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Sample records for rapamycin activity specificity

  1. Characterization of the cloned full-length and a truncated human target of rapamycin: Activity, specificity, and enzyme inhibition as studied by a high capacity assay

    International Nuclear Information System (INIS)

    Toral-Barza, Lourdes; Zhang Weiguo; Lamison, Craig; LaRocque, James; Gibbons, James; Yu, Ker

    2005-01-01

    The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (K m ) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 μM, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors

  2. Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, Christian R.; Zwartkruis, Fried J.; Vellenga, Edo; Coffer, Paul J.; Buitenhuis, Miranda

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic

  3. The antiaging activity and cerebral protection of rapamycin at micro-doses.

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    Qi, Haiyan; Su, Feng-Yun; Wan, Shan; Chen, Yongjie; Cheng, Yan-Qiong; Liu, Ai-Jun

    2014-11-01

    The immunosuppressant drug rapamycin was reported to have an antiaging activity, which was attributed to the TORC1 inhibition that inhibits cell proliferation and increases autophagy. However, rapamycin also exhibits a number of harmful adverse effects. Whether rapamycin can be developed into an antiaging agent remains unclear. We demonstrated that rapamycin at micro-doses (below the TORC1 inhibiting concentration) exhibits a cell-protective activity: (1) It protects cultured neurons against neurotoxin MPP(+) and H2O2. (2) It increases survival time of neuron in culture. (3) It maintains the nonproliferative state of cultured senescent human fibroblasts and prevents cell death induced by telomere dysfunction. (4) In animal models, it decreased the cerebral infarct sizes induced by acute ischemia and dramatically extended the life span of stroke prone spontaneously hypertensive rats (SHR-SPs). We propose that rapamycin at micro-dose can be developed into an antiaging agent with a novel mechanism. © 2014 John Wiley & Sons Ltd.

  4. Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

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    Joseph W Briggs

    Full Text Available Activation of the unfolded protein response (UPR in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.

  5. A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity.

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    Umeda, Nobuhiro; Ueno, Tasuku; Pohlmeyer, Christopher; Nagano, Tetsuo; Inoue, Takanari

    2011-01-12

    We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

  6. Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor.

    Science.gov (United States)

    Bastidas, Robert J; Shertz, Cecelia A; Lee, Soo Chan; Heitman, Joseph; Cardenas, Maria E

    2012-03-01

    The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis.

  7. Rapamycin Exerts Antifungal Activity In Vitro and In Vivo against Mucor circinelloides via FKBP12-Dependent Inhibition of Tor

    Science.gov (United States)

    Bastidas, Robert J.; Shertz, Cecelia A.; Lee, Soo Chan; Heitman, Joseph

    2012-01-01

    The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis. PMID:22210828

  8. Enhanced antitumor activity of 3-bromopyruvate in combination with rapamycin in vivo and in vitro.

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    Zhang, Qi; Pan, Jing; Lubet, Ronald A; Komas, Steven M; Kalyanaraman, Balaraman; Wang, Yian; You, Ming

    2015-04-01

    3-Bromopyruvate (3-BrPA) is an alkylating agent and a well-known inhibitor of energy metabolism. Rapamycin is an inhibitor of the serine/threonine protein kinase mTOR. Both 3-BrPA and rapamycin show chemopreventive efficacy in mouse models of lung cancer. Aerosol delivery of therapeutic drugs for lung cancer has been reported to be an effective route of delivery with little systemic distribution in humans. In this study, 3-BrPA and rapamycin were evaluated in combination for their preventive effects against lung cancer in mice by aerosol treatment, revealing a synergistic ability as measured by tumor multiplicity and tumor load compared treatment with either single-agent alone. No evidence of liver toxicity was detected by monitoring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. To understand the mechanism in vitro experiments were performed using human non-small cell lung cancer (NSCLC) cell lines. 3-BrPA and rapamycin also synergistically inhibited cell proliferation. Rapamycin alone blocked the mTOR signaling pathway, whereas 3-BrPA did not potentiate this effect. Given the known role of 3-BrPA as an inhibitor of glycolysis, we investigated mitochondrial bioenergetics changes in vitro in 3-BrPA-treated NSCLC cells. 3-BrPA significantly decreased glycolytic activity, which may be due to adenosine triphosphate (ATP) depletion and decreased expression of GAPDH. Our results demonstrate that rapamycin enhanced the antitumor efficacy of 3-BrPA, and that dual inhibition of mTOR signaling and glycolysis may be an effective therapeutic strategy for lung cancer chemoprevention. ©2015 American Association for Cancer Research.

  9. An approach to analyse the specific impact of rapamycin on mRNA-ribosome association

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    Jaquier-Gubler Pascale

    2008-08-01

    Full Text Available Abstract Background Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background. Methods We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out. For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation. Results High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug. Conclusion The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of

  10. Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

    International Nuclear Information System (INIS)

    Morrow, Peter W.; Tung, H.Y. Lim; Hemmings, Hugh C.

    2004-01-01

    Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A 1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD 4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA

  11. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development

    OpenAIRE

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-01-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and express...

  12. Rapamycin Synergizes with Cisplatin in Antiendometrial Cancer Activation by Improving IL-27–Stimulated Cytotoxicity of NK Cells

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    Wen-Jie Zhou

    2018-01-01

    Full Text Available Natural killer (NK cell function is critical for controlling initial tumor growth and determining chemosensitivity of the tumor. A synergistic relationship between rapamycin and cisplatin in uterine endometrial cancer (UEC in vitro has been reported, but the mechanism and the combined therapeutic strategy for endometrial cancer (EC are still unknown. We found a positive correlation between the level of IL-27 and the differentiated stage of UEC. The increase of IL-27 in uterine endometrial cancer cell (UECC lines (Ishikawa, RL95-2 and KLE led to a high cytotoxic activity of NK cells to UECC in the co-culture system. Exposure with rapamycin enhanced the cytotoxicity of NK cells by upregulating the expression of IL-27 in UECC and IL-27 receptors (IL-27Rs: WSX-1 and gp130 on NK cells and further restricted the growth of UEC in Ishikawa-xenografted nude mice. In addition, treatment with rapamycin resulted in an increased autophagy level of UECC, and IL-27 enhanced this ability of rapamycin. Cisplatin-mediated NK cells' cytotoxic activity and anti-UEC activation were independent of IL-27; however, the combination of rapamycin and cisplatin led to a higher cytotoxic activity of NK cells, smaller UEC volume and longer survival rate in vivo. These results suggest that rapamycin and cisplatin synergistically activate the cytotoxicity of NK cells and inhibit the progression of UEC in both an IL-27–dependent and –independent manner. This provides a scientific basis for potential rapamycin-cisplatin combined therapeutic strategies targeted to UEC, especially for the patients with low differentiated stage or abnormally low level of IL-27.

  13. Rapamycin and Glucose-Target of Rapamycin (TOR) Protein Signaling in Plants*

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    Xiong, Yan; Sheen, Jen

    2012-01-01

    Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs. PMID:22134914

  14. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    International Nuclear Information System (INIS)

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-01-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-α (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity

  15. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development.

    Science.gov (United States)

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-10-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm. © FASEB.

  16. GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin.

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    Schepetilnikov, Mikhail; Makarian, Joelle; Srour, Ola; Geldreich, Angèle; Yang, Zhenbiao; Chicher, Johana; Hammann, Philippe; Ryabova, Lyubov A

    2017-04-03

    Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR ROP2 physically interacts with and, when GTP-bound, activates TOR in vitro TOR activation in response to auxin is abolished in ROP-deficient rop2 rop6 ROP4 RNAi plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin. © 2017 The Authors.

  17. Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway

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    Kai Gao

    2015-01-01

    Full Text Available The Wnt/β-catenin signaling pathway plays a crucial role in neural development, axonal guidance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/β-catenin signaling pathway after spinal cord injury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of β-catenin protein, ca-spase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/β-catenin signaling pathway. Rapamycin increased the levels of β-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental findings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/β-catenin signaling pathway after spinal cord injury.

  18. Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis.

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    Ren, Jiafa; Li, Jianzhong; Feng, Ye; Shu, Bingyan; Gui, Yuan; Wei, Wei; He, Weichun; Yang, Junwei; Dai, Chunsun

    2017-08-01

    Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor -/- macrophages stimulated fibroblast activation less efficiently than that from TGF-β1-pretreated Rictor +/+ macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Rapamycin Influences the Efficiency of Fertilization and Development in the Mouse: A Role for Autophagic Activation

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    Geun-Kyung Lee

    2016-08-01

    Full Text Available The mammalian target of rapamycin (mTOR regulates cellular processes such as cell growth, metabolism, transcription, translation, and autophagy. Rapamycin is a selective inhibitor of mTOR, and induces autophagy in various systems. Autophagy contributes to clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified-warmed mouse oocytes show acute increases in autophagy during warming, and suggested that it is a natural response to cold stress. In this follow-up study, we examined whether the modulation of autophagy influences survival, fertilization, and developmental rates of vitrified-warmed mouse oocytes. We used rapamycin to enhance autophagy in metaphase II (MII oocytes before and after vitrification. The oocytes were then subjected to in vitro fertilization (IVF. The fertilization and developmental rates of vitrified-warmed oocytes after rapamycin treatment were significantly lower than those for control groups. Modulation of autophagy with rapamycin treatment shows that rapamycin-induced autophagy exerts a negative influence on fertilization and development of vitrified-warmed oocytes.

  20. Paradoxical effects of rapamycin on experimental house dust mite-induced asthma.

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    Karin Fredriksson

    Full Text Available The mammalian target of rapamycin (mTOR modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.

  1. Mammalian target of rapamycin complex 1 activation is required for the stimulation of human skeletal muscle protein synthesis by essential amino acids.

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    Dickinson, Jared M; Fry, Christopher S; Drummond, Micah J; Gundermann, David M; Walker, Dillon K; Glynn, Erin L; Timmerman, Kyle L; Dhanani, Shaheen; Volpi, Elena; Rasmussen, Blake B

    2011-05-01

    The relationship between mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis during instances of amino acid surplus in humans is based solely on correlational data. Therefore, the goal of this study was to use a mechanistic approach specifically designed to determine whether increased mTORC1 activation is requisite for the stimulation of muscle protein synthesis following L-essential amino acid (EAA) ingestion in humans. Examination of muscle protein synthesis and signaling were performed on vastus lateralis muscle biopsies obtained from 8 young (25 ± 2 y) individuals who were studied prior to and following ingestion of 10 g of EAA during 2 separate trials in a randomized, counterbalanced design. The trials were identical except during 1 trial, participants were administered a single oral dose of a potent mTORC1 inhibitor (rapamycin) prior to EAA ingestion. In response to EAA ingestion, an ~60% increase in muscle protein synthesis was observed during the control trial, concomitant with increased phosphorylation of mTOR (Ser(2448)), ribosomal S6 kinase 1 (Thr(389)), and eukaryotic initiation factor 4E binding protein 1 (Thr(37/46)). In contrast, prior administration of rapamycin completely blocked the increase in muscle protein synthesis and blocked or attenuated activation of mTORC1-signaling proteins. The inhibition of muscle protein synthesis and signaling was not due to differences in either extracellular or intracellular amino acid availability, because these variables were similar between trials. These data support a fundamental role for mTORC1 activation as a key regulator of human muscle protein synthesis in response to increased EAA availability. This information will be useful in the development of evidence-based nutritional therapies targeting mTORC1 to counteract muscle wasting associated with numerous clinical conditions.

  2. Activated mammalian target of rapamycin is a potential therapeutic target in gastric cancer

    International Nuclear Information System (INIS)

    Xu, Da-zhi; Sun, Xiao-wei; Guan, Yuan-xiang; Li, Yuan-fang; Lin, Tong-yu; Geng, Qi-rong; Tian, Ying; Cai, Mu-yan; Fang, Xin-juan; Zhan, You-qing; Zhou, Zhi-wei; Li, Wei; Chen, Ying-bo

    2010-01-01

    The mammalian target of rapamycin (mTOR) plays a key role in cellular growth and homeostasis. The purpose of our present study is to investigate the expression of activated mTOR (p-mTOR) in gastric cancer patients, their prognostic significance and the inhibition effect of RAD001 on tumor growth and to determine whether targeted inhibition of mTOR could be a potential therapeutic strategy for gastric cancer. The expression of p-mTOR was detected in specimens of 181 gastric cancers who underwent radical resection (R0) by immunohistochemistry. The correlation of p-mTOR expression to clinicopathologic features and survival of gastric cancer was studied. We also determined the inhibition effect of RAD001 on tumor growth using BGC823 and AGS human gastric cancer cell lines. Immunostaining for p-mTOR was positive in 93 of 181 (51.4%) gastric cancers, closely correlated with lymph node status and pTNM stage. Patients with p-mTOR positive showed significantly shorter disease-free survival (DFS) and overall survival (OS) rates than those with p-mTOR-negative tumors in univariable analyses, and there was a trend toward a correlation between p-mTOR expression and survival in multivariable analyses. RAD001 markedly inhibited dose-dependently proliferation of human gastric carcinoma cells by down-regulating expression of p70s6k, p-p70s6k, C-myc, CyclinD1 and Bcl-2, up-regulating expression of P53. In gastric cancer, p-mTOR is a potential therapeutic target and RAD001 was a promising treatment agent with inducing cell cycle arrest and apoptosis by down-regulating expression of C-myc, CyclinD1 and Bcl-2, up-regulating expression of P53

  3. Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma.

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    Peponi, Evangelia; Drakos, Elias; Reyes, Guadalupe; Leventaki, Vasiliki; Rassidakis, George Z; Medeiros, L Jeffrey

    2006-12-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

  4. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

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    Karl Andrew Rodriguez

    2014-11-01

    Full Text Available Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24mg/kg (14 ppm rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS, heat shock factor 1 (HSF1, and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

  5. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

  6. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system.

    Science.gov (United States)

    Arriola Apelo, Sebastian I; Neuman, Joshua C; Baar, Emma L; Syed, Faizan A; Cummings, Nicole E; Brar, Harpreet K; Pumper, Cassidy P; Kimple, Michelle E; Lamming, Dudley W

    2016-02-01

    Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA-approved drug rapamycin has been shown to promote lifespan and delay age-related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long-term prophylactic use of rapamycin as a therapy for age-related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR-containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA-approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA-approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  7. Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice

    NARCIS (Netherlands)

    Korsheninnikova, E.; van der Zon, G. C. M.; Voshol, P. J.; Janssen, G. M.; Havekes, L. M.; Grefhorst, A.; Kuipers, F.; Reijngoud, D. -J.; Romijn, J. A.; Ouwens, D. M.; Maassen, J. A.

    2006-01-01

    Aims/hypothesis Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. Materials and methods Chronic hepatic

  8. Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice

    NARCIS (Netherlands)

    Korsheninnikova, E.; van der Zon, G. C. M.; Voshol, P. J.; Janssen, G. M.; Havekes, L. M.; Grefhorst, A.; Kuipers, F.; Reijngoud, D.-J.; Romijn, J. A.; Ouwens, D. M.; Maassen, J. A.

    2006-01-01

    Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. Chronic hepatic steatosis and hepatic insulin resistance

  9. Rapamycin prevents drug seeking via disrupting reconsolidation of reward memory in rats.

    Science.gov (United States)

    Lin, Jue; Liu, Lingqi; Wen, Quan; Zheng, Chunming; Gao, Yang; Peng, Shuxian; Tan, Yalun; Li, Yanqin

    2014-01-01

    The maladaptive drug memory developed between the drug-rewarding effect and environmental cues contributes to difficulty in preventing drug relapse. Established reward memories can be disrupted by pharmacologic interventions following their reactivation. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, has been proved to be involved in various memory consolidation. However, it is less well characterized in drug memory reconsolidation. Using a conditioned place preference (CPP) procedure, we examined the effects of systemically administered rapamycin on reconsolidation of drug memory in rats. We found that systemically administered rapamycin (0.1 or 10 mg/kg, i.p.) after re-exposure to drug-paired environment, dose dependently decreased the expression of CPP 1 d later, and the effect lasted for up to 14 d and could not be reversed by a priming injection of morphine. The effect of rapamycin on morphine-associated memory was specific to drug-paired context, and rapamycin had no effect on subsequent CPP expression when rats were exposed to saline-paired context or homecage. These results indicated that systemic administration of rapamycin after memory reactivation can persistently inhibit the drug seeking behaviour via disruption of morphine memory reconsolidation in rats. Additionally, the effect of rapamycin on memory reconsolidation was reproduced in cocaine CPP and alcohol CPP. Furthermore, rapamycin did not induce conditioned place aversion and had no effect on locomotor activity and anxiety behaviour. These findings suggest that rapamycin could erase the acquired drug CPP in rats, and that mTOR activity plays an important role in drug reconsolidation and is required for drug relapse.

  10. Etude par génétique inverse du gène codant la protéine TARGET OF RAPAMYCIN d'Arabidopsis thaliana (AtTOR), l'homologue d'une kinase contrôlant la croissance cellulaire chez les eucaryotes

    OpenAIRE

    Menand, Benoit

    2002-01-01

    TOR (target of rapamycin) protein kinases were identified in yeast, mammals and Drosophila as central controllers of cell growth. Thu, G1 to S phases progression through the cell cycle is blocked by rapamycin, a drug which specifically inhibits TOR activity by forming a ternary complex with the peptidyl-prolyl isomerase FKBP12 (FK506 and rapamycin binding protein), and the FKBP-rapamycin binding domain (FRB) of TOR proteins. This work presents the study, the Arabidopsis homologue of yeast and...

  11. Rapamycin inhibits mTOR/p70S6K activation in CA3 region of the hippocampus of the rat and impairs long term memory.

    Science.gov (United States)

    Lana, D; Di Russo, J; Mello, T; Wenk, G L; Giovannini, M G

    2017-01-01

    The present study was aimed at establishing whether the mTOR pathway and its downstream effector p70S6K in CA3 pyramidal neurons are under the modulation of the cholinergic input to trigger the formation of long term memories, similar to what we demonstrated in CA1 hippocampus. We performed in vivo behavioral experiments using the step down inhibitory avoidance test in adult Wistar rats to evaluate memory formation under different conditions. We examined the effects of rapamycin, an inhibitor of mTORC1 formation, scopolamine, a muscarinic receptor antagonist or mecamylamine, a nicotinic receptor antagonist, on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition was conducted 30min after i.c.v. injection of rapamycin. Recall testing was performed 1h, 4h or 24h after acquisition. We found that (1) mTOR and p70S6K activation in CA3 pyramidal neurons were involved in long term memory formation; (2) rapamycin significantly inhibited mTOR and of p70S6K activation at 4h, and long term memory impairment 24h after acquisition; (3) scopolamine impaired short but not long term memory, with an early increase of mTOR/p70S6K activation at 1h followed by stabilization at longer times; (4) mecamylamine and scopolamine co-administration impaired short term memory at 1h and 4h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1h and 4h; (5) mecamylamine and scopolamine treatment did not impair long term memory formation; (6) unexpectedly, rapamycin increased mTORC2 activation in microglial cells. Our results demonstrate that in CA3 pyramidal neurons the mTOR/p70S6K pathway is under the modulation of the cholinergic system and is involved in long-term memory encoding, and are consistent with the hypothesis that the CA3 region of the hippocampus is involved in memory mechanisms based on rapid, one-trial object-place learning and recall. Furthermore, our results are in accordance with previous reports that selective

  12. HPV-16 E7 expression up-regulates phospholipase D activity and promotes rapamycin resistance in a pRB-dependent manner.

    Science.gov (United States)

    Rabachini, Tatiana; Boccardo, Enrique; Andrade, Rubiana; Perez, Katia Regina; Nonogaki, Suely; Cuccovia, Iolanda Midea; Villa, Luisa Lina

    2018-04-27

    Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role. PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures. HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin. This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.

  13. Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1).

    Science.gov (United States)

    Shibutani, Shusaku; Okazaki, Hana; Iwata, Hiroyuki

    2017-11-03

    The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

    Science.gov (United States)

    Nishitani, Shinobu; Horie, Mayumi; Ishizaki, Sonoko; Yano, Hirohisa

    2013-01-01

    Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy. PMID:24312415

  15. Branched chain amino acid suppresses hepatocellular cancer stem cells through the activation of mammalian target of rapamycin.

    Directory of Open Access Journals (Sweden)

    Shinobu Nishitani

    Full Text Available Differentiation of cancer stem cells (CSCs into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR leads to CSC survival, the effect of branched chain amino acids (BCAAs, an mTOR complex 1 (mTORC1 activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb. mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2 or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy.

  16. Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin.

    Science.gov (United States)

    Butler, Corwin R; Boychuk, Jeffery A; Smith, Bret N

    2017-01-01

    Following traumatic brain injury (TBI), treatment with rapamycin suppresses mammalian (mechanistic) target of rapamycin (mTOR) activity and specific components of hippocampal synaptic reorganization associated with altered cortical excitability and seizure susceptibility. Reemergence of seizures after cessation of rapamycin treatment suggests, however, an incomplete suppression of epileptogenesis. Hilar inhibitory interneurons regulate dentate granule cell (DGC) activity, and de novo synaptic input from both DGCs and CA3 pyramidal cells after TBI increases their excitability but effects of rapamycin treatment on the injury-induced plasticity of interneurons is only partially described. Using transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed in the somatostatinergic subset of hilar inhibitory interneurons, we tested the effect of daily systemic rapamycin treatment (3 mg/kg) on the excitability of hilar inhibitory interneurons after controlled cortical impact (CCI)-induced focal brain injury. Rapamycin treatment reduced, but did not normalize, the injury-induced increase in excitability of surviving eGFP+ hilar interneurons. The injury-induced increase in response to selective glutamate photostimulation of DGCs was reduced to normal levels after mTOR inhibition, but the postinjury increase in synaptic excitation arising from CA3 pyramidal cell activity was unaffected by rapamycin treatment. The incomplete suppression of synaptic reorganization in inhibitory circuits after brain injury could contribute to hippocampal hyperexcitability and the eventual reemergence of the epileptogenic process upon cessation of mTOR inhibition. Further, the cell-selective effect of mTOR inhibition on synaptic reorganization after CCI suggests possible mechanisms by which rapamycin treatment modifies epileptogenesis in some models but not others.

  17. Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

    International Nuclear Information System (INIS)

    Sharpe, Laura J.; Brown, Andrew J.

    2008-01-01

    As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2

  18. The effect of the immunophilin ligands rapamycin and FK506 on proliferation of mast cells and other hematopoietic cell lines.

    Science.gov (United States)

    Hultsch, T; Martin, R; Hohman, R J

    1992-01-01

    The immunosuppressive drugs FK506 and cyclosporin A have an identical spectrum of activities with respect to IgE receptor (Fc epsilon RI)-mediated exocytosis from mast cells and T cell receptor-mediated transcription of IL-2. These findings suggest a common step in receptor-mediated signal transduction leading to exocytosis and transcription and imply that immunosuppressive drugs target specific signal transduction pathways, rather than specific cell types. This hypothesis is supported by studies on the effect of rapamycin on IL-3 dependent proliferation of the rodent mast cell line PT18. Rapamycin inhibits proliferation of PT18 cells, achieving a plateau of 80% inhibition at 1 nM. This inhibition is prevented in a competitive manner by FK506, a structural analogue of rapamycin. Proliferation of rat basophilic leukemia cells and WEHI-3 cells was also inhibited, at doses comparable to those shown previously to inhibit IL-2-dependent proliferation of cytotoxic T lymphocyte line (CTLL) cells. In contrast, proliferation of A-431 cells, a epidermoid cell line, was not affected by rapamycin. DNA histograms indicate that complexes formed between the rapamycin-FK506-binding protein (FKBP) and rapamycin arrest-proliferating PT18 cells in the G0/G1-phase. It is concluded that FKBP-rapamycin complexes may inhibit proliferative signals emanating from IL-3 receptors, resulting in growth arrest of cytokine-dependent, hematopoietic cells. PMID:1384815

  19. A Signal-On Fluorosensor Based on Quench-Release Principle for Sensitive Detection of Antibiotic Rapamycin

    Directory of Open Access Journals (Sweden)

    Hee-Jin Jeong

    2015-03-01

    Full Text Available An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose “Q’-body”, which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body technology. We constructed rapamycin Q’-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q’-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

  20. A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.

    Science.gov (United States)

    Jeong, Hee-Jin; Itayama, Shuya; Ueda, Hiroshi

    2015-03-26

    An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

  1. The mTOR inhibitor rapamycin has limited acute anticonvulsant effects in mice.

    Directory of Open Access Journals (Sweden)

    Adam L Hartman

    Full Text Available The mammalian target of rapamycin (mTOR pathway integrates signals from different nutrient sources, including amino acids and glucose. Compounds that inhibit mTOR kinase activity such as rapamycin and everolimus can suppress seizures in some chronic animal models and in patients with tuberous sclerosis. However, it is not known whether mTOR inhibitors exert acute anticonvulsant effects in addition to their longer term antiepileptogenic effects. To gain insights into how rapamycin suppresses seizures, we investigated the anticonvulsant activity of rapamycin using acute seizure tests in mice.Following intraperitoneal injection of rapamycin, normal four-week-old male NIH Swiss mice were evaluated for susceptibility to a battery of acute seizure tests similar to those currently used to screen potential therapeutics by the US NIH Anticonvulsant Screening Program. To assess the short term effects of rapamycin, mice were seizure tested in ≤ 6 hours of a single dose of rapamycin, and for longer term effects of rapamycin, mice were tested after 3 or more daily doses of rapamycin.The only seizure test where short-term rapamycin treatment protected mice was against tonic hindlimb extension in the MES threshold test, though this protection waned with longer rapamycin treatment. Longer term rapamycin treatment protected against kainic acid-induced seizure activity, but only at late times after seizure onset. Rapamycin was not protective in the 6 Hz or PTZ seizure tests after short or longer rapamycin treatment times. In contrast to other metabolism-based therapies that protect in acute seizure tests, rapamycin has limited acute anticonvulsant effects in normal mice.The efficacy of rapamycin as an acute anticonvulsant agent may be limited. Furthermore, the combined pattern of acute seizure test results places rapamycin in a third category distinct from both fasting and the ketogenic diet, and which is more similar to drugs acting on sodium channels.

  2. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    International Nuclear Information System (INIS)

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-01-01

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  3. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  4. Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eun Joo [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Sowers, Anastasia; Thetford, Angela [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); McKay-Corkum, Grace; Chung, Su I. [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Mitchell, James B. [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Citrin, Deborah E., E-mail: citrind@mail.nih.gov [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States)

    2016-11-15

    Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. Methods and Materials: C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or a control diet 2 days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-time polymerase chain reaction. Senescence was assessed by staining for β-galactosidase activity. Results: Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116 days to 156 days (P=.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9 ± 11.8 μg per lung; irradiation plus rapamycin, 21.4 ± 6.0 μg per lung; P=.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16 weeks (3-fold reduction at 16 weeks, P<.001). Conclusions: Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.

  5. Drosophila insulin and target of rapamycin (TOR pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

    Directory of Open Access Journals (Sweden)

    Parisi Federica

    2011-09-01

    Full Text Available Abstract Background Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Results Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Conclusions Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At

  6. Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo.

    Science.gov (United States)

    Parisi, Federica; Riccardo, Sara; Daniel, Margaret; Saqcena, Mahesh; Kundu, Nandini; Pession, Annalisa; Grifoni, Daniela; Stocker, Hugo; Tabak, Esteban; Bellosta, Paola

    2011-09-27

    Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways

  7. Disease Evolution and Response to Rapamycin in Activated Phosphoinositide 3-Kinase δ Syndrome: The European Society for Immunodeficiencies-Activated Phosphoinositide 3-Kinase δ Syndrome Registry

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    Maria Elena Maccari

    2018-03-01

    Full Text Available Activated phosphoinositide 3-kinase (PI3K δ Syndrome (APDS, caused by autosomal dominant mutations in PIK3CD (APDS1 or PIK3R1 (APDS2, is a heterogeneous primary immunodeficiency. While initial cohort-descriptions summarized the spectrum of clinical and immunological manifestations, questions about long-term disease evolution and response to therapy remain. The prospective European Society for Immunodeficiencies (ESID-APDS registry aims to characterize the disease course, identify outcome predictors, and evaluate treatment responses. So far, 77 patients have been recruited (51 APDS1, 26 APDS2. Analysis of disease evolution in the first 68 patients pinpoints the early occurrence of recurrent respiratory infections followed by chronic lymphoproliferation, gastrointestinal manifestations, and cytopenias. Although most manifestations occur by age 15, adult-onset and asymptomatic courses were documented. Bronchiectasis was observed in 24/40 APDS1 patients who received a CT-scan compared with 4/15 APDS2 patients. By age 20, half of the patients had received at least one immunosuppressant, but 2–3 lines of immunosuppressive therapy were not unusual before age 10. Response to rapamycin was rated by physician visual analog scale as good in 10, moderate in 9, and poor in 7. Lymphoproliferation showed the best response (8 complete, 11 partial, 6 no remission, while bowel inflammation (3 complete, 3 partial, 9 no remission and cytopenia (3 complete, 2 partial, 9 no remission responded less well. Hence, non-lymphoproliferative manifestations should be a key target for novel therapies. This report from the ESID-APDS registry provides comprehensive baseline documentation for a growing cohort that will be followed prospectively to establish prognostic factors and identify patients for treatment studies.

  8. Nitrogen-responsive Regulation of GATA Protein Family Activators Gln3 and Gat1 Occurs by Two Distinct Pathways, One Inhibited by Rapamycin and the Other by Methionine Sulfoximine*

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    Georis, Isabelle; Tate, Jennifer J.; Cooper, Terrance G.; Dubois, Evelyne

    2011-01-01

    Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin. PMID:22039046

  9. Nitrogen-responsive regulation of GATA protein family activators Gln3 and Gat1 occurs by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine.

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    Georis, Isabelle; Tate, Jennifer J; Cooper, Terrance G; Dubois, Evelyne

    2011-12-30

    Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

  10. Rapamycin: An InhibiTOR of Aging Emerges From the Soil of Easter Island.

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    Arriola Apelo, Sebastian I; Lamming, Dudley W

    2016-07-01

    Rapamycin (sirolimus) is a macrolide immunosuppressant that inhibits the mechanistic target of rapamycin (mTOR) protein kinase and extends lifespan in model organisms including mice. Although rapamycin is an FDA-approved drug for select indications, a diverse set of negative side effects may preclude its wide-scale deployment as an antiaging therapy. mTOR forms two different protein complexes, mTORC1 and mTORC2; the former is acutely sensitive to rapamycin whereas the latter is only chronically sensitive to rapamycin in vivo. Over the past decade, it has become clear that although genetic and pharmacological inhibition of mTORC1 extends lifespan and delays aging, inhibition of mTORC2 has negative effects on mammalian health and longevity and is responsible for many of the negative side effects of rapamycin. In this review, we discuss recent advances in understanding the molecular and physiological effects of rapamycin treatment, and we discuss how the use of alternative rapamycin treatment regimens or rapamycin analogs has the potential to mitigate the deleterious side effects of rapamycin treatment by more specifically targeting mTORC1. Although the side effects of rapamycin are still of significant concern, rapid progress is being made in realizing the revolutionary potential of rapamycin-based therapies for the treatment of diseases of aging. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

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    Kenneth R. Watterson

    2012-03-01

    Full Text Available Activation of mammalian target of rapamycin 1 (mTORC1 by nutrients, insulin and leptin leads to appetite suppression (anorexia. Contrastingly, increased AMP-activated protein kinase (AMPK activity by ghrelin promotes appetite (orexia. However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K- and protein kinase B (PKB-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

  12. mTORC1 activity as a determinant of cancer risk--rationalizing the cancer-preventive effects of adiponectin, metformin, rapamycin, and low-protein vegan diets.

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    McCarty, Mark F

    2011-10-01

    Increased plasma levels of adiponectin, metformin therapy of diabetes, rapamycin administration in transplant patients, and lifelong consumption of low-protein plant-based diets have all been linked to decreased risk for various cancers. These benefits may be mediated, at least in part, by down-regulated activity of the mTORC1 complex, a key regulator of protein translation. By boosting the effective availability of the translation initiator eIF4E, mTORC1 activity promotes the translation of a number of "weak" mRNAs that code for proteins, often up-regulated in cancer, that promote cellular proliferation, invasiveness, and angiogenesis, and that abet cancer promotion and chemoresistance by opposing apoptosis. Measures which inhibit eIF4E activity, either directly or indirectly, may have utility not only for cancer prevention, but also for the treatment of many cancers in which eIF4E drives malignancy. Since eIF4E is overexpressed in many cancers, strategies which target eIF4E directly--some of which are now being assessed clinically--may have the broadest efficacy in this regard. Many of the "weak" mRNAs coding for proteins that promote malignant behavior or chemoresistance are regulated transcriptionally by NF-kappaB and/or Stat3, which are active in a high proportion of cancers; thus, regimens concurrently targeting eIF4E, NF-kappaB, and Stat3 may suppress these proteins at both the transcriptional and translational levels, potentially achieving a very marked reduction in their expression. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Differential Role of Rapamycin in Epidermis-Induced IL-15-IGF-1 Secretion via Activation of Akt/mTORC2.

