, Jose M.; Rodriguez, Luis F.; Anglada, Guillem; Estalella, Robert; Torrelles, Jose, M.; Marti, Josep; Pena, Miriam; Ayala, Sandra; Curiel, Salvador; Noriega-Crespo, Alberto

    1994-01-01

    HH 80-81 are two optically visible Herbig-Haro (HH) objects located about 5 minutes south of their exciting source IRAS 18162-2048. Displaced symmetrically to the north of this luminous IRAS source, a possible HH counterpart was recently detected as a radio continuum source with the very large array (VLA). This radio source, HH 80 North, has been proposed to be a member of the Herbig-Haro class since its centimeter flux density, angular size, spectral index, and morphology are all similar to those of HH 80. However, no object has been detected at optical wavelengths at the position of HH 80 North, possibly because of high extinction, and the confirmation of the radio continuum source as an HH object has not been possible. In the prototypical Herbig-Haro objects HH 1 and 2, ammonia emission has been detected downstream of the flow in both objects. This detection has been intepreted as a result of an enhancement in the ammonia emission produced by the radiation field of the shock associated with the HH object. In this Letter we report the detection of the (1,1) and (2,2) inversion transitions of ammonia downstream HH 80 North. This detection gives strong suppport to the interpretation of HH 80 North as a heavily obscured HH object. In addition, we suggest that ammonia emission may be a tracer of embedded Herbig-Haro objects in other regions of star formation. A 60 micrometer IRAS source could be associated with HH 80 North and with the ammonia condensation. A tentative explanation for the far-infrared emission as arising in dust heated by their optical and UV radiation of the HH object is presented.

  4. Early Decision: Effector and Effector Memory T Cell Differentiation in Chronic Infection

    Science.gov (United States)

    Opata, Michael M.; Stephens, Robin

    2013-01-01

    As effector memory T cells (Tem) are the predominant population elicited by chronic parasitic infections, increasing our knowledge of their function, survival and derivation, as phenotypically and functionally distinct from central memory and effector T cells will be critical to vaccine development for these diseases. In some infections, memory T cells maintain increased effector functions, however; this may require the presence of continued antigen, which can also lead to T cell exhaustion. Alternatively, in the absence of antigen, only the increase in the number of memory cells remains, without enhanced functionality as central memory. In order to understand the requirement for antigen and the potential for longevity or protection, the derivation of each type of memory must be understood. A thorough review of the data establishes the existence of both memory (Tmem) precursors and effector T cells (Teff) from the first hours of an immune response. This suggests a new paradigm of Tmem differentiation distinct from the proposition that Tmem only appear after the contraction of Teff. Several signals have been shown to be important in the generation of memory T cells, such as the integrated strength of “signals 1-3” of antigen presentation (antigen receptor, co-stimulation, cytokines) as perceived by each T cell clone. Given that these signals integrated at antigen presentation cells have been shown to determine the outcome of Teff and Tmem phenotypes and numbers, this decision must be made at a very early stage. It would appear that the overwhelming expansion of effector T cells and the inability to phenotypically distinguish memory T cells at early time points has masked this important decision point. This does not rule out an effect of repeated stimulation or chronic inflammatory milieu on populations generated in these early stages. Recent studies suggest that Tmem are derived from early Teff, and we suggest that this includes Tem as well as Tcm. Therefore, we

  5. Active membrane cholesterol as a physiological effector.

    Science.gov (United States)

    Lange, Yvonne; Steck, Theodore L

    2016-09-01

    Sterols associate preferentially with plasma membrane sphingolipids and saturated phospholipids to form stoichiometric complexes. Cholesterol in molar excess of the capacity of these polar bilayer lipids has a high accessibility and fugacity; we call this fraction active cholesterol. This review first considers how active cholesterol serves as an upstream regulator of cellular sterol homeostasis. The mechanism appears to utilize the redistribution of active cholesterol down its diffusional gradient to the endoplasmic reticulum and mitochondria, where it binds multiple effectors and directs their feedback activity. We have also reviewed a broad literature in search of a role for active cholesterol (as opposed to bulk cholesterol or lipid domains such as rafts) in the activity of diverse membrane proteins. Several systems provide such evidence, implicating, in particular, caveolin-1, various kinds of ABC-type cholesterol transporters, solute transporters, receptors and ion channels. We suggest that this larger role for active cholesterol warrants close attention and can be tested easily.

  6. Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges

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    Humira eSonah

    2016-02-01

    Full Text Available Effector proteins are mostly secretory proteins that stimulate plant infection by manipulating the host response. Identifying fungal effector proteins and understanding their function is of great importance in efforts to curb losses to plant diseases. Recent advances in high-throughput sequencing technologies have facilitated the availability of several fungal genomes and thousands of transcriptomes. As a result, the growing amount of genomic information has provided great opportunities to identify putative effector proteins in different fungal species. There is little consensus over the annotation and functionality of effector proteins, and mostly small secretory proteins are considered as effector proteins, a concept that tends to overestimate the number of proteins involved in a plant-pathogen interaction. With the characterization of Avr genes, criteria for computational prediction of effector proteins are becoming more efficient. There are hundreds of tools available for the identification of conserved motifs, signature sequences and structural features in the proteins. Many pipelines and online servers, which combine several tools, are made available to perform genome-wide identification of effector proteins. In this review, available tools and pipelines, their strength and limitations for effective identification of fungal effector proteins are discussed. We also present an exhaustive list of classically secreted proteins along with their key conserved motifs found in 12 common plant pathogens (11 fungi and one oomycete through an analytical pipeline.

  7. Microbial production of scleroglucan and downstream processing

    Directory of Open Access Journals (Sweden)

    Natalia Alejandra Castillo

    2015-10-01

    Full Text Available Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong global market, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a beta-1,3-beta-1,6-glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length and/or molecular weight depending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery, cosmetic and pharmaceutical products, biocompatible materials, etc., and biomedical (immunoceutical, antitumor, etc. applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high EPS concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selected strains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined.

  8. Microbial production of scleroglucan and downstream processing

    Science.gov (United States)

    Castillo, Natalia A.; Valdez, Alejandra L.; Fariña, Julia I.

    2015-01-01

    Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong global market, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a β-1,3-β-1,6-glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length, and/or molecular weight depending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery, cosmetic and pharmaceutical products, biocompatible materials, etc.), and biomedical (immunoceutical, antitumor, etc.) applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high exopolysaccharide concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selected strains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined. PMID:26528259

  9. Tissue specific heterogeneity in effector immune cell response

    Directory of Open Access Journals (Sweden)

    Saba eTufail

    2013-08-01

    Full Text Available Post pathogen invasion, migration of effector T-cell subsets to specific tissue locations is of prime importance for generation of robust immune response. Effector T cells are imprinted with distinct ‘homing codes’ (adhesion molecules and chemokine receptors during activation which regulate their targeted trafficking to specific tissues. Internal cues in the lymph node microenvironment along with external stimuli from food (vitamin A and sunlight (vitamin D3 prime dendritic cells, imprinting them to play centrestage in the induction of tissue tropism in effector T cells. B cells as well, in a manner similar to effector T cells, exhibit tissue tropic migration. In this review, we have focused on the factors regulating the generation and migration of effector T cells to various tissues alongwith giving an overview of tissue tropism in B cells.

  10. Type III effector-mediated processes in Salmonella infection.

    Science.gov (United States)

    van der Heijden, Joris; Finlay, B Brett

    2012-06-01

    Salmonella is one of the most successful bacterial pathogens that infect humans in both developed and developing countries. In order to cause infection, Salmonella uses type III secretion systems to inject bacterial effector proteins into host cells. In the age of antibiotic resistance, researchers have been looking for new strategies to reduce Salmonella infection. To understand infection and to analyze type III secretion as a potential therapeutic target, research has focused on identification of effectors, characterization of effector functions and how they contribute to disease. Many effector-mediated processes have been identified that contribute to infection but thus far no specific treatment has been found. In this perspective we discuss our current understanding of effector-mediated processes and discuss new techniques and approaches that may help us to find a solution to this worldwide problem.

  11. Carbonic Anhydrases Function in Anther Cell Differentiation Downstream of the Receptor-Like Kinase EMS1.

    Science.gov (United States)

    Huang, Jian; Li, Zhiyong; Biener, Gabriel; Xiong, Erhui; Malik, Shikha; Eaton, Nathan; Zhao, Catherine Z; Raicu, Valerica; Kong, Hongzhi; Zhao, Dazhong

    2017-06-01

    Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana Here, we report the identification of β-carbonic anhydrases (βCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with βCA proteins. Loss of function of βCA genes caused defective tapetal cell differentiation, while overexpression of βCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates βCA1 at four sites, resulting in increased βCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of βCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. βCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of βCA in controlling cell differentiation and provide insights into the posttranslational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation. © 2017 American Society of Plant Biologists. All rights reserved.

  12. A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib.

    Science.gov (United States)

    Sun, Tianliang; Yang, Lida; Kaur, Harmandeep; Pestel, Jenny; Looso, Mario; Nolte, Hendrik; Krasel, Cornelius; Heil, Daniel; Krishnan, Ramesh K; Santoni, Marie-Josée; Borg, Jean-Paul; Bünemann, Moritz; Offermanns, Stefan; Swiercz, Jakub M; Worzfeld, Thomas

    2017-01-02

    Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration. © 2017 Sun et al.

  13. Protective Effector Cells of the Recombinant Asp f3 Anti-Aspergillosis Vaccine

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    Diana eDiaz-Arevalo

    2012-08-01

    Full Text Available An Aspergillus fumigatus vaccine based on recombinant Asp f3 protein has the potential to prevent aspergillosis in humans, a devastating fungal disease that is the prime obstacle to the success of hematopoietic cell transplantation. This vaccine protects cortisone acetate (CA-immunosuppressed mice from invasive pulmonary aspergillosis via CD4+ T cell mediators. Aside from these mediators, the nature of downstream fungicidal effectors is not well understood. Neutrophils and macrophages protect immunocompetent individuals from invasive fungal infections, and selective neutrophil depletion rendered mice susceptible to aspergillosis whereas macrophage depletion failed to increase fungal susceptibility. We investigated the effect of neutrophil depletion on rAsp f3 vaccine protection, and explored differences in pathophysiology and susceptibility between CA-immunosuppression and neutrophil depletion. In addition to being protective under CA-immunosuppression, the vaccine also had a protective effect in neutrophil-depleted mice. However, in non-immunized mice, a ten-fold higher conidial dose was required to induce similar susceptibility to infection with neutrophil-depletion than with CA- immunosuppression. The lungs of non-immunized neutrophil-depleted mice became invaded by a patchy dense mycelium with highly branched hyphae, and the peribronchial inflammatory infiltrate consisted mainly of CD3+ T cells and largely lacked macrophages. In contrast, lungs of non-immunized CA-immunosuppressed mice were more evenly scattered with short hyphal elements. With rAsp f3-vaccination, the lungs were largely clear of fungal burden under either immunosuppressive condition. We conclude that neutrophils, although substantial for innate antifungal protection of immunocompetent hosts, are not the relevant effectors for rAsp f3-vaccine derived protection of immunosuppressed hosts. It is therefore more likely that macrophages represent the crucial effectors of the r