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    Bai, Yang; Xu, Rui; Zhang, Xueyuan; Zhang, Xiaorong; Hu, Xiaohong; Li, Yashu; Li, Haisheng; Liu, Meixi; Huang, Zhenggen; Yan, Rongshuai; He, Weifeng; Luo, Gaoxing; Wu, Jun

    2017-01-01

    Backgroud/Aims: The effects of rapamycin (RPM) on wound healing have been previously studied. However, reciprocal contradictory data have been reported, and the underlying mechanism remains unclear. This study aims to uncover differential role of RPM in regulation of wound healing and explore the possible mechanism. C57BL/6J mice and epidermal cells were treated with different doses of RPM. The wound re-epithelialization was observed by hematoxylin and eosin (HE) staining. The expression of IL-15 and IGF-1 were detected by immunohistochemistry and quantitative real-time PCR. Epidermal cell survival was determined by CCK-8 assays. Moreover, the mTORC1 and mTORC2 pathway were examined by western blot analysis. This study showed that differential doses of RPM could lead to separate consequences in epidermis. Histological analyses showed that low-dose RPM promoted wound healing, and enhanced the expression of IL-15 and IGF-1. Furthermore, western blot analysis showed that the effect of low-dose RPM in epidermis were not through mTORC1 pathway. Instead, activation of the Akt/mTORC2 pathway was involved in low-dose RPM-induced IL-15 and IGF-1 production in epidermis, while high-dose RPM inhibited the expression of IL-15 and IGF-1 and the activity of mTORC1 and mTORC2 pathway. This study for the first time demonstrated that RPM-mediated wound healing was dose-dependent. © 2017 The Author(s). Published by S. Karger AG, Basel.

  14. Biphasic Rapamycin Effects in Lymphoma and Carcinoma Treatment.

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    Liu, Yang; Pandeswara, Srilakshmi; Dao, Vinh; Padrón, Álvaro; Drerup, Justin M; Lao, Shunhua; Liu, Aijie; Hurez, Vincent; Curiel, Tyler J

    2017-01-15

    mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1-8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)-mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell-dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L + CD8 + central memory T cells in EL4 challenge, but without clinical benefit. LD rapamycin significantly enhanced DD treatment efficacy, but DD plus LD rapamycin treatment effects were independent of antitumor immunity. Instead, rapamycin upregulated EL4 IL2 receptor in vitro and in vivo, facilitating direct DD tumor cell killing. LD rapamycin augmented DD efficacy against B16 melanoma and a human B-cell lymphoma, but not against human Jurkat T-cell lymphoma or ID8agg ovarian cancer cells. Treatment effects correlated with IL2R expression, but mechanisms in some tumors were not fully defined. Overall, our data define a distinct, biphasic mechanisms of action of mTOR inhibition at doses that are clinically exploitable, including in T-cell lymphomas. Cancer Res; 77(2); 520-31. ©2016 AACR. ©2016 American Association for Cancer Research.

  15. Systemic inhibition of mTOR kinase via rapamycin disrupts consolidation and reconsolidation of auditory fear memory.

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    Mac Callum, Phillip E; Hebert, Mark; Adamec, Robert E; Blundell, Jacqueline

    2014-07-01

    The mammalian target of rapamycin (mTOR) kinase is a critical regulator of mRNA translation and is known to be involved in various long lasting forms of synaptic and behavioural plasticity. However, information concerning the temporal pattern of mTOR activation and susceptibility to pharmacological intervention during both consolidation and reconsolidation of long-term memory (LTM) remains scant. Male C57BL/6 mice were injected systemically with rapamycin at various time points following conditioning or retrieval in an auditory fear conditioning paradigm, and compared to vehicle (and/or anisomycin) controls for subsequent memory recall. Systemic blockade of mTOR with rapamycin immediately or 12h after training or reactivation impairs both consolidation and reconsolidation of an auditory fear memory. Further behavioural analysis revealed that the enduring effects of rapamycin on reconsolidation are dependent upon reactivation of the memory trace. Rapamycin, however, has no effect on short-term memory or the ability to retrieve an established fear memory. Collectively, our data suggest that biphasic mTOR signalling is essential for both consolidation and reconsolidation-like activities that contribute to the formation, re-stabilization, and persistence of long term auditory-fear memories, while not influencing other aspects of the memory trace. These findings also provide evidence for a cogent treatment model for reducing the emotional strength of established, traumatic memories analogous to those observed in acquired anxiety disorders such as posttraumatic stress disorder (PTSD) and specific phobias, through pharmacologic blockade of mTOR using systemic rapamycin following reactivation. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Tomato FK506 Binding Protein 12KD (FKBP12 mediates the interaction between rapamycin and Target of Rapamycin (TOR

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    Fangjie Xiong

    2016-11-01

    Full Text Available Target of Rapamycin (TOR signaling is an important regulator in multiple organisms including yeast, plants and animals. However, the TOR signaling in plants is much less understood as compared to that in yeast and animals. TOR kinase can be efficiently suppressed by rapamycin in the presence of functional FK506 Binding Protein 12KD (FKBP12 in yeast and animals. In most examined higher plants rapamycin fails to inhibit TOR kinase due to the non-functional FKBP12. Here we find that tomato plants showed obvious growth inhibition when treated with rapamycin and the inhibitory phenotype is similar to suppression of TOR causing by active-site TOR inhibitors (asTORis such as KU63794, AZD8055 and Torin1. The chemical genetic assays using TOR inhibitors and heterologous expressing SlFKBP12 in Arabidopsis indicated that the TOR signaling is functional in tomato. The protein gel shifting and TOR inhibitors combination assays showed that SlFKBP12 can mediate the interaction between rapamycin and TOR. Furthermore, comparative expression profiling analysis between treatments with rapamycin and KU63794 identified highly overlapped Differentially Expressed Genes (DEGs which are involved in many anabolic and catabolic processes, such as photosynthesis, cell wall restructuring, and senescence in tomato. These observations suggest that SlFFBP12 is functional in tomato. The results provided basic information of TOR signaling in tomato, and also some new insights into how TOR controls plant growth and development through reprogramming the transcription profiles

  17. Tomato FK506 Binding Protein 12KD (FKBP12) Mediates the Interaction between Rapamycin and Target of Rapamycin (TOR).

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    Xiong, Fangjie; Dong, Pan; Liu, Mei; Xie, Gengxin; Wang, Kai; Zhuo, Fengping; Feng, Li; Yang, Lu; Li, Zhengguo; Ren, Maozhi

    2016-01-01

    Target of Rapamycin (TOR) signaling is an important regulator in multiple organisms including yeast, plants, and animals. However, the TOR signaling in plants is much less understood as compared to that in yeast and animals. TOR kinase can be efficiently suppressed by rapamycin in the presence of functional FK506 Binding Protein 12 KD (FKBP12) in yeast and animals. In most examined higher plants rapamycin fails to inhibit TOR kinase due to the non-functional FKBP12. Here we find that tomato plants showed obvious growth inhibition when treated with rapamycin and the inhibitory phenotype is similar to suppression of TOR causing by active-site TOR inhibitors (asTORis) such as KU63794, AZD8055, and Torin1. The chemical genetic assays using TOR inhibitors and heterologous expressing SlFKBP12 in Arabidopsis indicated that the TOR signaling is functional in tomato. The protein gel shifting and TOR inhibitors combination assays showed that SlFKBP12 can mediate the interaction between rapamycin and TOR. Furthermore, comparative expression profile analysis between treatments with rapamycin and KU63794 identified highly overlapped Differentially Expressed Genes (DEGs) which are involved in many anabolic and catabolic processes, such as photosynthesis, cell wall restructuring, and senescence in tomato. These observations suggest that SlFFBP12 is functional in tomato. The results provided basic information of TOR signaling in tomato, and also some new insights into how TOR controls plant growth and development through reprogramming the transcription profiles.

  18. Characterization of the metabolic phenotype of rapamycin-treated CD8+ T cells with augmented ability to generate long-lasting memory cells.

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    Shan He

    Full Text Available BACKGROUND: Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized. DESIGN AND METHODS: Using lymphocytic choriomeningitis virus (LCMV peptide gp33-specific CD8(+ T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo. RESULTS: Antigen-activated CD8(+ T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS. These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells. CONCLUSIONS: Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

  19. Novel synergistic antitumor effects of rapamycin with bortezomib on hepatocellular carcinoma cells and orthotopic tumor model

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    Wang Cun

    2012-05-01

    Full Text Available Abstract Background Despite recent advances in the treatment of hepatocellular carcinoma (HCC, the chemotherapy efficacy against HCC is still unsatisfactory. The mammalian target of rapamycin (mTOR has been emerged as an important cancer therapeutic target. However, HCC cells often resistant to rapamycin because of the paradoxical activation of Akt by rapamycin. In this study, we investigated whether bortezomib could enhance the antitumor effects of rapamycin. Methods The effects of rapamycin and bortezomib on HCC proliferation, apoptosis, migration, and invasiveness in vitro were assessed by CCK-8 analysis, flow cytometry, Hoechst 33342 staining and transwell assays, respectively. Total and phosphorylated protein levels of Akt were detected by Western blotting. The effects of rapamycin and/or bortezomib on the mRNA expression levels of p53, p27, p21 and Bcl-2 family in HCCLM3 cells were evaluated by RT-PCR. The roles of rapamycin and bortezomib on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. The effects of rapamycin and bortezomib on cell proliferation and apoptosis in vivo were test by PCNA and TUNEL staining. Results Bortezomib synergized with rapamycin to reduce cell growth, induce apoptosis, and inhibit cell mobility in vitro. Further mechanistic studies showed that bortezomib inhibited rapamycin-induced phosphorylated Akt, which in turn enhanced apoptosis of HCC cell lines. The alteration of the mRNA expression of cell cycle inhibitors p53, p27, p21 and apoptosis associated genes Bcl-2, Bax were also involved in the synergistic antitumor effects of rapamycin and bortezomib. P53 inhibitor PFT-α significantly attenuate the effect of rapamycin and bortezomib on cell apoptosis, which indicated that the pro-apoptotic effect of rapamycin and bortezomib may be p53-dependent. Treatment of HCCLM3-R bearing nude mice with rapamycin and bortezomib significantly enhanced tumor growth

  20. Beneficial role of rapamycin in experimental autoimmune myositis.

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    Nicolas Prevel

    Full Text Available We developed an experimental autoimmune myositis (EAM mouse model of polymyositis where we outlined the role of regulatory T (Treg cells. Rapamycin, this immunosuppressant drug used to prevent rejection in organ transplantation, is known to spare Treg. Our aim was to test the efficacy of rapamycin in vivo in this EAM model and to investigate the effects of the drug on different immune cell sub-populations.EAM is induced by 3 injections of myosin emulsified in CFA. Mice received rapamycin during 25 days starting one day before myosin immunization (preventive treatment, or during 10 days following the last myosin immunization (curative treatment.Under preventive or curative treatment, an increase of muscle strength was observed with a parallel decrease of muscle inflammation, both being well correlated (R(2 = -0.645, p<0.0001. Rapamycin induced a general decrease in muscle of CD4 and CD8 T cells in lymphoid tissues, but spared B cells. Among T cells, the frequency of Treg was increased in rapamycin treated mice in draining lymph nodes (16.9 ± 2.2% vs. 9.3 ± 1.4%, p<0.001, which were mostly activated regulatory T cells (CD62L(lowCD44(high: 58.1 ± 5.78% vs. 33.1 ± 7%, treated vs. untreated, p<0.001. In rapamycin treated mice, inhibition of proliferation (Ki-67(+ is more important in effector T cells compared to Tregs cells (p<0.05. Furthermore, during preventive treatment, rapamycin increased the levels of KLF2 transcript in CD44(low CD62L(high naive T cell and in CD62L(low CD44(high activated T cell.Rapamycin showed efficacy both as curative and preventive treatment in our murine model of experimental myositis, in which it induced an increase of muscle strength with a parallel decrease in muscle inflammation. Rapamycin administration was also associated with a decrease in the frequency of effector T cells, an increase in Tregs, and, when administered as preventive treatment, an upregulation of KFL2 in naive and activated T cells.

  1. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

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    Seung Eun Lee

    2014-05-01

    Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

  2. Prognostic significance and therapeutic potential of the activation of anaplastic lymphoma kinase/protein kinase B/mammalian target of rapamycin signaling pathway in anaplastic large cell lymphoma

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    Gao, Ju; Yin, Minzhi; Zhu, Yiping; Gu, Ling; Zhang, Yanle; Li, Qiang; Jia, Cangsong; Ma, Zhigui

    2013-01-01

    Activation of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway has been demonstrated to be involved in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated tumorigenesis in anaplastic large cell lymphoma (ALCL) and correlated with unfavorable outcome in certain types of other cancers. However, the prognostic value of AKT/mTOR activation in ALCL remains to be fully elucidated. In the present study, we aim to address this question from a clinical perspective by comparing the expressions of the AKT/mTOR signaling molecules in ALCL patients and exploring the therapeutic significance of targeting the AKT/mTOR pathway in ALCL. A cohort of 103 patients with ALCL was enrolled in the study. Expression of ALK fusion proteins and the AKT/mTOR signaling phosphoproteins was studied by immunohistochemical (IHC) staining. The pathogenic role of ALK fusion proteins and the therapeutic significance of targeting the ATK/mTOR signaling pathway were further investigated in vitro study with an ALK + ALCL cell line and the NPM-ALK transformed BaF3 cells. ALK expression was detected in 60% of ALCLs, of which 79% exhibited the presence of NPM-ALK, whereas the remaining 21% expressed variant-ALK fusions. Phosphorylation of AKT, mTOR, 4E-binding protein-1 (4E-BP1), and 70 kDa ribosomal protein S6 kinase polypeptide 1 (p70S6K1) was detected in 76%, 80%, 91%, and 93% of ALCL patients, respectively. Both phospho-AKT (p-AKT) and p-mTOR were correlated to ALK expression, and p-mTOR was closely correlated to p-AKT. Both p-4E-BP1 and p-p70S6K1 were correlated to p-mTOR, but were not correlated to the expression of ALK and p-AKT. Clinically, ALK + ALCL occurred more commonly in younger patients, and ALK + ALCL patients had a much better prognosis than ALK-ALCL cases. However, expression of p-AKT, p-mTOR, p-4E-BP1, or p-p70S6K1 did not have an impact on the clinical outcome. Overexpression of NPM-ALK in a nonmalignant murine pro-B lymphoid cell line, BaF3, induced the

  3. Bone growth during rapamycin therapy in young rats

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    He Yu-Zhu

    2009-01-01

    Full Text Available Abstract Background Rapamycin is an effective immunosuppressant widely used to maintain the renal allograft in pediatric patients. Linear growth may be adversely affected in young children since rapamycin has potent anti-proliferative and anti-angiogenic properties. Methods Weanling three week old rats were given rapamycin at 2.5 mg/kg daily by gavage for 2 or 4 weeks and compared to a Control group given equivalent amount of saline. Morphometric measurements and biochemical determinations for serum calcium, phosphate, iPTH, urea nitrogen, creatinine and insulin-growth factor I (IGF-I were obtained. Histomorphometric analysis of the growth plate cartilage, in-situ hybridization experiments and immunohistochemical studies for various proteins were performed to evaluate for chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption. Results At the end of the 2 weeks, body and tibia length measurements were shorter after rapamycin therapy associated with an enlargement of the hypertrophic zone in the growth plate cartilage. There was a decrease in chondrocyte proliferation assessed by histone-4 and mammalian target of rapamycin (mTOR expression. A reduction in parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP and an increase in Indian hedgehog (Ihh expression may explain in part, the increase number of hypertrophic chondrocytes. The number of TRAP positive multinucleated chondro/osteoclasts declined in the chondro-osseous junction with a decrease in the receptor activator of nuclear factor kappa β ligand (RANKL and vascular endothelial growth factor (VEGF expression. Although body and tibial length remained short after 4 weeks of rapamycin, changes in the expression of chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption which were significant after 2 weeks of rapamycin improved at the end of 4 weeks. Conclusion When given to young rats, 2 weeks of rapamycin

  4. Intrathecal administration of rapamycin inhibits the phosphorylation of DRG Nav1.8 and attenuates STZ-induced painful diabetic neuropathy in rats.

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    He, Wan-You; Zhang, Bin; Xiong, Qing-Ming; Yang, Cheng-Xiang; Zhao, Wei-Cheng; He, Jian; Zhou, Jun; Wang, Han-Bing

    2016-04-21

    The mammalian target of rapamycin (mTOR) is a key regulator of mRNA translation and protein synthesis, and it is specifically inhibited by rapamycin. In chronic pain conditions, mTOR-mediated local protein synthesis is crucial for neuronal hyperexcitability and synaptic plasticity. The tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8 plays a major role in action potential initiation and propagation and cellular excitability in DRG (dorsal root ganglion) neurons. In this study, we investigated if mTOR modulates the phosphorylation of Nav1.8 that is associated with neuronal hyperexcitability and behavioral hypersensitivity in STZ-induced diabetic rats. Painful diabetic neuropathy (PDN) was induced in Sprague-Dawley rats by intraperitoneal injection with streptozotocin (STZ) at 60mg/kg. After the onset of PDN, the rats received daily intrathecal administrations of rapamycin (1μg, 3μg, or 10μg/day) for 7 days; other diabetic rats received the same volumes of dimethyl sulfoxide (DMSO). Herein, we demonstrate a marked increase in protein expression of total mTOR and phospho-mTOR (p-mTOR) together with the up-regulation of phosphor-Nav1.8 (p-Nav1.8) prior to the mechanical withdrawal threshold reaching a significant reduction in dorsal root ganglions (DRGs). Furthermore, the intrathecal administration of rapamycin, inhibiting the activity of mTOR, suppressed the phosphorylation of DRG Nav1.8, reduced the TTX-R current density, heightened the voltage threshold for activation and lowered the voltage threshold for inactivation and relieved mechanical hypersensitivity in diabetic rats. An intrathecal injection (i.t.) of rapamycin inhibited the phosphorylation and enhanced the functional availability of DRG Nav1.8 attenuated STZ-induced hyperalgesia. These results suggest that rapamycin is a potential therapeutic intervention for clinical PDN. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Potential use of rapamycin in HIV infection

    DEFF Research Database (Denmark)

    Donia, Marco; McCubrey, James A; Bendtzen, Klaus

    2010-01-01

    The strong need for the development of alternative anti-HIV agents is primarily due to the emergence of strain-resistant viruses, the need for sustained adherence to complex treatment regimens and the toxicity of currently used antiviral drugs. This review analyzes proof of concept studies...... indicating that the immunomodulatory drug rapamycin (RAPA) possesses anti-HIV properties both in vitro and in vivo that qualifies it as a potential new anti-HIV drug. It represents a literature review of published studies that evaluated the in vitro and in vivo activity of RAPA in HIV. RAPA represses HIV-1...... replication in vitro through different mechanisms including, but not limited, to down regulation of CCR5. In addition RAPA synergistically enhances the anti-HIV activity of entry inhibitors such as vicriviroc, aplaviroc and enfuvirtide in vitro. RAPA also inhibits HIV-1 infection in human peripheral blood...

  6. Inhibition of repopulation is not a determining factor for the radiosensitizing effects of rapamycin

    International Nuclear Information System (INIS)

    Sarkaria, J.N.; Carlson, B.L.; Mladek, A.C.

    2003-01-01

    The mammalian target of rapamycin (mTOR) is a key downstream effector of the PI3K-Akt signaling pathway, and we have previously shown that inhibition of mTOR by rapamycin significantly enhances the efficacy of prolonged fractionated radiation in U87 glioma cells grown as xenografts or spheroids. To test whether inhibition of repopulation between radiation fractions contributes to the sensitizing effects of rapamycin, the efficacy of our previous protracted radiation schedule was compared with an accelerated regimen in U87 spheroids. Regrowth of individual spheroids was tracked over time following treatment with either accelerated or protracted radiation in the presence or absence of rapamycin. As in our previous studies, treatment with 10 nM rapamycin significantly increased the time required for U87 spheroids to regrow to 10 times their original volume (22 ± 2 days [mean ± 95% CI]) compared to control (7 ± 1 days). Regrowth after protracted radiation (2 Gy every 3 days x 4; 9 ± 2 days)did not significantly differ from control treatment, while accelerated radiation (2 Gy every 4 hours x 4) modestly delayed spheroid regrowth (12 ± 2 days). Specific to our model, the relatively small difference in regrowth time between the two radiation fractionation schedules suggests that repopulation is not a major detrimental factor in the protracted radiation schedule. Interestingly, the combination of rapamycin with either protracted or accelerated RT significantly enhanced the efficacy of the radiation with regrowth times of 31 ± 4 days and 29 ± 4 days, respectively. Consistent with this in vitro data, preliminary results from an animal study suggest that treatment with a rapamycin analog and daily radiation is as effective as protracted radiation/ rapamycin schedules. Thus, any effects of rapamycin on repopulation in our model systems do not contribute significantly to the sensitizing effects of rapamycin

  7. Suppression of Th17-polarized airway inflammation by rapamycin.

    Science.gov (United States)

    Joean, Oana; Hueber, Anja; Feller, Felix; Jirmo, Adan Chari; Lochner, Matthias; Dittrich, Anna-Maria; Albrecht, Melanie

    2017-11-10

    Because Th17-polarized airway inflammation correlates with poor control in bronchial asthma and is a feature of numerous other difficult-to-treat inflammatory lung diseases, new therapeutic approaches for this type of airway inflammation are necessary. We assessed different licensed anti-inflammatory agents with known or expected efficacy against Th17-polarization in mouse models of Th17-dependent airway inflammation. Upon intravenous transfer of in vitro derived Th17 cells and intranasal challenge with the corresponding antigen, we established acute and chronic murine models of Th17-polarised airway inflammation. Consecutively, we assessed the efficacy of methylprednisolone, roflumilast, azithromycin, AM80 and rapamycin against acute or chronic Th17-dependent airway inflammation. Quantifiers for Th17-associated inflammation comprised: bronchoalveolar lavage (BAL) differential cell counts, allergen-specific cytokine and immunoglobulin secretion, as well as flow cytometric phenotyping of pulmonary inflammatory cells. Only rapamycin proved effective against acute Th17-dependent airway inflammation, accompanied by increased plasmacytoid dendritic cells (pDCs) and reduced neutrophils as well as reduced CXCL-1 levels in BAL. Chronic Th17-dependent airway inflammation was unaltered by rapamycin treatment. None of the other agents showed efficacy in our models. Our results demonstrate that Th17-dependent airway inflammation is difficult to treat with known agents. However, we identify rapamycin as an agent with inhibitory potential against acute Th17-polarized airway inflammation.

  8. Potential use of rapamycin in HIV infection

    DEFF Research Database (Denmark)

    Donia, Marco; McCubrey, James A; Bendtzen, Klaus

    2010-01-01

    The strong need for the development of alternative anti-HIV agents is primarily due to the emergence of strain-resistant viruses, the need for sustained adherence to complex treatment regimens and the toxicity of currently used antiviral drugs. This review analyzes proof of concept studies......, the evidence presented in this review suggests that RAPA may be a useful drug that should be evaluated for the prevention and treatment of HIV-1 infection....... indicating that the immunomodulatory drug rapamycin (RAPA) possesses anti-HIV properties both in vitro and in vivo that qualifies it as a potential new anti-HIV drug. It represents a literature review of published studies that evaluated the in vitro and in vivo activity of RAPA in HIV. RAPA represses HIV-1...

  9. Phosphorylation of translation factors in response to anoxia in turtles, Trachemys scripta elegans: role of the AMP-activated protein kinase and target of rapamycin signalling pathways.

    Science.gov (United States)

    Rider, Mark H; Hussain, Nusrat; Dilworth, Stephen M; Storey, Kenneth B

    2009-12-01

    Long-term survival of oxygen deprivation by animals with well-developed anoxia tolerance depends on multiple biochemical adaptations including strong metabolic rate depression. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the suppression of protein synthesis that occurs when turtles experience anoxic conditions. AMPK activity and the phosphorylation state of ribosomal translation factors were measured in liver, heart, red muscle and white muscle of red-eared slider turtles (Trachemys scripta elegans) subjected to 20 h of anoxic submergence. AMPK activity increased twofold in white muscle of anoxic turtles compared with aerobic controls but remained unchanged in liver and red muscle, whereas in heart AMPK activity decreased by 40%. Immunoblotting with phospho-specific antibodies revealed that eukaryotic elongation factor-2 phosphorylation at the inactivating Thr56 site increased six- and eightfold in red and white muscles from anoxic animals, respectively, but was unchanged in liver and heart. The phosphorylation state of the activating Thr389 site of p70 ribosomal protein S6 kinase was reduced under anoxia in red muscle and heart but was unaffected in liver and white muscle. Exposure to anoxia decreased 40S ribosomal protein S6 phosphorylation in heart and promoted eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) dephosphorylation in red muscle, but surprisingly increased 4E-BP1 phosphorylation in white muscle. The changes in phosphorylation state of translation factors suggest that organ-specific patterns of signalling and response are involved in achieving the anoxia-induced suppression of protein synthesis in turtles.

  10. Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats I: Magnetic resonance imaging.

    Science.gov (United States)

    van Vliet, Erwin A; Otte, Willem M; Wadman, Wytse J; Aronica, Eleonora; Kooij, Gijs; de Vries, Helga E; Dijkhuizen, Rick M; Gorter, Jan A

    2016-01-01

    The mammalian target of rapamycin (mTOR) pathway has received increasing attention as a potential antiepileptogenic target. Treatment with the mTOR inhibitor rapamycin after status epilepticus reduces the development of epilepsy in a rat model. To study whether rapamycin mediates this effect via restoration of blood-brain barrier (BBB) dysfunction, contrast-enhanced magnetic resonance imaging (CE-MRI) was used to determine BBB permeability throughout epileptogenesis. Imaging was repeatedly performed until 6 weeks after kainic acid-induced status epilepticus in rapamycin (6 mg/kg for 6 weeks starting 4 h after SE) and vehicle-treated rats, using gadobutrol as contrast agent. Seizures were detected using video monitoring in the week following the last imaging session. Gadobutrol leakage was widespread and extensive in both rapamycin and vehicle-treated epileptic rats during the acute phase, with the piriform cortex and amygdala as the most affected regions. Gadobutrol leakage was higher in rapamycin-treated rats 4 and 8 days after status epilepticus compared to vehicle-treated rats. However, during the chronic epileptic phase, gadobutrol leakage was lower in rapamycin-treated epileptic rats along with a decreased seizure frequency. This was confirmed by local fluorescein staining in the brains of the same rats. Total brain volume was reduced by this rapamycin treatment regimen. The initial slow recovery of BBB function in rapamycin-treated epileptic rats indicates that rapamycin does not reduce seizure activity by a gradual recovery of BBB integrity. The reduced BBB leakage during the chronic phase, however, could contribute to the decreased seizure frequency in post-status epilepticus rats treated with rapamycin. Furthermore, the data show that CE-MRI (using step-down infusion with gadobutrol) can be used as biomarker for monitoring the effect of drug therapy in rats. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.

  11. Activation of phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin pathway and response to everolimus in endocrine receptor-positive metastatic breast cancer – A retrospective pilot analysis and viewpoint

    Directory of Open Access Journals (Sweden)

    Jyoti Bajpai

    2017-01-01

    Full Text Available Introduction: Biomarkers predictive of response to mechanistic target of rapamycin (mTOR inhibitor, everolimus, in endocrine receptor (ER-positive metastatic breast cancer (MBC are a work in progress. We evaluated the feasibility of directly measuring mTOR activity and phosphatase and tensin homolog (PTEN expression and correlating their expression with response and survival. Materials and Methods: MBC patients who received everolimus with endocrine therapy (ET after progression on an aromatase inhibitor and had adequate tissue preservation for estimation of mTOR activity and PTEN expression were selected for analysis from a prospectively maintained database. Progression-free survival (PFS and overall survival (OS were estimated by Kaplan–Meier method, and correlation between mTOR activity and PTEN expression with survival was done by log-rank test. Results: Thirteen ER-positive MBC patients were available for analysis. PTEN expression was lost in 11/13 (84.6% patients and retained in 2/13 patients (15.4%. mTOR activity was absent in four patients (30.7%, weak in six patients (46.1%, and moderate in 3 patients (23.2%. Median PFS for the entire population was 2.5 months while median OS was not reached. Patients with an absent mTOR activity showed a longer PFS (5 vs. 1.5 vs. 2 months than those with weak and moderate activity, respectively (P = 0.043. There was no correlation between loss of PTEN expression and PFS. Conclusions: Measurement of direct mTOR activity in patients with MBC receiving everolimus/ET combination appears feasible. Absent mTOR activity may predict for longer PFS with everolimus-ET combination and requires further study.

  12. Differential effects of rapamycin and dexamethasone in mouse models of established allergic asthma.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Mushaben

    Full Text Available The mammalian target of rapamycin (mTOR plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4-6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR, inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4(+ T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4(+ T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established.

  13. Rapamycin sensitizes T-ALL cells to dexamethasone-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Mu Dezhi

    2010-11-01

    Full Text Available Abstract Background Glucocorticoid (GC resistance is frequently seen in acute lymphoblastic leukemia of T-cell lineage (T-ALL. In this study we investigate the potential and mechanism of using rapamycin to restore the sensitivity of GC-resistant T-ALL cells to dexamethasone (Dex treatment. Methods Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide (MTT assay. Fluorescence-activated cell sorting (FACS analysis was used to analyze apoptosis and cell cycles. Western blot analysis was performed to test the expression of the downstream effector proteins of mammalian target of rapamycin (mTOR, the cell cycle regulatory proteins, and apoptosis associated proteins. Results 10 nM rapamycin markedly increased GC sensitivity in GC-resistant T-ALL cells and this effect was mediated, at least in part, by inhibition of mTOR signaling pathway. Cell cycle arrest was associated with modulation of G1-S phase regulators. Both rapamycin and Dex can induce up-regulation of cyclin-dependent kinase (CDK inhibitors of p21 and p27 and co-treatment of rapamycin with Dex resulted in a synergistic induction of their expressions. Rapamycin did not obviously affect the expression of cyclin A, whereas Dex induced cyclin A expression. Rapamycin prevented Dex-induced expression of cyclin A. Rapamycin had a stronger inhibition of cyclin D1 expression than Dex. Rapamycin enhanced GC-induced apoptosis and this was not achieved by modulation of glucocorticoid receptor (GR expression, but synergistically up-regulation of pro-apoptotic proteins like caspase-3, Bax, and Bim, and down-regulation of anti-apoptotic protein of Mcl-1. Conclusion Our data suggests that rapamycin can effectively reverse GC resistance in T-ALL and this effect is achieved by inducing cell cycles arrested at G0/G1 phase and activating the intrinsic apoptotic program. Therefore, combination of mTOR inhibitor rapamycin with GC containing protocol might be an attracting

  14. Target of Rapamycin (TOR) Regulates Growth in Response to Nutritional Signals.

    Science.gov (United States)

    Weisman, Ronit

    2016-10-01

    All organisms can respond to the availability of nutrients by regulating their metabolism, growth, and cell division. Central to the regulation of growth in response to nutrient availability is the target of rapamycin (TOR) signaling that is composed of two structurally distinct complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2). The TOR genes were first identified in yeast as target of rapamycin, a natural product of a soil bacterium, which proved beneficial as an immunosuppressive and anticancer drug and is currently being tested for a handful of other pathological conditions including diabetes, neurodegeneration, and age-related diseases. Studies of the TOR pathway unraveled a complex growth-regulating network. TOR regulates nutrient uptake, transcription, protein synthesis and degradation, as well as metabolic pathways, in a coordinated manner that ensures that cells grow or cease growth in response to nutrient availability. The identification of specific signals and mechanisms that stimulate TOR signaling is an active and exciting field of research that has already identified nitrogen and amino acids as key regulators of TORC1 activity. The signals, as well as the cellular functions of TORC2, are far less well understood. Additional open questions in the field concern the relationships between TORC1 and TORC2, as well as the links with other nutrient-responsive pathways. Here I review the main features of TORC1 and TORC2, with a particular focus on yeasts as model organisms.

  15. Table of specific activities of selected isotopes

    International Nuclear Information System (INIS)

    Shipley, G.

    The bulk of this publication consists of a table of the half-lives, decay modes, and specific activities of isotopes selected for their particular interest to the Environmental Health and Safety Department, LBL. The specific activities were calculated with a PDP 9/15 computer. Also included in the report is a table of stable isotopes, the Th and U decay chains, a chart of the nuclides for elements 101 through 106, the heavy element region of the periodic table, and a specific activity monograph. 5 figures, 2 tables

  16. Enzyme specific activity in functionalized nanoporous supports

    International Nuclear Information System (INIS)

    Lei Chenghong; Soares, Thereza A; Shin, Yongsoon; Liu Jun; Ackerman, Eric J

    2008-01-01

    Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (P LD ) in functionalized nanoporous supports so that the enzyme immobilization efficiency (I e , defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH 2 - and HOOC-functionalized mesoporous silica (300 A, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing P LD . With decreasing P LD , I e of GOX in FMS increased from 150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing P LD . With increasing P LD , the corresponding I e of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high P LD , consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high P LD and may promote a more favorable confinement environment that enhances the OPH activity

  17. A hexane fraction of guava Leaves (Psidium guajava L.) induces anticancer activity by suppressing AKT/mammalian target of rapamycin/ribosomal p70 S6 kinase in human prostate cancer cells.

    Science.gov (United States)

    Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik; Cho, Somi K; Ahn, Kwang Seok

    2012-03-01

    This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer.

  18. Restoration of Corticosteroid Sensitivity in Chronic Obstructive Pulmonary Disease by Inhibition of Mammalian Target of Rapamycin.

    Science.gov (United States)

    Mitani, Akihisa; Ito, Kazuhiro; Vuppusetty, Chaitanya; Barnes, Peter J; Mercado, Nicolas

    2016-01-15

    Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.

  19. IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes

    DEFF Research Database (Denmark)

    Marzec, Michal; Liu, Xiaobin; Kasprzycka, Monika

    2008-01-01

    We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as ...

  20. Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons.

    Science.gov (United States)

    Coutinho-Budd, Jaeda C; Snider, Samuel B; Fitzpatrick, Brendan J; Rittiner, Joseph E; Zylka, Mark J

    2013-09-08

    Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. These systems are based on rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphatase (Inp54p) system to deplete PIP2 in nociceptive dorsal root ganglia (DRG) neurons. We genetically targeted membrane-tethered CFP-FRBPLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRBPLF knockin mouse. In a second knockin mouse line, we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha (CGRPα) locus. We hypothesized that after intercrossing these mice, rapamycin treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in CGRP+ DRG neurons. In control experiments with cell lines, rapamycin induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and subsequent depletion of PIP2, as measured with a PIP2 biosensor. However, rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in FRBPLF-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP2-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRBPLF in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRBPLF with endogenous FKBP12. Taken together, our data indicate that these knockin mice cannot be used to inducibly deplete PIP2 in DRG neurons. Moreover, our data suggest that high levels of endogenous FKBP12 could compete for binding to FRBPLF, hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12.

  1. Are invertebrates relevant models in ageing research? Focus on the effects of rapamycin on TOR.

    Science.gov (United States)

    Erdogan, Cihan Suleyman; Hansen, Benni Winding; Vang, Ole

    2016-01-01

    Ageing is the organisms increased susceptibility to death, which is linked to accumulated damage in the cells and tissues. Ageing is a complex process regulated by crosstalk of various pathways in the cells. Ageing is highly regulated by the Target of Rapamycin (TOR) pathway activity. TOR is an evolutionary conserved key protein kinase in the TOR pathway that regulates growth, proliferation and cell metabolism in response to nutrients, growth factors and stress. Comparing the ageing process in invertebrate model organisms with relatively short lifespan with mammals provides valuable information about the molecular mechanisms underlying the ageing process faster than mammal systems. Inhibition of the TOR pathway activity via either genetic manipulation or rapamycin increases lifespan profoundly in most invertebrate model organisms. This contribution will review the recent findings in invertebrates concerning the TOR pathway and effects of TOR inhibition by rapamycin on lifespan. Besides some contradictory results, the majority points out that rapamycin induces longevity. This suggests that administration of rapamycin in invertebrates is a promising tool for pursuing the scientific puzzle of lifespan prolongation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Blocking mammalian target of rapamycin (mTOR) improves neuropathic pain evoked by spinal cord injury.

    Science.gov (United States)

    Wang, Xiaoping; Li, Xiaojia; Huang, Bin; Ma, Shuai

    2016-01-01

    Spinal cord injury (SCI) is an extremely serious type of physical trauma observed in clinics. Neuropathic pain resulting from SCI has a lasting and significant impact on most aspects of daily life. Thus, a better understanding of the molecular pathways responsible for the cause of neuropathic pain observed in SCI is important to develop effective therapeutic agents and treatment strategies. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that is well known for its critical roles in regulating protein synthesis and growth. Furthermore, compelling evidence supports the notion that widespread dysregulation of mTOR and its downstream pathways are involved in neuropathic pain. Thus, in this study we specifically examined the underlying mechanisms by which mTOR and its signaling pathways are involved in SCI-evoked neuropathic pain in a rat model. Overall, we demonstrated that SCI increased the protein expression of p-mTOR, and mTORmediated- phosphorylation of 4E-binding protein 4 (4E-BP1) and p70 ribosomal S6 protein kinase 1 (S6K1) in the superficial dorsal horn of the spinal cord. Also, we showed that blocking spinal mTOR by intrathecal injection of rapamycin significantly inhibited pain responses induced by mechanical and thermal stimulation. In addition, blocking spinal phosphatidylinositide 3-kinase (p-PI3K) pathway significantly attenuated activities of p-mTOR pathways as well as mechanical and thermal hyperalgesia in SCI rats. Moreover, blocking mTOR and PI3K decreased the enhanced levels of substance P and calcitonin gene-related peptide (CGRP) in the dorsal horn of SCI rats. We revealed specific signaling pathways leading to SCI-evoked neuropathic pain, including the activation of PI3K, mTOR and its downstream signaling pathways. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of neuropathic pain often observed in patients with SCI.

  3. TORC1 Inhibition by Rapamycin Promotes Antioxidant Defences in a Drosophila Model of Friedreich's Ataxia.

    Directory of Open Access Journals (Sweden)

    Pablo Calap-Quintana

    Full Text Available Friedreich's ataxia (FRDA, the most common inherited ataxia in the Caucasian population, is a multisystemic disease caused by a significant decrease in the frataxin level. To identify genes capable of modifying the severity of the symptoms of frataxin depletion, we performed a candidate genetic screen in a Drosophila RNAi-based model of FRDA. We found that genetic reduction in TOR Complex 1 (TORC1 signalling improves the impaired motor performance phenotype of FRDA model flies. Pharmacologic inhibition of TORC1 signalling by rapamycin also restored this phenotype and increased the lifespan and ATP levels. Furthermore, rapamycin reduced the altered levels of malondialdehyde + 4-hydroxyalkenals and total glutathione of the model flies. The rapamycin-mediated protection against oxidative stress is due in part to an increase in the transcription of antioxidant genes mediated by cap-n-collar (Drosophila ortholog of Nrf2. Our results suggest that autophagy is indeed necessary for the protective effect of rapamycin in hyperoxia. Rapamycin increased the survival and aconitase activity of model flies subjected to high oxidative insult, and this improvement was abolished by the autophagy inhibitor 3-methyladenine. These results point to the TORC1 pathway as a new potential therapeutic target for FRDA and as a guide to finding new promising molecules for disease treatment.

  4. Production of high specific activity silicon-32

    International Nuclear Information System (INIS)

    Phillips, D.R.; Brzezinski, M.A.

    1998-01-01

    This is the final report of a three-year, Laboratory Directed Research and Development Project (LDRD) at Los Alamos National Laboratory (LANL). There were two primary objectives for the work performed under this project. The first was to take advantage of capabilities and facilities at Los Alamos to produce the radionuclide 32 Si in unusually high specific activity. The second was to combine the radioanalytical expertise at Los Alamos with the expertise at the University of California to develop methods for the application of 32 Si in biological oceanographic research related to global climate modeling. The first objective was met by developing targetry for proton spallation production of 32 Si in KCl targets and chemistry for its recovery in very high specific activity. The second objective was met by developing a validated field-useable, radioanalytical technique, based upon gas-flow proportional counting, to measure the dynamics of silicon uptake by naturally occurring diatoms

  5. TOR (target of rapamycin) is a key regulator of triacylglycerol accumulation in microalgae.

    Science.gov (United States)

    Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan

    2016-01-01

    Most microalgae abundantly accumulate lipid droplets (LDs) containing triacylglycerols (TAGs) under several stress conditions, but the underlying molecular mechanism of this accumulation remains unclear. In a recent study, we found that inhibition of TOR (target of rapamycin), a highly conserved protein kinase of eukaryotes, by rapamycin resulted in TAG accumulation in microalgae, indicating that TOR negatively regulates TAG accumulation. Here, we show that formation of intracellular LDs and TAG accumulation were also induced in the unicellular green alga Chlamydomonas reinhardtii after exposure to Torin1 or AZD8055, which are novel TOR inhibitors that inhibit TOR activity in a manner different from rapamycin. These results supported quite well our previous conclusion that TOR is a central regulator of TAG accumulation in microalgae.

  6. Arylesterase Phenotype-Specific Positive Association Between Arylesterase Activity and Cholinesterase Specific Activity in Human Serum

    Directory of Open Access Journals (Sweden)

    Yutaka Aoki

    2014-01-01

    Full Text Available Context: Cholinesterase (ChE specific activity is the ratio of ChE activity to ChE mass and, as a biomarker of exposure to cholinesterase inhibitors, has a potential advantage over simple ChE activity. Objective: To examine the association of several potential correlates (serum arylesterase/paraoxonase activity, serum albumin, sex, age, month of blood collection, and smoking with plasma ChE specific activity. Methods: We analyzed data from 195 cancer-free controls from a nested case-control study, accounting for potential confounding. Results: Arylesterase activity had an independent, statistically significant positive association with ChE specific activity, and its magnitude was the greatest for the arylesterase phenotype corresponding to the QQ PON1192 genotype followed by phenotypes corresponding to QR and RR genotypes. Serum albumin was positively associated with ChE specific activity. Conclusions: Plasma arylesterase activity was positively associated with plasma ChE specific activity. This observation is consistent with protection conferred by a metabolic phenotype resulting in reduced internal dose.

  7. Descending serotonergic facilitation mediated by spinal 5-HT3 receptors engages spinal rapamycin-sensitive pathways in the rat

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    Asante, Curtis O.; Dickenson, Anthony H.

    2010-01-01

    We have recently reported the importance of spinal rapamycin-sensitive pathways in maintaining persistent pain-like states. A descending facilitatory drive mediated through spinal 5-HT3 receptors (5-HT3Rs) originating from superficial dorsal horn NK1-expressing neurons and that relays through the parabrachial nucleus and the rostroventral medial medulla to act on deep dorsal horn neurons is known be important in maintaining these pain-like states. To determine if spinal rapamycin-sensitive pathways are activated by a descending serotonergic drive, we investigated the effects of spinally administered rapamycin on responses of deep dorsal horn neurons that had been pre-treated with the selective 5-HT3R antagonist ondansetron. We also investigated the effects of spinally administered cell cycle inhibitor (CCI)-779 (a rapamycin ester analogue) on deep dorsal horn neurons from rats with carrageenan-induced inflammation of the hind paw. Unlike some other models of persistent pain, this model does not involve an altered 5-HT3R-mediated descending serotonergic drive. We found that the inhibitory effects of rapamycin were significantly reduced for neuronal responses to mechanical and thermal stimuli when the spinal cord was pre-treated with ondansetron. Furthermore, CCI-779 was found to be ineffective in attenuating spinal neuronal responses to peripheral stimuli in carrageenan-treated rats. Therefore, we conclude that 5-HT3R-mediated descending facilitation is one requirement for activation of rapamycin-sensitive pathways that contribute to persistent pain-like states. PMID:20709148

  8. The rapamycin-regulated gene expression signature determines prognosis for breast cancer

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    Tsavachidis Spiridon

    2009-09-01

    Full Text Available Abstract Background Mammalian target of rapamycin (mTOR is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes may also be used to simulate a biologic process or effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results Colony formation and sulforhodamine B (IC50 in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI, of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%. In the Miller dataset, RMI did not correlate with tumor size or lymph node status. High (>75th percentile RMI was significantly associated with longer survival (P = 0.015. On multivariate analysis, RMI (P = 0.029, tumor size (P = 0.015 and lymph node status (P = 0.001 were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41. In the Wang dataset, RMI predicted time to disease relapse (P = 0.009. Conclusion Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment.

  9. Prenatal Mechanistic Target of Rapamycin Complex 1 (m TORC1) Inhibition by Rapamycin Treatment of Pregnant Mice Causes Intrauterine Growth Restriction and Alters Postnatal Cardiac Growth, Morphology, and Function.

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    Hennig, Maria; Fiedler, Saskia; Jux, Christian; Thierfelder, Ludwig; Drenckhahn, Jörg-Detlef

    2017-08-04

    Fetal growth impacts cardiovascular health throughout postnatal life in humans. Various animal models of intrauterine growth restriction exhibit reduced heart size at birth, which negatively influences cardiac function in adulthood. The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient and growth factor availability with cell growth, thereby regulating organ size. This study aimed at elucidating a possible involvement of mTORC1 in intrauterine growth restriction and prenatal heart growth. We inhibited mTORC1 in fetal mice by rapamycin treatment of pregnant dams in late gestation. Prenatal rapamycin treatment reduces mTORC1 activity in various organs at birth, which is fully restored by postnatal day 3. Rapamycin-treated neonates exhibit a 16% reduction in body weight compared with vehicle-treated controls. Heart weight decreases by 35%, resulting in a significantly reduced heart weight/body weight ratio, smaller left ventricular dimensions, and reduced cardiac output in rapamycin- versus vehicle-treated mice at birth. Although proliferation rates in neonatal rapamycin-treated hearts are unaffected, cardiomyocyte size is reduced, and apoptosis increased compared with vehicle-treated neonates. Rapamycin-treated mice exhibit postnatal catch-up growth, but body weight and left ventricular mass remain reduced in adulthood. Prenatal mTORC1 inhibition causes a reduction in cardiomyocyte number in adult hearts compared with controls, which is partially compensated for by an increased cardiomyocyte volume, resulting in normal cardiac function without maladaptive left ventricular remodeling. Prenatal rapamycin treatment of pregnant dams represents a new mouse model of intrauterine growth restriction and identifies an important role of mTORC1 in perinatal cardiac growth. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  10. Effects of rapamycin treatment after controlled cortical impact injury on neurogenesis and synaptic reorganization in the mouse dentate gyrus

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    Corwin R Butler

    2015-11-01

    Full Text Available Post-traumatic epilepsy (PTE is one consequence of traumatic brain injury (TBI. A prominent cell signaling pathway activated in animal models of both TBI and epilepsy is the mammalian target of rapamycin (mTOR. Inhibition of mTOR with rapamycin has shown promise as a potential modulator of epileptogenesis in several animal models of epilepsy, but cellular mechanisms linking mTOR expression and epileptogenesis are unclear. In this study, the role of mTOR in modifying functional hippocampal circuit reorganization after focal TBI induced by controlled cortical impact was investigated. Rapamycin (3 or 10 mg/kg, an inhibitor of mTOR signaling, was administered by intraperitoneal injection beginning on the day of injury and continued daily until tissue collection. Relative to controls, rapamycin treatment reduced dentate granule cell area in the hemisphere ipsilateral to the injury two weeks post-injury. Brain injury resulted in a significant increase in doublecortin immunolabeling in the dentate gyrus ipsilateral to the injury, indicating increased neurogenesis shortly after TBI. Rapamycin treatment prevented the increase in doublecortin labeling, with no overall effect on Fluoro-Jade B staining in the ipsilateral hemisphere, suggesting that rapamycin treatment reduced posttraumatic neurogenesis but did not prevent cell loss after injury. At later times post-injury (8-13 weeks, evidence of mossy fiber sprouting and increased recurrent excitation of dentate granule cells was detected, which were attenuated by rapamycin treatment. Rapamycin treatment also diminished seizure prevalence relative to vehicle-treated controls after TBI. Collectively, these results support a role for adult neurogenesis in PTE development and suggest that suppression of epileptogenesis by mTOR inhibition includes effects on post-injury neurogenesis.

  11. Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility.

    Science.gov (United States)

    Sormani, Rodnay; Yao, Lei; Menand, Benoît; Ennar, Najla; Lecampion, Cécile; Meyer, Christian; Robaglia, Christophe

    2007-06-01

    The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants. Here we show that none of the FKBPs from the model plant Arabidopsis (AtFKBPs) is able to form a ternary complex with the FRB domain of AtTOR in the presence of rapamycin in a two hybrid system. An antibody has been raised against the AtTOR protein and binding of recombinant yeast ScFKBP12 to native Arabidopsis TOR in the presence of rapamycin was demonstrated in pull-down experiments. Transgenic lines expressing ScFKBP12 were produced and were found to display a rapamycin-dependent reduction of the primary root growth and a lowered accumulation of high molecular weight polysomes. These results further strengthen the idea that plant resistance to rapamycin evolved as a consequence of mutations in plant FKBP proteins. The production of rapamycin-sensitive plants through the expression of the ScFKBP12 protein illustrates the conservation of the TOR pathway in eukaryotes. Since AtTOR null mutants were found to be embryo lethal 1, transgenic ScFKBP12 plants will provide an useful tool for the post-embryonic study of plant TOR functions. This work also establish for the first time a link between TOR activity and translation in plant cells.

  12. Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility

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    Meyer Christian

    2007-06-01

    Full Text Available Abstract Background The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants. Results Here we show that none of the FKBPs from the model plant Arabidopsis (AtFKBPs is able to form a ternary complex with the FRB domain of AtTOR in the presence of rapamycin in a two hybrid system. An antibody has been raised against the AtTOR protein and binding of recombinant yeast ScFKBP12 to native Arabidopsis TOR in the presence of rapamycin was demonstrated in pull-down experiments. Transgenic lines expressing ScFKBP12 were produced and were found to display a rapamycin-dependent reduction of the primary root growth and a lowered accumulation of high molecular weight polysomes. Conclusion These results further strengthen the idea that plant resistance to rapamycin evolved as a consequence of mutations in plant FKBP proteins. The production of rapamycin-sensitive plants through the expression of the ScFKBP12 protein illustrates the conservation of the TOR pathway in eukaryotes. Since AtTOR null mutants were found to be embryo lethal 1, transgenic ScFKBP12 plants will provide an useful tool for the post-embryonic study of plant TOR functions. This work also establish for the first time a link between TOR activity and translation in plant cells

  13. Synthesis of I-125 labeled photoaffinity rapamycin analogs

    International Nuclear Information System (INIS)

    Shu, A.Y.L.; Yamashita, D.S.; Holt, D.A.; Heys, J.R.

    1996-01-01

    Two no-carrier-added 125 I-labelled photoaffinity rapamycin analogs were prepared: 7-demethoxy-7-(4-azido-3- 125 I-benzyloxy) rapamycin and its C 28 -C 29 seco analog. The key reactions of the synthesis were substitution of the C 7 methoxyl of rapamycin with 4-azido-3-tributylstannylbenzyloxy group, exchange of tributyltin with 125 I using Na 125 I and Chloramine-T, and a ZnCl 2 mediated retro-Aldol cleavage of the C 28 -C 29 bond of rapamycin. (author)

  14. Rapamycin Eye Drops Suppress Lacrimal Gland Inflammation In a Murine Model of Sjögren's Syndrome

    Science.gov (United States)

    Shah, Mihir; Edman, Maria C.; Reddy Janga, Srikanth; Yarber, Frances; Meng, Zhen; Klinngam, Wannita; Bushman, Jonathan; Ma, Tao; Liu, Siyu; Louie, Stan; Mehta, Arjun; Ding, Chuanqing; MacKay, J. Andrew; Hamm-Alvarez, Sarah F.

    2017-01-01

    Purpose To evaluate the efficacy of topical rapamycin in treating autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome. Methods We developed rapamycin in a poly(ethylene glycol)-distearoyl phosphatidylethanolamine (PEG-DSPE) micelle formulation to maintain solubility. Rapamycin or PEG-DSPE eye drops (vehicle) were administered in a well-established Sjögren's syndrome disease model, the male nonobese diabetic (NOD) mice, twice daily for 12 weeks starting at 8 weeks of age. Mouse tear fluid was collected and tear Cathepsin S, a putative tear biomarker for Sjögren's syndrome, was measured. Lacrimal glands were retrieved for histological evaluation, and quantitative real-time PCR of genes associated with Sjögren's syndrome pathogenesis. Tear secretion was measured using phenol red threads, and corneal fluorescein staining was used to assess corneal integrity. Results Lymphocytic infiltration of lacrimal glands from rapamycin-treated mice was significantly (P = 0.0001) reduced by 3.8-fold relative to vehicle-treated mice after 12 weeks of treatment. Rapamycin, but not vehicle, treatment increased tear secretion and decreased corneal fluorescein staining after 12 weeks. In rapamycin-treated mice, Cathepsin S activity was significantly reduced by 3.75-fold in tears (P eye. PMID:28122086

  15. The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain.

    Science.gov (United States)

    Edwards, Sarah R; Wandless, Thomas J

    2007-05-04

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.

  16. Synergistic Effect of Rapamycin and Metformin Against Age-Dependent Oxidative Stress in Rat Erythrocytes.

    Science.gov (United States)

    Singh, Abhishek Kumar; Garg, Geetika; Singh, Sandeep; Rizvi, Syed Ibrahim

    2017-10-01

    Erythrocytes are particularly vulnerable toward age-dependent oxidative stress-mediated damage. Caloric restriction mimetics (CRMs) may provide a novel strategy for the maintenance of redox balance as well as effective treatment of age-associated diseases. Herein, we have investigated the beneficial effect of cotreatment with CRM-candidate drugs, rapamycin (an immunosuppressant drug and inhibitor of mammalian target of rapamycin) and metformin (an antidiabetic biguanide and activator of adenosine monophosphate kinase), against aging-induced oxidative stress in erythrocytes and plasma of aging rats. Male Wistar rats of age 4 (young) and 24 months (old) were coexposed to rapamycin (0.5 mg/kg body weight [b.w.]) and metformin (300 mg/kg b.w.), and data were compared with the response of rats receiving an independent exposure to these chemicals at similar doses. The exposure of individual candidate drugs significantly reversed the age-dependent alterations in the endpoints associated with oxidative stress such as reactive oxygen species, ferric reducing ability of plasma, malondialdehyde, reduced glutathione, plasma membrane redox system, plasma protein carbonyl, and acetyl cholinesterase in erythrocytes and plasma of aging rats. However, the cotreatment with rapamycin and metformin showed a significant augmented effect compared with individual drug interventions on reversal of these age-dependent biomarkers of oxidative stress, suggesting a synergistic response. Thus, the findings open up further possibilities for the design of new combinatorial therapies to prevent oxidative stress- and age-associated health problems.

  17. Surface Engineering of Porous Silicon Microparticles for Intravitreal Sustained Delivery of Rapamycin

    Science.gov (United States)

    Nieto, Alejandra; Hou, Huiyuan; Moon, Sang Woong; Sailor, Michael J.; Freeman, William R.; Cheng, Lingyun

    2015-01-01

    Purpose. To understand the relationship between rapamycin loading/release and surface chemistries of porous silicon (pSi) to optimize pSi-based intravitreal delivery system. Methods. Three types of surface chemical modifications were studied: (1) pSi-COOH, containing 10-carbon aliphatic chains with terminal carboxyl groups grafted via hydrosilylation of undecylenic acid; (2) pSi-C12, containing 12-carbon aliphatic chains grafted via hydrosilylation of 1-dodecene; and (3) pSiO2-C8, prepared by mild oxidation of the pSi particles followed by grafting of 8-hydrocarbon chains to the resulting porous silica surface via a silanization. Results. The efficiency of rapamycin loading follows the order (micrograms of drug/milligrams of carrier): pSiO2-C8 (105 ± 18) > pSi-COOH (68 ± 8) > pSi-C12 (36 ± 6). Powder X-ray diffraction data showed that loaded rapamycin was amorphous and dynamic drug-release study showed that the availability of the free drug was increased by 6-fold (compared with crystalline rapamycin) by using pSiO2-C8 formulation (P = 0.0039). Of the three formulations in this study, pSiO2-C8-RAP showed optimal performance in terms of simultaneous release of the active drug and carrier degradation, and drug-loading capacity. Released rapamycin was confirmed with the fingerprints of the mass spectrometry and biologically functional as the control of commercial crystalline rapamycin. Single intravitreal injections of 2.9 ± 0.37 mg pSiO2-C8-RAP into rabbit eyes resulted in more than 8 weeks of residence in the vitreous while maintaining clear optical media and normal histology of the retina in comparison to the controls. Conclusions. Porous silicon–based rapamycin delivery system using the pSiO2-C8 formulation demonstrated good ocular compatibility and may provide sustained drug release for retina. PMID:25613937

  18. Surface engineering of porous silicon microparticles for intravitreal sustained delivery of rapamycin.

    Science.gov (United States)

    Nieto, Alejandra; Hou, Huiyuan; Moon, Sang Woong; Sailor, Michael J; Freeman, William R; Cheng, Lingyun

    2015-01-22

    To understand the relationship between rapamycin loading/release and surface chemistries of porous silicon (pSi) to optimize pSi-based intravitreal delivery system. Three types of surface chemical modifications were studied: (1) pSi-COOH, containing 10-carbon aliphatic chains with terminal carboxyl groups grafted via hydrosilylation of undecylenic acid; (2) pSi-C12, containing 12-carbon aliphatic chains grafted via hydrosilylation of 1-dodecene; and (3) pSiO2-C8, prepared by mild oxidation of the pSi particles followed by grafting of 8-hydrocarbon chains to the resulting porous silica surface via a silanization. The efficiency of rapamycin loading follows the order (micrograms of drug/milligrams of carrier): pSiO2-C8 (105 ± 18) > pSi-COOH (68 ± 8) > pSi-C12 (36 ± 6). Powder X-ray diffraction data showed that loaded rapamycin was amorphous and dynamic drug-release study showed that the availability of the free drug was increased by 6-fold (compared with crystalline rapamycin) by using pSiO2-C8 formulation (P = 0.0039). Of the three formulations in this study, pSiO2-C8-RAP showed optimal performance in terms of simultaneous release of the active drug and carrier degradation, and drug-loading capacity. Released rapamycin was confirmed with the fingerprints of the mass spectrometry and biologically functional as the control of commercial crystalline rapamycin. Single intravitreal injections of 2.9 ± 0.37 mg pSiO2-C8-RAP into rabbit eyes resulted in more than 8 weeks of residence in the vitreous while maintaining clear optical media and normal histology of the retina in comparison to the controls. Porous silicon-based rapamycin delivery system using the pSiO2-C8 formulation demonstrated good ocular compatibility and may provide sustained drug release for retina. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

  19. Rapamycin suppresses brain aging in senescence-accelerated OXYS rats.

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    Kolosova, Nataliya G; Vitovtov, Anton O; Muraleva, Natalia A; Akulov, Andrey E; Stefanova, Natalia A; Blagosklonny, Mikhail V

    2013-06-01

    Cellular and organismal aging are driven in part by the MTOR (mechanistic target of rapamycin) pathway and rapamycin extends life span inC elegans, Drosophila and mice. Herein, we investigated effects of rapamycin on brain aging in OXYS rats. Previously we found, in OXYS rats, an early development of age-associated pathological phenotypes similar to several geriatric disorders in humans, including cerebral dysfunctions. Behavioral alterations as well as learning and memory deficits develop by 3 months. Here we show that rapamycin treatment (0.1 or 0.5 mg/kg as a food mixture daily from the age of 1.5 to 3.5 months) decreased anxiety and improved locomotor and exploratory behavior in OXYS rats. In untreated OXYS rats, MRI revealed an increase of the area of hippocampus, substantial hydrocephalus and 2-fold increased area of the lateral ventricles. Rapamycin treatment prevented these abnormalities, erasing the difference between OXYS and Wister rats (used as control). All untreated OXYS rats showed signs of neurodegeneration, manifested by loci of demyelination. Rapamycin decreased the percentage of animals with demyelination and the number of loci. Levels of Tau and phospho-Tau (T181) were increased in OXYS rats (compared with Wistar). Rapamycin significantly decreased Tau and inhibited its phosphorylation in the hippocampus of OXYS and Wistar rats. Importantly, rapamycin treatment caused a compensatory increase in levels of S6 and correspondingly levels of phospo-S6 in the frontal cortex, indicating that some downstream events were compensatory preserved, explaining the lack of toxicity. We conclude that rapamycin in low chronic doses can suppress brain aging.

  20. Blocking mammalian target of rapamycin (mTOR improves neuropathic pain evoked by spinal cord injury

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    Wang Xiaoping

    2016-01-01

    Full Text Available Spinal cord injury (SCI is an extremely serious type of physical trauma observed in clinics. Neuropathic pain resulting from SCI has a lasting and significant impact on most aspects of daily life. Thus, a better understanding of the molecular pathways responsible for the cause of neuropathic pain observed in SCI is important to develop effective therapeutic agents and treatment strategies. Mammalian target of rapamycin (mTOR is a serine/threonine protein kinase that is well known for its critical roles in regulating protein synthesis and growth. Furthermore, compelling evidence supports the notion that widespread dysregulation of mTOR and its downstream pathways are involved in neuropathic pain. Thus, in this study we specifically examined the underlying mechanisms by which mTOR and its signaling pathways are involved in SCI-evoked neuropathic pain in a rat model. Overall, we demonstrated that SCI increased the protein expression of p-mTOR, and mTORmediated- phosphorylation of 4E–binding protein 4 (4E-BP1 and p70 ribosomal S6 protein kinase 1 (S6K1 in the superficial dorsal horn of the spinal cord. Also, we showed that blocking spinal mTOR by intrathecal injection of rapamycin significantly inhibited pain responses induced by mechanical and thermal stimulation. In addition, blocking spinal phosphatidylinositide 3-kinase (p-PI3K pathway significantly attenuated activities of p-mTOR pathways as well as mechanical and thermal hyperalgesia in SCI rats. Moreover, blocking mTOR and PI3K decreased the enhanced levels of substance P and calcitonin gene-related peptide (CGRP in the dorsal horn of SCI rats. We revealed specific signaling pathways leading to SCI-evoked neuropathic pain, including the activation of PI3K, mTOR and its downstream signaling pathways. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of neuropathic pain often observed in patients with SCI.

  1. Rapamycin targeting mTOR and hedgehog signaling pathways blocks human rhabdomyosarcoma growth in xenograft murine model

    Energy Technology Data Exchange (ETDEWEB)

    Kaylani, Samer Z. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Xu, Jianmin; Srivastava, Ritesh K. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, Bethesda (United States); Pressey, Joseph G. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)

    2013-06-14

    Graphical abstract: Intervention of poorly differentiated RMS by rapamycin: In poorly differentiated RMS, rapamycin blocks mTOR and Hh signaling pathways concomitantly. This leads to dampening in cell cycle regulation and induction of apoptosis. This study provides a rationale for the therapeutic intervention of poorly differentiated RMS by treating patients with rapamycin alone or in combination with other chemotherapeutic agents. -- Highlights: •Rapamycin abrogates RMS tumor growth by modulating proliferation and apoptosis. •Co-targeting mTOR/Hh pathways underlie the molecular basis of effectiveness. •Reduction in mTOR/Hh pathways diminish EMT leading to reduced invasiveness. -- Abstract: Rhabdomyosarcomas (RMS) represent the most common childhood soft-tissue sarcoma. Over the past few decades outcomes for low and intermediate risk RMS patients have slowly improved while patients with metastatic or relapsed RMS still face a grim prognosis. New chemotherapeutic agents or combinations of chemotherapies have largely failed to improve the outcome. Based on the identification of novel molecular targets, potential therapeutic approaches in RMS may offer a decreased reliance on conventional chemotherapy. Thus, identification of effective therapeutic agents that specifically target relevant pathways may be particularly beneficial for patients with metastatic and refractory RMS. The PI3K/AKT/mTOR pathway has been found to be a potentially attractive target in RMS therapy. In this study, we provide evidence that rapamycin (sirolimus) abrogates growth of RMS development in a RMS xenograft mouse model. As compared to a vehicle-treated control group, more than 95% inhibition in tumor growth was observed in mice receiving parenteral administration of rapamycin. The residual tumors in rapamycin-treated group showed significant reduction in the expression of biomarkers indicative of proliferation and tumor invasiveness. These tumors also showed enhanced apoptosis

  2. FOXO3a reactivation mediates the synergistic cytotoxic effects of rapamycin and cisplatin in oral squamous cell carcinoma cells

    International Nuclear Information System (INIS)

    Fang Liang; Wang Huiming; Zhou Lin; Yu Da

    2011-01-01

    FOXO3a, a well-known transcriptional regulator, controls a wide spectrum of biological processes. The Phosphoinositide-3-kinase (PI3K)/Akt signaling pathway inactivates FOXO3a via phosphorylation-induced nuclear exclusion and degradation. A loss or gain of FOXO3a activity has been correlated with efficiency of chemotherapies in various cancers including oral squamous cell carcinoma (OSCC). Therefore, in the current study, we have investigated the FOXO3a activity modulating and antitumor effects of rapamycin and cisplatin in OSCC cells. Cisplatin inhibited proliferation and induced apoptosis in a dose-dependent way in OSCC Tca8113 cells. Rapamycin alone had no effect on cell proliferation and apoptosis. Rapamycin downregulated the expression of S-phase kinase associated protein-2 (Skp2) and increased the FOXO3a protein stability but induced the upregulation of feedback Akt activation-mediated FOXO3a phosphorylation. Cisplatin decreased the phosphorylation of FOXO3a via Akt inhibition. Rapamycin combined with cisplatin as its feedback Akt activation inhibitor revealed the most dramatic FOXO3a nuclear localization and reactivation with the prevention of its feedback loop and exposed significant synergistic effects of decreased cell proliferation and increased apoptosis in vitro and decreased tumor size in vivo. Furthermore, the downstream effects of FOXO3a reactivation were found to be accumulation of p27 and Bim. In conclusion, rapamycin/cisplatin combination therapy boosts synergistic antitumor effects through the significant FOXO3a reactivation in OSCC cells. These results may represent a novel mechanism by which rapamycin/cisplatin combination therapy proves to be a potent molecular-targeted strategy for OSCC.