  14. Plant Photosynthetic Responses During Insect Effector-Triggered Plant Susceptibility and Immunity.

    Science.gov (United States)

    Gramig, Greta G; Harris, Marion O

    2015-06-01

    Gall-inducing insects are known for altering source-sink relationships within plants. Changes in photosynthesis may contribute to this phenomenon. We investigated photosynthetic responses in wheat [Triticum aestivum L. (Poaceae: Triticeae)] seedlings attacked by the Hessian fly [Mayetiola destructor (Say) (Diptera: Cecidomyiidae], which uses a salivary effector-based strategy to induce a gall nutritive tissue in susceptible plants. Resistant plants have surveillance systems mediated by products of Resistance (R) genes. Detection of a specific salivary effector triggers downstream responses that result in a resistance that kills neonate larvae. A 2 × 2 factorial design was used to study maximum leaf photosynthetic assimilation and stomatal conductance rates. The plant treatments were-resistant or susceptible wheat lines expressing or not expressing the H13 resistance gene. The insect treatments were-no attack (control) or attack by larvae killed by H13 gene-mediated resistance. Photosynthesis was measured for the second and third leaves of the seedling, the latter being the only leaf directly attacked by larvae. We predicted effector-based attack would trigger increases in photosynthetic rates in susceptible but not resistant plants. For susceptible plants, attack was associated with increases (relative to controls) in photosynthesis for the third but not the second leaf. For resistant plants, attack was associated with increases in photosynthesis for both the second and third leaves. Mechanisms underlying the increases appeared to differ. Resistant plants exhibited responses suggesting altered source-sink relationships. Susceptible plants exhibited responses suggesting a mechanism other than altered source-sink relationships, possibly changes in water relations that contributed to increased stomatal conductance.

  15. A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths.

    Science.gov (United States)

    Boudinot, Pierre; Zou, Jun; Ota, Tatsuya; Buonocore, Francesco; Scapigliati, Giuseppe; Canapa, Adriana; Cannon, John; Litman, Gary; Hansen, John D

    2014-09-01

    The recent availability of both robust transcriptome and genome resources for coelacanth (Latimeria chalumnae) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods.

  16. A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths

    Science.gov (United States)

    Boudinot, Pierre; Zou, Jun; Ota, Tatsuya; Buonocore, Francesco; Scapigliati, Giuseppe; Canapa, Adriana; Cannon, John; Litman, Gary; Hansen, John D.

    2014-01-01

    The recent availability of both robust transcriptome and genome resources for coelacanth (Latimeria chalumnae) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods.

  17. Interplay between Rab27a effectors in pancreatic β-cells

    Institute of Scientific and Technical Information of China (English)

    Mami Yamaoka; Toshimasa Ishizaki; Toshihide Kimura

    2015-01-01

    The small GTPase Rab27a is a member of the Rab familythat is involved in membrane trafficking in various kindsof cells. Rab27a has GTP- and GDP-bound forms, andtheir interconversion regulates intracellular signalingpathways. Typically, only a GTP-bound GTPase binds itsspecific effectors with the resulting downstream signalscontrolling specific cellular functions. We previouslyidentified novel Rab27a-interacting proteins. Surprisingly,some of these proteins interacted with GDP-boundRab27a. The present study reviews recent progressin our understanding of the roles of Rab27a and itseffectors in the secretory process. In pancreatic β-cells,GTP-bound Rab27a regulates insulin secretion at the preexocytoticstages via its GTP-specific effectors such asExophilin8/Slac2-c/MyRIP and Slp4/Granuphilin. Glucosestimulation causes insulin exocytosis. Glucose stimulationalso converts Rab27a from its GTP- to its GDP-boundform. GDP-bound Rab27a interacts with GDP-specificeffectors and controls endocytosis of the secretorymembrane. Thus, Rab27a cycling between GTP- andGDP-bound forms synchronizes with the recycling ofsecretory membrane to re-use the membrane and keepthe β-cell volume constant.

  18. DOWNSTREAM ECOCIDE FROM UPSTREAM WATER PIRACY

    Directory of Open Access Journals (Sweden)

    Miah Muhammad Adel

    2012-01-01

    Full Text Available Upstream India and downstream Bangladesh share more than 50 international rivers. India has set up water diversion constructions in more than 50% of these rivers, the largest one being on the Bangladesh’s northwest upon the Ganges River, puts Bangladesh’s Gangetic ecosystem at stake. In some border rivers, India has set up groins on her side of river banks. Also, Indian side pumps Bangladesh river water stealthily from border-rivers. Further, India is constructing another dam and reservoir upstream on the Barak River on the northeast of Bangladesh. Furthermore, India has chalked out a grand plan for river networking. Exploration has been made to assess the degree of the ecosystem degradation both inland and on the coast due to all water diversion constructions around the border, except for the Tipaimukh Dam in which case estimation of projected ecosystem degradation has been mentioned. Finally, Indian grand plan of river networking plan has been briefly touched upon. Site visitations, observations, surveys, measurements and interviews of professionals were made in the project country. Relevant literatures on this issue were reviewed in electronic and print databases. Related published articles in electronic and print media were systematically searched following the key words for the case. Finally, both electronic and print news media have been closely followed to know the latest developments on this issue. The reduced flow of the Ganges in Bangladesh has caused scarcity of fresh water, species endangerment and extinction, obstruction to livestock raising, loss of livelihoods, people’s displacement, changes in crop production, reduction in navigable routes, extreme weather, increased flood occurrences, scarcity of potable water, groundwater contamination, reduction in coastal sediment deposition, deterioration of the Ganges water quality and inland intrusion of saline water front. Water diversion constructions in other rivers have

  19. TRAF5 is a downstream target of MAVS in antiviral innate immune signaling.

    Directory of Open Access Journals (Sweden)

    Eric D Tang

    Full Text Available The recognition of nucleic acids by the innate immune system during viral infection results in the production of type I interferons and the activation of antiviral immune responses. The RNA helicases RIG-I and MDA-5 recognize distinct types of cytosolic RNA species and signal through the mitochondrial protein MAVS to stimulate the phosphorylation and activation of the transcription factors IRF3 and IRF7, thereby inducing type I interferon expression. Alternatively, the activation of NF-kappaB leads to proinflammatory cytokine production. The function of MAVS is dependent on both its C-terminal transmembrane (TM domain and N-terminal caspase recruitment domain (CARD. The TM domain mediates MAVS dimerization in response to viral RNA, allowing the CARD to bind to and activate the downstream effector TRAF3. Notably, dimerization of the MAVS CARD alone is sufficient to activate IRF3, IRF7, and NF-kappaB. However, TRAF3-deficient cells display only a partial reduction in interferon production in response to RNA virus infection and are not defective in NF-kappaB activation. Here we find that the related ubiquitin ligase TRAF5 is a downstream target of MAVS that mediates both IRF3 and NF-kappaB activation. The TM domain of MAVS allows it to dimerize and thereby associate with TRAF5 and induce its ubiquitination in a CARD-dependent manner. Also, NEMO is recruited to the dimerized MAVS CARD domain in a TRAF3 and TRAF5-dependent manner. Thus, our findings reveal a possible function for TRAF5 in mediating the activation of IRF3 and NF-kappaB downstream of MAVS through the recruitment of NEMO. TRAF5 may be a key molecule in the innate response against viral infection.

  20. Bioprospecting open reading frames for peptide effectors.

    Science.gov (United States)

    Xiong, Ling; Scott, Charles

    2014-01-01

    Recent successes in the development of small-molecule antagonists of protein-protein interactions designed based on co-crystal structures of peptides bound to their biological targets confirm that short peptides derived from interacting proteins can be high-value ligands for pharmacologic validation of targets and for identification of druggable sites. Evolved sequence space is likely to be enriched for interacting peptides, but identifying minimal peptide effectors within genomic sequence can be labor intensive. Here we describe the use of incremental truncation to diversify genetic material on the scale of open reading frames into comprehensive libraries of constituent peptides. The approach is capable of generating peptides derived from both continuous and discontinuous sequence elements, and is compatible with the expression of free linear or backbone cyclic peptides, with peptides tethered to amino- or carboxyl-terminal fusion partners or with the expression of peptides displayed within protein scaffolds (peptide aptamers). Incremental truncation affords a valuable source of molecular diversity to interrogate the druggable genome or evaluate the therapeutic potential of candidate genes.

  1. Hippo pathway effector Yap promotes cardiac regeneration.

    Science.gov (United States)

    Xin, Mei; Kim, Yuri; Sutherland, Lillian B; Murakami, Masao; Qi, Xiaoxia; McAnally, John; Porrello, Enzo R; Mahmoud, Ahmed I; Tan, Wei; Shelton, John M; Richardson, James A; Sadek, Hesham A; Bassel-Duby, Rhonda; Olson, Eric N

    2013-08-20

    The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process.

  2. TAL effectors specificity stems from negative discrimination.

    Directory of Open Access Journals (Sweden)

    Basile I M Wicky

    Full Text Available Transcription Activator-Like (TAL effectors are DNA-binding proteins secreted by phytopathogenic bacteria that interfere with native cellular functions by binding to plant DNA promoters. The key element of their architecture is a domain of tandem-repeats with almost identical sequences. Most of the polymorphism is located at two consecutive amino acids termed Repeat Variable Diresidue (RVD. The discovery of a direct link between the RVD composition and the targeted nucleotide allowed the design of TAL-derived DNA-binding tools with programmable specificities that revolutionized the field of genome engineering. Despite structural data, the molecular origins of this specificity as well as the recognition mechanism have remained unclear. Molecular simulations of the recent crystal structures suggest that most of the protein-DNA binding energy originates from non-specific interactions between the DNA backbone and non-variable residues, while RVDs contributions are negligible. Based on dynamical and energetic considerations we postulate that, while the first RVD residue promotes helix breaks--allowing folding of TAL as a DNA-wrapping super-helix--the second provides specificity through a negative discrimination of matches. Furthermore, we propose a simple pharmacophore-like model for the rationalization of RVD-DNA interactions and the interpretation of experimental findings concerning shared affinities and binding efficiencies. The explanatory paradigm presented herein provides a better comprehension of this elegant architecture and we hope will allow for improved designs of TAL-derived biotechnological tools.

  3. Effector T cell differentiation: are master regulators of effector T cells still the masters?

    Science.gov (United States)

    Wang, Chao; Collins, Mary; Kuchroo, Vijay K

    2015-12-01

    Effector CD4 T cell lineages have been implicated as potent inducers of autoimmune diseases. Tbet, Gata3 and Rorgt are master transcriptional regulators of Th1, Th2 and Th17 lineages respectively and promote the distinct expression of signature cytokines. Significant progress has been made in understanding the transcriptional network that drives CD4 T cell differentiation, revealing novel points of regulation mediated by transcription factors, cell surface receptors, cytokines and chemokines. Epigenetic modifications and metabolic mediators define the transcriptional landscape in which master transcription factors operate and collaborate with a network of transcriptional modifiers to guide lineage specification, plasticity and function.

  4. Actin dynamics shape microglia effector functions.

    Science.gov (United States)

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

  5. Downstream Evolution of Longitudinal Embedded Vortices with Helical Structure

    DEFF Research Database (Denmark)

    Velte, Clara Marika; Okulov, Valery; Hansen, Martin Otto Laver

    2009-01-01

    In the present work the downstream development of device induced vortices with helical symmetry embedded in wall bounded flow on a bump is studied with the aid of Stereoscopic Particle Image Velocimetry (SPIV). The downstream evolution of characteristic parameters of helical vortices is studied...

  6. Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

    Science.gov (United States)

    Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

    2015-01-01

    The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery.

  7. Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires.

    Science.gov (United States)

    Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

    2016-02-01

    Infection by the human pathogen Legionella pneumophila relies on the translocation of ∼ 300 virulence proteins, termed effectors, which manipulate host cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species and predicted their effector repertoires using a previously validated machine learning approach. This analysis identified 5,885 predicted effectors. The effector repertoires of different Legionella species were found to be largely non-overlapping, and only seven core effectors were shared by all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from the natural protozoan hosts of these species. Furthermore, we detected numerous new conserved effector domains and discovered new domain combinations, which allowed the inference of as yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.

  8. An intelligent end-effector for a rehabilitation robot.

    Science.gov (United States)

    Gosine, R G; Harwin, W S; Furby, L J; Jackson, R D

    1989-01-01

    A UMI RTX robot, modified with limited end-effector sensors and a restricted but effective vision system, is currently used in a developmental education setting for severely physically disabled children. The low physical and cognitive abilities of the children involved in the project require a semi-autonomous robot with environmental sensing capability to operate in a task oriented mode. A variety of low-cost sensors including proximity, distance, force and slip sensors, have been investigated for integration in end-effectors for the RTX robot. The sensors employed on a modified end-effector are detailed and experimental results are presented. A design for an end-effector with integrated sensors is discussed. The integration of the sensor information into a high-level, task-oriented programming language is detailed and examples of high-level control sequences using sensor inputs are presented. Finally, the development of intelligent gripping strategies based on sensor information is discussed.

  9. Characterization of the largest effector gene cluster of Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Thomas Brefort

    2014-07-01

    Full Text Available In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

  10. Characterization of the largest effector gene cluster of Ustilago maydis.

    Science.gov (United States)

    Brefort, Thomas; Tanaka, Shigeyuki; Neidig, Nina; Doehlemann, Gunther; Vincon, Volker; Kahmann, Regine

    2014-07-01

    In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

  11. Functional heterogeneity of human effector CD8+ T cells.

    Science.gov (United States)

    Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

    2012-02-09

    Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

  12. A dock and coalesce mechanism driven by hydrophobic interactions governs Cdc42 binding with its effector protein ACK.

    Science.gov (United States)

    Tetley, George J N; Mott, Helen R; Cooley, R Neil; Owen, Darerca

    2017-07-07

    Cdc42 is a Rho-family small G protein that has been widely studied for its role in controlling the actin cytoskeleton and plays a part in several potentially oncogenic signaling networks. Similar to most other small G proteins, Cdc42 binds to many downstream effector proteins to elicit its cellular effects. These effector proteins all engage the same face of Cdc42, the conformation of which is governed by the activation state of the G protein. Previously, the importance of individual residues in conferring binding affinity has been explored for residues within Cdc42 for three of its Cdc42/Rac interactive binding (CRIB) effectors, activated Cdc42 kinase (ACK), p21-activated kinase (PAK), and Wiskott-Aldrich syndrome protein (WASP). Here, in a complementary study, we have used our structure of Cdc42 bound to ACK via an intrinsically disordered ACK region to guide an analysis of the Cdc42 interface on ACK, creating a panel of mutant proteins with which we can now describe the complete energetic landscape of the Cdc42-binding site on ACK. Our data suggest that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. These residues are centered on the CRIB region, with the complete binding region anchored at each end by hydrophobic interactions. These findings suggest that ACK adopts a dock and coalesce binding mechanism with Cdc42. In contrast to other CRIB-family effectors and indeed other intrinsically disordered proteins, hydrophobic residues likely drive Cdc42-ACK binding. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. BMX acts downstream of PI3K to promote colorectal cancer cell survival and pathway inhibition sensitizes to the BH3 mimetic ABT-737.

    Science.gov (United States)

    Potter, Danielle S; Kelly, Paul; Denneny, Olive; Juvin, Veronique; Stephens, Len R; Dive, Caroline; Morrow, Christopher J

    2014-02-01

    Evasion of apoptosis is a hallmark of cancer, and reversing this process by inhibition of survival signaling pathways is a potential therapeutic strategy. Phosphoinositide 3-kinase (PI3K) signaling can promote cell survival and is upregulated in solid tumor types, including colorectal cancer (CRC), although these effects are context dependent. The role of PI3K in tumorigenesis combined with their amenability to specific inhibition makes them attractive drug targets. However, we observed that inhibition of PI3K in HCT116, DLD-1, and SW620 CRC cells did not induce apoptotic cell death. Moreover, these cells were relatively resistant to the Bcl-2 homology domain 3 (BH3) mimetic ABT-737, which directly targets the Bcl-2 family of apoptosis regulators. To test the hypothesis that PI3K inhibition lowers the apoptotic threshold without causing apoptosis per se, PI3K inhibitors were combined with ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis by 2.3- to 4.5-fold and reduced expression levels of MCL-1, the resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4- to 2.4-fold in CRC cells with small interfering RNA-depleted MCL-1, indicative of additional sensitizing mechanisms. The observation that ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR, implicated a novel PI3K-dependant pathway. To elucidate this, an RNA interference (RNAi) screen of potential downstream effectors of PI3K signaling was conducted, which demonstrated that knockdown of the TEC kinase BMX sensitized to ABT-737. This suggests that BMX is an antiapoptotic downstream effector of PI3K, independent of AKT.

  14. BMX Acts Downstream of PI3K to Promote Colorectal Cancer Cell Survival and Pathway Inhibition Sensitizes to the BH3 Mimetic ABT-737

    Directory of Open Access Journals (Sweden)

    Danielle S. Potter

    2014-02-01

    Full Text Available Evasion of apoptosis is a hallmark of cancer, and reversing this process by inhibition of survival signaling pathways is a potential therapeutic strategy. Phosphoinositide 3-kinase (PI3K signaling can promote cell survival and is upregulated in solid tumor types, including colorectal cancer (CRC, although these effects are context dependent. The role of PI3K in tumorigenesis combined with their amenability to specific inhibition makes them attractive drug targets. However, we observed that inhibition of PI3K in HCT116, DLD-1, and SW620 CRC cells did not induce apoptotic cell death. Moreover, these cells were relatively resistant to the Bcl-2 homology domain 3 (BH3 mimetic ABT-737, which directly targets the Bcl-2 family of apoptosis regulators. To test the hypothesis that PI3K inhibition lowers the apoptotic threshold without causing apoptosis per se, PI3K inhibitors were combined with ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis by 2.3- to 4.5-fold and reduced expression levels of MCL-1, the resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4- to 2.4-fold in CRC cells with small interfering RNA-depleted MCL-1, indicative of additional sensitizing mechanisms. The observation that ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR, implicated a novel PI3K-dependant pathway. To elucidate this, an RNA interference (RNAi screen of potential downstream effectors of PI3K signaling was conducted, which demonstrated that knockdown of the TEC kinase BMX sensitized to ABT-737. This suggests that BMX is an antiapoptotic downstream effector of PI3K, independent of AKT.

  15. MATHEMATICAL MODELLING OF DEGRADATION AND FLUVIAL PROCESS DOWNSTREAM RESERVOIRS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To research into the problem of degradation and fluvial process downstream reservoirs and its influence on flood control and navigation, a 1-D mathematical model of degradation and fluvial process downstream the reservoir was established in this paper. The non-equilibrium transport of non-uniform suspended load, the non-uniform bedload transport and bed material sorting were considered in the model. Some techniques were suggested for some problems in calculation, such as the effective suspended load carrying capacity of the different reaches of bed materials, the coefficient of suspended load carrying capacity, the recovering coefficient of carrying capacity, the mixed layer thickness, the bedload transport width, bifurcation and confluence of main and branch channel, and the distribution of deposition and erosion along the cross section, etc. The model was tested by the data of degradation downstream the Danjiangkou reservoir on the Hanjiang River and the data of degradation downstream the Gezhouba Project on the Yangtze River.

  16. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    Energy Technology Data Exchange (ETDEWEB)

    Daber,R.; Stayrook, S.; Rosenberg, A.; Lewis, M.

    2007-01-01

    The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

  17. Target selection biases from recent experience transfer across effectors.

    Science.gov (United States)

    Moher, Jeff; Song, Joo-Hyun

    2016-02-01

    Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions.

  18. MATHEMATICAL MODEL OF RIVER BED CHANGE DOWNSTREAM OF XIAOLANGDI RESERVOIR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A mathematical model of river bed change downstream of the Xiaolangdi Reservoir was developed based on the most recent achievement of sediment theory in the Yellow River. The model was verified by the comparison of computed results and measured data from 1986 to 1996. Numerical prediction of the erosion and deposition downstream of the Xiaolangdi Reservoir in its first operation year was carried out, and a series of suggestions were given for reservoir operation mode in its early operation period.

  19. Downstream-based Scheduling for Energy Conservation in Green EPONs

    KAUST Repository

    Chen, Shen

    2012-05-01

    Maximizing the optical network unit’s (ONU) sleep time is an effective approach for achieving maximum energy conservation in green Ethernet passive optical networks (EPONs). While overlapping downstream and upstream ONU transmissions can maximize the ONU sleep time, it jeopardizes the quality of service (QoS) performance of the network, especially for downstream traffic in case the overlapping is based on the upstream time slot. In this paper, we study the downstream traffic performance in green EPONs under the limited service discipline and the upstream-based overlapped time window. Specifically, we first derive the expected mean packet delay, and then present a closed-form expression of the ONU sleep time, setting identical upstream/downstream transmission cycle times based on a maximum downstream traffic delay re-quirement. With the proposed system model, we present a novel downstream bandwidth allocation scheme for energy conservation in green EPONs. Simulation results verify the proposed model and highlight the advantages of our scheme over conventional approaches.

  20. Phytopathogen effectors subverting host immunity: different foes, similar battleground.

    Science.gov (United States)

    Dou, Daolong; Zhou, Jian-Min

    2012-10-18

    Phytopathogenic bacteria, fungi, and oomycetes invade and colonize their host plants through distinct routes. These pathogens secrete diverse groups of effector proteins that aid infection and establishment of different parasitic lifestyles. Despite this diversity, a comparison of different plant-pathogen systems has revealed remarkable similarities in the host immune pathways targeted by effectors from distinct pathogen groups. Immune signaling pathways mediated by pattern recognition receptors, phytohormone homeostasis or signaling, defenses associated with host secretory pathways and pathogen penetrations, and plant cell death represent some of the key processes controlling disease resistance against diverse pathogens. These immune pathways are targeted by effectors that carry a wide range of biochemical functions and are secreted by completely different pathogen groups, suggesting that these pathways are a common battleground encountered by many plant pathogens. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Effector and suppressor T cells in celiac disease.