  3. Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog.

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    Huang, He; Gao, Ping; Zhao, Qi; Hu, Hai-Feng

    2018-03-01

    In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis. Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  4. Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1.

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    Gokhan Demirkan

    Full Text Available Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR complex 1 (mTORC1, which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2 affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

  5. Distribution measurement of 60Co target radioactive specific activity

    International Nuclear Information System (INIS)

    Li Xingyan; Chen Zigen; Ren Min

    1994-01-01

    Radioactive specific activity distribution of cobalt 60 target by irradiation in HFETR is a key parameter. With the collimate principle, the under water measurement device and conversion coefficient which is get by experiments, and the radioactive specific activity distribution is obtained. The uncertainty of measurement is less than 10%

  6. Effect of ultrasonic specific energy on waste activated sludge ...

    African Journals Online (AJOL)

    The effect of ultrasonic specific energy on waste activated sludge (WAS) solubilization and enzyme activity was investigated in this study. Experimental results showed that the increase of ultrasonic specific energy in the range of 0 - 90000 kJ/kg dried sludge (DS) benefited WAS particle size reduction and the solubilization ...

  7. Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

    Science.gov (United States)

    LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

    2014-01-01

    Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

  8. Prevention of age-related macular degeneration-like retinopathy by rapamycin in rats.

    Science.gov (United States)

    Kolosova, Nataliya G; Muraleva, Natalia A; Zhdankina, Anna A; Stefanova, Natalia A; Fursova, Anzhela Z; Blagosklonny, Mikhail V

    2012-08-01

    Age-related macular degeneration, a neurodegenerative and vascular retinal disease, is the most common cause of blindness in the Western countries. Evidence accumulates that target of rapamycin is involved in aging and age-related diseases, including neurodegeneration. The target of rapamycin inhibitor, rapamycin, suppresses the senescent cell phenotype and extends life span in diverse species, including mice. Rapamycin decreases senescence-associated phenotypes in retinal pigment epithelial cells in culture. Herein, we investigated the effect of rapamycin on spontaneous retinopathy in senescence-accelerated OXYS rats, an animal model of age-related macular degeneration. Rats were treated with either 0.1 or 0.5 mg/kg rapamycin, which was given orally as a food mixture. In a dose-dependent manner, rapamycin decreased the incidence and severity of retinopathy. Rapamycin improved some (but not all) histological abnormalities associated with retinopathy. Thus, in retinal pigment epithelial cell layers, rapamycin decreased nuclei heterogeneity and normalized intervals between nuclei. In photoreceptor cells, associated neurons, and radial glial cells, rapamycin prevented nuclear and cellular pyknosis. More important, rapamycin prevented destruction of ganglionar neurons in the retina. Rapamycin did not exert any adverse effects on the retina in control disease-free Wistar rats. Taken together, our data suggest the therapeutic potential of rapamycin for treatment and prevention of retinopathy. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. Efficacy of Polyphenon E, Red Ginseng, and Rapamycin on Benzo(apyrene-Induced Lung Tumorigenesis in A/J Mice

    Directory of Open Access Journals (Sweden)

    Ying Yan

    2006-01-01

    Full Text Available The objective of this investigation was to determine the efficacy of several novel agents in preventing lung tumorigenesis in mice. We evaluated polyphenon E, red ginseng, and rapamycin in A/J mice treated with the tobacco-specific carcinogen benzo(apyrene for their ability to inhibit pulmonary adenoma formation and growth. We found that treatment with polyphenon E exhibited a significant reduction on both tumor multiplicity and tumor load (tumor multiplicity × tumor volume in a dose-dependent fashion. Polyphenon E (2% wt/wt in the diet reduced tumor multiplicity by 46% and tumor load by 94%. This result provided key evidence in support of a phase II clinical chemoprevention trial of lung cancer. Administration of red ginseng in drinking water decreased tumor multiplicity by 36% and tumor load by 70%. The mammalian target of rapamycin inhibitor rapamycin showed significant efficacy against lung tumor growth in the tumor progression protocol and reduced tumor load by 84%. The results of these investigations demonstrate that polyphenon E, red ginseng, and rapamycin significantly inhibit pulmonary adenoma formation and growth in A/J mice.

  10. Disruption of mammalian target of rapamycin complex 1 in macrophages decreases chemokine gene expression and atherosclerosis

    NARCIS (Netherlands)

    Ai, Ding; Jiang, Hongfeng; Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Ganda, Anjali; Abramowicz, Sandra; Welch, Carrie; Almazan, Felicidad; Zhu, Yi; Miller, Yury I.; Tall, Alan R.

    2014-01-01

    The mammalian target of rapamycin complex 1 inhibitor, rapamycin, has been shown to decrease atherosclerosis, even while increasing plasma low-density lipoprotein levels. This suggests an antiatherogenic effect possibly mediated by the modulation of inflammatory responses in atherosclerotic plaques.

  11. GSK-3/Rb12 Pathway as a Novel Target of Rapamycin in Prostate Cancer

    National Research Council Canada - National Science Library

    Litovchick, Larissa

    2005-01-01

    .... Rapamycin exerts its effects through inhibition of mammalian Target of Rapamycin (mTOR) protein kinase resulting in a decreased expression of a subset of proteins essential for cell cycle progression...

  12. Age Specifics of Cognitive Activity Development in Preschool Age

    OpenAIRE

    Klopotova E.E.; Samkova I.A.

    2017-01-01

    This paper present results of the research on the specifics of cognitive activity development in preschool children. The hypothesis tested was that content and dynamic components of cognitive activity reveal themselves in a different way depending on the stage of preschool childhood. The authors reviewed the diagnostic tools suitable for studying cognitive activity in preschoolers and selected the techniques. The research proved that content and dynamic components of cognitive activity have t...

  13. PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

    2015-10-23

    p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

  14. Platelet monoamine oxidase: specific activity and turnover number in headache

    International Nuclear Information System (INIS)

    Summers, K.M.; Brown, G.K.; Craig, I.W.; Peatfield, R.; Rose, F.C.

    1982-01-01

    Monoamine oxidase turnover numbers (molecules of substrate converted to product per minute per active site) have been calculated for the human platelet enzyme using [ 3 H]pargyline. Headache patients with high and low monoamine oxidase specific activities relative to controls were found to have turnover numbers very close to those for controls. This finding suggests that their specific activities vary because of differences in the concentration of active monoamine oxidase molecules, rather than differences in the ability of those enzyme molecules to catalyse the deamination reaction. (Auth.)

  15. Experimental study on active specific immunotherapy modified with irradiation

    International Nuclear Information System (INIS)

    Imanaka, Kazufumi; Ogawa, Yasuhiro; Gose, Kyuhei; Imajo, Yoshinari; Kumura, Shuji

    1982-01-01

    We have already reported that the effectiveness of active specific immunotherapy using irradiated tumor cells and infiltrating mononuclear cells which were separated from the topical tumor tissue 7 days after irradiation of 2,000 rad in experimental study. The present study was designed to investigate the effect of non-specific immunopotentiator PS-K combined with active specific immunotherapy. Female C3H/He mice aged 12 weeks were inoculated 4 x 10 6 MM 46 tumor cells in the right hind paws and received local electron irradiation with the dose of 3,000 rad on the 5th day after irradiation. Active specific immunotherapy was performed on the 12th day, and daily dose of 200 mg/kg of PS-K was injected intraperitoneally from the 6th day to the 10th day. The inhibition of the tumor growth and the elongation of survival period were noted in the group which received active specific immunotherapy combined with non-specific immunopotentiator, PS-K compared with the active specific immunotherapy alone. (author)

  16. Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses

    DEFF Research Database (Denmark)

    Procaccini, Claudio; De Rosa, Veronica; Galgani, Mario

    2012-01-01

    The sensing by T cells of metabolic and energetic changes in the microenvironment can determine the differentiation, maturation, and activation of these cells. Although it is known that mammalian target of rapamycin (mTOR) gauges nutritonal and energetic signals in the extracellular milieu, it is...

  17. Rapamycin-based inducible translocation systems for studying phagocytosis.

    Science.gov (United States)

    Bohdanowicz, Michal; Fairn, Gregory D

    2011-01-01

    Phagocytosis is an immune receptor-mediated process whereby cells engulf large particles. The process is dynamic and requires several localized factors acting in concert with and sequentially after the engagement of immune receptors to envelope the particle. Once the particle is internalized, the nascent -phagosome undergoes a series of events leading to its maturation to the microbicidal phagolysosome. Investigating these dynamic and temporally controlled series of events in live cells requires noninvasive methods. The ability to rapidly recruit the proteins of interest to the sites of phagocytosis or to nascent phagosomes would help dissect the regulatory mechanisms involved during phagocytosis. Here, we describe a general approach to express in RAW264.7 murine macrophages, a genetically encoded rapamycin--induced heterodimerization system. In the presence of rapamycin, tight association between FK506-binding protein (FKBP) and FKBP rapamycin-binding protein (FRB) is observed. Based on this principle, a synthetic system consisting of a targeting domain attached to FKBP can recruit a protein of interest fused to FRB upon the addition of rapamycin. Previously, this technique has been used to target lipid-modifying enzymes and small GTPases to the phagosome or plasma membrane. The recruitment of the FRB module can be monitored by fluorescent microscopy if a fluorescent protein is fused to the FRB sequence. While the focus of this chapter is on phagocytic events, this method can be employed to study any organelle of interest when the appropriate targeting sequence is used.

  18. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    International Nuclear Information System (INIS)

    Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

    2014-01-01

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor flox/flox mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor flox/flox mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function

  19. Specific activity measurement of 64Cu: A comparison of methods

    International Nuclear Information System (INIS)

    Mastren, Tara; Guthrie, James; Eisenbeis, Paul; Voller, Tom; Mebrahtu, Efrem; Robertson, J. David; Lapi, Suzanne E.

    2014-01-01

    Effective specific activity of 64 Cu (amount of radioactivity per µmol metal) is important in order to determine purity of a particular 64 Cu lot and to assist in optimization of the purification process. Metal impurities can affect effective specific activity and therefore it is important to have a simple method that can measure trace amounts of metals. This work shows that ion chromatography (IC) yields similar results to ICP mass spectrometry for copper, nickel and iron contaminants in 64 Cu production solutions. - Highlights: • Comparison of TETA titration, ICP mass spectrometry, and ion chromatography to measure specific activity. • Validates ion chromatography by using ICP mass spectrometry as the “gold standard”. • Shows different types and amounts of metal impurities present in 64 Cu

  20. Specific cesium activity in freshwater fish and the size effect

    International Nuclear Information System (INIS)

    Kulikov, A.O.; Ryabov, I.N.; USSR Academy of Sciences, Moscow

    1992-01-01

    The specific Cs-137 activity of muscle tissues of silver carp (Hypophthalmichthys molitrix) from the cooling pond of the Chernobyl nuclear power plant caught in 1987 and 1988 increased almost linearly with fish weight ('size effect') in contrast to liver tissue, whose specific activity remained independent of weight. A kinetic model for uptake and excretion was developed to describe the size effect in muscle tissue by introducing a weight-dependent Cs biological half-time to fish. Similar size effects of specific Cs-137 activity were also found for other species of fish from cooling pond, but were primarily attributed to changes in feeding habits with increasing weight of fish rather than to metabolic changes in feeding habits with both of muscle and liver tissue increased with fish weight for those species in contrast to silver carp. (author). 12 refs.; 12 figs.; 1 tab

  1. An approach for activity-based DEVS model specification

    DEFF Research Database (Denmark)

    Alshareef, Abdurrahman; Sarjoughian, Hessam S.; Zarrin, Bahram

    2016-01-01

    Creation of DEVS models has been advanced through Model Driven Architecture and its frameworks. The overarching role of the frameworks has been to help develop model specifications in a disciplined fashion. Frameworks can provide intermediary layers between the higher level mathematical models...... and their corresponding software specifications from both structural and behavioral aspects. Unlike structural modeling, developing models to specify behavior of systems is known to be harder and more complex, particularly when operations with non-trivial control schemes are required. In this paper, we propose specifying...... activity-based behavior modeling of parallel DEVS atomic models. We consider UML activities and actions as fundamental units of behavior modeling, especially in the presence of recent advances in the UML 2.5 specifications. We describe in detail how to approach activity modeling with a set of elemental...

  2. Preparation of [35S]sulfobromophthalein of high specific activity

    International Nuclear Information System (INIS)

    Kurisu, H.; Nilprabhassorn, P.; Wolkoff, A.W.

    1989-01-01

    Study of the hepatocyte transport mechanism of organic anions such as bilirubin and sulfobromophthalein has been limited by the relatively low specific activities of these ligands. [ 3 H]Bilirubin and [ 35 S]sulfobromophthalein have been available with specific activities of only approximately 100 mCi/mmol. We now report a relatively simple procedure to prepare [ 35 S]sulfobromophthalein at a specific activity of approximately 3000 mCi/mmol. This compound is radiochemically pure and serves as a tracer for authentic sulfobromophthalein as judged by chromatography, hepatocyte uptake, metabolism, and biliary excretion. Use of this material as a photoaffinity probe and as a transported ligand may permit dissection and understanding of its transport mechanism

  3. Synthesis of high specific activity tritium labelled compounds

    International Nuclear Information System (INIS)

    Parent, P.

    1986-01-01

    Tritiated methyl iodide of high specific activity is synthetized by Fischer-Tropsch reaction of tritium with carbon monoxide, tritiated methanol obtained is reacted with hydriodic acid. It is used for the synthesis of S-adenosyl L-methionine 3 H-methyl and of diazepam 3 H-methyl derivatives. Synthesis of 3-PPP 3 H: (hydroxy-3 phenyl)-3N-n propyl [ 3 H-2.3] piperidine [ 3 H-2.3] with a specific activity of 4.25 T Bq/mM (115 Ci/mM) and of baclofene 3 H with a specific activity of 0.925 TBq (25 Ci/mM) are also described [fr

  4. Intraperitoneal alpha-radioimmunotherapy in mice using different specific activities

    DEFF Research Database (Denmark)

    Elgqvist, Jörgen; Andersson, Håkan; Haglund, Elin

    2009-01-01

    The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At.......The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At....

  5. N-tritioacetoxyphthalimide: A new high specific activity tritioacetylating reagent

    International Nuclear Information System (INIS)

    Saljoughian, M.; Morimoto, Hiromi; Than, Chit

    1996-01-01

    The authors' aim was to develop a nonvolatile, stable, and facile tritioacetylating reagent and to demonstrate its use on simple peptides. Accordingly, the authors made the synthesis of high specific activity N-(tritioacetoxy) derivatives of succinimide, phthalimide, and naphthalimide a major focus. As the preferred approach, N-(tritioacetoxy)phthalimide was prepared by radical dehalogenation of N-(iodoacetoxy)phthalimide using high specific activity tributyltin tritide. This tritiated acetylation reagent was characterized by 3 H and 1 H NMR spectroscopy and by radio-HPLC. Efficacy of the reagent was investigated by tritioacetylation of several peptides at their N-terminal amino group. 26 refs., 1 fig

  6. Preparation of tritiated thymidine of high specific activity

    International Nuclear Information System (INIS)

    Ivan'kova, E.K.; Sidorov, G.V.; Myasoedov, N.F.

    1981-01-01

    Optimum conditions for the reaction are determined; and conditions for reaction component separation on resins of Dowex-1x8 and APA-8p (HCOO - , elution with ammonium formate) are optimized. It is established that the transition from thymine preparations with the specific activity of 0.15 and 1.5 TBq/mmol to the preparation with the specific activity of 3.25 TBq/mmol brings about the reduction in the desoxyribosylation reaction rate and the decrease in the thymidine yield from 85-90 to 65% [ru

  7. 50 CFR 635.32 - Specifically authorized activities.

    Science.gov (United States)

    2010-10-01

    ... specific criteria for selection, and the application deadline. Complete applications, including all... information provided on the applications and their ability to meet the selection criteria as published in the... approved food bank networks; or chartering arrangements. Such activities must be authorized in writing and...

  8. The progesterone-induced enhancement of object recognition memory consolidation involves activation of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways in the dorsal hippocampus

    Science.gov (United States)

    Orr, Patrick T.; Rubin, Amanda J.; Fan, Lu; Kent, Brianne A.; Frick, Karyn M.

    2012-01-01

    Although much recent work has elucidated the biochemical mechanisms underlying the modulation of memory by 17β-estradiol, little is known about the signaling events through which progesterone (P) regulates memory. We recently demonstrated that immediate post-training infusion of P into the dorsal hippocampus enhances object recognition memory consolidation in young ovariectomized female mice (Orr et al., 2009). The goal of the present study was to identify the biochemical alterations that might underlie this mnemonic enhancement. We hypothesized that the P-induced enhancement of object recognition would be dependent on activation of the ERK and mTOR pathways. In young ovariectomized mice, we found that bilateral dorsal hippocampal infusion of P significantly increased levels of phospho-p42 ERK and the mTOR substrate S6K in the dorsal hippocampus 5 minutes after infusion. Phospho-p42 ERK levels were downregulated 15 minutes after infusion and returned to baseline 30 minutes after infusion, suggesting a biphasic effect of P on ERK activation. Dorsal hippocampal ERK and mTOR activation were necessary for P to facilitate memory consolidation, as suggested by the fact that inhibitors of both pathways infused into the dorsal hippocampus immediately after training blocked the P-induced enhancement of object recognition. Collectively, these data provide the first demonstration that the ability of P to enhance memory consolidation depends on the rapid activation of cell signaling and protein synthesis pathways in the dorsal hippocampus. PMID:22265866

  9. Rapamycin causes growth arrest and inhibition of invasion in human chondrosarcoma cells.

    Science.gov (United States)

    Song, Jian; Wang, Xiaobo; Zhu, Jiaxue; Liu, Jun

    2016-01-01

    Chondrosarcoma is a highly malignant tumor that is characterized by a potent capacity to invade locally and cause distant metastasis and notable for its lack of response to conventional chemotherapy or radiotherapy. Rapamycin, the inhibitor of mammalian target of rapamycin (mTOR), is a valuable drug with diverse clinical applications and regulates many cellular processes. However, the effects of rapamycin on cell growth and invasion of human chondrosarcoma cells are not well known. We determined the effect of rapamycin on cell proliferation, cell cycle arrest and invasion by using MTS, flow cytometry and invasion assays in two human chondrosarcoma cell lines, SW1353 and JJ012. Cell cycle regulatory and invasion-related genes' expression analysis was performed by quantitative RT-PCR (qRT-PCR). We also evaluated the effect of rapamycin on tumor growth by using mice xenograph models. Rapamycin significantly inhibited the cell proliferation, induced cell cycle arrest and decreased the invasion ability of human chondrosarcoma cells. Meanwhile, rapamycin modulated the cell cycle regulatory and invasion-related genes' expression. Furthermore, the tumor growth of mice xenograph models with human chondrosarcoma cells was significantly inhibited by rapamycin. These results provided further insight into the role of rapamycin in chondrosarcoma. Therefore, rapamycin targeted therapy may be a potential treatment strategy for chondrosarcoma.

  10. Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1-Harnessed SAINT-Based Lipid Carrier System.

    Directory of Open Access Journals (Sweden)

    Ganesh Ram R Visweswaran

    Full Text Available Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes.

  11. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    International Nuclear Information System (INIS)

    Highsmith, R.F.; Gallaher, M.J.

    1986-01-01

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125 I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

  12. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    Energy Technology Data Exchange (ETDEWEB)

    Highsmith, R.F.; Gallaher, M.J.

    1986-03-05

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive /sup 125/I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

  13. Gene program-specific regulation of PGC-1{alpha} activity

    DEFF Research Database (Denmark)

    Schmidt, Søren F; Mandrup, Susanne

    2011-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1 α (PGC-1α) activation coordinates induction of the hepatic fasting response through coactivation of numerous transcription factors and gene programs. In the June 15, 2011, issue of Genes & Development, Lustig and colleagues (pp....... 1232-1244) demonstrated that phosphorylation of PGC-1α by the p70 ribosomal protein S6 kinase 1 (S6K1) specifically interfered with the interaction between PGC-1α and HNF4α in liver and blocked the coactivation of the gluconeogenic target genes. This demonstrates how independent fine-tuning of gene...

  14. Novel strategies for ultrahigh specific activity targeted nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Dong

    2012-12-13

    We have developed novel strategies optimized for preparing high specific activity radiolabeled nanoparticles, targeting nuclear imaging of low abundance biomarkers. Several compounds have been labeled with F-18 and Cu-64 for radiolabeling of SCK-nanoparticles via Copper(I) catalyzed or copper-free alkyne-azide cyclolization. Novel strategies have been developed to achieve ultrahigh specific activity with administrable amount of dose for human study using copper-free chemistry. Ligands for carbonic anhydrase 12 (CA12), a low abundance extracellular biomarker for the responsiveness of breast cancer to endocrine therapie, have been labeled with F-18 and Cu-64, and one of them has been evaluated in animal models. The results of this project will lead to major improvements in the use of nanoparticles in nuclear imaging and will significantly advance their potential for detecting low abundance biomarkers of medical importance.

  15. Accelerator Production and Separations for High Specific Activity Rhenium-186

    Energy Technology Data Exchange (ETDEWEB)

    Jurisson, Silvia S. [Univ. of Missouri, Columbia, MO (United States); Wilbur, D. Scott [Univ. of Washington, Seattle, WA (United States)

    2016-04-01

    Tungsten and osmium targets were evaluated for the production of high specific activity rhenium-186. Rhenium-186 has potential applications in radiotherapy for the treatment of a variety of diseases, including targeting with monoclonal antibodies and peptides. Methods were evaluated using tungsten metal, tungsten dioxide, tungsten disulfide and osmium disulfide. Separation of the rhenium-186 produced and recycling of the enriched tungsten-186 and osmium-189 enriched targets were developed.

  16. Specific classification of financial analysis of enterprise activity

    Directory of Open Access Journals (Sweden)

    Synkevych Nadiia I.

    2014-01-01

    Full Text Available Despite the fact that one can find a big variety of classifications of types of financial analysis of enterprise activity, which differ with their approach to classification and a number of classification features and their content, in modern scientific literature, their complex comparison and analysis of existing classification have not been done. This explains urgency of this study. The article studies classification of types of financial analysis of scientists and presents own approach to this problem. By the results of analysis the article improves and builds up a specific classification of financial analysis of enterprise activity and offers classification by the following features: objects, subjects, goals of study, automation level, time period of the analytical base, scope of study, organisation system, classification features of the subject, spatial belonging, sufficiency, information sources, periodicity, criterial base, method of data selection for analysis and time direction. All types of financial analysis significantly differ with their inherent properties and parameters depending on the goals of financial analysis. The developed specific classification provides subjects of financial analysis of enterprise activity with a possibility to identify a specific type of financial analysis, which would correctly meet the set goals.

  17. Rapamycin attenuates bleomycin-induced pulmonary fibrosis in rats and the expression of metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 in lung tissue.

    Science.gov (United States)

    Jin, Xiaoguang; Dai, Huaping; Ding, Ke; Xu, Xuefeng; Pang, Baosen; Wang, Chen

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is the most common and devastating form of interstitial lung disease (ILD) in the clinic. There is no effective therapy except for lung transplantation. Rapamycin is an immunosuppressive drug with potent antifibrotic activity. The purpose of this study was to examine the effects of rapamycin on bleomycin-induced pulmonary fibrosis in rats and the relation to the expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Sprague-Dawley rats were treated with intratracheal injection of 0.3 ml of bleomycin (5 mg/kg) in sterile 0.9% saline to make the pulmonary fibrosis model. Rapamycin was given at a dose of 0.5 mg/kg per gavage, beginning one day before bleomycin instillation and once daily until animal sacrifice. Ten rats in each group were sacrificed at 3, 7, 14, 28 and 56 days after bleomycin administration. Alveolitis and pulmonary fibrosis were semi-quantitatively assessed after HE staining and Masson staining under an Olympus BX40 microscope with an IDA-2000 Image Analysis System. Type I and III collagen fibers were identified by Picro-sirius-polarization. Hydroxyproline content in lung tissue was quantified by a colorimetric-based spectrophotometric assay, MMP-9 and TIMP-1 were detected by immunohistochemistry and by realtime quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Bleomycin induced alveolitis and pulmonary fibrosis of rats was inhibited by rapamycin. Significant inhibition of alveolitis and hydroxyproline product were demonstrated when daily administration of rapamycin lasted for at least 14 days. The inhibitory efficacy on pulmonary fibrosis was unremarkable until rapamycin treatment lasted for at least 28 days (P pulmonary fibrosis, which is associated with decreased expression of MMP-9 and TIMP-1.

  18. Effects of ketamine administration on mTOR and reticulum stress signaling pathways in the brain after the infusion of rapamycin into prefrontal cortex.

    Science.gov (United States)

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Michels, Monique; Abatti, Mariane; Sonai, Beatriz; Dal Pizzol, Felipe; Carvalho, André F; Quevedo, João

    2017-04-01

    Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-D-aspartate (NMDA) receptor antagonists. A relationship between mTOR kinase and the endoplasmic reticulum (ER) stress pathway, also known as the unfolded protein response (UPR) has been shown. We evaluate the effects of ketamine administration on the mTOR signaling pathway and proteins of UPR in the prefrontal cortex (PFC), hippocampus, amygdala and nucleus accumbens, after the inhibiton of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol), or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). The immunocontent of mTOR, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), eukaryotic elongation factor 2 kinase (eEF2K) homologous protein (CHOP), PKR-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1) - alpha were determined in the brain. The mTOR levels were reduced in the rapamycin group treated with saline and ketamine in the PFC; p4EBP1 levels were reduced in the rapamycin group treated with ketamine in the PFC and nucleus accumbens; the levels of peEF2K were increased in the PFC in the vehicle group treated with ketamine and reduced in the rapamycin group treated with ketamine. The PERK and IRE1-alpha levels were decreased in the PFC in the rapamycin group treated with ketamine. Our results suggest that mTOR signaling inhibition by rapamycin could be involved, at least in part, with the mechanism of action of ketamine; and the ketamine antidepressant on ER stress pathway could be also mediated by mTOR signaling pathway in certain brain structures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. SEVERAL ASPECTS REGARDING THE SPECIFIC ACTIVITIES FROM MUREŞ DEFILE

    Directory of Open Access Journals (Sweden)

    George-Bogdan TOFAN

    2013-12-01

    Full Text Available The geographical location, as well as the natural conditions (the relief’s morphometry, less than favourable climatic conditions, as well as the presence of shallow soils played a deciding role in developing some activities characteristic to mountain areas, mainly represented by forestry and animal husbandry, with peaks and lows caused by social and historical factors that also affected the population of the area. Agriculture became one of the most important components of the defile’s economy, and still remains the main source of nourishment and income for a rather significant part of the population. When it comes to industry, it developed based on the extraction and exploitation of the area’s natural riches (construction rocks, mineral waters, timber, which are then incorporated into the economic circuit. The tertiary activities, in a strong correlation with the territory’s specificity, are less representative, trade being the one activity that stands out (timber, mineral water, construction rocks.

  20. ASPECTS OF SEASONALITY TOURISTIC ACTIVITY SPECIFIC TO MAMAIA STATION

    Directory of Open Access Journals (Sweden)

    Mariana C. JUGANARU

    2017-05-01

    Full Text Available The study of phenomena and social-economic processes under the aspect of their evolution in time, mainly on a short term or intra-annual represents a preoccupation at a micro and macroeconomic level. For the tourism operators, this process includes knowing the touristic market and the anticipations of its evolution, as an important condition for taking decisions in their activity. The aim of this work is to analyze the touristic activity according to seasonality in Mamaia station, using qualitative and quantitative research methods. The study is important through the aspects that emphasize the specific evolution of the touristic activity from this station. For this aim, a database was formed by the monthly values of three indicators of the touristic activity (number of arrivals, number of overnights and the average duration of the stay from the period 2010-2016, using a series of statistic and econometric instruments. The results of the research can be proved by the units that maintain or are connected to the touristic activity, but also to the local administration, in making up the attenuation strategy of the touristic activity concerning the seasonality of Mamaia. Also, the work is a case study for the work with the students (especially, for tourism economy, applied statistics in tourism and marketing.

  1. Longitudinal imaging studies of tumor microenvironment in mice treated with the mTOR inhibitor rapamycin.

    Directory of Open Access Journals (Sweden)

    Keita Saito

    Full Text Available Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII by electron paramagnetic resonance imaging (EPRI and magnetic resonance imaging (MRI to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3 and measurements of tumor pO(2 and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2 levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2<10 mm Hg in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

  2. Fundamental Activity Constraints Lead to Specific Interpretations of the Connectome.

    Directory of Open Access Journals (Sweden)

    Jannis Schuecker

    2017-02-01

    Full Text Available The continuous integration of experimental data into coherent models of the brain is an increasing challenge of modern neuroscience. Such models provide a bridge between structure and activity, and identify the mechanisms giving rise to experimental observations. Nevertheless, structurally realistic network models of spiking neurons are necessarily underconstrained even if experimental data on brain connectivity are incorporated to the best of our knowledge. Guided by physiological observations, any model must therefore explore the parameter ranges within the uncertainty of the data. Based on simulation results alone, however, the mechanisms underlying stable and physiologically realistic activity often remain obscure. We here employ a mean-field reduction of the dynamics, which allows us to include activity constraints into the process of model construction. We shape the phase space of a multi-scale network model of the vision-related areas of macaque cortex by systematically refining its connectivity. Fundamental constraints on the activity, i.e., prohibiting quiescence and requiring global stability, prove sufficient to obtain realistic layer- and area-specific activity. Only small adaptations of the structure are required, showing that the network operates close to an instability. The procedure identifies components of the network critical to its collective dynamics and creates hypotheses for structural data and future experiments. The method can be applied to networks involving any neuron model with a known gain function.

  3. Acridinium esters as high-specific-activity labels in immunoassay

    International Nuclear Information System (INIS)

    Weeks, I.; Beheshti, I.; McCapra, F.; Campbell, A.K.; Woodhead, J.S.

    1983-01-01

    A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample

  4. Production of N-13 labeled compounds with high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kazutoshi; Sasaki, Motoji; Yoshida, Yuichiro; Haradahira, Terushi; Inoue, Osamu [National Inst. of Radiological Sciences, Chiba (Japan)

    1997-03-01

    Nitrogen-13 was produced by irradiating ultra pure water saturated with a pure gas (N2, O2, He, H2) with 18 MeV protons. Ion species generated by irradiation were analyzed with radio ion chromatography systems. An automated equipment was developed to synthesize anhydrous (13N)NH3 as a synthetic precursor and (13N)p-nitrophenyl carbamate ((13N)NPC) as a model compound, using the (13N)NH3. The radiochemical yield and specific activity of (13N)NPC was high enough to carry out the receptor study with PET. (author)

  5. Low Specific Activity materials concepts are being reevaluated

    International Nuclear Information System (INIS)

    Rawl, R.R.

    1993-01-01

    Many types of radioactive low-level waste are classified, packaged, and transported as Low-Specific Activity (LSA) material. The transportation regulations allow LSA materials to be shipped in economical packagings and, under certain conditions, waives compliance with other detailed requirements such as labeling. The fundamental concepts which support the LSA category are being thoroughly reevaluated to determine the defensibility of the provisions. A series of national and international events are leading to the development of new dose models which are likely to fundamentally change the ways these materials are defined. Similar basis changes are likely for the packaging requirements applicable to these materials

  6. Brassinosteriod Insensitive 2 (BIN2) acts as a downstream effector of the Target of Rapamycin (TOR) signaling pathway to regulate photoautotrophic growth in Arabidopsis.