    Science.gov (United States)

    Mazzarella, Giuseppe

    2015-06-28

    Celiac disease (CD) is a T-cell mediated immune disease in which gliadin-derived peptides activate lamina propria effector CD4+ T cells. This activation leads to the release of cytokines, compatible with a Th1-like pattern, which play a crucial role in the pathogenesis of CD, controlling many aspects of the inflammatory immune response. Recent studies have shown that a novel subset of effector T cells, characterized by expression of high levels of IL-17A, termed Th17 cells, plays a pathogenic role in CD. While these effector T cell subsets produce proinflammatory cytokines, which cause substantial tissue injury in vivo in CD, recent studies have suggested the existence of additional CD4(+) T cell subsets with suppressor functions. These subsets include type 1 regulatory T cells and CD25(+)CD4(+) regulatory T cells, expressing the master transcription factor Foxp3, which have important implications for disease progression.

  2. Effector proteins that modulate plant--insect interactions.

    Science.gov (United States)

    Hogenhout, Saskia A; Bos, Jorunn I B

    2011-08-01

    Insect herbivores have highly diverse life cycles and feeding behaviors. They establish close interactions with their plant hosts and suppress plant defenses. Chewing herbivores evoke characteristic defense responses distinguishable from general mechanical damage. In addition, piercing-sucking hemipteran insects display typical feeding behavior that suggests active suppression of plant defense responses. Effectors that modulate plant defenses have been identified in the saliva of these insects. Tools for high-throughput effector identification and functional characterization have been developed. In addition, in some insect species it is possible to silence gene expression by RNAi. Together, this technological progress has enabled the identification of insect herbivore effectors and their targets that will lead to the development of novel strategies for pest resistances in plants.

  3. Structural Insights into Rab27 Recruitment by its Effectors

    Science.gov (United States)

    M. G. Chavas, Leonard; Ihara, Kentaro; Kawasaki, Masato; Wakatsuki, Soichi

    An increasing number of Rab GTPases associated with partial dysfunction has been linked to several human diseases characterized by a diminution in vesicle transport. Due to its direct implication in human disorders, the Rab27 subfamily is considered as a standard for vesicle docking studies. By which mechanism Rab27 effectors distinguish among the pool of Rab GTPases? What is the underneath machinery rendering the interaction of eleven distinct effectors specific of Rab27 when compared to other Rabs of the secretory pathway? By solving the X-ray structures of Rab27, both in its inactive form and active form bound to the effector protein Slp2-a, attempts have been given to unravel the molecular basis of regulation of the delivering process of vesicles to fusion by the Rab27 subfamily.

  4. Salvador-warts-hippo signaling promotes Drosophila posterior follicle cell maturation downstream of notch.

    Science.gov (United States)

    Polesello, Cédric; Tapon, Nicolas

    2007-11-06

    The Salvador Warts Hippo (SWH) network limits tissue size in Drosophila and vertebrates [1]. Decreased SWH pathway activity gives rise to excess proliferation and reduced apoptosis. The core of the SWH network is composed of two serine/threonine kinases Hippo (Hpo) and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki) [1]. Two band 4.1 related proteins, Merlin (Mer) and Expanded (Ex), have been proposed to act upstream of Hpo, which in turn activates Wts ([1] for review). Wts phosphorylates and inhibits Yki, repressing the expression of Yki target genes [2-4]. Recently, several planar cell polarity (PCP) genes have been implicated in the SWH network in growth control [5-8]. Here, we show that, during oogenesis, the core components of the SWH network are required in posterior follicle cells (PFCs) competent to receive the Gurken (Grk)/TGFalpha signal emitted by the oocyte to control body axis formation. Our results suggest that the SWH network controls the expression of Hindsight, the downstream effector of Notch, required for follicle cell mitotic cycle-endocycle switch. The PCP members of the SWH network are not involved in this process, indicating that signaling upstream of Hpo varies according to developmental context.

  5. Identification of new secreted effectors in Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Geddes, Kaoru; Worley, Micah; Niemann, George; Heffron, Fred

    2005-10-01

    A common theme in bacterial pathogenesis is the secretion of bacterial products that modify cellular functions to overcome host defenses. Gram-negative bacterial pathogens use type III secretion systems (TTSSs) to inject effector proteins into host cells. The genes encoding the structural components of the type III secretion apparatus are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. A strategy was developed to identify virulence factors secreted by Salmonella enterica serovar Typhimurium into the host cell cytoplasm. We constructed a transposon, which we refer to as mini-Tn5-cycler, to generate translational fusions between Salmonella chromosomal genes and a fragment of the calmodulin-dependent adenylate cyclase gene derived from Bordetella pertussis (cyaA'). In-frame fusions to bacterial proteins that are secreted into the eukaryotic cell cytoplasm were identified by high levels of cyclic AMP in infected cells. The assay was sufficiently sensitive that a single secreted fusion could be identified among several hundred that were not secreted. This approach identified three new effectors as well as seven that have been previously characterized. A deletion of one of the new effectors, steA (Salmonella translocated effector A), attenuated virulence. In addition, SteA localizes to the trans-Golgi network in both transfected and infected cells. This approach has identified new secreted effector proteins in Salmonella and will likely be useful for other organisms, even those in which genetic manipulation is more difficult.

  6. Identification of Anaplasma marginale type IV secretion system effector proteins.

    Directory of Open Access Journals (Sweden)

    Svetlana Lockwood

    Full Text Available BACKGROUND: Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS. The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now. RESULTS: By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141 of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system. CONCLUSIONS: The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.

  7. Development and testing of the cooling coil cleaning end effector

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, K.I.; Mullen, O.D.; Powell, M.R.; Daly, D.S.; Engel, D.W.

    1997-09-30

    The Retrieval Process Development and Enhancement (KPD{ampersand}E) program has developed and tested an end effector to support the waste retrieval mission at the Idaho National Engineering and Environmental Laboratory (INEEL). The end effector was developed specifically to remove a sticky waste material from the cooling coils in the High Level Liquid Waste (HLLW) tank, and to vacuum up a sediment layer that has settled beneath the cooling coils. An extensive testing program was conducted in the hydraulic test bed (HTB) at the Pacific Northwest National Laboratory (PNNL) to evaluate the performance of the end effector under simulated in-tank conditions. A mock up of the cooling coils was installed in the test bed tank, and simulated waste materials were included to represent the sticky waste on the tubes and the particulate waste settled beneath them. The testing program focused on assessing long-duration mining strategies for cleaning the cooling coils and removing the particulate waste forms. The report describes the results of the end effector testing program at PNNL. Section 2 describes the physical characteristics of the HLLW tanks, including the layout of the cooling coils, and it also describes what is known of the waste forms in the tanks. Section 3 describes the cleaning and retrieval strategy that was used in developing the end effector design. Section 4 describes the cooling coil mockup in the hydraulic test bed. Section 5 discusses the rationale used in selecting the simulants for the tarry waste and particulate waste forms. Section 6 describes the tests that were performed to evaluate cleaning of the cooling coils and retrieval of the particulate simulant. Section 7 summarizes the cleaning and retrieval tests, assesses the relative importance of cleaning the cooling coils and retrieving the particulate waste, and suggests modifications that would simplify the end effector design.

  8. Yersinia type III effectors perturb host innate immune responses

    Science.gov (United States)

    Pha, Khavong; Navarro, Lorena

    2016-01-01

    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

  9. Yersinia type Ⅲ effectors perturb host innate immune responses

    Institute of Scientific and Technical Information of China (English)

    Khavong Pha; Lorena Navarro

    2016-01-01

    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type Ⅲ secretion system(T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp.(Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gramnegative bacteria that share in common a 70 kb virulence plasmid which encodes the T3 SS. Translocation of the Yersinia effector proteins(YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector

  10. Common themes in the design and function of bacterial effectors.

    Science.gov (United States)

    Galán, Jorge E

    2009-06-18

    Central to the biology of many pathogenic bacteria are a number of specialized machines, known as type III, type IV, or type VI protein secretion systems. These machines have specifically evolved to deliver bacterial effector proteins into host cells with the capacity to modulate a variety of cellular functions. The identification of the biochemical activities of many effector proteins, coupled with a better understanding of their potential contribution to pathogenesis, has revealed common themes in the evolutionary design and function of these remarkable bacterial proteins.

  11. Visual End-Effector Position Error Compensation for Planetary Robotics

    Science.gov (United States)

    Bajracharya, Max; DiCicco, Matthew; Backes, Paul; Nickels, Kevin

    2007-01-01

    This paper describes a vision-guided manipulation algorithm that improves arm end-effector positioning to subpixel accuracy and meets the highly restrictive imaging and computational constraints of a planetary robotic flight system. Analytical, simulation-based, and experimental analyses of the algorithm's effectiveness and sensitivity to camera and arm model error is presented along with results on several prototype research systems and 'ground-in-the-loop' technology experiments on the Mars Exploration Rover (MER) vehicles. A computationally efficient and robust subpixel end-effector fiducial detector that is instrumental to the algorithm's ability to achieve high accuracy is also described along with its validation results on MER data.

  12. Nanorobotic end-effectors: Design, fabrication, and in situ characterization

    Science.gov (United States)

    Fan, Zheng

    Nano-robotic end-effectors have promising applications for nano-fabrication, nano-manufacturing, nano-optics, nano-medical, and nano-sensing; however, low performances of the conventional end-effectors have prevented the widespread utilization of them in various fields. There are two major difficulties in developing the end-effectors: their nano-fabrication and their advanced characterization in the nanoscale. Here we introduce six types of end-effectors: the nanotube fountain pen (NFP), the super-fine nanoprobe, the metal-filled carbon nanotube (m CNT)-based sphere-on-pillar (SOP) nanoantennas, the tunneling nanosensor, and the nanowire-based memristor. The investigations on the NFP are focused on nano-fluidics and nano-fabrications. The NFP could direct write metallic "inks" and fabricating complex metal nanostructures from 0D to 3D with a position servo control, which is critically important to future large-scale, high-throughput nanodevice production. With the help of NFP, we could fabricate the end-effectors such as super-fine nanoprobe and m CNT-based SOP nanoantennas. Those end-effectors are able to detect local flaws or characterize the electrical/mechanical properties of the nanostructure. Moreover, using electron-energy-loss-spectroscopy (EELS) technique during the operation of the SOP optical antenna opens a new basis for the application of nano-robotic end-effectors. The technique allows advanced characterization of the physical changes, such as carrier diffusion, that are directly responsible for the device's properties. As the device was coupled with characterization techniques of scanning-trasmission-electron-microscopy (STEM), the development of tunneling nanosensor advances this field of science into quantum world. Furthermore, the combined STEM-EELS technique plays an important role in our understanding of the memristive switching performance in the nanowire-based memristor. The developments of those nano-robotic end-effectors expend the study

  13. Visual End-Effector Position Error Compensation for Planetary Robotics

    Science.gov (United States)

    Bajracharya, Max; DiCicco, Matthew; Backes, Paul; Nickels, Kevin

    2007-01-01

    This paper describes a vision-guided manipulation algorithm that improves arm end-effector positioning to subpixel accuracy and meets the highly restrictive imaging and computational constraints of a planetary robotic flight system. Analytical, simulation-based, and experimental analyses of the algorithm's effectiveness and sensitivity to camera and arm model error is presented along with results on several prototype research systems and 'ground-in-the-loop' technology experiments on the Mars Exploration Rover (MER) vehicles. A computationally efficient and robust subpixel end-effector fiducial detector that is instrumental to the algorithm's ability to achieve high accuracy is also described along with its validation results on MER data.

  14. Bacterial toxin effector membrane targeting: Outside in, then back again.

    Directory of Open Access Journals (Sweden)

    Brett eGeissler

    2012-05-01

    Full Text Available Pathogenic bacteria utilize multiple approaches to mediate their toxicity to eukaryotic cells. Dedicated protein machines deposit toxic effectors directly inside the host, whereas secreted toxins must enter cells independently of other bacterial components. Regardless of how they reach the cytosol, these toxic proteins must accurately identify their intracellular target before they can manipulate the host cell to benefit their associated bacteria. Within eukaryotic cells, individual targeting motifs and post-translational modifications spatially regulate host proteins. This review focuses on the strategies employed by bacterial effectors to associate with a frequently targeted location within eukaryotic cells, the plasma membrane.

  15. How do filamentous pathogens deliver effector proteins into plant cells?

    Directory of Open Access Journals (Sweden)

    Benjamin Petre

    2014-02-01

    Full Text Available Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens.

  16. How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells?

    Science.gov (United States)

    Petre, Benjamin; Kamoun, Sophien

    2014-01-01

    Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens. PMID:24586116

  17. HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus

    Science.gov (United States)

    Fusco, Dahlene N.; Pratt, Henry; Kandilas, Stephen; Cheon, Scarlett Se Yun; Lin, Wenyu; Cronkite, D. Alex; Basavappa, Megha; Jeffrey, Kate L.; Anselmo, Anthony; Sadreyev, Ruslan; Yapp, Clarence; Shi, Xu; O'Sullivan, John F.; Gerszten, Robert E.; Tomaru, Takuya; Yoshino, Satoshi; Satoh, Tetsurou; Chung, Raymond T.

    2017-01-01

    Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral

  18. Cdc42 Effector Protein 2 (XCEP2 is required for normal gastrulation and contributes to cellular adhesion in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Nelson Richard W

    2004-10-01

    Full Text Available Abstract Background Rho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis. Results In a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis, a gene encoding a Rho GTPase effector protein (Xenopus Cdc42 effector protein 2, or XCEP2 was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell

  19. Effector profiles distinguish formae speciales of Fusarium oxysporum.

    Science.gov (United States)

    van Dam, Peter; Fokkens, Like; Schmidt, Sarah M; Linmans, Jasper H J; Kistler, H Corby; Ma, Li-Jun; Rep, Martijn

    2016-11-01

    Formae speciales (ff.spp.) of the fungus Fusarium oxysporum are often polyphyletic within the species complex, making it impossible to identify them on the basis of conserved genes. However, sequences that determine host-specific pathogenicity may be expected to be similar between strains within the same forma specialis. Whole genome sequencing was performed on strains from five different ff.spp. (cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici). In each genome, genes for putative effectors were identified based on small size, secretion signal, and vicinity to a "miniature impala" transposable element. The candidate effector genes of all genomes were collected and the presence/absence patterns in each individual genome were clustered. Members of the same forma specialis turned out to group together, with cucurbit-infecting strains forming a supercluster separate from other ff.spp. Moreover, strains from different clonal lineages within the same forma specialis harbour identical effector gene sequences, supporting horizontal transfer of genetic material. These data offer new insight into the genetic basis of host specificity in the F. oxysporum species complex and show that (putative) effectors can be used to predict host specificity in F. oxysporum. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  20. Structure and evolution of barley powdery mildew effector candidates

    DEFF Research Database (Denmark)

    Pedersen, Carsten; Themaat, Emiel Ver Loren van; McGuffin, Liam J.;

    2012-01-01

    Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute...

  1. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation

    NARCIS (Netherlands)

    Jensen, K.D.C.; Wang, Y.; Tait Wonjo, E.D.; Shastri, A.J.; Hu, K.; Cornel, L.; Boedec, E.; Ong, Y.C.; Chien, Y.H.; Hunter, C.A.; Boothroyd, J.C.; Saeij, J.P.J.

    2011-01-01

    European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major g

  2. Plasmodium cellular effector mechanisms and the hepatic microenvironment

    Science.gov (United States)

    Frevert, Ute; Krzych, Urszula

    2015-01-01

    Plasmodium falciparum malaria remains one of the most serious health problems globally. Immunization with attenuated parasites elicits multiple cellular effector mechanisms capable of eliminating Plasmodium liver stages. However, malaria liver stage (LS) immunity is complex and the mechanisms effector T cells use to locate the few infected hepatocytes in the large liver in order to kill the intracellular LS parasites remain a mystery to date. Here, we review our current knowledge on the behavior of CD8 effector T cells in the hepatic microvasculature, in malaria and other hepatic infections. Taking into account the unique immunological and lymphogenic properties of the liver, we discuss whether classical granule-mediated cytotoxicity might eliminate infected hepatocytes via direct cell contact or whether cytokines might operate without cell–cell contact and kill Plasmodium LSs at a distance. A thorough understanding of the cellular effector mechanisms that lead to parasite death hence sterile protection is a prerequisite for the development of a successful malaria vaccine to protect the 40% of the world’s population currently at risk of Plasmodium infection. PMID:26074888

  3. Type IV secretion system of Brucella spp. and its effectors.

    Science.gov (United States)

    Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

    2015-01-01

    Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis.

  4. Identification and characterization of novel effectors of Cladosporium fulvum

    NARCIS (Netherlands)

    Ökmen, B.

    2013-01-01

    In order to establish disease, plant pathogenic fungi deliver effectors in the apoplastic space surrounding host cells as well as into host cells themselves to manipulate host physiology in favour of their own growth. Cladosporium fulvum is a non-obligate biotrophic fungus causing leaf mould disease

  5. How to conquer a tomato plant? Fusarium oxysporum effector targets

    NARCIS (Netherlands)

    de Sain, M.

    2016-01-01

    Pathogens secrete small proteins, called effectors, to alter the environment in their host to facilitate infection. The causal agent of Fusarium wilt on tomato, Fusarium oxysporum f. sp. lycopersici (Fol), secretes these proteins in the xylem sap of infected plants and hence they have been called Si

  6. Differential potencies of effector genes in adult Drosophila.

    Science.gov (United States)

    Thum, Andreas S; Knapek, Stephan; Rister, Jens; Dierichs-Schmitt, Eva; Heisenberg, Martin; Tanimoto, Hiromu

    2006-09-10

    The GAL4/UAS gene expression system in Drosophila has been crucial in revealing the behavioral significance of neural circuits. Transgene products that block neurotransmitter release and induce cell death have been proved to inhibit neural function powerfully. Here we compare the action of the five effector genes shibire(ts1), Tetanus toxin light chain (TNT), reaper, Diphtheria toxin A-chain (DTA), and inwardly rectifying potassium channel (Kir2.1) and show differences in their efficiency depending on the target cells and the timing of induction. Specifically, effectors blocking neuronal transmission or excitability led to adult-induced paralysis more efficiently than those causing cell ablation. We contrasted these differential potencies in adult to their actions during development. Furthermore, we induced TNT expression in the adult mushroom bodies. In contrast to the successful impairment in short-term olfactory memory by shibire(ts1), adult TNT expression in the same set of cells did not lead to any obvious impairment. Altogether, the efficiency of effector genes depends on properties of the targeted neurons. Thus, we conclude that the selection of the appropriate effector gene is critical for evaluating the function of neural circuits.

  7. Type IV Secretion System of Brucella spp. and its Effectors

    Directory of Open Access Journals (Sweden)

    Yuehua eKe

    2015-10-01

    Full Text Available Brucella spp. cause brucellosis in domestic and wild animals. They are intracellular bacterial pathogens and used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we will discuss roles of Brucella VirB T4SS and in more detail of all 15 identified effectors, which may be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells, suggesting that it plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. So, we listed some key molecular events in the intracellular life cycle of Brucella potentially targeted by the VirB T4SS effectors. Elucidating functions of the effectors secreted will be crucial to clarifying mechanism of T4SS during infection. Studying the effectors secreted by Brucella spp. might provide insights into the mechanisms by which the bacteria hijack the host signaling pathways, which help us to develop better vaccines and therapies against brucellosis.

  8. Targeting DNA double-strand breaks with TAL effector nucleases.

    Science.gov (United States)

    Christian, Michelle; Cermak, Tomas; Doyle, Erin L; Schmidt, Clarice; Zhang, Feng; Hummel, Aaron; Bogdanove, Adam J; Voytas, Daniel F

    2010-10-01

    Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

  9. Structure and evolution of barley powdery mildew effector candidates

    DEFF Research Database (Denmark)

    Pedersen, Carsten; Themaat, Emiel Ver Loren van; McGuffin, Liam J.;

    2012-01-01

    Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute...... requirement to suppress or avoid host immunity if it is to survive and cause disease....

  10. Experimental investigation of unsteady fan flow interaction with downstream struts

    Science.gov (United States)

    Ng, W. F.; Obrien, W. F.; Olsen, T. L.

    1986-07-01

    In the present study of the unsteady pressure field produced on fan rotor blades by interaction with downstream struts, a single stage, low speed axial-flow fan was instrumented with blade-mounted high frequency pressure transducers. In addition, stationary pressure problems were used to map out the flowfield. Fluctuating pressure measurements are presented for blade midspan and 85-percent span on both the suction and pressure surfaces of the rotor blades at several positions of the downstream struts, and for two different flow coefficients. The strut is found to produce an effect on the unsteady pressure field on the rotor blades; this effect exceeds that due to the stator at design rotor-stator-strut spacing, but it rapidly declines as the struts are moved downstream.

  11. Study of Scour Downstream Different Shapes of Culverts

    Directory of Open Access Journals (Sweden)

    Ibrahem Adel Al Hafed

    2013-04-01

    Full Text Available In this research a laboratory study was carried out to investigate the scour phenomenon in sandy soil beds downstream different shapes of culverts, Four   shapes of culverts such as circular, ellipse, rectangular and square were used. So, a best hydraulic section was used in rectangular and ellipse shapes. The study includes the measurement and comparison of maximum scour depth and length of scour hole downstream these different shapes of culverts. Also, the distribution of soil parts was studied. It was used five discharges for each one of culvert.              Laboratory results of this study showed that the minimum depth of scour in the same discharge occurred downstream ellipse culvert and then rectangular culvert and then square culvert and the last circular culvert.  