    Science.gov (United States)

    Xiong, Fangjie; Zhang, Rui; Meng, Zhigang; Deng, Kexuan; Que, Yumei; Zhuo, Fengping; Feng, Li; Guo, Sundui; Datla, Raju; Ren, Maozhi

    2017-01-01

    The components of the target of rapamycin (TOR) signaling pathway have been well characterized in heterotrophic organisms from yeast to humans. However, because of rapamycin insensitivity, embryonic lethality in tor null mutants and a lack of reliable ways of detecting TOR protein kinase in higher plants, the key players upstream and downstream of TOR remain largely unknown in plants. Using engineered rapamycin-sensitive Binding Protein 12-2 (BP12-2) plants, the present study showed that combined treatment with rapamycin and active-site TOR inhibitors (asTORis) results in synergistic inhibition of TOR activity and plant growth in Arabidopsis. Based on this system, we revealed that TOR signaling plays a crucial role in modulating the transition from heterotrophic to photoautotrophic growth in Arabidopsis. Ribosomal protein S6 kinase 2 (S6K2) was identified as a direct downstream target of TOR, and the growth of TOR-suppressed plants could be rescued by up-regulating S6K2. Systems, genetic, and biochemical analyses revealed that Brassinosteriod Insensitive 2 (BIN2) acts as a novel downstream effector of S6K2, and the phosphorylation of BIN2 depends on TOR-S6K2 signaling in Arabidopsis. By combining pharmacological with genetic and biochemical approaches, we determined that the TOR-S6K2-BIN2 signaling pathway plays important roles in regulating the photoautotrophic growth of Arabidopsis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  7. Active site mutations change the cleavage specificity of neprilysin.

    Directory of Open Access Journals (Sweden)

    Travis Sexton

    Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

  8. The Rapamycin-Binding Domain of the Protein Kinase mTOR is a Destabilizing Domain*

    Science.gov (United States)

    Edwards, Sarah R.; Wandless, Thomas J.

    2013-01-01

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to ten-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retain the ability to inhibit mTOR, albeit with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wildtype FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. PMID:17350953

  9. Chemically induced and light-independent cryptochrome photoreceptor activation.

    Science.gov (United States)

    Rosenfeldt, Gesa; Viana, Rafael Muñoz; Mootz, Henning D; von Arnim, Albrecht G; Batschauer, Alfred

    2008-01-01

    The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP-rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.

  10. Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation.

    Science.gov (United States)

    Liu, Jiayan; Seibold, Steve A; Rieke, Caroline J; Song, Inseok; Cukier, Robert I; Smith, William L

    2007-06-22

    The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

  11. Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha; Piatek, Marek J.; Bangarusamy, Dhinoth Kumar; Mahfouz, Magdy M.

    2013-01-01

    The development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects. © 2013 Springer-Verlag.

  12. Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha

    2013-10-01

    The development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects. © 2013 Springer-Verlag.

  13. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Shan, Tizhong; Liang, Xinrong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2014-09-12

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

  14. SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

    International Nuclear Information System (INIS)

    Zhu Shanshan; Zhang Hong; Matunis, Michael J.

    2006-01-01

    SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification

  15. Inhibition of Mammalian Target of Rapamycin Complex 1 Attenuates Salt-Induced Hypertension and Kidney Injury in Dahl Salt-Sensitive Rats.

    Science.gov (United States)

    Kumar, Vikash; Wollner, Clayton; Kurth, Theresa; Bukowy, John D; Cowley, Allen W

    2017-10-01

    The goal of the present study was to explore the protective effects of mTORC1 (mammalian target of rapamycin complex 1) inhibition by rapamycin on salt-induced hypertension and kidney injury in Dahl salt-sensitive (SS) rats. We have previously demonstrated that H 2 O 2 is elevated in the kidneys of SS rats. The present study showed a significant upregulation of renal mTORC1 activity in the SS rats fed a 4.0% NaCl for 3 days. In addition, renal interstitial infusion of H 2 O 2 into salt-resistant Sprague Dawley rats for 3 days was also found to stimulate mTORC1 activity independent of a rise of arterial blood pressure. Together, these data indicate that the salt-induced increases of renal H 2 O 2 in SS rats activated the mTORC1 pathway. Daily administration of rapamycin (IP, 1.5 mg/kg per day) for 21 days reduced salt-induced hypertension from 176.0±9.0 to 153.0±12.0 mm Hg in SS rats but had no effect on blood pressure salt sensitivity in Sprague Dawley treated rats. Compared with vehicle, rapamycin reduced albumin excretion rate in SS rats from 190.0±35.0 to 37.0±5.0 mg/d and reduced the renal infiltration of T lymphocytes (CD3 + ) and macrophages (ED1 + ) in the cortex and medulla. Renal hypertrophy and cell proliferation were also reduced in rapamycin-treated SS rats. We conclude that enhancement of intrarenal H 2 O 2 with a 4.0% NaCl diet stimulates the mTORC1 pathway that is necessary for the full development of the salt-induced hypertension and kidney injury in the SS rat. © 2017 American Heart Association, Inc.

  16. Mammalian target of rapamycin is required for phrenic long-term facilitation following severe but not moderate acute intermittent hypoxia.

    Science.gov (United States)

    Dougherty, Brendan J; Fields, Daryl P; Mitchell, Gordon S

    2015-09-01

    Phrenic long-term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). Distinct cell-signaling cascades give rise to pLTF depending on the severity of hypoxemia within hypoxic episodes. Moderate AIH (mAIH; three 5-min episodes, PaO2 ∼35-55 mmHG) elicits pLTF by a serotonin (5-HT)-dependent mechanism that requires new synthesis of brain-derived neurotrophic factor (BDNF), activation of its high-affinity receptor (TrkB), and ERK MAPK signaling. In contrast, severe AIH (sAIH; three 5-min episodes, PaO2 ∼25-30 mmHG) elicits pLTF by an adenosine-dependent mechanism that requires new TrkB synthesis and Akt signaling. Although both mechanisms require spinal protein synthesis, the newly synthesized proteins are distinct, as are the neurochemicals inducing plasticity (serotonin vs. adenosine). In many forms of neuroplasticity, new protein synthesis requires translational regulation via mammalian target of rapamycin (mTOR) signaling. Since Akt regulates mTOR activity, we hypothesized that mTOR activity is necessary for sAIH- but not mAIH-induced pLTF. Phrenic nerve activity in anesthetized, paralyzed, and ventilated rats was recorded before, during, and 60 min after mAIH or sAIH. Rats were pretreated with intrathecal injections of 20% DMSO (vehicle controls) or rapamycin (0.1 mM, 12 μl), a selective mTOR complex 1 inhibitor. Consistent with our hypothesis, rapamycin blocked sAIH- but not mAIH-induced pLTF. Thus spinal mTOR activity is required for adenosine-dependent (sAIH) but not serotonin-dependent (mAIH) pLTF, suggesting that distinct mechanisms regulate new protein synthesis in these forms of spinal neuroplasticity. Copyright © 2015 the American Physiological Society.

  17. Rapamycin Induces Heme Oxygenase-1 in Liver but Inhibits Bile Flow Recovery after Ischemia

    NARCIS (Netherlands)

    Kist, Alwine; Wakkie, Joris; Madu, Max; Versteeg, Ruth; ten Berge, Judith; Nikolic, Andrej; Nieuwenhuijs, Vincent B.; Porte, Robert J.; Padbury, Robert T. A.; Barritt, Greg J.

    Background/Aims. Rapamycin, which is employed in the management of patients undergoing liver surgery, induces the synthesis of heme oxygenase-1 (HO-1) in some non-liver cell types. The aim was to investigate whether rapamycin can induce HO-1 expression in the liver, and to test the effects of

  18. Religious Activities and Suicide Prevention: A Gender Specific Analysis

    Directory of Open Access Journals (Sweden)

    Steven Stack

    2018-04-01

    Full Text Available The present analysis contributes to the existing literature on religion and suicide in three interrelated ways: (1 providing an analysis of suicide completions whereas most research is based on non-lethal levels of suicidality; (2 assessing the relationship with concrete individual level data on completed suicides instead of aggregated data marked by the ecological fallacy issue; and (3 providing gender specific analyses to determine if the relationship is gendered. METHODS. Data come from the U.S. Public Health Service, National Mortality Followback Survey. They refer to 16,795 deaths including 1385 suicides. Significant others of the deceased were interviewed to measure all variables. The dependent variable is a binary variable where 1 = death by suicide and 0 = all other causes. The central independent variable is an index of religious activities. Controls are included for five categories of confounders (1 psychiatric morbidity; (2 help-seeking behavior; (3 Opportunity factors such as firearms; (4 social integration; and (5 demographics. RESULTS. Multivariate logistic regression analysis determined that controlling for 16 predictors of suicide, a one unit increase in religious activities reduced the odds of a suicide death by 17% for males and by 15% for females. The difference in coefficients is not significant (Z = 0.51. Other significant predictors of suicide deaths included suicide ideation (OR = 8.87, males, OR = 11.48, females and firearm availability (OR = 4.21, males, OR = 2.83, females. DISCUSSION. Religious activities were found to lower suicide risk equally for both men and women. Further work is needed to assess pathways, including suicide ideation, between religious activities and lowered suicide risk. This is the first U.S. based study to test for a gendered association between religion and suicide at the individual level of analysis.

  19. Application-specific architectures of CMOS monolithic active pixel sensors

    Energy Technology Data Exchange (ETDEWEB)

    Szelezniak, Michal [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France)]. E-mail: michal.szelezniak@ires.in2p3.fr; Besson, Auguste [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Claus, Gilles; Colledani, Claude; [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Degerli, Yavuz [CEA Saclay, DAPNIA, Gif-sur-Yvette Cedex (France); Deptuch, Grzegorz [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Deveaux, Michael [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); GSI, Planckstrasse 1, Darmstadt 64291 (Germany); Dorokhov, Andrei [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Dulinski, Wojciech [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Fourches, Nicolas [CEA Saclay, DAPNIA, Gif-sur-Yvette Cedex (France); Goffe, Mathieu [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Grandjean, Damien; Guilloux, Fabrice [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Heini, Sebastien [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France)]|[GSI, Planckstrasse 1, Darmstadt 64291 (Germany); Himmi, Abdelkader [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Hu, Christine [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France); Jaaskelainen, Kimmo; Li, Yan; Lutz, Pierre; Orsini, Fabienne [CEA Saclay, DAPNIA, Gif-sur-Yvette Cedex (France); Pellicioli, Michel; Shabetai, Alexandre; Valin, Isabelle; Winter, Marc [Institute de Recherches Subatomiques, 23 rue du Loess, Strasbourg 67037 Cedex 02 (France)

    2006-11-30

    Several development directions intended to adapt and optimize monolithic active pixel sensors for specific applications are presented in this work. The first example, compatible with the STAR microvertex upgrade, is based on a simple two-transistor pixel circuitry. It is suited for a long integration time, room-temperature operation and minimum power dissipation. In another approach for this application, a specific readout method is proposed, allowing optimization of the integration time independently of the full frame-readout time. The circuit consists of an in-pixel front-end voltage amplifier, with a gain on the order of five, followed by two analog memory cells. The extended version of this scheme, based on the implementation of more memory cells per pixel, is the solution considered for the outer layers of a microvertex detector at the international linear collider. For the two innermost layers, a circuit allowing fast frame scans together with on-line, on-chip data sparsification is proposed. The first results of this prototype demonstrate that the fixed pattern dispersion is reduced below a noise level of 15 e{sup -}, allowing the use of a single comparator or a low-resolution ADC per pixel column. A common element for most of the mentioned readout schemes is a low-noise, low power consumption, layout efficient in-pixel amplifier. A review of possible solutions for this element together with some experimental results is presented.

  20. Rapamycin preconditioning attenuates transient focal cerebral ischemia/reperfusion injury in mice.

    Science.gov (United States)

    Yin, Lele; Ye, Shasha; Chen, Zhen; Zeng, Yaoying

    2012-12-01

    Rapamycin, an mTOR inhibitor and immunosuppressive agent in clinic, has protective effects on traumatic brain injury and neurodegenerative diseases. But, its effects on transient focal ischemia/reperfusion disease are not very clear. In this study, we examined the effects of rapamycin preconditioning on mice treated with middle cerebral artery occlusion/reperfusion operation (MCAO/R). We found that the rapamycin preconditioning by intrahippocampal injection 20 hr before MCAO/R significantly improved the survival rate and longevity of mice. It also decreased the neurological deficit score, infracted areas and brain edema. In addition, rapamycin preconditioning decreased the production of NF-κB, TNF-α, and Bax, but not Bcl-2, an antiapoptotic protein in the ischemic area. From these results, we may conclude that rapamycin preconditioning attenuate transient focal cerebral ischemia/reperfusion injury and inhibits apoptosis induced by MCAO/R in mice.

  1. Priming anticancer active specific immunotherapy with dendritic cells.

    Science.gov (United States)

    Mocellin, Simone

    2005-06-01

    Dendritic cells (DCs) probably represent the most powerful naturally occurring immunological adjuvant for anticancer vaccines. However, the initial enthusiasm for DC-based vaccines is being tempered by clinical results not meeting expectations. The partial failure of current vaccine formulations is explained by the extraordinary complexity of the immune system, which makes the task of exploiting the potential of such a biotherapeutic approach highly challenging. Clinical findings obtained in humans so far indicate that the immune system can be actively polarized against malignant cells by means of DC-based active specific immunotherapy, and that in some cases this is associated with tumor regression. This implies that under some unique circumstances, the naturally 'dormant' immune effectors can actually be employed as endogenous weapons against malignant cells. Only the thorough understanding of DC biology and tumor-host immune system interactions will allow researchers to reproduce, in a larger set of patients, the cellular/molecular conditions leading to an effective immune-mediated eradication of cancer.

  2. AATR an ionospheric activity indicator specifically based on GNSS measurements

    Science.gov (United States)

    Juan, José Miguel; Sanz, Jaume; Rovira-Garcia, Adrià; González-Casado, Guillermo; Ibáñez, D.; Perez, R. Orus

    2018-03-01

    This work reviews an ionospheric activity indicator useful for identifying disturbed periods affecting the performance of Global Navigation Satellite System (GNSS). This index is based in the Along Arc TEC Rate (AATR) and can be easily computed from dual-frequency GNSS measurements. The AATR indicator has been assessed over more than one Solar Cycle (2002-2017) involving about 140 receivers distributed world-wide. Results show that it is well correlated with the ionospheric activity and, unlike other global indicators linked to the geomagnetic activity (i.e. DST or Ap), it is sensitive to the regional behaviour of the ionosphere and identifies specific effects on GNSS users. Moreover, from a devoted analysis of different Satellite Based Augmentation System (SBAS) performances in different ionospheric conditions, it follows that the AATR indicator is a very suitable mean to reveal whether SBAS service availability anomalies are linked to the ionosphere. On this account, the AATR indicator has been selected as the metric to characterise the ionosphere operational conditions in the frame of the European Space Agency activities on the European Geostationary Navigation Overlay System (EGNOS). The AATR index has been adopted as a standard tool by the International Civil Aviation Organization (ICAO) for joint ionospheric studies in SBAS. In this work we explain how the AATR is computed, paying special attention to the cycle-slip detection, which is one of the key issues in the AATR computation, not fully addressed in other indicators such as the Rate Of change of the TEC Index (ROTI). After this explanation we present some of the main conclusions about the ionospheric activity that can extracted from the AATR values during the above mentioned long-term study. These conclusions are: (a) the different spatial correlation related with the MOdified DIP (MODIP) which allows to clearly separate high, mid and low latitude regions, (b) the large spatial correlation in mid

  3. Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

    Science.gov (United States)

    Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

    2017-10-02

    Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional

  4. Rapamycin up-regulates triglycerides in hepatocytes by down-regulating Prox1.

    Science.gov (United States)

    Kwon, Sora; Jeon, Ji-Sook; Kim, Su Bin; Hong, Young-Kwon; Ahn, Curie; Sung, Jung-Suk; Choi, Inho

    2016-02-27

    Although the prolonged use of rapamycin may cause unwanted side effects such as hyperlipidemia, the underlying mechanism remains unknown. Prox1 is a transcription factor responsible for the development of several tissues including lymphatics and liver. There is growing evidences that Prox1 participates in metabolism in addition to embryogenesis. However, whether Prox1 is directly related to lipid metabolism is currently unknown. HepG2 human hepatoma cells were treated with rapamycin and total lipids were analyzed by thin layer chromatography. The effect of rapamycin on the expression of Prox1 was determined by western blotting. To investigate the role of Prox1 in triglycerides regulation, siRNA and overexpression system were employed. Rapamycin was injected into mice for 2 weeks and total lipids and proteins in liver were measured by thin layer chromatography and western blot analysis, respectively. Rapamycin up-regulated the amount of triglyceride and down-regulated the expression of Prox1 in HepG2 cells by reducing protein half-life but did not affect its transcript. The loss-of-function of Prox1 was coincident with the increase of triglycerides in HepG2 cells treated with rapamycin. The up-regulation of triglycerides by rapamycin in HepG2 cells reverted to normal levels by the compensation of Prox1 using the overexpression system. Rapamycin also down-regulated Prox1 expression but increased triglycerides in mouse liver. This study suggests that rapamycin can increase the amount of triglycerides by down-regulating Prox1 expression in hepatocytes, which means that the mammalian target of rapamycin (mTOR) signaling is important for the regulation of triglycerides by maintaining Prox1 expression.

  5. Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

    Directory of Open Access Journals (Sweden)

    Angèle Nalbandian

    Full Text Available Mutations in the valosin containing protein (VCP gene cause hereditary Inclusion body myopathy (hIBM associated with Paget disease of bone (PDB, frontotemporal dementia (FTD, more recently termed multisystem proteinopathy (MSP. Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem

  6. FK506 Binding Protein Mediates Glioma Cell Growth and Sensitivity to Rapamycin Treatment by Regulating NF-κB Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Wei Jiang

    2008-03-01

    Full Text Available FK506 binding protein 5 (FKBP5 belongs to a family of immunophilins named for their ability to bind immunosuppressive drugs, also known as peptidyl-prolyl cis-trans isomerases, and also with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The roles of FKBP5 in glioma cells were then examined. We found that cell growth was suppressed after FKBP5 expression was inhibited by short interfering RNA transfection and enhanced by FKBP5 overexpression. Electrophoretic mobility shift assay showed that nuclear factor-kappa B (NF-κB and DNA binding was enhanced by FKBP5 overexpression. The expression level of I-kappa B alpha and phosphorylated NF-κB was regulated by the expression of FKBP5. These data suggest that FKBP5 is involved in NF-κB pathway activation in glioma cells. In addition, FKBP5 overexpression in rapamycin-sensitive U87 cells blocked the cells' response to rapamycin treatment, whereas rapamycin-resistant glioma cells, both PTEN-positive and -negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, suggesting that the FKBP5 regulates glioma cell response to rapamycin treatment. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance through regulating signal transduction of the NF-κB pathway.

  7. RelEx: Visualization for Actively Changing Overlay Network Specifications.

    Science.gov (United States)

    Sedlmair, M; Frank, A; Munzner, T; Butz, A

    2012-12-01

    We present a network visualization design study focused on supporting automotive engineers who need to specify and optimize traffic patterns for in-car communication networks. The task and data abstractions that we derived support actively making changes to an overlay network, where logical communication specifications must be mapped to an underlying physical network. These abstractions are very different from the dominant use case in visual network analysis, namely identifying clusters and central nodes, that stems from the domain of social network analysis. Our visualization tool RelEx was created and iteratively refined through a full user-centered design process that included a full problem characterization phase before tool design began, paper prototyping, iterative refinement in close collaboration with expert users for formative evaluation, deployment in the field with real analysts using their own data, usability testing with non-expert users, and summative evaluation at the end of the deployment. In the summative post-deployment study, which entailed domain experts using the tool over several weeks in their daily practice, we documented many examples where the use of RelEx simplified or sped up their work compared to previous practices.

  8. Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Liao, Hsien-Ching; Chen, Mei-Yu

    2012-02-24

    The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.

  9. Rapamycin protects kidney against ischemia reperfusion injury through recruitment of NKT cells.

    Science.gov (United States)

    Zhang, Chao; Zheng, Long; Li, Long; Wang, Lingyan; Li, Liping; Huang, Shang; Gu, Chenli; Zhang, Lexi; Yang, Cheng; Zhu, Tongyu; Rong, Ruiming

    2014-08-19

    NKT cells play a protective role in ischemia reperfusion (IR) injury, of which the trafficking in the body and recruitment in injured organs can be influenced by immunosuppressive therapy. Therefore, we investigated the effects of rapamycin on kidneys exposed to IR injury in early stage and on trafficking of NKT cells in a murine model. Balb/c mice were subjected to kidney 30 min ischemia followed by 24 h reperfusion. Rapamycin (2.5 ml/kg) was administered by gavage daily, starting 1 day before the operation. Renal function and histological changes were assessed. The proportion of NKT cells in peripheral blood, spleen and kidney was detected by flow cytometry. The chemokines and corresponding receptor involved in NKT cell trafficking were determined by RT-PCR and flow cytometry respectively. Rapamycin significantly improved renal function and ameliorated histological injury. In rapamycin-treated group, the proportion of NKT cells in spleen was significantly decreased but increased in peripheral blood and kidney. In addition, the CXCR3+ NKT cell in the kidney increased remarkably in the rapamycin-treated group. The chemokines, CXCL9 and CXCL10, as the ligands of CXCR3, were also increased in the rapamycin-treated kidney. Rapamycin may recruit NKT cells from spleen to the IR-induced kidney to ameliorate renal IR injury in the early stage.

  10. Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts.

    Science.gov (United States)

    Schachner, Thomas; Oberhuber, Alexander; Zou, Yping; Tzankov, Alexandar; Ott, Harald; Laufer, Günther; Bonatti, Johannes

    2005-02-01

    Rapamycin is an immunosuppressive agent with marked antiproliferative properties and is effective in reducing in stent restenosis and vein graft neointimal hyperplasia. Apoptosis is one mechanism counterbalancing cellular proliferation. We therefore investigated the role of apoptosis in rapamycin treated vein grafts in a mouse model. C57BL6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a cuff technique. In the treatment group 200 microg of rapamycin were applied locally in pluronic gel. The control group did not receive local treatment. Vein grafts were harvested at 4 weeks postoperatively and underwent morphometric analysis as well as immunohistochemical analysis for apoptosis (TUNEL). In grafted veins without treatment (controls) neointimal thickness was 50 (12-58) microm at 4 weeks postoperatively. In 200 microg rapamycin treated grafts the neointimal thickness was 17 (5-55) microm. Rapamycin treated vein grafts showed a significantly increased rate of apoptosis in the adventitia as compared with controls (P=0.032). In the neointima the apoptosis rate was lower in both groups with no significant difference between rapamycin treated grafts and controls. We conclude that treatment of experimental vein grafts with rapamycin is associated with an increased apoptosis rate in the vascular wall and a trend towards reduction of neointimal hyperplasia. These results suggest that apoptosis may be a beneficial antiproliferative component for the treatment of vein graft disease.

  11. Inhibition of Akt enhances the chemopreventive effects of topical rapamycin in mouse skin

    Science.gov (United States)

    Dickinson, Sally E; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R.; Liu, Zhonglin; Barber, Christie; Rusche, Jadrian J.; Petricoin, Emmanuel; Calvert, Valerie; Einspahr, Janine G.; Dickinson, Jesse; Stratton, Steven P.; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M.; Dong, Zigang; Alberts, David S.; Bowden, G. Timothy

    2016-01-01

    The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

  12. Mechanistic Target of Rapamycin-Independent Antidepressant Effects of (R)-Ketamine in a Social Defeat Stress Model.

    Science.gov (United States)

    Yang, Chun; Ren, Qian; Qu, Youge; Zhang, Ji-Chun; Ma, Min; Dong, Chao; Hashimoto, Kenji

    2018-01-01

    The role of the mechanistic target of rapamycin (mTOR) signaling in the antidepressant effects of ketamine is controversial. In addition to mTOR, extracellular signal-regulated kinase (ERK) is a key signaling molecule in prominent pathways that regulate protein synthesis. (R)-Ketamine has a greater potency and longer-lasting antidepressant effects than (S)-ketamine. Here we investigated whether mTOR signaling and ERK signaling play a role in the antidepressant effects of two enantiomers. The effects of mTOR inhibitors (rapamycin and AZD8055) and an ERK inhibitor (SL327) on the antidepressant effects of ketamine enantiomers in the chronic social defeat stress (CSDS) model (n = 7 or 8) and on those of ketamine enantiomers in these signaling pathways in mouse brain regions were examined. The intracerebroventricular infusion of rapamycin or AZD8055 blocked the antidepressant effects of (S)-ketamine, but not (R)-ketamine, in the CSDS model. Furthermore, (S)-ketamine, but not (R)-ketamine, significantly attenuated the decreased phosphorylation of mTOR and its downstream effector, ribosomal protein S6 kinase, in the prefrontal cortex of susceptible mice after CSDS. Pretreatment with SL327 blocked the antidepressant effects of (R)-ketamine but not (S)-ketamine. Moreover, (R)-ketamine, but not (S)-ketamine, significantly attenuated the decreased phosphorylation of ERK and its upstream effector, mitogen-activated protein kinase/ERK kinase, in the prefrontal cortex and hippocampal dentate gyrus of susceptible mice after CSDS. This study suggests that mTOR plays a role in the antidepressant effects of (S)-ketamine, but not (R)-ketamine, and that ERK plays a role in (R)-ketamine's antidepressant effects. Thus, it is unlikely that the activation of mTOR signaling is necessary for antidepressant actions of (R)-ketamine. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  13. [Health care activity in a headache-specific clinic].

    Science.gov (United States)

    Garcia-Escrivà, A; Asensio-Asensio, M; López-Hernández, N; González-Aznar, O J; Oliver-Navarrete, C; Alvarez-Saúco, M; Pampliega-Pérez, A

    It is reckoned that headaches affect, at least once a year, around 90% of the population. The socioeconomic repercussion occasioned by this malady justifies the appearance in recent years of headache units. To conduct a descriptive epidemiological and health care study of the activity carried out in a headache-specific clinic. All the relevant points from the histories of patients who visited our surgery over a period of two years were collected prospectively and consecutively. The different types of headaches were classified according to the 1988 IHS criteria. Both the symptomatic and the preventive treatment were analysed. In all, a total of 866 patients were found; 691 (79.8%) were females and the mean age was 39.8 +/- 15.9 years (range: 6-90 years); 208 (24%) had a history of migraine in the family; 399 (49.9%) were diagnosed as suffering from migraine: 256 (64.2%) had migraine without aura, 152 (19%) were diagnosed as having tension-type headache, and 218 (27.3%) presented chronic daily headache (CDH). The most frequently used symptomatic treatments were NSAI drugs (36.7%) and triptanes (28.4%). Amitriptyline (47.7%), beta-blockers (14.5%) and calcium antagonists (11.3%) were the main drugs used as preventive treatment. After several years' operation of our Headache Unit, we thought there was a need to analyse the population seen in the visits. The fact that the majority of our patients were middle-aged females matched our expectations. Although most of the patients were diagnosed as suffering from M, we also want to highlight the high proportion of cases of CDH, above all associated with the abuse of analgesics.

  14. Is active participation in specific sport activities linked with back pain?

    DEFF Research Database (Denmark)

    Mogensen, A.M.; Gausel, AM; Wedderkopp, Niels

    2007-01-01

    A cross-sectional survey of 439 children/adolescents aged 12-13, living in Odense, Denmark, in the year 2001. To investigate (1) if there is any difference in back pain reporting among those practising specific sports as compared with non-performers and (2) if there is an association between...... specific kinds of sports and self-reported back problems. Back pain is a common complaint in young people and physical inactivity is generally thought to contribute to this. However, some specific sport activities may be detrimental or beneficial to the spine. Information was collected through a semi......-structured interview, a physical examination, and a questionnaire. Associations for back pain, low back pain, mid back pain and neck pain in the preceding month were investigated in relation to specific sports. Associations were controlled for body mass index, puberty stage and sex. There was no association between...

  15. Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.

    Science.gov (United States)

    Ruwanpura, Saleela M; McLeod, Louise; Dousha, Lovisa F; Seow, Huei J; Alhayyani, Sultan; Tate, Michelle D; Deswaerte, Virginie; Brooks, Gavin D; Bozinovski, Steven; MacDonald, Martin; Garbers, Christoph; King, Paul T; Bardin, Philip G; Vlahos, Ross; Rose-John, Stefan; Anderson, Gary P; Jenkins, Brendan J

    2016-12-15

    The potent immunomodulatory cytokine IL-6 is consistently up-regulated in human lungs with emphysema and in mouse emphysema models; however, the mechanisms by which IL-6 promotes emphysema remain obscure. IL-6 signals using two distinct modes: classical signaling via its membrane-bound IL-6 receptor (IL-6R), and trans-signaling via a naturally occurring soluble IL-6R. To identify whether IL-6 trans-signaling and/or classical signaling contribute to the pathogenesis of emphysema. We used the gp130 F/F genetic mouse model for spontaneous emphysema and cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various methods including in vivo lung function and stereology, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and location of IL-6 signaling-related genes and proteins were measured, and the levels of IL-6 and related proteins in sera from emphysematous mice and patients were also assessed. Lung tissues from patients with emphysema, and from spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by excessive production of soluble IL-6R. Genetic blockade of IL-6 trans-signaling in emphysema mouse models and therapy with the IL-6 trans-signaling antagonist sgp130Fc ameliorated emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6 trans-signaling-driven emphysematous changes in the lung correlated with mechanistic target of rapamycin complex 1 hyperactivation, and treatment of emphysema mouse models with the mechanistic target of rapamycin complex 1 inhibitor rapamycin attenuated emphysematous changes. Collectively, our data reveal that specific targeting of IL-6 trans-signaling may represent a novel treatment strategy for emphysema.

  16. Serotonin induces memory-like, rapamycin-sensitive hyperexcitability in sensory axons of aplysia that contributes to injury responses.

    Science.gov (United States)

    Weragoda, Ramal M S; Walters, Edgar T

    2007-09-01

    The induction of long-term facilitation (LTF) of synapses of Aplysia sensory neurons (SNs) by serotonin (5-HT) has provided an important mechanistic model of memory, but little is known about other long-term effects of 5-HT on sensory properties. Here we show that crushing peripheral nerves results in long-term hyperexcitability (LTH) of the axons of these nociceptive SNs that requires 5-HT activity in the injured nerve. Serotonin application to a nerve segment induces local axonal (but not somal) LTH that is inhibited by 5-HT-receptor antagonists. Blockade of crush-induced axonal LTH by an antagonist, methiothepin, provides evidence for mediation of this injury response by 5-HT. This is the first demonstration in any axon of neuromodulator-induced LTH, a phenomenon potentially important for long-lasting pain. Methiothepin does not reduce axonal LTH induced by local depolarization, so 5-HT is not required for all forms of axonal LTH. Serotonin-induced axonal LTH is expressed as reduced spike threshold and increased repetitive firing, whereas depolarization-induced LTH involves only reduced threshold. Like crush- and depolarization-induced LTH, 5-HT-induced LTH is blocked by inhibiting protein synthesis. Blockade by rapamycin, which also blocks synaptic LTF, is interesting because the eukaryotic protein kinase that is the target of rapamycin (TOR) has a conserved role in promoting growth by stimulating translation of proteins required for translation. Rapamycin sensitivity suggests that localized increases in translation of proteins that promote axonal conduction and excitability at sites of nerve injury may be regulated by the same signals that increase translation of proteins that promote neuronal growth.

  17. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    International Nuclear Information System (INIS)

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

    2015-01-01

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs

  18. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Song, Sang Heon [Department of Internal Medicine, Pusan National University Hospital, Busan (Korea, Republic of); Shin, Hwa Kyoung [Department of Anatomy, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

    2015-08-14

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

  19. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    International Nuclear Information System (INIS)

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

  20. Ketamine Exhibits Different Neuroanatomical Profile After Mammalian Target of Rapamycin Inhibition in the Prefrontal Cortex: the Role of Inflammation and Oxidative Stress.