  12. Genome-scale identification of Legionella pneumophila effectors using a machine learning approach.

    Directory of Open Access Journals (Sweden)

    David Burstein

    2009-07-01

    Full Text Available A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine

  13. Downstream Processability of Crystal Habit-Modified Active Pharmaceutical Ingredient

    DEFF Research Database (Denmark)

    Pudasaini, Nawin; Upadhyay, Pratik Pankaj; Parker, Christian Richard

    2017-01-01

    Efficient downstream processing of active pharmaceutical ingredients (APIs) can depend strongly on their particulate properties, such as size and shape distributions. Especially in drug products with high API content, needle-like crystal habit of an API may show compromised flowability and tablet......Efficient downstream processing of active pharmaceutical ingredients (APIs) can depend strongly on their particulate properties, such as size and shape distributions. Especially in drug products with high API content, needle-like crystal habit of an API may show compromised flowability...

  14. Transition duct with divided upstream and downstream portions

    Energy Technology Data Exchange (ETDEWEB)

    McMahan, Kevin Weston; LeBegue, Jeffrey Scott; Maldonado, Jaime Javier; Dillard, Daniel Jackson; Flanagan, James Scott

    2015-07-14

    Turbine systems are provided. In one embodiment, a turbine system includes a transition duct comprising an inlet, an outlet, and a duct passage extending between the inlet and the outlet and defining a longitudinal axis, a radial axis, and a tangential axis. The outlet of the transition duct is offset from the inlet along the longitudinal axis and the tangential axis. The duct passage includes an upstream portion extending from the inlet and a downstream portion extending from the outlet. The turbine system further includes a rib extending from an outer surface of the duct passage, the rib dividing the upstream portion and the downstream portion.

  15. Anchors for effectors: subversion of phosphoinositide lipids by Legionella

    Directory of Open Access Journals (Sweden)

    Hubert eHilbi

    2011-04-01

    Full Text Available The facultative intracellular pathogen Legionella pneumophila replicates in free-living amoebae and macrophages within a distinct compartment, the Legionella-containing vacuole (LCV. LCV formation involves phosphoinositide (PI glycerolipids, which are key factors controlling vesicle trafficking pathways and membrane dynamics of eukaryotic cells. To govern the interactions with host cells, L. pneumophila employs the Icm/Dot type IV secretion system and more than 250 translocated effector proteins that presumably subvert host signaling and vesicle trafficking pathways. Some of the effector proteins anchor through distinct PIs to the cytosolic face of LCVs and promote the interaction with host vesicles and organelles, catalyze guanine nucleotide exchange of small GTPases, or bind to PI-metabolizing enzymes, such as OCRL1. The PI 5-phosphatase OCRL1 and its Dictyostelium homologue Dd5P4 restrict intracellular growth of L. pneumophila. Moreover, OCRL1/Dd5P4, PI 3-kinases (PI3Ks and PI4KIIIβ regulate LCV formation and localization of the effector protein SidC, which selectively decorates the LCV membrane. SidC or its 20 kDa P4C fragment are robust and specific probes for phosphatidylinositol-4-phosphate, and SidC can be targeted to purify intact LCVs by immuno-magnetic separation. Taken together, bacterial PI-binding effectors as well as host PIs and PI-modulating enzymes play a pivotal role for intracellular replication of L. pneumophila, and the PI-binding effectors are valuable tools for the analysis of eukaryotic PI lipids.

  16. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks.

    Science.gov (United States)

    Liu, Shuxi; Wang, Yue; Worley, Paul F; Mattson, Mark P; Gaiano, Nicholas

    2015-05-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here, we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling.

  17. Operation and maintenance manual for the common video end effector system (CVEE) system 6260

    Energy Technology Data Exchange (ETDEWEB)

    Pardini, A.F., Westinghouse Hanford

    1996-07-24

    This document defines the requirements for the operation,maintenance, and storage of the Common Video End Effector System (CVEE) used with the video end effectors as part of the Light Duty Utility Arm (LDUA) system.

  18. Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

    Science.gov (United States)

    Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

    2014-02-01

    During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

  19. A do-it-yourself protocol for simple transcription activator-like effector assembly

    Directory of Open Access Journals (Sweden)

    Uhde-Stone Claudia

    2013-01-01

    Full Text Available Abstract Background TALEs (transcription activator-like effectors are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, long set-up time and specific training requirements. Results We streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases and TALE-TFs (TALE-transcription factors under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target. Conclusions We established and validated a do-it-yourself system that enables individual researchers to assemble TALENs and TALE-TFs within 2 days. The simplified TALE assembly combined with multiple choices of vectors will facilitate the broad use of TALE technology.

  20. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants.

    Science.gov (United States)

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

  1. Flow diagnostics downstream of a tribladed rotor model

    DEFF Research Database (Denmark)

    Naumov, I. V.; Rahmanov, V. V.; Okulov, Valery

    2012-01-01

    This paper presents results of a study of vortex wake structures and measurements of instantaneous 3D velocity fields downstream of a triblade turbine model. Two operation modes of flow around the rotor with different tip speed ratios were tested. Initially the wake structures were visualized and...

  2. Extreme wave phenomena in down-stream running modulated waves

    NARCIS (Netherlands)

    Andonowati, A.; Karjanto, N.; van Groesen, Embrecht W.C.

    Modulational, Benjamin-Feir, instability is studied for the down-stream evolution of surface gravity waves. An explicit solution, the soliton on finite background, of the NLS equation in physical space is used to study various phenomena in detail. It is shown that for sufficiently long modulation

  3. Modeling downstream fining in sand-bed rivers. II: Application

    Science.gov (United States)

    Wright, S.; Parker, G.

    2005-01-01

    In this paper the model presented in the companion paper, Wright and Parker (2005) is applied to a generic river reach typical of a large, sand-bed river flowing into the ocean in order to investigate the mechanisms controlling longitudinal profile development and downstream fining. Three mechanisms which drive downstream fining are studied: a delta prograding into standing water, sea-level rise, and tectonic subsidence. Various rates of sea-level rise (typical of the late Holocene) and tectonic subsidence are modeled in order to quantify their effects on the degree of profile concavity and downstream fining. Also, several other physical mechanisms which may affect fining are studied, including the relative importance of the suspended versus bed load, the effect of the loss of sediment overbank, and the influence of the delta bottom slope. Finally, sensitivity analysis is used to show that the grain-size distribution at the interface between the active layer and substrate has a significant effect on downstream fining. ?? 2005 International Association of Hydraulic Engineering and Research.

  4. Patents and Downstream Innovation Suppresion - Facts or Fiction?

    DEFF Research Database (Denmark)

    Howells, John

    Merges and Nelson have proposed that pioneer patents of "broad" scope (where the claimed scope is typically broader than that strictly justified by the invention) enable their owners to "block" or "hold-up" downstream innovation.[1] They claim to have illustrated this thesis in such important cas...

  5. Patents and Downstream Innovation Suppression - Facts or Fiction?

    DEFF Research Database (Denmark)

    Howells, John

    Merges and Nelson have proposed that pioneer patents have enabled their owners to 'block' or 'hold-up' downstream innovation in cases as important as the car, radio, aircraft and electric lighting (Merges and Nelson 1990, ; Merges and Nelson 1994). Merges and Nelson use their work to question the...

  6. Patents and Downstream Innovation Suppression - Facts or Fiction?

    DEFF Research Database (Denmark)

    Howells, John

    Merges and Nelson have proposed that pioneer patents have enabled their owners to 'block' or 'hold-up' downstream innovation in cases as important as the car, radio, aircraft and electric lighting (Merges and Nelson 1990, ; Merges and Nelson 1994). Merges and Nelson use their work to question...

  7. Downstream processing of Isochrysis galbana: a step towards microalgal biorefinery

    NARCIS (Netherlands)

    Gilbert-López, B.; Mendiola, J.A.; Fontecha, J.; Broek, van den L.A.M.; Sijtsma, L.; Cifuentes, A.; Herrero, M.; Ibáñez, E.

    2015-01-01

    An algae-based biorefinery relies on the efficient use of algae biomass through its fractionation of several valuable/bioactive compounds that can be used in industry. If this biorefinery includes green platforms as downstream processing technologies able to fulfill the requirements of green

  8. Downstream processing of Isochrysis galbana: a step towards microalgal biorefinery

    NARCIS (Netherlands)

    Gilbert-López, B.; Mendiola, J.A.; Fontecha, J.; Broek, van den L.A.M.; Sijtsma, L.; Cifuentes, A.; Herrero, M.; Ibáñez, E.

    2015-01-01

    An algae-based biorefinery relies on the efficient use of algae biomass through its fractionation of several valuable/bioactive compounds that can be used in industry. If this biorefinery includes green platforms as downstream processing technologies able to fulfill the requirements of green chemist

  9. DNS and RANS Simulation of Dispersion Downstream of an Obstacle

    Science.gov (United States)

    2008-12-01

    DNS AND RANS SIMULATION OF DISPERSION DOWNSTREAM OF AN OBSTACLE Riccardo Rossi*, Gianluca Iaccarino** * Laboratorio di Termofluidodinamica...ORGANIZATION NAME(S) AND ADDRESS(ES) Laboratorio di Termofluidodinamica Computazionale, Seconda Facolt‘a di Ingegneria di Forl‘ý Universit‘a di Bologna

  10. Downstream processing of monoclonal antibodies--application of platform approaches.

    Science.gov (United States)

    Shukla, Abhinav A; Hubbard, Brian; Tressel, Tim; Guhan, Sam; Low, Duncan

    2007-03-15

    This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.

  11. Extreme wave phenomena in down-stream running modulated waves

    NARCIS (Netherlands)

    Andonowati,; Karjanto, N.; Groesen, van E.

    2006-01-01

    Modulational, Benjamin-Feir, instability is studied for the down-stream evolution of surface gravity waves. An explicit solution, the soliton on finite background, of the NLS equation in physical space is used to study various phenomena in detail. It is shown that for sufficiently long modulation le

  12. Interactive Learning-driven Innovation in Upstream-Downstream Relations

    DEFF Research Database (Denmark)

    Machikita, Tomohiro; Ueki, Yasushi

    2012-01-01

    This paper presents a simple framework of the innovations that result from interfirm learning through exchanges of engineers in upstream-downstream relations within a production chain. To examine the framework, we empirically investigate the impact of mutual knowledge exchanges on product and pro...