    Science.gov (United States)

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Danielski, Lucineia G; Petronilho, Fabricia; Carvalho, André F; Quevedo, João

    2017-09-01

    Studies indicated that mammalian target of rapamycin (mTOR), oxidative stress, and inflammation are involved in the pathophysiology of major depressive disorder (MDD). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been identified as a novel MDD therapy; however, the antidepressant mechanism is not fully understood. In addition, the effects of ketamine after mTOR inhibition have not been fully investigated. In the present study, we examined the behavioral and biochemical effects of ketamine in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens after inhibition of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol) or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). Immobility was assessed in forced swimming tests, and then oxidative stress parameters and inflammatory markers were evaluated in the brain and periphery. mTOR activation in the PFC was essential to ketamine's antidepressant-like effects. Ketamine increased lipid damage in the PFC, hippocampus, and amygdala. Protein carbonyl was elevated in the PFC, amygdala, and NAc after ketamine administration. Ketamine also increased nitrite/nitrate in the PFC, hippocampus, amygdala, and NAc. Myeloperoxidase activity increased in the hippocampus and NAc after ketamine administration. The activities of superoxide dismutase and catalase were reduced after ketamine administration in all brain areas studied. Inhibition of mTOR signaling pathways by rapamycin in the PFC was required to protect against oxidative stress by reducing damage and increasing antioxidant enzymes. Finally, the TNF-α level was increased in serum by ketamine; however, the rapamycin plus treatment group was not able to block this increase. Activation of mTOR in the PFC is involved in the antidepressant-like effects of ketamine; however, the inhibition of this pathway was able to protect certain brain areas against

  1. Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation

    OpenAIRE

    Takahashi, Hironobu; Wang, Yuwei; Grainger, David W.

    2010-01-01

    Fibrous encapsulation of surgically implant devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering...

  2. Beyond AICA Riboside: In Search of New Specific AMP-activated Protein Kinase Activators

    Science.gov (United States)

    Guigas, Bruno; Sakamoto, Kei; Taleux, Nellie; Reyna, Sara M.; Musi, Nicolas; Viollet, Benoit; Hue, Louis

    2010-01-01

    Summary 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects. PMID:18798311

  3. Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

    Directory of Open Access Journals (Sweden)

    Rachel S. Lee

    2011-01-01

    Full Text Available The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.

  4. Rapamycin delays growth of Wnt-1 tumors in spite of suppression of host immunity

    International Nuclear Information System (INIS)

    Svirshchevskaya, Elena V; Mariotti, Jacopo; Wright, Mollie H; Viskova, Natalia Y; Telford, William; Fowler, Daniel H; Varticovski, Lyuba

    2008-01-01

    Rapamycin, an inhibitor of mammalian target of Rapamycin (mTOR), is an immunosuppressive agent that has anti-proliferative effects on some tumors. However, the role of Rapamycin-induced immune suppression on tumor progression has not been examined. We developed a transplantation model for generation of mammary tumors in syngeneic recipients that can be used to address the role of the immune system on tumor progression. We examined the effect of Rapamycin on the immune system and growth of MMTV-driven Wnt-1 mammary tumors which were transplanted into irradiated and bone marrow-reconstituted, or naïve mice. Rapamycin induced severe immunosuppression and significantly delayed the growth of Wnt-1 tumors. T cell depletion in spleen and thymus and reduction in T cell cytokine secretion were evident within 7 days of therapy. By day 20, splenic but not thymic T cell counts, and cytokine secretion recovered. We determined whether adoptive T cell therapy enhances the anti-cancer effect using ex vivo generated Rapamycin-resistant T cells. However, T cell transfer during Rapamycin therapy did not improve the outcome relative to drug therapy alone. Thus, we could not confirm that suppression of T cell immunity contributes to tumor growth in this model. Consistent with suppression of the mTOR pathway, decreased 4E-BP1, p70 S6-kinase, and S6 protein phosphorylation correlated with a decrease in Wnt-1 tumor cell proliferation. Rapamycin has a direct anti-tumor effect on Wnt-1 breast cancer in vivo that involves inhibition of the mTOR pathway at doses that also suppress host immune responses

  5. Significance of specific activity and a possible universal unit for its definition

    International Nuclear Information System (INIS)

    Svoboda, K.

    1985-01-01

    The growing importance of specific activity is reviewed. It concerns especially surface phenomena, toxicity, labelling of radiopharmaceuticals, isotope exchange, enzymatic and pharmacological ligand-receptor reactions. The present state of evaluating the specific activity is analyzed. Introduction of the coefficient Dsub(CF) (deviation from true carrier free state) is proposed as a possibility for universal declaration of the specific activity. (author)

  6. Activity Specific Knowledge Characteristics in the Internationalization Process

    DEFF Research Database (Denmark)

    Søberg, Peder Veng

    2012-01-01

    /methodology/approach – The paper presents a framework primarily based on knowledge management theory, which is illustrated in relation to interesting cases of four companies that are global leaders. Findings – An R&D knowledge gap still exists in China and India. Differences across business activities exist in terms......Purpose – The purpose of this paper is to investigate differences in the characteristics of knowledge, which is very important for the internationalization of different business activities. In particular, the focus is on internationalization in emerging markets such as China and India. Design...... of the characteristics of the knowledge, which is most important for the internationalization in emerging markets within multinational corporations (MNCs). The most important knowledge for the internationalization of R&D activities is more tacit than it is for manufacturing activities and international purchasing...

  7. Transport of uranium concentrates: low specific activity versus logistic complexity

    International Nuclear Information System (INIS)

    Dias, Pedro L.S.; Macedo, Eclesio F.; Carvalho, Leonardo B.; Carvalho, Renata R.

    2011-01-01

    This paper describes the case of radioactive material transport, according to pertinent documentation - nuclear material specifically in the form op ammonium diuranate, produced by Industrias Nucleares do Brasil S.A. - from the mine and physic-chemical processing at Caetite, Bahia, to the port of Salvador, state of Bahia, approaching the radiological protection aspects

  8. A comprehensive review of everolimus clinical reports: a new mammalian target of rapamycin inhibitor.

    Science.gov (United States)

    Gurk-Turner, Cheryle; Manitpisitkul, Wana; Cooper, Matthew

    2012-10-15

    As new immunosuppressive agents are introduced to the market, clinicians are faced with the daunting task of sifting through the published literature to decide the value that the agent will add to their own practice. We often must extrapolate information provided through study in other solid-organ transplantation populations than our specific area of interest as we interpret the results and outcomes. With these challenges in mind, this compilation of published work for the newest mammalian target of rapamycin inhibitor everolimus (Certican; Novartis Pharmaceuticals, Hanover, NJ) (Zortress; Novartis Pharmaceuticals, Basel, Switzerland) is intended to provide a concise but thorough presentation of available literature so that the reader who may be unfamiliar with the agent can make their own judgment. Both Ovid and PubMed search engines were queried with a particular focus on high-impact articles noted in the Web of Science or Citation Index. Work described solely in abstract or case report form was excluded, as well as meta-analyses or those that were editorial or commentary in nature. Included were publications presented using the English language that described adult human subjects who received a heart, lung, kidney, or liver allograft. The goal of this strategy was to allow for the inclusion of pertinent literature in an unbiased fashion. Tables are provided that outline trial specific information, leaving a discussion of major outcomes to the text of the review.

  9. Sensitivity and Specificity of Hypnosis Effects on Gastric Myoelectrical Activity

    Science.gov (United States)

    Enck, Paul; Weimer, Katja; Muth, Eric R.; Zipfel, Stephan; Martens, Ute

    2013-01-01

    Objectives The effects of hypnosis on physiological (gastrointestinal) functions are incompletely understood, and it is unknown whether they are hypnosis-specific and gut-specific, or simply unspecific effects of relaxation. Design Sixty-two healthy female volunteers were randomly assigned to either a single session of hypnotic suggestion of ingesting an appetizing meal and an unappetizing meal, or to relax and concentrate on having an appetizing or unappetizing meal, while the electrogastrogram (EGG) was recorded. At the end of the session, participants drank water until they felt full, in order to detect EGG-signal changes after ingestion of a true gastric load. During both conditions participants reported their subjective well-being, hunger and disgust at several time points. Results Imagining eating food induced subjective feelings of hunger and disgust as well as changes in the EGG similar to, but more pronounced than those seen with a real gastric water load during both hypnosis and relaxation conditions. These effects were more pronounced when imagining an appetizing meal than with an unappetizing meal. There was no significant difference between the hypnosis and relaxation conditions. Conclusion Imagination with and without hypnosis exhibits similar changes in subjective and objective measures in response to imagining an appetizing and an unappetizing food, indicating high sensitivity but low specificity. PMID:24358287

  10. Ileal brake activation: macronutrient-specific effects on eating behavior?

    NARCIS (Netherlands)

    Avesaat, van M.; Troost, F.J.; Ripken, D.; Hendriks, H.F.; Masclee, A.A.M.

    2015-01-01

    Background:Activation of the ileal brake, by infusing lipid directly into the distal part of the small intestine, alters gastrointestinal (GI) motility and inhibits food intake. The ileal brake effect on eating behavior of the other macronutrients is currently unknown.Objective:The objective of this

  11. Ileal brake activation: Macronutrient-specific effects on eating behavior?

    NARCIS (Netherlands)

    Avesaat, M. van; Troost, F.J.; Ripken, D.; Hendriks, H.F.; Aam, M.

    2015-01-01

    BACKGROUND: Activation of the ileal brake, by infusing lipid directly into the distal part of the small intestine, alters gastrointestinal (GI) motility and inhibits food intake. The ileal brake effect on eating behavior of the other macronutrients is currently unknown. OBJECTIVE: The objective of

  12. Location and activity specific site-management for military locations

    NARCIS (Netherlands)

    Maring, L.; Hulst, M. van; Meuken, D.

    2009-01-01

    pace is limited in the Netherlands and military activities, that may cause nuisance or environmental hazards, should therefore be considered and evaluated during the use of military locations. The last few years TNO and Deltares have worked on a research program on environmental effects due to

  13. The antidepressant sertraline inhibits translation initiation by curtailing mammalian target of rapamycin signaling.

    Science.gov (United States)

    Lin, Chen-Ju; Robert, Francis; Sukarieh, Rami; Michnick, Stephen; Pelletier, Jerry

    2010-04-15

    Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner. (c) 2010 AACR.

  14. Immunomodulatory activity of andrographolide on macrophage activation and specific antibody response

    Science.gov (United States)

    Wang, Wei; Wang, Jing; Dong, Sheng-fu; Liu, Chun-hong; Italiani, Paola; Sun, Shu-hui; Xu, Jing; Boraschi, Diana; Ma, Shi-ping; Qu, Di

    2010-01-01

    Aim: To investigate the immunomodulatory effects of andrographolide on both innate and adaptive immune responses. Methods: Andrographolide (10 μg/mL in vitro or 1 mg/kg in vivo) was used to modulate LPS-induced classical activated (M1) or IL-4-induced alternative activated (M2) macrophages in vitro and humor immune response to HBsAg in vivo. Cytokine gene expression profile (M1 vs M2) was measured by real-time PCR, IL-12/IL-10 level was detected by ELISA, and surface antigen expression was evaluated by flow cytometry, whereas phosphorylation level of ERK 1/2 and AKT was determined by Western blot. The level of anti-HBs antibodies in HBsAg immunized mice was detected by ELISA, and the number of HBsAg specific IL-4-producing splenocyte was enumerated by ELISPOT. Results: Andrographolide treatment in vitro attenuated either LPS or IL-4 induced macrophage activation, inhibited both M1 and M2 cytokines expression and decreased IL-12/IL-10 ratio (the ratio of M1/M2 polarization). Andrographolide down-regulated the expression of mannose receptor (CD206) in IL-4 induced macrophages and major histocompability complex/costimulatory molecules (MHC I, CD40, CD80, CD86) in LPS-induced macrophages. Correspondingly, anti-HBs antibody production and the number of IL-4-producing splenocytes were reduced by in vivo administration of andrographolide. Reduced phosphorylation levels of ERK1/2 and AKT were observed in macrophages treated with andrographolide. Conclusion: Andrographolide can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and Ag-specific antibody production. MAPK and PI3K signaling pathways may participate in the mechanisms of andrographolide regulating macrophage activation and polarization. PMID:20139902

  15. Detection of activated platelets using activation-specific monoclonal antibody (SZ-51) in clinical disorders

    International Nuclear Information System (INIS)

    Wu Guoxin; Li Fugang; Li Jianyong; Ruan Changgeng

    1991-10-01

    A direct test for activated platelets in whole blood was developed by radioimmunoassay with 125 I labeled SZ-51, an antibody specific for an α-granule membrane protein (GMP-140) that associates with the platelet surface during secretion. The assay had sufficient sensitivity to detect as few as 2% activated platelets. In 50 normal subjects, minimal GMP-140 molecules per platelet were expressed on the surface of circulating platelets. Ten patients undergoing cardiopulmonary bypass had transiently increased expression of GMP-140 molecules during the bypass procedure, especially at the end of bypass. Evaluation of 18 patients with epidemic hemorrhagic fever (EHF) has shown that the number of GMP-140 molecules on the platelet surface was closely related to the four different phases of EHF. In six patients suffered from acute myocardial infarction (AMI), the number of GMP-140 molecules changed with the procession of AMI and the highest occurred 48 h after AMI. The GMP-140 molecules were also increased in patients with asthma attack (n = 14), but not in patients with idiopathic thrombocytopenic purpura (n = 11) and diabetic mellitus (n = 48). Taken together, these studies suggest that activated platelet can be reliably measured in whole blood using radiolabeled SZ-51 antibody and the detection of activated platelets is potentially useful in identifying patients with certain thrombotic disorders and others

  16. Specific activity of 129I in environmental samples

    International Nuclear Information System (INIS)

    Ravi, P.M.; Iyer, M.R.; Bhat, I.S.; Somasundaram, S.; Subramanian, M.S.

    1988-01-01

    129 I finds its way into the environment as a result of man-made nuclear operations. It is also formed by the interaction of cosmic rays with xenon isotopes and spontaneous fission of naturally occurring uranium. 129 I and stable 127 I contents of thyroid, milk, seaweed and aplysia cell samples collected from around a fuel reprocessing plant were estimated by neutron activation analysis method. The annual 129 I intake of an individual works out to be about 0.3 Bq as compared to the natural radioactivity content in human body of about 5000 Bq. (author). 3 tabs

  17. Pharmacokinetics of orally administered low-dose rapamycin in healthy dogs.

    Science.gov (United States)

    Larson, Jeanne C; Allstadt, Sara D; Fan, Timothy M; Khanna, Chand; Lunghofer, Paul J; Hansen, Ryan J; Gustafson, Daniel L; Legendre, Alfred M; Galyon, Gina D; LeBlanc, Amy K; Martin-Jimenez, Tomas

    2016-01-01

    To determine the pharmacokinetics of orally administered rapamycin in healthy dogs. 5 healthy purpose-bred hounds. The study consisted of 2 experiments. In experiment 1, each dog received rapamycin (0.1 mg/kg, PO) once; blood samples were obtained immediately before and at 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours after administration. In experiment 2, each dog received rapamycin (0.1 mg/kg, PO) once daily for 5 days; blood samples were obtained immediately before and at 3, 6, 24, 27, 30, 48, 51, 54, 72, 75, 78, 96, 96.5, 97, 98, 100, 102, 108, 120, 144, and 168 hours after the first dose. Blood rapamycin concentration was determined by a validated liquid chromatography-tandem mass spectrometry assay. Pharmacokinetic parameters were determined by compartmental and noncompartmental analyses. Mean ± SD blood rapamycin terminal half-life, area under the concentration-time curve from 0 to 48 hours after dosing, and maximum concentration were 38.7 ± 12.7 h, 140 ± 23.9 ng•h/mL, and 8.39 ± 1.73 ng/mL, respectively, for experiment 1, and 99.5 ± 89.5 h, 126 ± 27.1 ng•h/mL, and 5.49 ± 1.99 ng/mL, respectively, for experiment 2. Pharmacokinetic parameters for rapamycin after administration of 5 daily doses differed significantly from those after administration of 1 dose. Results indicated that oral administration of low-dose (0.1 mg/kg) rapamycin to healthy dogs achieved blood concentrations measured in nanograms per milliliter. The optimal dose and administration frequency of rapamcyin required to achieve therapeutic effects in tumor-bearing dogs, as well as toxicity after chronic dosing, need to be determined.

  18. Suppression of AKT phosphorylation restores rapamycin-based synthetic lethality in SMAD4-defective pancreatic cancer cells.

    Science.gov (United States)

    Le Gendre, Onica; Sookdeo, Ayisha; Duliepre, Stephie-Anne; Utter, Matthew; Frias, Maria; Foster, David A

    2013-05-01

    mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-β synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-β signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-β and cytotoxic in the absence of serum. However, if TGF-β signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-β in colon and breast cancer cell lines. Because genetic dysregulation of TGF-β signaling is commonly observed in pancreatic cancers-with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-β. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-β signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-β signaling. ©2013 AACR.

  19. Overexpression of TOR (target of rapamycin) inhibits cell proliferation in Dictyostelium discoideum.

    Science.gov (United States)

    Swer, Pynskhem Bok; Mishra, Himanshu; Lohia, Rakhee; Saran, Shweta

    2016-05-01

    TOR (target of rapamycin) protein kinase acts as a central controller of cell growth and development of an organism. Present study was undertaken to find the expression pattern and role of TOR during growth and development of Dictyostelium discoideum. Failures to generate either knockout and/or knockdown mutants indicate that interference with its levels led to cellular defects. Thus, the effects of TOR (DDB_G0281569) overexpression specifically, cells expressing Dd(Δ211-TOR)-Eyfp mutant was analyzed. Elevated expression of (Δ211-TOR)-Eyfp reduced both cell size and cell proliferation. DdTOR was found to be closer to fungus. mRNA level of TOR was found maximally in the freshly starved/aggregate cells that gradually declined. This was also strengthened by the expression patterns observed by in situ and the analysis of β-galactosidase reporter driven by the putative TOR promoter. The TOR protein was found to be highest at the aggregate stage. The fusion protein, (Δ211-TOR)-Eyfp was localized to the cell membrane, cytosol, and the nucleus. We suggest, DdTOR to be an essential protein and high TOR expression inhibits cell proliferation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Mammalian target of rapamycin inhibition in polycystic kidney disease: From bench to bedside

    Directory of Open Access Journals (Sweden)

    Hyun-Jung Kim

    2012-09-01

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is the most common life-threatening hereditary disease in the USA resulting in chronic kidney disease and the need for dialysis and transplantation. Approximately 85% of cases of ADPKD are caused by a mutation in the Pkd1 gene that encodes polycystin-1, a large membrane receptor. The Pkd1 gene mutation results in abnormal proliferation in tubular epithelial cells, which plays a crucial role in cyst development and/or growth in PKD. Activation of the proliferative mammalian target of rapamycin (mTOR signaling pathway has been demonstrated in polycystic kidneys from rodents and humans. mTOR inhibition with sirolimus or everolimus decreases cysts in most animal models of PKD including Pkd1 and Pkd2 gene deficient orthologous models of human disease. On the basis of animal studies, human studies were undertaken. Two large randomized clinical trials published in the New England Journal of Medicine of everolimus or sirolimus in ADPKD patients were very unimpressive and associated with a high side-effect profile. Possible reasons for the unimpressive nature of the human studies include their short duration, the high drop-out rate, suboptimal dosing, lack of randomization of “fast” and “slow progressors” and the lack of correlation between kidney size and kidney function in ADPKD. The future of mTOR inhibition in ADPKD is discussed.

  1. A software architectural framework specification for neutron activation analysis

    International Nuclear Information System (INIS)

    Preston, J.A.; Grant, C.N.

    2013-01-01

    Neutron Activation Analysis (NAA) is a sensitive multi-element nuclear analytical technique that has been routinely applied by research reactor (RR) facilities to environmental, nutritional, health related, geological and geochemical studies. As RR facilities face calls to increase their research output and impact, with existing or reducing budgets, automation of NAA offers a possible solution. However, automation has many challenges, not the least of which is a lack of system architecture standards to establish acceptable mechanisms for the various hardware/software and software/software interactions among data acquisition systems, specialised hardware such as sample changers, sample loaders, and data processing modules. This lack of standardization often results in automation hardware and software being incompatible with existing system components, in a facility looking to automate its NAA operations. This limits the availability of automation to a few RR facilities with adequate budgets or in-house engineering resources. What is needed is a modern open system architecture for NAA, that provides the required set of functionalities. This paper describes such an 'architectural framework' (OpenNAA), and portions of a reference implementation. As an example of the benefits, calculations indicate that applying this architecture to the compilation and QA steps associated with the analysis of 35 elements in 140 samples, with 14 SRM's, can reduce the time required by over 80 %. The adoption of open standards in the nuclear industry has been very successful over the years in promoting interchangeability and maximising the lifetime and output of nuclear measurement systems. OpenNAA will provide similar benefits within the NAA application space, safeguarding user investments in their current system, while providing a solid path for development into the future. (author)

  2. Negative Effects of Chronic Rapamycin Treatment on Behavior in a Mouse Model of Fragile X Syndrome

    Directory of Open Access Journals (Sweden)

    Rachel M. Saré

    2018-01-01

    Full Text Available Fragile X syndrome (FXS, the most common form of inherited intellectual disability, is also highly associated with autism spectrum disorders (ASD. It is caused by expansion of a CGG repeat sequence on the X chromosome resulting in silencing of the FMR1 gene. This is modeled in the mouse by deletion of Fmr1 (Fmr1 KO. Fmr1 KO mice recapitulate many of the behavioral features of the disorder including seizure susceptibility, hyperactivity, impaired social behavior, sleep problems, and learning and memory deficits. The mammalian target of rapamycin pathway (mTORC1 is upregulated in Fmr1 KO mice and is thought to be important for the pathogenesis of this disorder. We treated Fmr1 KO mice chronically with an mTORC1 inhibitor, rapamycin, to determine if rapamycin treatment could reverse behavioral phenotypes. We performed open field, zero maze, social behavior, sleep, passive avoidance, and audiogenic seizure testing. We found that pS6 was upregulated in Fmr1 KO mice and normalized by rapamycin treatment, but, except for an anxiogenic effect, it did not reverse any of the behavioral phenotypes examined. In fact, rapamycin treatment had an adverse effect on sleep and social behavior in both control and Fmr1 KO mice. These results suggest that targeting the mTOR pathway in FXS is not a good treatment strategy and that other pathways should be considered.

  3. Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae.

    Science.gov (United States)

    Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Sone, Toshiyuki; Era, Atsuko; Miyagishima, Shin-Ya; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan

    2015-10-01

    Most microalgae produce triacylglycerol (TAG) under stress conditions such as nitrogen depletion, but the underlying molecular mechanism remains unclear. In this study, we focused on the role of target of rapamycin (TOR) in TAG accumulation. TOR is a serine/threonine protein kinase that is highly conserved and plays pivotal roles in nitrogen and other signaling pathways in eukaryotes. We previously constructed a rapamycin-susceptible Cyanidioschyzon merolae, a unicellular red alga, by expressing yeast FKBP12 protein to evaluate the results of TOR inhibition (Imamura et al. in Biochem Biophys Res Commun 439:264-269, 2013). By using this strain, we here report that rapamycin-induced TOR inhibition results in accumulation of cytoplasmic lipid droplets containing TAG. Transcripts for TAG synthesis-related genes, such as glycerol-3-phosphate acyltransferase and acyl-CoA:diacylglycerol acyltransferase (DGAT), were increased by rapamycin treatment. We also found that fatty acid synthase-dependent de novo fatty acid synthesis was required for the accumulation of lipid droplets. Induction of TAG and up-regulation of DGAT gene expression by rapamycin were similarly observed in the unicellular green alga, Chlamydomonas reinhardtii. These results suggest the general involvement of TOR signaling in TAG accumulation in divergent microalgae.

  4. Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin-independent pathway.

    Science.gov (United States)

    Coëffier, Moïse; Claeyssens, Sophie; Bôle-Feysot, Christine; Guérin, Charlène; Maurer, Brigitte; Lecleire, Stéphane; Lavoinne, Alain; Donnadieu, Nathalie; Cailleux, Anne-Françoise; Déchelotte, Pierre

    2013-02-01

    Glutamine modulates duodenal protein metabolism in fasted healthy humans, but its effects in a fed state remain unknown. We aimed to assess the effects of either glutamine or an isonitrogenous protein mixture on duodenal protein metabolism in humans in the fed state. Twenty-four healthy volunteers were randomly included in 2 groups. Each volunteer was studied on 2 occasions in a random order and received, during 5 h, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹; both groups) that mimicked a carbohydrate fed state or maltodextrins with glutamine (group 1) or an isonitrogenous (22.4 mg N · kg⁻¹ · h⁻¹) protein powder (group 2). Simultaneously, a continuous intravenous infusion of ¹³C-leucine and ²H₅-phenylalanine (both 9 μmol · kg⁻¹ · h⁻¹) was performed. Endoscopic duodenal biopsies were taken. Leucine and phenylalanine enrichments were assessed by using gas chromatography-mass spectrometry in duodenal proteins and the intracellular free amino acids pool to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates and macroarrays, respectively. The FSR and proteasome activity were not different after the glutamine supply compared with after maltodextrins alone. In contrast, the FSR increased (1.7-fold increase; P protein-powder delivery without modification of total proteasome activity. The protein powder increased insulinemia, PI3 kinase, and erk phosphorylation but did not affect the mammalian target of rapamycin (mTOR) pathway and mitogen-activated protein kinase signal-integrating kinase 1 phosphorylation. A trend for an increase of eukaryotic translation initiation factor 4E phosphorylation was observed (P = 0.07). In the carbohydrate fed state, enteral proteins but not glutamine increased duodenal protein synthesis through an mTOR independent pathway in humans.

  5. Site-Specific Modification Using the 2′-Methoxyethyl Group Improves the Specificity and Activity of siRNAs

    Directory of Open Access Journals (Sweden)

    Xinyun Song

    2017-12-01

    Full Text Available Rapid progress has been made toward small interfering RNA (siRNA-based therapy for human disorders, but rationally optimizing siRNAs for high specificity and potent silencing remains a challenge. In this study, we explored the effect of chemical modification at the cleavage site of siRNAs. We found that modifications at positions 9 and 10 markedly reduced the silencing potency of the unmodified strand of siRNAs but were well tolerated by the modified strand. Intriguingly, addition of the 2′-methoxyethyl (MOE group at the cleavage site improved both the specificity and silencing activity of siRNAs by facilitating the oriented RNA-induced silencing complex (RISC loading of the modified strand. Furthermore, we combined MOE modifications at positions 9 and 10 of one strand together with 2′-O-methylation (OMe at position 14 of the other strand and found a synergistic effect that improved the specificity of siRNAs. The surprisingly beneficial effect of the combined modification was validated using siRNA-targeting endogenous gene intercellular adhesion molecule 1 (ICAM1. We found that the combined modifications eliminated its off-target effects. In conclusion, we established effective strategies to optimize siRNAs using site-specific MOE modifications. The findings may allow the creation of superior siRNAs for therapy in terms of activity and specificity.

  6. Predictive capabilities of the specific activity hypothesis for Cs and Zn in freshwater systems

    International Nuclear Information System (INIS)

    Seelye, J.G.

    1975-01-01

    Predictions of radioisotope concentrations in components of aquatic systems have been attempted using the specific activity concept, an approach that seems theoretically sound. A comprehensive examination of the specific activities of 134 Cs and 65 Zn in the components of a freshwater system, over a 10 month period, was conducted to evaluate the specific activity hypothesis under applied conditions. This study was designed to provide comparisons of predicted and observed specific activities and to test the equivalence of specific activities between all components of the system. One dose of radioisotopes was added to the system in this study and even after 10 months these radioisotopes were not distributed similarly to the stable isotopes. This suggests that the time necessary to reach a specific activity equilibrium might be a matter of years rather than months. More importantly, in natural systems, where the radioisotope addition is continuous a specific activity equilibrium may never be achieved. These things plus the non-conservative nature of the 134 Cs and 65 Zn predicted concentrations indicates that the use of the specific activity concept for predicting radioisotope concentrations of Cs and Zn in freshwater systems is questionable. A more rigorous approach must be used, considering isotope transfer rates between components and the complexity of the system. Problems with statistical comparisons of derived variables, such as specific activities, are discussed and were considered in interpreting the results of this study

  7. Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

    Science.gov (United States)

    Yeh, Lee-Chuan C; Ma, Xiuye; Ford, Jeffery J; Adamo, Martin L; Lee, John C

    2013-08-01

    Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation. Copyright © 2013 Wiley Periodicals, Inc.

  8. The Inhibitory Effect of Rapamycin on Toll Like Receptor 4 and Interleukin 17 in the Early Stage of Rat Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Ruichao Yu

    2016-02-01

    Full Text Available Background/Aims: There is increasing evidence showing that innate immune responses and inflammatory processes play an important role in the development and progression of diabetic nephropathy (DN. The potential effect of innate immunity in the early stage of DN is still unclear. Toll-Like-Receptor 4 (TLR4 is vigorously involved in the progress of kidney diseases in a sterile environment. The activation of the interleukin 17 (IL-17 pathway produces inflammatory cytokines, appearing in various kidney diseases. Unfortunately the relationship between TLR4 and IL-17 has not been investigated in diabetic nephropathy to date. The aim of this study is to investigate whether mammalian target of rapamycin (mTOR inhibition may be dependent on TLR4 signaling and the pro-inflammatory factor IL-17 to delay the progression of DN. Methods: Streptozotocin (STZ-induced diabetic rats were randomly assigned to 3 experimental groups: a diabetic nephropathy group (DN, n = 6; and a diabetic nephropathy treated with rapamycin group (Rapa, n = 6 and a control group (Control, n =6. Body weight, fasting blood sugar, and 24h urine albumin were assessed at week 2, week 4 and week 8. Renal tissues were harvested for H&E, PAS staining, as well as an immunohistochemistry assay for TLR4 and IL-17. TLR4 quantitative expression was measured by Western-Blot analysis and RT-PCR. Results: Our results demonstrated that the expression of both TLR4 and IL-17 were upregulated in early stage DN and reduced by rapamycin. TLR4 and IL-17 both increased and positively related to 24h urinary albumin and kidney/weight ratio. However, neither TLR4 nor IL-17 made a significant difference on fasting blood sugar. Conclusions: Taken together, our results confirm and extend previous studies identifying the significance of the TLR4 and Th17 pathways in development of early stage DN. Furthermore, we suggest this overexpression of TLR4 might be involved in the immunopathogenesis of DN through

  9. Pharmacokinetics of orally administered low-dose rapamycin in healthy dogs: A pilot study

    Science.gov (United States)

    Larson, Jeanne C.; Allstadt, Sara D.; Fan, Timothy M.; Khanna, Chand; Lunghofer, Paul J.; Hansen, Ryan J.; Gustafson, Daniel L.; Legendre, Alfred M.; Galyon, Gina D.; LeBlanc, Amy K.; Martin-Jimenez, Tomas

    2017-01-01

    Objective To determine the pharmacokinetics of orally administered rapamycin in healthy dogs. Animals 5 healthy purpose-bred hounds. Procedures The study consisted of 2 experiments. In experiment 1, each dog received rapamycin (0.1 mg/kg, PO) once; blood samples were obtained immediately before and at 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours after administration. In experiment 2, each dog received (0.1 mg/kg, PO) once daily for 5 days; blood samples were obtained immediately before and at 3, 6, 24, 27, 30, 48, 51, 54, 72, 75, 78, 96, 96.5, 97, 98, 100, 102, 108, 120, 144, and 168 hours after the first dose. Blood rapamycin concentration was determined by a validated liquid chromatography-tandem mass spectrometry assay. Pharmacokinetic parameters were determined by compartmental and non-compartmental analyses. Results Mean ± SD blood rapamycin terminal half-life, area under the concentration-time curve from 0 to 48 hours after dosing, and maximum concentration were 38.7 ± 12.7 h, 140 ± 23.9 ng•h/mL, and 8.39 ± 1.73 ng/mL, respectively, for experiment 1, and 99.5 ± 89.5 h, 126 ± 27.1 ng•h/mL, and 5.49 ± 1.99 ng/mL, respectively, for experiment 2. Pharmacokinetic parameters for rapamycin after administration of 5 daily doses differed significantly from those after administration of 1 dose. Conclusions and Clinical Relevance Results indicated that oral administration of low-dose (0.1 mg/kg) rapamycin to healthy dogs achieved blood concentrations measured in ng/mL. The optimal dose and administration frequency of rapamcyin required to achieve therapeutic effects in tumor-bearing dogs, as well as toxicity after chronic dosing, needs to be determined. PMID:26709938

  10. Facile determination of the specific activity of carbonyl compounds reduced by tritiated borohydride

    Energy Technology Data Exchange (ETDEWEB)

    Avigad, G [Rutgers--the State Univ., Piscataway, NJ (USA)

    1979-12-01

    Three procedures are described for microliter samples of glucose 6-phosphate or lactose as model compounds. After the reduction with (/sup 3/H)-NaBH/sub 4/ and suitable treatment, specific activity is calculated from the ratios /sup 3/H activity/total phosphorus, /sup 3/H//sup 14/C activity, or /sup 3/H activity/galactoside concentration.