  13. Downstream-migrating fluvial point bars in the rock record

    Science.gov (United States)

    Ghinassi, Massimiliano; Ielpi, Alessandro; Aldinucci, Mauro; Fustic, Milovan

    2016-04-01

    Classical models developed for ancient fluvial point bars are based on the assumption that meander bends invariably increase their radius as meander-bend apices migrate in a direction transverse to the channel-belt axis (i.e., meander bend expansion). However, many modern meandering rivers are also characterized by down-valley migration of the bend apex, a mechanism that takes place without a significant change in meander radius and wavelength. Downstream-migrating fluvial point bars (DMFPB) are the dominant architectural element of these types of meander belts. Yet they are poorly known from ancient fluvial-channel belts, since their disambiguation from expansional point bars often requires fully-3D perspectives. This study aims to review DMFPB deposits spanning in age from Devonian to Holocene, and to discuss their main architectural and sedimentological features from published outcrop, borehole and 3D-seismic datasets. Fluvial successions hosting DMFPB mainly accumulated in low accommodation conditions, where channel belts were affected by different degrees of morphological (e.g., valleys) or tectonic (e.g., axial drainage of shortening basins) confinement. In confined settings, bends migrate downstream along the erosion-resistant valley flanks and little or no floodplain deposits are preserved. Progressive floor aggradation (e.g., valley filling) allow meander belts with DMFPB to decrease their degree of confinement. In less confined settings, meander bends migrate downstream mainly after impinging against older, erosion-resistant channel fill mud. By contrast, tectonic confinement is commonly associated with uplifted alluvial plains that prevented meander-bend expansion, in turn triggering downstream translation. At the scale of individual point bars, translational morphodynamics promote the preservation of downstream-bar deposits, whereas the coarser-grained upstream and central beds are less frequently preserved. However, enhanced preservation of upstream

  14. AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo.

    Science.gov (United States)

    Wang, Xiaowen; Li, Kai; Liu, Ling; Shi, Qiong; Song, Pu; Jian, Zhe; Guo, Sen; Wang, Gang; Li, Chunying; Gao, Tianwen

    2015-09-15

    Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR -129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that -129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with -129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than -129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo.

  15. D-type Cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells.

    Science.gov (United States)

    Zhang, Qian; Sakamoto, Kazuhito; Wagner, Kay-Uwe

    2014-01-25

    In response to the ligand-mediated activation of cytokine receptors, cells decide whether to proliferate or to undergo differentiation. D-type Cyclins (Cyclin D1, D2, or D3) and their associated Cyclin-dependent kinases (CDK4, CDK6) connect signals from cytokines to the cell cycle machinery, and they propel cells through the G1 restriction point and into the S phase, after which growth factor stimulation is no longer essential to complete cell division. D-type Cyclins are upregulated in many human malignancies including breast cancer to promote an uncontrolled proliferation of cancer cells. After summarizing important aspects of the cytokine-mediated transcriptional regulation and the posttranslational modification of D-type Cyclins, this review will highlight the physiological significance of these cell cycle regulators during normal mammary gland development as well as the initiation and promotion of breast cancer. Although the vast majority of published reports focus almost exclusively on the role of Cyclin D1 in breast cancer, we summarize here previous and recent findings that demonstrate an important contribution of the remaining two members of this Cyclin family, in particular Cyclin D3, for the growth of ErbB2-associated breast cancer cells in humans and in mouse models. New data from genetically engineered models as well as the pharmacological inhibition of CDK4/6 suggest that targeting the combined functions of D-type Cyclins could be a suitable strategy for the treatment of ErbB2-positive and potentially other types of breast cancer.

  16. Functional Validation of H2 Relaxin, and its Downstream Effectors, as Mediators, Therapeutic Targets and Potential Biomarkers of Prostate Cancer Progression

    Science.gov (United States)

    2011-06-01

    Endocr Rev 2004; 25: 205-34. 20. Samuel CS, Tian H, Zhao L, Amento EP. Relaxin is a key mediator of prostate growth and male reproductive tract...Prostatic Dis 2005; 8: 119-26. 31. Bathgate RA, Samuel CS, Burazin TC, Gundlach AL, Tregear GW. Relaxin: new peptides, receptors and novel actions...R, Beckett LA, Deitch AD, de Vere White RW. (2004). A modified yeast assay used on archival samples of localized prostate cancer tissue improves the

  17. Antifolate/folate-activated HGF/c-Met signalling pathways in mouse kidneys-the putative role of their downstream effectors in cross-talk with androgen receptor.

    Science.gov (United States)

    Dudkowska, Magdalena; Bajer, Seweryn; Jaworski, Tomasz; Zielińska, Joanna; Manteuffel-Cymborowska, Małgorzata; Grzelakowska-Sztabert, Barbara

    2009-03-01

    This in vivo study of mouse kidneys was focused on the identification of protein mediators involved in the cross-talk between two signalling pathways. One pathway was triggered by testosterone via an androgen receptor, AR, and the other induced by CB 3717/folate via HGF, and its membrane receptor c-Met. Sequential activation of these pathways leads to a drastic decrease of testosterone-induced ornithine decarboxylase, ODC, expression. We proved that CB 3717/folate-induced ODC expression is Akt-dependent. CB 3717/folate activates Akt and ERK1/2 kinases, PTEN phosphatase and also up-regulates cyclin D2 and PCNA, but decreases GSK3beta and cyclin D1 protein levels. Testosterone activation of AR induces GSK3beta and PTEN. Results of the sequential activation of the studied signalling pathways suggest that Akt, GSK3beta and possibly ERK1/2 kinases may participate in the negative cross-talk and attenuation of AR transactivity, while the involvement of PTEN and cyclin D1 seems to be doubtful.

  18. A novel virtual hub approach for multisource downstream service integration

    Science.gov (United States)

    Previtali, Mattia; Cuca, Branka; Barazzetti, Luigi

    2016-08-01

    A large development of downstream services is expected to be stimulated starting from earth observations (EO) datasets acquired by Copernicus satellites. An important challenge connected with the availability of downstream services is the possibility for their integration in order to create innovative applications with added values for users of different categories level. At the moment, the world of geo-information (GI) is extremely heterogeneous in terms of standards and formats used, thus preventing a facilitated access and integration of downstream services. Indeed, different users and data providers have also different requirements in terms of communication protocols and technology advancement. In recent years, many important programs and initiatives have tried to address this issue even on trans-regional and international level (e.g. INSPIRE Directive, GEOSS, Eye on Earth and SEIS). However, a lack of interoperability between systems and services still exists. In order to facilitate the interaction between different downstream services, a new architectural approach (developed within the European project ENERGIC OD) is proposed in this paper. The brokering-oriented architecture introduces a new mediation layer (the Virtual Hub) which works as an intermediary to bridge the gaps linked to interoperability issues. This intermediation layer de-couples the server and the client allowing a facilitated access to multiple downstream services and also Open Data provided by national and local SDIs. In particular, in this paper an application is presented integrating four services on the topic of agriculture: (i) the service given by Space4Agri (providing services based on MODIS and Landsat data); (ii) Gicarus Lab (providing sample services based on Landsat datasets) and (iii) FRESHMON (providing sample services for water quality) and services from a several regional SDIs.

  19. Downstream migrating antidunes or in-phase waves?

    Science.gov (United States)

    Núñez González, Francisco

    2014-05-01

    Late back in the beginning of the 20th century, Gilbert observed bedforms that migrated in opposite direction to flow. Since this feature was remarkable and inverse to the behavior of dunes (most often observed in rivers and flumes), he called the new species antidunes. Subsequent researchers identified other characteristic attributes of the new species, and it was later commonly accepted that a defining characteristic of antidunes was that undulations of bed and water profiles were roughly in-phase. Due to its generality, such definition has given place to some ambiguities, particularly when dealing with bedforms close to the critical-supercritical transition, as occurs with bedforms with bed and water profiles roughly in-phase but migrating downstream. Such bedforms are described by different researchers, but they are not always classified as antidunes. Some sedimentologists argue that given the depositional pattern of such streamwise migrating forms is different to that of upstream-migrating antidunes, the more generic term "in-phase waves" should be applied to consider them as a different class. The lack of a stability field for 2D downstream-migrating antidunes in the classical theoretical study of Kennedy in the early sixties, has also contributed to some confusion. According to such theoretical diagram, downstream-migrating antidunes could only exist being 3D, but empirical evidences -even from Kennedy- contradict this outcome. In this work, such results and other morphodynamic features of downstream-migrating antidunes will be discussed, in light of experimental data and a simple hydraulic analysis of the direction of movement of antidunes. An open question will be left to debate about the appropriateness of classifying downstream-migrating in-phase waves as antidunes, and it will be emphasized that finding consensus between different disciplines involved with the study of bedforms will be advantageous.

  20. Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

    NARCIS (Netherlands)

    Kombrink, Anja; Rovenich, Hanna; Shi, Xiaoqian; Rojas-Padilla, Eduardo; Berg-Velthuis, van den Grardy; Domazakis, Emmanouil; Jonge, De Ronnie; Valkenburg, Dirk-Jan; Sánchez-Vallet, Andrea; Seidl, Michael F.; Thomma, Bart P.H.J.

    2017-01-01

    Chitin-binding LysM effectors contribute to virulence of various plant pathogenic fungi that are causal agents of foliar diseases. Here, we report on LysM effectors of the soil-borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are

  1. Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

    Science.gov (United States)

    Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

  2. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M;

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...... sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could...

  3. Design and Implementation of Multifunctional Automatic Drilling End Effector

    Science.gov (United States)

    Wang, Zhanxi; Qin, Xiansheng; Bai, Jing; Tan, Xiaoqun; Li, Jing

    2017-03-01

    In order to realize the automatic drilling in aircraft assembly, a drilling end effector is designed by integrating the pressure unit, drilling unit, measurement unit, control system and frame structure. In order to reduce the hole deviation, this paper proposes a vertical normal adjustment program based on 4 laser distance sensors. The actual normal direction of workpiece surface can be calculated through the sensors measurements, and then robot posture is adjusted to realize the hole deviation correction. A base detection method is proposed to detect and locate the hole automatically by using the camera and the reference hole. The experiment results show that the position accuracy of the system is less than 0.3mm, and the normal precision is less than 0.5°. The drilling end effector and robot can greatly improve the efficiency of the aircraft parts and assembly quality, and reduce the product development cycle.

  4. Identification of Novel Type III Effectors Using Latent Dirichlet Allocation

    Directory of Open Access Journals (Sweden)

    Yang Yang

    2012-01-01

    Full Text Available Among the six secretion systems identified in Gram-negative bacteria, the type III secretion system (T3SS plays important roles in the disease development of pathogens. T3SS has attracted a great deal of research interests. However, the secretion mechanism has not been fully understood yet. Especially, the identification of effectors (secreted proteins is an important and challenging task. This paper adopts machine learning methods to identify type III secreted effectors (T3SEs. We extract features from amino acid sequences and conduct feature reduction based on latent semantic information by using latent Dirichlet allocation model. The experimental results on Pseudomonas syringae data set demonstrate the good performance of the new methods.