  11. Nuclear PIM1 confers resistance to rapamycin-impaired endothelial proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Walpen, Thomas; Kalus, Ina [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Schwaller, Juerg [Department of Biomedicine, University of Basel, 4031 Basel (Switzerland); Peier, Martin A. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Battegay, Edouard J. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland); Humar, Rok, E-mail: Rok.Humar@usz.ch [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Pim1{sup -/-} endothelial cell proliferation displays increased sensitivity to rapamycin. Black-Right-Pointing-Pointer mTOR inhibition by rapamycin enhances PIM1 cytosolic and nuclear protein levels. Black-Right-Pointing-Pointer Truncation of Pim1 beyond serine 276 results in nuclear localization of the kinase. Black-Right-Pointing-Pointer Nuclear PIM1 increases endothelial proliferation independent of rapamycin. -- Abstract: The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumor growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1{sup -/-} cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation

  12. Paradigmenwechsel in der Anti-Aging-Medizin: Hormesis, Target-of-Rapamycin-Komplex und erste Anti-Aging-Pillen // Paradigm Shift in Anti-Aging Medicine: Hormesis, Target of Rapamycin Complex and First Human Anti-Aging-Pills

    Directory of Open Access Journals (Sweden)

    Römmler A

    2016-01-01

    Full Text Available Studies in model organisms have shown that some drugs and lifestyle practices (calorie-restricted diets, regular exercise, e.g. can extend life and health span and protect against the onset of age-related chronic diseases by targeting physiological pathways.brA common mode of action was found via mTOR (mechanistic Target Of Rapamycin pathway signalling. This intracellular protein kinase complex plays a key role in stimulating anabolic and cell growth promoting processes, while inhibiting autophagy. On the other hand, downregulation results in antiproliferative, anticancer and intensive cell-repairing effects leading to life and health span extension and stress resistance. The mTOR complex regulates such basic cell activities and integrates signals from nutrition sensing, energy metabolism, insulin and growth factors, stress and hypoxia.brImportantly, mTOR can be inhibited by some molecules and their analogs (rapamycin, resveratrol, metformin, e.g., which are released naturally from plants, yeast or bacteria to protect against natural enemies. Its dosage resembles an adaptive hormetic response relationship, as high concentrations are toxic and mild doses are associated with anticancer and antiaging effects. This opens up new avenues for their use as „anti-aging pills“ in humans.brRecent human data suggest that metformin, rapamycin and other mTOR-inhibitors could delay heart disease, cancer, cognitive decline and improve survival time in people with diabetes mellitus. In addition, response to influenca vaccine was enhanced by rapamycin in adults with immunosenescence, indicating beneficial anti-aging effects in the elderly.br“Treat aging” is an actual call to recognize aging as an indication appropriate for clinical trials and treatments, as it was recently approved by the Federal Drug Administration (FDA USA. p bKurzfassung/b: Die ansteigende Morbidität und Invalidität in alternden Industrienationen stößt an die Grenzen der Ressourcen

  13. On the radiochemical purity of elementary 35S with high specific activity

    International Nuclear Information System (INIS)

    Todorovsky, D.S.; Kostadinov, K.N.; Efremova, Yu.N.

    1979-01-01

    Radiochemical composition and chemical changes with increasing storage time of benzene solutions and of solid species of elementary 35 S with high specific activity are studied. The dependence of the stability on the specific activity and the radioactive concentration is shown and some tentative limits are given for permissible storage periods. (author)

  14. Eukaryotic initiation factor 2α--a downstream effector of mammalian target of rapamycin--modulates DNA repair and cancer response to treatment.

    Directory of Open Access Journals (Sweden)

    Liron Tuval-Kochen

    Full Text Available In an effort to circumvent resistance to rapamycin--an mTOR inhibitor--we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal--an inhibitor of eIF2α dephosphorylation--decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal--the phosphomimetic eIF2α variant--S51D--increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor--vorinostat. Finally, the catalytic competitive inhibitor of mTOR--Ku-0063794--increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for

  15. A situation-specific theory of Midlife Women's Attitudes Toward Physical Activity (MAPA).

    Science.gov (United States)

    Im, Eun-Ok; Stuifbergen, Alexa K; Walker, Lorraine

    2010-01-01

    This paper presents a situation specific theory-the Midlife Women's Attitudes Toward Physical Activity (MAPA) theory-that explains how women's attitudes toward physical activity influence their participation in physical activity. Using the integrative approach of Im, the theory was developed based on the Attitude, Social Influence, and Self Efficacy Model; a review of the related literature; and a study of women's attitudes toward physical activity. As a situation-specific theory, the MAPA theory can be linked easily to nursing practice and research projects related to physical activity in midlife women, especially interventions aimed at increasing midlife women's participation in physical activity. Copyright 2010 Mosby, Inc. All rights reserved.

  16. Preparation of high specific activity labelled triiodothyronine (T3) for radioimmunoassay

    International Nuclear Information System (INIS)

    Pillai, M.R.A.; Nagvekar, U.H.; Desai, C.N.; Mani, R.S.

    1981-01-01

    A method standardized for the preparation of high specific activity labelled triiodothyronine (T 3 ) is discussed. Iodine-125 labelled T 3 with a specific activity of 3 mCi μg was prepared by iodinating 3,5-diiodothyronine (T 2 ) and purifying it over Sephadex G-25 gel. Radochemical purity and stability evaluations were done by paper chromatography. Specific activity of the labelled T 3 prepared was estimated by the self-displacement method. The use of this high specific activity labelled T 3 in radioimmunoassay increased the sensitivity considerably. The advantage of this procedure is that the specific activity of labelled T 3 formed is independent of reaction yield and labelled T 3 yield. (author)

  17. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C

    2011-01-01

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types......, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active u......PA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems...

  18. Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy.

    Science.gov (United States)

    Foltz, Steven J; Luan, Junna; Call, Jarrod A; Patel, Ankit; Peissig, Kristen B; Fortunato, Marisa J; Beedle, Aaron M

    2016-01-01

    Secondary dystroglycanopathies are a subset of muscular dystrophy caused by abnormal glycosylation of α-dystroglycan (αDG). Loss of αDG functional glycosylation prevents it from binding to laminin and other extracellular matrix receptors, causing muscular dystrophy. Mutations in a number of genes, including FKTN (fukutin), disrupt αDG glycosylation. We analyzed conditional Fktn knockout (Fktn KO) muscle for levels of mTOR signaling pathway proteins by Western blot. Two cohorts of Myf5-cre/Fktn KO mice were treated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) for 4 weeks and evaluated for changes in functional and histopathological features. Muscle from 17- to 25-week-old fukutin-deficient mice has activated mTOR signaling. However, in tamoxifen-inducible Fktn KO mice, factors related to Akt/mTOR signaling were unchanged before the onset of dystrophic pathology, suggesting that Akt/mTOR signaling pathway abnormalities occur after the onset of disease pathology and are not causative in early dystroglycanopathy development. To determine any pharmacological benefit of targeting mTOR signaling, we administered RAPA daily for 4 weeks to Myf5/Fktn KO mice to inhibit mTORC1. RAPA treatment reduced fibrosis, inflammation, activity-induced damage, and central nucleation, and increased muscle fiber size in Myf5/Fktn KO mice compared to controls. RAPA-treated KO mice also produced significantly higher torque at the conclusion of dosing. These findings validate a misregulation of mTOR signaling in dystrophic dystroglycanopathy skeletal muscle and suggest that such signaling molecules may be relevant targets to delay and/or reduce disease burden in dystrophic patients.

  19. Synthesis of [diene-"1"4C] curcumin at high specific activity

    International Nuclear Information System (INIS)

    Filer, Crist N.; Lacy, James M.; Wright, Christopher

    2016-01-01

    An efficient method is described to label curcumin with "1"4C at high specific activity. - Highlights: • This paper describes the synthesis of ["1"4C] Curcumin at the highest specific activity and total activity amount yet reported. • The "1"4C label was installed in the diene framework of Curcumin. • This paper also describes the characterization of ["1"4C] Curcumin by HPLC and mass spectrometry.

  20. Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

    Science.gov (United States)

    Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

    2013-09-01

    Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

  1. Quantitation of fibroblast activation protein (FAP-specific protease activity in mouse, baboon and human fluids and organs

    Directory of Open Access Journals (Sweden)

    Fiona M. Keane

    2014-01-01

    Full Text Available The protease fibroblast activation protein (FAP is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

  2. Managing Complexity in Activity Specifications by Separation of Concerns and Reusability

    Directory of Open Access Journals (Sweden)

    Peter Forbrig

    2016-10-01

    Full Text Available The specification of activities of the different stakeholders is an important activity for software development. Currently, a lot of specification languages like task models, activity diagrams, state charts, and business specifications are used to document the results of the analysis of the domain in most projects. The paper discusses the aspect of reusability by considering generic submodels. This approach increases the quality of models. Additionally, the separation of concerns of cooperation and individual work by subject-oriented specifications is discussed. It will be demonstrated how task models can be used to support subject-oriented specification by so called team models and role models in a more precise way than S-BPM specifications. More precise restrictions on instances of roles can be specified.

  3. Rapamycin doses sufficient to extend lifespan do not compromise muscle mitochondrial content or endurance

    DEFF Research Database (Denmark)

    Widlund, Anne Lykkegaard; Vang, Ole; Ye, Lan

    2013-01-01

    mitochondrial transcripts were decreased, particularly in the highly oxidative soleus muscle, we found no consistent change in mitochondrial DNA or protein levels. In agreement with the lack of change in mitochondrial components, rapamycin-treated mice had endurance equivalent to that of untreated controls...

  4. Rapamycin blocks the antidepressant effect of ketamine in task-dependent manner

    Czech Academy of Sciences Publication Activity Database

    Holubová, Kristína; Kletečková, Lenka; Škurlová, Martina; Říčný, J.; Stuchlík, Aleš; Valeš, Karel

    2016-01-01

    Roč. 233, č. 11 (2016), s. 2077-2097 ISSN 0033-3158 R&D Projects: GA MZd(CZ) NT13403 Institutional support: RVO:67985823 Keywords : ketamine * rapamycin * antidepressants * anxiety * cognitive deficit * bulbectomy * mTOR * BDNF Subject RIV: FH - Neurology Impact factor: 3.308, year: 2016

  5. The Immunosuppressive drug – Rapamycin – Electroanalytical Sensing Using Boron- Doped Diamond electrode

    International Nuclear Information System (INIS)

    Stanković, Dalibor M.; Kalcher, Kurt

    2015-01-01

    Graphical abstract: Display Omitted -- Abstract: This paper presents for the first time the study of electrochemical behavior of well known immunosuppressant drug – rapamycin (sirolimus) using boron-doped diamond electrode. Rapamycin provided single and oval-shaped oxidation peak at +1.1 V vs. Ag/AgCl electrode in Britton–Robinson buffer solution at pH 3 confirming highly irreversible behavior of analyte at boron-doped diamond electrode. A differential pulse voltammetry was used for quantification of tested drug under the optimum experimental conditions. The calibration curve was linear over the range from 0.5 to 19.5 μM (R 2 = 0.9976) with detection limit of 0.22 μM. Repeatability of ten successfully measurements of three different concentrations (5, 10 and 15 μM) was 2.5, 1.9 and 1,7 %, respectively. Influence of most common biomolecules presented in urine samples was evaluated. The suggested analytical methodology was successfully applied for determination of rapamycin in four urine samples with excellent recoveries. The developed approach could be beneficial in analysis of rapamycin in biological samples using boron-doped diamond electrode as up-to-date electrochemical sensor and could represent inexpensive analytical alternative to separation methods

  6. Rapamycin potentiates cytotoxicity by docetaxel possibly through downregulation of Survivin in lung cancer cells

    Directory of Open Access Journals (Sweden)

    Li Hui

    2011-03-01

    Full Text Available Abstract Background To elucidate whether rapamycin, the inhibitor of mTOR (mammalian target of rapamycin, can potentiate the cytotoxic effect of docetaxel in lung cancer cells and to probe the mechanism underlying such enhancement. Methods Lung cancer cells were treated with docetaxel and rapamycin. The effect on the proliferation of lung cancer cells was evaluated using the MTT method, and cell apoptosis was measured by flow cytometry. Protein expression and level of phosphorylation were assayed using Western Blot method. Results Co-treatment of rapamycin and docetaxel was found to favorably enhance the cytotoxic effect of docetaxel in four lung cancer cell lines. This tumoricidal boost is associated with a reduction in the expression and phosphorylation levels of Survivin and ERK1/2, respectively. Conclusion The combined application of mTOR inhibitor and docetaxel led to a greater degree of cancer cell killing than that by either compound used alone. Therefore, this combination warrants further investigation in its suitability of serving as a novel therapeutic scheme for treating advanced and recurrent lung cancer patients.

  7. Prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in active surveillance patients.

    Science.gov (United States)

    Iremashvili, Viacheslav; Barney, Shane L; Manoharan, Murugesan; Kava, Bruce R; Parekh, Dipen J; Punnen, Sanoj

    2016-04-01

    To analyze the association between prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in prostate cancer patients on active surveillance, and to study the effect of prediagnostic prostate-specific antigen values on the predictive performance of prostate-specific antigen velocity and prostate-specific antigen doubling time. The study included 137 active surveillance patients with two or more prediagnostic prostate-specific antigen levels measured over a period of at least 3 months. Two sets of analyses were carried out. First, the association between prostate-specific antigen kinetics calculated using only the prediagnostic prostate-specific antigen values and the risk of biopsy progression was studied. Second, using the same cohort of patients, the predictive value of prostate-specific antigen kinetics calculated using only post-diagnostic prostate-specific antigens and compared with that of prostate-specific antigen kinetics based on both pre- and post-diagnostic prostate-specific antigen levels was analyzed. Of 137 patients included in the analysis, 37 (27%) had biopsy progression over a median follow-up period of 3.2 years. Prediagnostic prostate-specific antigen velocity of more than 2 ng/mL/year and 3 ng/mL/year was statistically significantly associated with the risk of future biopsy progression. However, after adjustment for baseline prostate-specific antigen density, these associations were no longer significant. None of the tested prostate-specific antigen kinetics based on combined pre- and post-diagnostic prostate-specific antigen values were statistically significantly associated with the risk of biopsy progression. Historical prediagnostic prostate-specific antigens seems to be not clinically useful in patients diagnosed with low-risk prostate cancer on active surveillance. © 2016 The Japanese Urological Association.

  8. Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue

    International Nuclear Information System (INIS)

    Ogawa, Y.; Imanaka, K.; Ashida, C.; Takashima, H.; Imajo, Y.; Kimura, S.

    1983-01-01

    Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue was studied on the transplanted MM46 tumor of female C3H/He mice after radiotherapy. MM46 tumor cells were inoculated into the right hind paws of mice. On the 5th day, irradiation with the dose irradiated tumor tissue (2000 rad on the fifth day), were injected into the left hind paws of the tumor-bearing mice. Effectiveness of this active specific immunotherapy against tumor was evaluated by the regression of tumor and survival rate of mice. Tumor was markedly regressed and survival rate was significantly increased by the active specific immunitherapy

  9. A suggested revision to the specific activity limit for tritiated water transported as LSA-II

    International Nuclear Information System (INIS)

    Nandakumar, A.N.

    2003-01-01

    Tritiated water of specific activity not greater than 0.8 TBq L -1 is classified as LSA-II. This paper demonstrates by some simple calculations that the dose that may result from an accident involving tritiated water of this specific activity is very low and suggests that even if the specific activity limit of tritiated water which may be transported as LSA-II is raised above 0.8 TBq kg -1 , the resulting dose in accident conditions would not be unacceptable. (author)

  10. Co-delivery of rapamycin- and piperine-loaded polymeric nanoparticles for breast cancer treatment.

    Science.gov (United States)

    Katiyar, Sameer S; Muntimadugu, Eameema; Rafeeqi, Towseef Amin; Domb, Abraham J; Khan, Wahid

    2016-09-01

    P-glycoprotein (P-gp) efflux is the major cause of multidrug resistance (MDR) in tumors when using anticancer drugs, moreover, poor bioavailability of few drugs is also due to P-gp efflux in the gut. Rapamycin (RPM) is in the clinical trials for breast cancer treatment, but its P-gp substrate property leads to poor oral bioavailability and efficacy. The objective of this study is to formulate and evaluate nanoparticles of RPM, along with a chemosensitizer (piperine, PIP) for improved oral bioavailability and efficacy. Poly(d,l-lactide-co-glycolide) (PLGA) was selected as polymer as it has moderate MDR reversal activity, which may provide additional benefits. The nanoprecipitation method was used to prepare PLGA nanoparticles with particle size below 150 nm, loaded with both drugs (RPM and PIP). Prepared nanoparticles showed sustained in vitro drug release for weeks, with initial release kinetics of zero order with non-Fickian transport, subsequently followed by Higuchi kinetics with Fickian diffusion. An everted gut sac method was used to study the effect of P-gp efflux on drug transport. This reveals that the uptake of the RPM (P-gp substrate) has been increased in the presence of chemosensitizer. Pharmacokinetic studies showed better absorption profile of RPM from polymeric nanoparticles compared to its suspension counterpart and improved bioavailability of 4.8-folds in combination with a chemosensitizer. An in vitro cell line study indicates higher efficacy of nanoparticles compared to free drug solution. Results suggest that the use of a combination of PIP with RPM nanoparticles would be a promising approach in the treatment of breast cancer.

  11. Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.

    Science.gov (United States)

    Bhagwat, Shripad V; Gokhale, Prafulla C; Crew, Andrew P; Cooke, Andy; Yao, Yan; Mantis, Christine; Kahler, Jennifer; Workman, Jennifer; Bittner, Mark; Dudkin, Lorina; Epstein, David M; Gibson, Neil W; Wild, Robert; Arnold, Lee D; Houghton, Peter J; Pachter, Jonathan A

    2011-08-01

    The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clinically validated target. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metabolism, proliferation, and survival. Rapamycin and its analogues partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clinical utility in certain cancers. Here, we report the preclinical characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochemical IC(50) values of 22 nmol/L and 65 nmol/L, respectively. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kβ, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analogue of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K-AKT signaling. OSI-027 shows concentration-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clinical trials in cancer patients. ©2011 AACR

  12. Perceived correlates of domain-specific physical activity in rural adults in the Midwest.

    Science.gov (United States)

    Chrisman, Matthew; Nothwehr, Faryle; Yang, Jingzen; Oleson, Jacob

    2014-01-01

    In response to calls for more specificity when measuring physical activity, this study examined perceived correlates of this behavior in rural adults separately by the domain in which this behavior occurs (ie, home care, work, active living, and sport). A cross-sectional survey was completed by 407 adults from 2 rural towns in the Midwest. The questionnaire assessed the perceived social and physical environment, including neighborhood characteristics, as well as barriers to being active. The Kaiser Physical Activity Survey captured domain-specific activity levels. The response rate was 25%. Multiple regression analyses were conducted to examine the associations between social and physical environment factors and domain-specific physical activity. Having a favorable attitude toward using government funds for exercise and activity-friendly neighborhood characteristic were positively associated with active living. Friends encouraging exercise was positively associated with participation in sport. Barriers were inversely associated with active living and sport. Total physical activity was positively associated with workplace incentives for exercise, favorable policy attitudes toward supporting physical education in schools and supporting the use of government funds for biking trails, and it was inversely associated with barriers. There were no factors associated with physical activity in the domains of work or home care. Correlates of physical activity are unique to the domain in which this behavior occurs. Programs to increase physical activity in rural adults should target policy attitudes, neighborhood characteristics, and social support from friends while also working to decrease personal barriers to exercise. © 2014 National Rural Health Association.

  13. Evaluation of specific activity in the primary circuit of SMART-P

    International Nuclear Information System (INIS)

    Kim, Ah Young; Choi, Byung Seon; Kim, Seong Hoon; Yoon, Ju Hyeon; Zee, Sung Qunn

    2005-01-01

    SMART-P is a soluble boron free reactor, and the ammonia is used as a pH reagent. The titanium alloy, which has a high corrosion resistance, is chosen as a steam generator tube material. Despite these design features to achieve the corrosion reduction, it is expected that SMART-P exhibits a relatively high specific activity in the coolant due to the lack of purification during the power operation. The main reason for the high specific activity is the activation and transportation of the corrosion products that released from the primary circuit surfaces. The objective of this work is to analyze the corrosion product activity in the primary circuit of SMART-P using a multi-region model, KORA. This model, which is incorporated with the mass and activity transport between the dissolved corrosion products in the coolant and the surface, describes the specific activity of corrosion products in coolant and on the surfaces according to the operation modes

  14. Experimental study of an active specific immunotherapy modified with irradiation, 2

    International Nuclear Information System (INIS)

    Imanaka, Kazufumi; Ogawa, Yasuhiro; Takashima, Hitoshi

    1982-01-01

    We had demonstrated in the former investigation that the strongest local infiltration of T-lymphocytes was observed in C3H/He mice transplanted MM46 at seven days after irradiation with the dose of 2,000 rads. This result indicated that the enhancement of the antigenicity of tumor cells was attained after low-dose-irradiation. We had also reported that specific active immunotherapy using low-dose-irradiated tumor cells and activated mononuclear cells after radiotherapy was effective on the elongation of survival period. In this paper, we studied whether the tumor cell inoculated after active specific immunotherapy was inhibited or not. Active specific immunotherapy using tumor cells and mononuclear cells was performed on female C3H/He mice aged 12 weeks in the left hind paws. Tumor cells and mononuclear cells were separated from the tumor tissue on the 12th day since inoculation of 5 x 10 6 of MM46 tumor cells which were irradiated with the dose of 2,000 rads of 3,000 rads by high energy electron beam on the fifth day. Seven days after active specific immunotherapy 1 x 10 5 or 1 x 10 6 of tumor cell were i noculated in the right hind paws of mice which received active specific immunotherapy. Anti-tumor effect was evaluated by the changes of tumor volume and survival rate. The tumor volume of the group which received active specific immunotherapy was smaller than that without active specific immunotherapy for about nine days since inoculation. Fifty-day survival rate was significantly higher in the group which received active specific immunotherapy compared with the group without immunotherapy (p < 0.01). (author)

  15. When a Step Is Not a Step! Specificity Analysis of Five Physical Activity Monitors.

    Directory of Open Access Journals (Sweden)

    Sandra O'Connell

    Full Text Available Physical activity is an essential aspect of a healthy lifestyle for both physical and mental health states. As step count is one of the most utilized measures for quantifying physical activity it is important that activity-monitoring devices be both sensitive and specific in recording actual steps taken and disregard non-stepping body movements. The objective of this study was to assess the specificity of five activity monitors during a variety of prescribed non-stepping activities.Participants wore five activity monitors simultaneously for a variety of prescribed activities including deskwork, taking an elevator, taking a bus journey, automobile driving, washing and drying dishes; functional reaching task; indoor cycling; outdoor cycling; and indoor rowing. Each task was carried out for either a specific duration of time or over a specific distance. Activity monitors tested were the ActivPAL micro™, NL-2000™ pedometer, Withings Smart Activity Monitor Tracker (Pulse O2™, Fitbit One™ and Jawbone UP™. Participants were video-recorded while carrying out the prescribed activities and the false positive step count registered on each activity monitor was obtained and compared to the video.All activity monitors registered a significant number of false positive steps per minute during one or more of the prescribed activities. The Withings™ activity performed best, registering a significant number of false positive steps per minute during the outdoor cycling activity only (P = 0.025. The Jawbone™ registered a significant number of false positive steps during the functional reaching task and while washing and drying dishes, which involved arm and hand movement (P < 0.01 for both. The ActivPAL™ registered a significant number of false positive steps during the cycling exercises (P < 0.001 for both.As a number of false positive steps were registered on the activity monitors during the non-stepping activities, the authors conclude that non

  16. Rapamycin enhances the anti-angiogenesis and anti-proliferation ability of YM155 in oral squamous cell carcinoma.

    Science.gov (United States)

    Li, Kong-Liang; Wang, Yu-Fan; Qin, Jia-Ruo; Wang, Feng; Yang, Yong-Tao; Zheng, Li-Wu; Li, Ming-Hua; Kong, Jie; Zhang, Wei; Yang, Hong-Yu

    2017-06-01

    YM155, a small molecule inhibitor of survivin, has been studied in many tumors. It has been shown that YM155 inhibited oral squamous cell carcinoma through promoting apoptosis and autophagy and inhibiting proliferation. It was found that YM155 also inhibited the oral squamous cell carcinoma-mediated angiogenesis through the inactivation of the mammalian target of rapamycin pathway. Rapamycin, a mammalian target of rapamycin inhibitor, played an important role in the proliferation and angiogenesis of oral squamous cell carcinoma cell lines. In our study, cell proliferation assay, transwell assay, tube formation assay, and western blot assay were used to investigate the synergistic effect of rapamycin on YM155 in oral squamous cell carcinoma. Either in vitro or in vivo, rapamycin and YM155 exerted a synergistic effect on the inhibition of survivin and vascular endothelial growth factor through mammalian target of rapamycin pathway. Overall, our results revealed that low-dose rapamycin strongly promoted the sensitivity of oral squamous cell carcinoma cell lines to YM155.

  17. When a Step Is Not a Step! Specificity Analysis of Five Physical Activity Monitors.

    Science.gov (United States)

    O'Connell, Sandra; ÓLaighin, Gearóid; Quinlan, Leo R

    2017-01-01

    Physical activity is an essential aspect of a healthy lifestyle for both physical and mental health states. As step count is one of the most utilized measures for quantifying physical activity it is important that activity-monitoring devices be both sensitive and specific in recording actual steps taken and disregard non-stepping body movements. The objective of this study was to assess the specificity of five activity monitors during a variety of prescribed non-stepping activities. Participants wore five activity monitors simultaneously for a variety of prescribed activities including deskwork, taking an elevator, taking a bus journey, automobile driving, washing and drying dishes; functional reaching task; indoor cycling; outdoor cycling; and indoor rowing. Each task was carried out for either a specific duration of time or over a specific distance. Activity monitors tested were the ActivPAL micro™, NL-2000™ pedometer, Withings Smart Activity Monitor Tracker (Pulse O2)™, Fitbit One™ and Jawbone UP™. Participants were video-recorded while carrying out the prescribed activities and the false positive step count registered on each activity monitor was obtained and compared to the video. All activity monitors registered a significant number of false positive steps per minute during one or more of the prescribed activities. The Withings™ activity performed best, registering a significant number of false positive steps per minute during the outdoor cycling activity only (P = 0.025). The Jawbone™ registered a significant number of false positive steps during the functional reaching task and while washing and drying dishes, which involved arm and hand movement (P positive steps during the cycling exercises (P positive steps were registered on the activity monitors during the non-stepping activities, the authors conclude that non-stepping physical activities can result in the false detection of steps. This can negatively affect the quantification of physical

  18. Activity and specificity of TRV-mediated gene editing in plants

    KAUST Repository

    Ali, Zahir; Abulfaraj, Aala A.; Piatek, Marek J.; Mahfouz, Magdy M.

    2015-01-01

    editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system

  19. A specific metabolic pattern related to the hallucinatory activity in schizophrenia

    International Nuclear Information System (INIS)

    Huret, J.D.; Martinot, J.L.; Lesur, A.; Mazoyer, B.; Pappata, S.; Syrota, A.; Baron, J.C.; Lemperiere, T.

    1988-01-01

    A clinical and PEI study using 18 F - fluorodesoxyglucose for measuring local cerebral glucose metabolism with the aim of showing a specific pattern related to the hallucinatory activity, is presented in schizophrenic patients all experiencing hallucinations or pseudo-halluccinations

  20. StrigoQuant: A genetically encoded biosensor for quantifying strigolactone activity and specificity

    KAUST Repository

    Samodelov, S. L.; Beyer, H. M.; Guo, X.; Augustin, M.; Jia, K.-P.; Baz, Lina Abdulkareem Ali; Ebenho  h, O.; Beyer, P.; Weber, W.; Al-Babili, Salim; Zurbriggen, M. D.

    2016-01-01

    into the stereoselectivity of strigolactone perception. Given the high specificity, sensitivity, dynamic range of activity, modular construction, ease of implementation, and wide applicability, the biosensor StrigoQuant will be useful in unraveling multiple levels

  1. Synthesis of glycolic acid-1-14C of high specific activity

    International Nuclear Information System (INIS)

    Ramamurthy, T.V.; Viswanathan, K.V.

    1987-01-01

    A simple procedure is described which efficiently converts traces of 14 C labelled cyanide present as a dilute solution into glycolic acid-1- 14 C with more than 85% radiochemical recovery and of high specific activity. (author)

  2. A non-specific biomarker of disease activity in HIV/AIDS patients ...

    African Journals Online (AJOL)

    Background: A general non-specific marker of disease activity that could alert the clinician and prompt further investiga- tion would be of ... laration of Helsinki, the National Health Act and the ..... CD8+ lymphocytes and neopterin are related to.

  3. Preparation of 80-82BrNa of high specific activity in the Chatillon pile

    International Nuclear Information System (INIS)

    Fisher, C.; Herczeg, C.; Laurent, H.

    1951-10-01

    The Szilard-Chalmers reaction was used to concentrate the 80,82 Br produced by pile irradiation of an organic bromide, solvent partition being carried out between benzene and H 2 S-saturated H-2O. Specific activities produced by various lengths of irradiation of bromoform, ethyl bromide, and ethylene bromide are tabulated. Bromoform is recommended for production since it gives the highest specific activity. The enrichment factor decreases with extended irradiation time

  4. A system of radiation monitoring, and methods and equipment for measuring water of low specific activity

    International Nuclear Information System (INIS)

    Sivintsev, Yu.V.

    1975-01-01

    The author considers criteria for the radiation protection of the population and the environment. He describes the role of procedures for monitoring waters of low specific activity in the framework of a system for ensuring the radiation safety of the population living near a nuclear power station. The main technical characteristics (background, efficiency, sensitivity) of the laboratory equipment for gamma spectrometric analysis of water samples of low specific activity are discussed. (author)

  5. Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation.

    Science.gov (United States)

    Takahashi, Hironobu; Wang, Yuwei; Grainger, David W

    2010-11-01

    Fibrous encapsulation of surgically implanted devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering RNA (siRNA) conjugated with branched polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knock-down led to an inhibition in fibroblast proliferation by 70% and simultaneous down-regulation in the expression of type I collagen in fibroblasts in vitro. These siRNA/bPEI complexes were released from poly(ethylene glycol) (PEG)-based hydrogel coatings surrounding model polymer implants in a subcutaneous rodent model in vivo. No significant reduction in fibrous capsule thickness and mTOR expression in the foreign body capsules were observed. The siRNA inefficacy in this in vivo implant model was attributed to siRNA dosing limitations in the gel delivery system, and lack of targeting ability of the siRNA complex specifically to fibroblasts. While in vitro data supported mTOR knock-down in fibroblast cultures, in vivo siRNA delivery must be further improved to produce clinically relevant effects on fibrotic encapsulation around implants. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells

    NARCIS (Netherlands)

    Avalos, Ana M.; Bilate, Angelina M.; Witte, Martin D.; Tai, Albert K.; He, Jiang; Frushicheva, Maria P.; Thill, Peter D.; Meyer-Wentrup, Friederike; Theile, Christopher S.; Chakraborty, Arup K.; Zhuang, Xiaowei; Ploegh, Hidde L.

    2014-01-01

    Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR.

  7. Ambulatory activity monitoring: Progress in measurement of activity, posture, and specific motion patterns in daily life

    NARCIS (Netherlands)

    J.B.J. Bussmann (Hans); U.W. Ebner-Priemer (Ulrich); J. Fahrenberg (Jochen)

    2009-01-01

    textabstractBehavior is central to psychology in almost any definition. Although observable activity is a core aspect of behavior, assessment strategies have tended to focus on emotional, cognitive, or physiological responses. When physical activity is assessed, it is done so mostly with

  8. Reported frequency of physical activity in a large epidemiological study: relationship to specific activities and repeatability over time

    Directory of Open Access Journals (Sweden)

    Reeves Gillian K

    2011-06-01

    Full Text Available Abstract Background How overall physical activity relates to specific activities and how reported activity changes over time may influence interpretation of observed associations between physical activity and health. We examine the relationships between various physical activities self-reported at different times in a large cohort study of middle-aged UK women. Methods At recruitment, Million Women Study participants completed a baseline questionnaire including questions on frequency of strenuous and of any physical activity. About 3 years later 589,896 women also completed a follow-up questionnaire reporting the hours they spent on a range of specific activities. Time spent on each activity was used to estimate the associated excess metabolic equivalent hours (MET-hours and this value was compared across categories of physical activity reported at recruitment. Additionally, 18,655 women completed the baseline questionnaire twice, at intervals of up to 4 years; repeatability over time was assessed using the weighted kappa coefficient (κweighted and absolute percentage agreement. Results The average number of hours per week women reported doing specific activities was 14.0 for housework, 4.5 for walking, 3.0 for gardening, 0.2 for cycling, and 1.4 for all strenuous activity. Time spent and the estimated excess MET-hours associated with each activity increased with increasing frequency of any or strenuous physical activity reported at baseline (tests for trend, P weighted = 0.71 for questionnaires administered less than 6 months apart, and 52% (κweighted = 0.51 for questionnaires more than 2 years apart. Corresponding values for any physical activity were 57% (κweighted = 0.67 and 47% (κweighted = 0.58. Conclusions In this cohort, responses to simple questions on the frequency of any physical activity and of strenuous activity asked at baseline were associated with hours spent on specific activities and the associated estimated excess MET

  9. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.