  5. Downstream Toll-like receptor signaling mediates adaptor-specific cytokine expression following focal cerebral ischemia

    Directory of Open Access Journals (Sweden)

    Bolanle Famakin

    2012-07-01

    Full Text Available Abstract Background Deletion of some Toll-like receptors (TLRs affords protection against cerebral ischemia, but disruption of their known major downstream adaptors does not. To determine whether compensation in the production of downstream effectors by one pathway when the other is disrupted can explain these findings, we examined cytokine/chemokine expression and inflammatory infiltrates in wild-type (WT, MyD88−/− and TRIF-mutant mice following permanent middle cerebral artery occlusion (pMCAO. Methods Cytokine/chemokine expression was measured with a 25-plex bead array in the serum and brains of all three groups of mice at baseline (no surgery/naïve and at 3 hours and 24 hours following pMCAO. Brain inflammatory and neutrophil infiltrates were examined 24 hours following pMCAO. Results IL-6, keratinocyte chemoattractant (KC, granulocyte colony-stimulating factor (G-CSF and IL-10 were significantly decreased in MyD88−/− mice compared to WT mice following pMCAO. Significantly, decreased levels of the neutrophil chemoattractants KC and G-CSF corresponded with a trend toward fewer neutrophils in the brains of MyD88−/− mice. IP-10 was significantly decreased when either pathway was disrupted. MIP-1α was significantly decreased in TRIF-mutant mice, consistent with TRIF-dependent production. MyD88−/− mice showed elevations of a number of Th2 cytokines, such as IL-13, at baseline, which became significantly decreased following pMCAO. Conclusions Both MyD88 and TRIF mediate pathway-specific cytokine production following focal cerebral ischemia. Our results also suggest a compensatory Th2-type skew at baseline in MyD88−/− mice and a paradoxical switch to a Th1 phenotype following focal cerebral ischemia. The MyD88 pathway directs the expression of neutrophil chemoattractants following cerebral ischemia.

  6. Common themes in the design and function of bacterial effectors

    OpenAIRE

    2009-01-01

    Central to the biology of many pathogenic bacteria are a number of specialized machines, known as type III, type IV or type VI protein secretion systems. These machines have specifically evolved to deliver bacterial “effector” proteins into host cells with the capacity to modulate a variety of cellular functions. The identification of the biochemical activities of many effector proteins, coupled with a better understanding of their potential contribution to pathogenesis, have revealed common ...

  7. Hacker Within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy

    OpenAIRE

    2016-01-01

    Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector protei...

  8. Designing and testing the activities of TAL effector nucleases.

    Science.gov (United States)

    Lin, Yanni; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have rapidly developed into a powerful tool for genome editing. To avoid labor-intensive and time-consuming experimental screening for active TALENs, a scoring system can help select optimal target sites. Here we describe a procedure to design active TALENs using a scoring system named Scoring Algorithm for Predicted TALEN Activity (SAPTA) and a method to test the activity of individual and pairs of TALENs.

  9. Mirror Sub-Assembly End-Effector Design

    Energy Technology Data Exchange (ETDEWEB)

    Butlin, B

    2007-01-08

    The Optic Assembly Building (OAB) is a facility where large optical mirror units are assembled and installed into Line Replaceable Units (LRUs) for deployment into the National Ignition Facility (NIF) laser system. The New Optics Insertion Device (NOID) is a powered jib crane specially designed to handle large optical assemblies. The NOID arm has three degrees of freedom. it can rotate about the vertical boom, travel up and down the boom, and extend away from and retract in towards the boom. The NOID is used to assist in the assembly of five types of Laser Mirror (LM) LRUs. These five LMs have been creatively named, LM4, LM5, LM6, LM7, and LM8. The LM4 and LM5 LRUs each contain four Mirror Sub-Assemblies (MSAs). The LM6, LM7, and LM8 LRUs each contain 2 MSAs. The MSAs are assembled apart from the LRU and are then installed in the LRU at the LM4-8 workstations. An MSA NOID End-Effector is required to interface with the MSAs and install them into the LRUs. The End-Effector must attach to the robo-hand on the end of the NOID arm. At the time the MSA NOID End-Effector was being designed the NOID, the LM4-5 workstation, and the LM6-8 workstation were already installed in the OAB. The LRUs and the MSAs designs were also complete. The MSA NOID End-Effector design had to work with the assembly equipment and LRU designs that were already in place.

  10. The Functions of Effector Proteins in Yersinia Virulence.

    Science.gov (United States)

    Zhang, Linglin; Mei, Meng; Yu, Chan; Shen, Wenwen; Ma, Lixin; He, Jiewang; Yi, Li

    2016-01-01

    Yersinia species are bacterial pathogens that can cause plague and intestinal diseases after invading into human cells through the Three Secretion System (TTSS). The effect of pathogenesis is mediated by Yersinia outer proteins (Yop) and manifested as down-regulation of the cytokine genes expression by inhibiting nuclear factor-κ-gene binding (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. In addition, its pathogenesis can also manipulate the disorder of host innate immune system and cell death such as apoptosis, pyroptosis, and autophagy. Among the Yersinia effector proteins, YopB and YopD assist the injection of other virulence effectors into the host cytoplasm, while YopE, YopH, YopJ, YopO, and YopT target on disrupting host cell signaling pathways in the host cytosols. Many efforts have been applied to reveal that intracellular proteins such as Rho-GTPase, and transmembrane receptors such as Toll-like receptors (TLRs) both play critical roles in Yersinia pathogenesis, establishing a connection between the pathogenic process and the signaling response. This review will mainly focus on how the effector proteins of Yersinia modulate the intrinsic signals in host cells and disturb the innate immunity of hosts through TTSS.

  11. Current activities of the Yersinia effector protein YopM.

    Science.gov (United States)

    Höfling, Sabrina; Grabowski, Benjamin; Norkowski, Stefanie; Schmidt, M Alexander; Rüter, Christian

    2015-05-01

    Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogen's virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

    Science.gov (United States)

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; Nakayasu, Ernesto S.; Staiger, Christopher J.

    2017-01-01

    Legionella pneumophila, the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen. PMID:28129393

  13. Turtle functions downstream of Cut in differentially regulating class specific dendrite morphogenesis in Drosophila.

    Directory of Open Access Journals (Sweden)

    Mikolaj J Sulkowski

    Full Text Available BACKGROUND: Dendritic morphology largely determines patterns of synaptic connectivity and electrochemical properties of a neuron. Neurons display a myriad diversity of dendritic geometries which serve as a basis for functional classification. Several types of molecules have recently been identified which regulate dendrite morphology by acting at the levels of transcriptional regulation, direct interactions with the cytoskeleton and organelles, and cell surface interactions. Although there has been substantial progress in understanding the molecular mechanisms of dendrite morphogenesis, the specification of class-specific dendritic arbors remains largely unexplained. Furthermore, the presence of numerous regulators suggests that they must work in concert. However, presently, few genetic pathways regulating dendrite development have been defined. METHODOLOGY/PRINCIPAL FINDINGS: The Drosophila gene turtle belongs to an evolutionarily conserved class of immunoglobulin superfamily members found in the nervous systems of diverse organisms. We demonstrate that Turtle is differentially expressed in Drosophila da neurons. Moreover, MARCM analyses reveal Turtle acts cell autonomously to exert class specific effects on dendritic growth and/or branching in da neuron subclasses. Using transgenic overexpression of different Turtle isoforms, we find context-dependent, isoform-specific effects on mediating dendritic branching in class II, III and IV da neurons. Finally, we demonstrate via chromatin immunoprecipitation, qPCR, and immunohistochemistry analyses that Turtle expression is positively regulated by the Cut homeodomain transcription factor and via genetic interaction studies that Turtle is downstream effector of Cut-mediated regulation of da neuron dendrite morphology. CONCLUSIONS/SIGNIFICANCE: Our findings reveal that Turtle proteins differentially regulate the acquisition of class-specific dendrite morphologies. In addition, we have established a

  14. chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation and stem cell self-renewal in Drosophila

    Science.gov (United States)

    Flaherty, Maria Sol; Salis, Pauline; Evans, Cory J.; Ekas, Laura A.; Marouf, Amine; Zavadil, Jiri; Banerjee, Utpal; Bach, Erika A.

    2010-01-01

    SUMMARY The Drosophila STAT transcription factor Stat92E regulates diverse functions, including organ development and stem cell self-renewal. However, the Stat92E functional effectors that mediate these processes are largely unknown. Here we show that chinmo is a cell-autonomous, downstream mediator of Stat92E that shares numerous functions with this protein. Loss of either gene results in malformed eyes and head capsules due to defects in eye progenitor cells. Hyperactivation of Stat92E or misexpression of Chinmo results in blood cell tumors. Both proteins are expressed in germline (GSCs) and cyst stem cells (CySCs) in the testis. While Stat92E is required for the self-renewal of both populations, chinmo is only required in CySCs, indicating that Stat92E regulates self-renewal in different stem cells through independent effectors. Like hyperactivated Stat92E, Chinmo misexpression in CySCs is sufficient to maintain GSCs non-autonomously. Chinmo is therefore a key effector of JAK/STAT signaling in a variety of developmental and pathological contexts. PMID:20412771

  15. Upstream-downstream cooperation approach in Guanting Reservoir watershed

    Institute of Scientific and Technical Information of China (English)

    YANG Zhi-feng; ZHANG Wen-guo

    2005-01-01

    A case study is introduced and discussed concerning water dispute of misuse and pollution between up- and down-stream parts.The relations between water usage and local industrial structures are analyzed. Results show it is important to change industrial structures of the target region along with controlling water pollution by technical and engineering methods. Three manners of upstream-downstream cooperation are presented and discussed based on the actual conditions of Guangting Reservoir watershed, Two typical scenarios are supposed and studied along with the local plan on water resources development. The best solution for this cooperation presents a good way to help the upstream developing in a new pattern of eco-economy.

  16. Dead zone area at the downstream flow of barrages

    Directory of Open Access Journals (Sweden)

    Mohamed F. Sauida

    2016-12-01

    Full Text Available Flow separation is a natural phenomenon encountered at some cases downstream of barrages. The main flow is divided into current and dead zone flows. The percentage area of dead zone flow must be taken into consideration downstream of barrages, due to its negative effect on flow characteristics. Experimental studies were conducted in the Hydraulic Research Institute (HRI, on a physical regulator model with five vents. Theoretically the separation zone is described as a part of an ellipse which is practically verified by plotting velocity vectors. The results show that the percentage area of dead zone to the area through length of separation depends mainly on the expansion ratio [channel width to width of opened vents], with maximum value of 81% for operated side gates. A statistical analysis was derived, to predict the percentage area of dead zone flow to the area through length of separation.

  17. Patents and Downstream Innovation Suppresion - Facts or Fiction?

    DEFF Research Database (Denmark)

    Howells, John

    Merges and Nelson have proposed that pioneer patents of "broad" scope (where the claimed scope is typically broader than that strictly justified by the invention) enable their owners to "block" or "hold-up" downstream innovation.[1] They claim to have illustrated this thesis in such important cases...... of development as aircraft, the car, radio and electric lighting. Merges and Nelson quite logically use their work to question the value of Kitch's prospect theory of patents,[2] a theory that emphasises that the social value of patents is that they enable the coordination of technological development....... This article re-examines Merges and Nelson's illustrative historical-empirical evidence and finds their thesis of