    2007-01-01

    To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Several FDA-approved nucleoside analogue drugs were screened for their potential bacteric......-specific manner. Therefore, nucleoside analogues have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria....

  10. Life review based on remembering specific positive events in active aging.

    Science.gov (United States)

    Latorre, José M; Serrano, Juan P; Ricarte, Jorge; Bonete, Beatriz; Ros, Laura; Sitges, Esther

    2015-02-01

    The aim of this study is to evaluate the effectiveness of life review (LR) based on specific positive events in non-depressed older adults taking part in an active aging program. Fifty-five older adults were randomly assigned to an experimental group or an active control (AC) group. A six-session individual training of LR based on specific positive events was carried out with the experimental group. The AC group undertook a "media workshop" of six sessions focused on learning journalistic techniques. Pre-test and post-test measures included life satisfaction, depressive symptoms, experiencing the environment as rewarding, and autobiographical memory (AM) scales. LR intervention decreased depressive symptomatology, improved life satisfaction, and increased specific memories. The findings suggest that practice in AM for specific events is an effective component of LR that could be a useful tool in enhancing emotional well-being in active aging programs, thus reducing depressive symptoms. © The Author(s) 2014.

  11. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity

    DEFF Research Database (Denmark)

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T

    2014-01-01

    sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable......Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms....... Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal...

  12. Benchmark studies of induced radioactivity produced in LHC materials, Part I: Specific activities.

    Science.gov (United States)

    Brugger, M; Khater, H; Mayer, S; Prinz, A; Roesler, S; Ulrici, L; Vincke, H

    2005-01-01

    Samples of materials which will be used in the LHC machine for shielding and construction components were irradiated in the stray radiation field of the CERN-EU high-energy reference field facility. After irradiation, the specific activities induced in the various samples were analysed with a high-precision gamma spectrometer at various cooling times, allowing identification of isotopes with a wide range of half-lives. Furthermore, the irradiation experiment was simulated in detail with the FLUKA Monte Carlo code. A comparison of measured and calculated specific activities shows good agreement, supporting the use of FLUKA for estimating the level of induced activity in the LHC.

  13. Benchmark studies of induced radioactivity produced in LHC materials, part I: Specific activities

    International Nuclear Information System (INIS)

    Brugger, M.; Khater, H.; Mayer, S.; Prinz, A.; Roesler, S.; Ulrici, L.; Vincke, H.

    2005-01-01

    Samples of materials which will be used in the LHC machine for shielding and construction components were irradiated in the stray radiation field of the CERN-EU high-energy reference field facility. After irradiation, the specific activities induced in the various samples were analysed with a high-precision gamma spectrometer at various cooling times, allowing identification of isotopes with a wide range of half-lives. Furthermore, the irradiation experiment was simulated in detail with the FLUKA Monte Carlo code. A comparison of measured and calculated specific activities shows good agreement, supporting the use of FLUKA for estimating the level of induced activity in the LHC. (authors)

  14. Tracer technique for measuring specific activity of 63 Ni, using 4πβ-γ

    International Nuclear Information System (INIS)

    Iwahara, A.

    1979-01-01

    The specific activity of a 6 3 Ni solution has been measured by an efficiency tracer technique using a 4 π β - γ coincidence system. 6 3 Ni was chosen. Because it's a very low energy pure beta emitter. Due to chemical compatibility and beta spectral shapes, 6 0 Co has been chosen as tracer. In the determination of 6 3 Ni, the specific activity. As the efficiency tracer techniques requires a previous knowledge of tracer activity, this has been measured by a conventional 4 π β -γ coincidence method. (author)

  15. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies.

    Science.gov (United States)

    Botkjaer, Kenneth A; Fogh, Sarah; Bekes, Erin C; Chen, Zhuo; Blouse, Grant E; Jensen, Janni M; Mortensen, Kim K; Huang, Mingdong; Deryugina, Elena; Quigley, James P; Declerck, Paul J; Andreasen, Peter A

    2011-08-15

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation. © The Authors Journal compilation © 2011 Biochemical Society

  16. Recurrent activity in higher order, modality non-specific brain regions

    DEFF Research Database (Denmark)

    Lou, Hans Olav Christensen; Joensson, Morten; Biermann-Ruben, Katja

    2011-01-01

    It has been proposed that the workings of the brain are mainly intrinsically generated recurrent neuronal activity, with sensory inputs as modifiers of such activity in both sensory and higher order modality non-specific regions. This is supported by the demonstration of recurrent neuronal activity...... in the visual system as a response to visual stimulation. In contrast recurrent activity has never been demonstrated before in higher order modality non-specific regions. Using magneto-encephalography and Granger causality analysis, we tested in a paralimbic network the hypothesis that stimulation may enhance...... causal recurrent interaction between higher-order, modality non-specific regions. The network includes anterior cingulate/medial prefrontal and posterior cingulate/medial parietal cortices together with pulvinar thalami, a network known to be effective in autobiographic memory retrieval and self...

  17. Differential responses of human regulatory T cells (Treg and effector T cells to rapamycin.

    Directory of Open Access Journals (Sweden)

    Laura Strauss

    Full Text Available BACKGROUND: The immunosuppressive drug rapamycin (RAPA promotes the expansion of CD4(+ CD25(highFoxp3(+ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-gamma chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. METHODOLOGY/PRINCIPAL FINDINGS: CD4(+CD25(+ and CD4(+CD25(neg T cells were isolated from PBMC of normal controls (n = 21 using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1-100 nM was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4(+CD25(high and CD4(+CD25(neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4(+CD25(neg or CD8(+CD25(neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4(+CD25(+ T cells in the presence of 1-100 nM RAPA (p<0.001. RAPA-expanded Treg were largely CD4(+CD25(highFoxp3(+ cells and were resistant to apoptosis, while CD4(+CD25(neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4(+CD25(neg cells. Activated Treg+/-RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway. CONCLUSIONS/SIGNIFICANCE: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.

  18. Extracurricular activities and the development of social skills in children with intellectual and specific learning disabilities.

    Science.gov (United States)

    Brooks, B A; Floyd, F; Robins, D L; Chan, W Y

    2015-07-01

    Children with intellectual disability and specific learning disabilities often lack age-appropriate social skills, which disrupts their social functioning. Because of the limited effectiveness of classroom mainstreaming and social skills training for these children, it is important to explore alternative opportunities for social skill acquisition. Participation in social activities is positively related to children's social adjustment, but little is known about the benefits of activity participation for children with intellectual and specific learning disabilities. This study investigated the association between frequency and type of social activity participation and the social competence of 8-11-year-old children with intellectual disability (n = 40) and specific learning disabilities (n = 53), in comparison with typically developing peers (n = 24). More time involved in unstructured activities, but not structured activities, was associated with higher levels of social competence for all children. This association was strongest for children with intellectual disability, suggesting that participation in unstructured social activities was most beneficial for these children. Future research on the quality of involvement is necessary to further understand specific aspects of unstructured activities that might facilitate social development. © 2014 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.

  19. Rapamycin-ameliorated diabetic symptoms involved in increasing adiponectin expression in diabetic mice on a high-fat diet.

    Science.gov (United States)

    Gong, Fang-Hua; Ye, Yan-Na; Li, Jin-Meng; Zhao, Hai-Yang; Li, Xiao-Kun

    2017-07-01

    Recent studies showed that rapamycin improved diabetic complications. Here, we investigated the metabolic effects of rapamycin in type 2 diabetes model (T2DM) mice. Mice were treated with a daily intraperitoneal injection of rapamycin at 2 mg/kg or vehicle only for 3 weeks and were maintained on a high-fat diet. The treated diabetic mice exhibited decreased body weight, blood glucose levels, and fat mass. FGF21 expression was suppressed in C57B/L6 mice, but adiponectin expression increased both in FGF21 KO and C57B/L6 mice. These results suggest that rapamycin may alleviate FGF21 resistance in mice on a high-fat diet. The reduction of adipose tissue mass of the diabetic mice may be due to the increased adiponectin. Copyright © 2017. Published by Elsevier Taiwan.

  20. Substrate specificity, regioselectivity and hydrolytic activity of lipases activated from Geotrichum sp

    Czech Academy of Sciences Publication Activity Database

    Stránský, Karel; Zarevúcka, Marie; Kejík, Z.; Wimmer, Zdeněk; Macková, M.; Demnerová, K.

    2007-01-01

    Roč. 34, č. 3 (2007), s. 209-216 ISSN 1369-703X R&D Projects: GA ČR GA203/04/0120 Institutional research plan: CEZ:AV0Z40550506 Keywords : biocatalysis * enzyme activity * gas chromatography * lipase * blackcurrant oil Subject RIV: CC - Organic Chemistry Impact factor: 1.872, year: 2007

  1. Chemical synthesis of high specific-activity [35S]adenosylhomocysteine

    International Nuclear Information System (INIS)

    Stern, P.H.; Hoffman, R.M.

    1986-01-01

    The study of the family of transmethylases, critical to normal cellular function and often altered in cancer, can be facilitated by the availability of a high specific-activity S-adenosylhomocysteine. The authors report the two-step preparation of [ 35 S]adenosylhomocysteine from [ 35 S]methionine at a specific activity of 1420 Ci/mmol in an overall yield of 24% by a procedure involving demethylation of the [ 35 S]methionine to [ 35 S]homocysteine followed by condensation with 5'-chloro-5'-deoxyadenosine. The ease of the reactions, ready availability and low cost of the reagents and high specific-activity and stability of the product make the procedure an attractive one with many uses, and superior to current methodology

  2. Peroxisome proliferator activated receptor alpha regulates a male-specific cytochrome P450 in mouse liver.

    Science.gov (United States)

    Jeffery, Brett; Choudhury, Agharul I; Horley, Neill; Bruce, Mary; Tomlinson, Simon R; Roberts, Ruth A; Gray, Tim J B; Barrett, David A; Shaw, P Nicholas; Kendall, David; Bell, David R

    2004-09-15

    We set out to find if the strain-specific, male-specific hepatic expression of Cyp4a protein in mouse was due to expression of Cyp4a12 and to understand the genetic basis for reported differences in expression. 12-Lauric acid hydroxylase (LAH) activity was found to show higher levels in male ddY, but not C57Bl/6, mouse liver microsomes. The expression of Cyp4a12 mRNA was studied using RNAase protection assays in male and female liver and kidney of nine mouse strains. Cyp4a12 was found to be highly expressed in male liver and kidney, but at much lower levels in female liver and kidney, in all strains studied. Western blotting with an antibody specific for Cyp4a12 confirmed that Cyp4a12 was expressed in a male specific fashion in C57Bl/6 mouse liver. RNAase protection analysis for Cyp4a10 and 14 in ddY mice revealed that neither of these genes showed male-specific expression. To further investigate genetic factors that control male-specific Cyp4a12 expression, PPARalpha+/+ and -/- mice were studied, showing that total P450 and 12-LAH activity was male-specific in +/+, but not -/- mice. RNAase protection assays were used to confirm that Cyp4a12 was lower in -/- mice. However, the male-specific Slp and MUP-1 genes retained hepatic male-specific levels of expression in +/+ and -/- mice, showing that the decrease in Cyp4a12 was not a general effect on male-specific expression. Thus, PPARalpha has a specific effect on constitutive expression of Cyp4a12.

  3. Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

    Directory of Open Access Journals (Sweden)

    Alexey Navdaev

    Full Text Available von Willebrand factor/ristocetin (vWF/R induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.

  4. Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

    Science.gov (United States)

    Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

    2014-01-01

    von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

  5. Sensory modality specificity of neural activity related to memory in visual cortex.

    Science.gov (United States)

    Gibson, J R; Maunsell, J H

    1997-09-01

    Previous studies have shown that when monkeys perform a delayed match-to-sample (DMS) task, some neurons in inferotemporal visual cortex are activated selectively during the delay period when the animal must remember particular visual stimuli. This selective delay activity may be involved in short-term memory. It does not depend on visual stimulation: both auditory and tactile stimuli can trigger selective delay activity in inferotemporal cortex when animals expect to respond to visual stimuli in a DMS task. We have examined the overall modality specificity of delay period activity using a variety of auditory/visual cross-modal and unimodal DMS tasks. The cross-modal DMS tasks involved making specific long-term memory associations between visual and auditory stimuli, whereas the unimodal DMS tasks were standard identity matching tasks. Delay activity existed in auditory/visual cross-modal DMS tasks whether the animal anticipated responding to visual or auditory stimuli. No evidence of selective delay period activation was seen in a purely auditory DMS task. Delay-selective cells were relatively common in one animal where they constituted up to 53% neurons tested with a given task. This was only the case for up to 9% of cells in a second animal. In the first animal, a specific long-term memory representation for learned cross-modal associations was observed in delay activity, indicating that this type of representation need not be purely visual. Furthermore, in this same animal, delay activity in one cross-modal task, an auditory-to-visual task, predicted correct and incorrect responses. These results suggest that neurons in inferotemporal cortex contribute to abstract memory representations that can be activated by input from other sensory modalities, but these representations are specific to visual behaviors.

  6. Virus-specific cytotoxic T cells in chronic active Epstein-Barr virus infection.

    Science.gov (United States)

    Shibayama, Haruna; Imadome, Ken-Ichi; Onozawa, Erika; Tsuzura, Akiho; Miura, Osamu; Koyama, Takatoshi; Arai, Ayako

    2017-01-01

    Chronic active Epstein-Barr virus infection (CAEBV) is a disease characterized by clonally proliferating and activated EBV-infected T or NK cells accompanied by chronic inflammation and T- or NK-cell neoplasms. However, the mechanism for developing CAEBV has not been clarified to date. Because the decreased number or inactivation of EBV-specific cytotoxic T lymphocytes (CTLs) resulted in the development of EBV-positive B-cell neoplasms, we investigated the number of CTLs in CAEBV patients using the tetrameric complexes of HLA-restricted EBV-specific peptides. Among the seven patients examined, EBV-specific CTLs were detected in the peripheral blood mononuclear cells (PBMCs) of four cases but were not detected in three cases. The ratio of EBV-specific CTLs in PBMCs tended to be higher in the patients with active disease than in those with inactive disease. In two patients in whom EBV-specific CTLs had not been detected, CTLs appeared after the eradication of EBV-infected T cells by allogeneic bone marrow transplantation. These results suggested that the failure of CTLs had a role in developing CAEBV, although the induction number and function of EBV-specific CTLs might vary in each patient.

  7. Comparison of four specific dynamic office chairs with a conventional office chair: impact upon muscle activation, physical activity and posture.

    Science.gov (United States)

    Ellegast, Rolf P; Kraft, Kathrin; Groenesteijn, Liesbeth; Krause, Frank; Berger, Helmut; Vink, Peter

    2012-03-01

    Prolonged and static sitting postures provoke physical inactivity at VDU workplaces and are therefore discussed as risk factors for the musculoskeletal system. Manufacturers have designed specific dynamic office chairs featuring structural elements which promote dynamic sitting and therefore physical activity. The aim of the present study was to evaluate the effects of four specific dynamic chairs on erector spinae and trapezius EMG, postures/joint angles and physical activity intensity (PAI) compared to those of a conventional standard office chair. All chairs were fitted with sensors for measurement of the chair parameters (backrest inclination, forward and sideward seat pan inclination), and tested in the laboratory by 10 subjects performing 7 standardized office tasks and by another 12 subjects in the field during their normal office work. Muscle activation revealed no significant differences between the specific dynamic chairs and the reference chair. Analysis of postures/joint angles and PAI revealed only a few differences between the chairs, whereas the tasks performed strongly affected the measured muscle activation, postures and kinematics. The characteristic dynamic elements of each specific chair yielded significant differences in the measured chair parameters, but these characteristics did not appear to affect the sitting dynamics of the subjects performing their office tasks. Copyright © 2011 Elsevier Ltd and The Ergonomics Society. All rights reserved.

  8. Development of acceptance specifications for low-activity waste from the Hanford tanks

    International Nuclear Information System (INIS)

    Cunnane, J.C.; Kier, P.H.; Brown, N.R.

    1997-01-01

    Low-activity products will be in the form of soldified waste and optional matrix and filler materials enclosed in sealed metal boxes. Acceptance specifications limit the physical characteristics of the containers, the chemical and physical characteristics of the waste form and other materials that may be in the container, the waste loading, and the radionuclide leaching characteristics of the waste form. The specifications are designed to ensure that low-activity waste products will be compatible with the driving regulatory and operational requirements and with existing production technologies

  9. A comparison between activities for non-specific esterases and esterproteases

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D

    1988-01-01

    Electrophoretic separation of non-specific esterases and esterproteases from kidney, lung, and liver have been carried out in polyacrylamide gels. By use of zone electrophoresis, isoelectric focusing, and 2-dimensional electrophoresis it was found that most of the esterprotease bands had the same...... localization in the gels as non-specific esterase bands. A number of esterase bands showed no activity towards the esterprotease substrates and a single kidney band possessed esterprotease activity only. Isozymes of the ES-6 and ES-9 zones showed sex dependent esterprotease reactions. In sections esterase...

  10. Effect of plant species on the specific activity of 65Zn and 54Mn

    International Nuclear Information System (INIS)

    Muraoka, T.; Neptune, A.M.L.

    1983-01-01

    The effect of five plant species on the specific activity of 65 Zn and 54 Mn is studied. Soybean (Glycine max (L.) Merril), bean (Phaseolus vulgaris, L.), rice (Oryza sativa, L.), wheat (Triticum aestivum, L.) and tomato (Lycopersium esculentum Mill) were grown in PV and TE soils labelled with 65 Zn and 54 Mn. The plants were harvested 30 days after seeding and specific activities of zinco and manganese were determined in the above ground part and in the roots. (M.A.C.) [pt

  11. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

    Science.gov (United States)

    Andrusiak, Matthew G.; Jin, Yishi

    2016-01-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

  12. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    Science.gov (United States)

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-08

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1-Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism.

    Science.gov (United States)

    Volovelsky, Oded; Cohen, Gili; Kenig, Ariel; Wasserman, Gilad; Dreazen, Avigail; Meyuhas, Oded; Silver, Justin; Naveh-Many, Tally

    2016-04-01

    Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6(p-/-) mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid-induced AKI. Uremic rpS6(p-/-) mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild-type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6. Copyright © 2016 by the American Society of Nephrology.

  14. Mycobacteria-specific cytokine responses as correlates of treatment response in active and latent tuberculosis.

    Science.gov (United States)

    Clifford, Vanessa; Tebruegge, Marc; Zufferey, Christel; Germano, Susie; Forbes, Ben; Cosentino, Lucy; McBryde, Emma; Eisen, Damon; Robins-Browne, Roy; Street, Alan; Denholm, Justin; Curtis, Nigel

    2017-08-01

    A biomarker indicating successful tuberculosis (TB) therapy would assist in determining appropriate length of treatment. This study aimed to determine changes in mycobacteria-specific antigen-induced cytokine biomarkers in patients receiving therapy for latent or active TB, to identify biomarkers potentially correlating with treatment success. A total of 33 adults with active TB and 36 with latent TB were followed longitudinally over therapy. Whole blood stimulation assays using mycobacteria-specific antigens (CFP-10, ESAT-6, PPD) were done on samples obtained at 0, 1, 3, 6 and 9 months. Cytokine responses (IFN-γ, IL-1ra, IL-2, IL-10, IL-13, IP-10, MIP-1β, and TNF-α) in supernatants were measured by Luminex xMAP immunoassay. In active TB cases, median IL-1ra (with CFP-10 and with PPD stimulation), IP-10 (CFP-10, ESAT-6), MIP-1β (ESAT-6, PPD), and TNF-α (ESAT-6) responses declined significantly over the course of therapy. In latent TB cases, median IL-1ra (CFP-10, ESAT-6, PPD), IL-2 (CFP-10, ESAT-6), and IP-10 (CFP-10, ESAT-6) responses declined significantly. Mycobacteria-specific cytokine responses change significantly over the course of therapy, and their kinetics in active TB differ from those observed in latent TB. In particular, mycobacteria-specific IL-1ra responses are potential correlates of successful therapy in both active and latent TB. Copyright © 2017 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  15. Proteins labelling with 125I and experimental determination of their specific activity

    International Nuclear Information System (INIS)

    Caro, R.A.; Ciscato, V.A.; Giacomini, S.M.V. de; Quiroga, S.; Radicella, R.

    1975-11-01

    A standardization of the labelling technique of proteins with 125 I and the control of the obtained products, principally their specific activities was performed, in order to utilize them correctly in radioimmunoassays. The quantities of chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3.6 and 9.6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. For these reasons the yields and the corresponding specific activities were determined from ascending chromatographies performed with 70 per cent methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labelled which the proposed method and the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 +- 4. (author) [es

  16. Comprehensive analysis of the specificity of transcription activator-like effector nucleases

    DEFF Research Database (Denmark)

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien

    2014-01-01

    A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within...... their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator......-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only...

  17. Enzymatic Activity of Free-Prostate-Specific Antigen (f-PSA) Is Not Required for Some of its Physiological Activities

    Science.gov (United States)

    Chadha, Kailash C.; Nair, Bindukumar B.; Chakravarthi, Srikant; Zhou, Rita; Godoy, Alejandro; Mohler, James L.; Aalinkeel, Ravikumar; Schwartz, Stanley A.; Smith, Gary J.

    2015-01-01

    BACKGROUND Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+. Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity PMID:21446007

  18. Production of high specific activity 123I for protein iodination for medical use

    International Nuclear Information System (INIS)

    Legoux, Y.; Cieur, M.; Crouzel, C.; Syrota, A.

    1985-01-01

    Iodine-123 is produced via xenon-133 by irradiation of a sodium iodide target with 108 MeV deuterons from the synchrocyclotron of IPN. The on-line production method is described. The specific activity of the iodine is determined by neutron activation analysis and by a radioimmunological method. The conditions labelling different proteins (insulin, angiotensin) are given and also the purification method to obtain a product ready for injection to patients. (author)

  19. Effects of oxytetracycline, tylosin, and amoxicillin antibiotics on specific methanogenic activity of anaerobic biomass

    OpenAIRE

    Mohammad Mehdi Amin; Hassan Hashemi; Afshin Ebrahimi; Asghar Ebrahimi

    2012-01-01

    Aims: The purpose of this study was to survey the antibiotics effects of oxytetracycline, tylosin, and amoxicillin on anerobic wastewater treatment process. Materials and Methods: To evaluate the inhibitory antibiotics amoxicillin, tetracycline, and tylosin on biomass activity, specific methanogenic activity (SMA) using anerobic biomass batch; into 120 ml vials: 30 ml biomass and 70 ml substrate including volatile fatty acids, mainly acetic acid and various concentrations of antibiotics we...

  20. Comparing Domain-Specific Physical Activity Efficacy Level between Turkish Adolescent Girls and Boys

    Science.gov (United States)

    Çatikkas, Fatih

    2017-01-01

    The adolescence period is a very critical developmental period for personality, socializing and promotion of physical activity. In this regard, the aim of this study was to compare domain-specific physical activity efficacy level between adolescent boys and girls. A total of 219 girls (body weight: 57.50 ± 10.44 kg, height: 160.30 ± 7.40 cm, age…

  1. Production of high specific activity /sup 123/I for protein iodination for medical use

    Energy Technology Data Exchange (ETDEWEB)

    Legoux, Y; Cieur, M [Paris-11 Univ., 91 - Orsay (France). Inst. de Physique Nucleaire; Goutheraud, R; Drouet, J [Centre National de Transfusion Sanguine, 75 - Paris (France); Crouzel, C; Syrota, A [CEA, 91 - Orsay (France). Service Hospitalier Frederic Joliot

    1985-01-01

    Iodine-123 is produced via xenon-133 by irradiation of a sodium iodide target with 108 MeV deuterons from the synchrocyclotron of IPN. The on-line production method is described. The specific activity of the iodine is determined by neutron activation analysis and by a radioimmunological method. The conditions labelling different proteins (insulin, angiotensin) are given and also the purification method to obtain a product ready for injection to patients.

  2. Mechanistic Target of Rapamycin Is a Novel Molecular Mechanism Linking Folate Availability and Cell Function.

    Science.gov (United States)

    Silva, Elena; Rosario, Fredrick J; Powell, Theresa L; Jansson, Thomas

    2017-07-01

    Folate deficiency has been linked to a wide range of disorders, including cancer, neural tube defects, and fetal growth restriction. Folate regulates cellular function mediated by its involvement in the synthesis of nucleotides, which are needed for DNA synthesis, and its function as a methyl donor, which is critical for DNA methylation. Here we review current data showing that folate sensing by mechanistic target of rapamycin (mTOR) constitutes a novel and distinct pathway by which folate modulates cell functions such as nutrient transport, protein synthesis, and mitochondrial respiration. The mTOR signaling pathway responds to growth factors and changes in nutrient availability to control cell growth, proliferation, and metabolism. mTOR exists in 2 complexes, mTOR complex (mTORC) 1 and mTORC2, which have distinct upstream regulators and downstream targets. Folate deficiency in pregnant mice caused a marked inhibition of mTORC1 and mTORC2 signaling in multiple maternal and fetal tissues, downregulation of placental amino acid transporters, and fetal growth restriction. In addition, folate deficiency in primary human trophoblast (PHT) cells resulted in inhibition of mTORC1 and mTORC2 signaling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells is independent of the accumulation of homocysteine and requires the proton-coupled folate transporter (PCFT; solute carrier 46A1). Furthermore, mTORC1 and mTORC2 regulate trophoblast folate uptake by modulating the cell surface expression of folate receptor α and the reduced folate carrier. These findings, which provide a novel link between folate availability and cell function, growth, and proliferation, may have broad biological significance given the critical role of folate in normal cell function and the multiple diseases that have been associated with decreased or excessive folate availability. Low maternal folate concentrations are linked to restricted fetal growth, and we

  3. Liver-Specific Activation of AMPK Prevents Steatosis on a High-Fructose Diet

    Directory of Open Access Journals (Sweden)

    Angela Woods

    2017-03-01

    Full Text Available AMP-activated protein kinase (AMPK plays a key role in integrating metabolic pathways in response to energy demand. We identified a mutation in the γ1 subunit (γ1D316A that leads to activation of AMPK. We generated mice with this mutation to study the effect of chronic liver-specific activation of AMPK in vivo. Primary hepatocytes isolated from these mice have reduced gluconeogenesis and fatty acid synthesis, but there is no effect on fatty acid oxidation compared to cells from wild-type mice. Liver-specific activation of AMPK decreases lipogenesis in vivo and completely protects against hepatic steatosis when mice are fed a high-fructose diet. Our findings demonstrate that liver-specific activation of AMPK is sufficient to protect against hepatic triglyceride accumulation, a hallmark of non-alcoholic fatty liver disease (NAFLD. These results emphasize the clinical relevance of activating AMPK in the liver to combat NAFLD and potentially other associated complications (e.g., cirrhosis and hepatocellular carcinoma.

  4. [Gender-specific manifestations of daily physical activity and sedentary behaviour in elderly people of Surgut].

    Science.gov (United States)

    Loginov, S I; Malkov, M N; Nikolayev, A Yu

    2017-01-01

    Objective of the study was to establish gender-specific characteristics of physical activity (PA) and sedentary behavior in elderly people living in Yugra North. 295 residents of Surgut (102 men aged 62,9±5,3 years, 35%; 193 women aged 61,9±3,8 years, 65%) were subject to a IPAQ-RU questionnaire. The study revealed the gender-specific differences in body length and mass, body mass and body fat indices. It was detected that more energy is spent on the housework and physical activity in the country (moderate-intensity physical activity for women and high-intensity one for men). The study data showed no statistically significant gender-specific differences in general physical activity. Sedentary behavior is more popular among men rather than women (2543 vs 2441 min/week). 47% of low-active men and 56% of women reported the sitting times of 6-9 hours per day, 42% - 9-12 hours per day. Actions need to be taken to increase physical activity which is low at the moment and decrease sedentary behavior which is currently on the high level.

  5. DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways

    Science.gov (United States)

    Mellström, Britt; Sahún, Ignasi; Ruiz-Nuño, Ana; Murtra, Patricia; Gomez-Villafuertes, Rosa; Savignac, Magali; Oliveros, Juan C.; Gonzalez, Paz; Kastanauskaite, Asta; Knafo, Shira; Zhuo, Min; Higuera-Matas, Alejandro; Errington, Michael L.; Maldonado, Rafael; DeFelipe, Javier; Jefferys, John G. R.; Bliss, Tim V. P.; Dierssen, Mara

    2014-01-01

    Changes in nuclear Ca2+ homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K+ channel interacting protein 3), is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory. PMID:24366545

  6. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    Science.gov (United States)

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

  7. Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

    Energy Technology Data Exchange (ETDEWEB)

    Miyagawa, Atsushi, E-mail: miyagawa.atsushi@nitech.ac.jp [RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-Ku, Nagoya 466-8555 (Japan); Totani, Kiichiro [Department of Materials and Life Science, Seikei University, Musashino, Tokyo 180-8633 (Japan); Matsuo, Ichiro [Department of Chemistry and Chemical Biology, Gunma University, Kiryu, Gunma 376-8515 (Japan); Ito, Yukishige, E-mail: yukito@riken.jp [RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); ERATO Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2010-12-17

    Research highlights: {yields} UGGT has a narrow donor specificity. {yields} UGGT gave several non-natural high-mannose-type glycans. {yields} G-II has a promiscuous activity as broad specificity hexosidase. -- Abstract: In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man{sub 9}GlcNAc{sub 2}, providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.

  8. Neural network versus activity-specific prediction equations for energy expenditure estimation in children.

    Science.gov (United States)

    Ruch, Nicole; Joss, Franziska; Jimmy, Gerda; Melzer, Katarina; Hänggi, Johanna; Mäder, Urs

    2013-11-01

    The aim of this study was to compare the energy expenditure (EE) estimations of activity-specific prediction equations (ASPE) and of an artificial neural network (ANNEE) based on accelerometry with measured EE. Forty-three children (age: 9.8 ± 2.4 yr) performed eight different activities. They were equipped with one tri-axial accelerometer that collected data in 1-s epochs and a portable gas analyzer. The ASPE and the ANNEE were trained to estimate the EE by including accelerometry, age, gender, and weight of the participants. To provide the activity-specific information, a decision tree was trained to recognize the type of activity through accelerometer data. The ASPE were applied to the activity-type-specific data recognized by the tree (Tree-ASPE). The Tree-ASPE precisely estimated the EE of all activities except cycling [bias: -1.13 ± 1.33 metabolic equivalent (MET)] and walking (bias: 0.29 ± 0.64 MET; P MET) and walking (bias: 0.61 ± 0.72 MET) and underestimated the EE of cycling (bias: -0.90 ± 1.18 MET; P MET, Tree-ASPE: 0.08 ± 0.21 MET) and walking (ANNEE 0.61 ± 0.72 MET, Tree-ASPE: 0.29 ± 0.64 MET) were significantly smaller in the Tree-ASPE than in the ANNEE (P < 0.05). The Tree-ASPE was more precise in estimating the EE than the ANNEE. The use of activity-type-specific information for subsequent EE prediction equations might be a promising approach for future studies.

  9. Rapid analysis method for the determination of 14C specific activity in irradiated graphite.

    Directory of Open Access Journals (Sweden)

    Vidmantas Remeikis

    Full Text Available 14C is one of the limiting radionuclides used in the categorization of radioactive graphite waste; this categorization is crucial in selecting the appropriate graphite treatment/disposal method. We propose a rapid analysis method for 14C specific activity determination in small graphite samples in the 1-100 μg range. The method applies an oxidation procedure to the sample, which extracts 14C from the different carbonaceous matrices in a controlled manner. Because this method enables fast online measurement and 14C specific activity evaluation, it can be especially useful for characterizing 14C in irradiated graphite when dismantling graphite moderator and reflector parts, or when sorting radioactive graphite waste from decommissioned nuclear power plants. The proposed rapid method is based on graphite combustion and the subsequent measurement of both CO2 and 14C, using a commercial elemental analyser and the semiconductor detector, respectively. The method was verified using the liquid scintillation counting (LSC technique. The uncertainty of this rapid method is within the acceptable range for radioactive waste characterization purposes. The 14C specific activity determination procedure proposed in this study takes approximately ten minutes, comparing favorably to the more complicated and time consuming LSC method. This method can be potentially used to radiologically characterize radioactive waste or used in biomedical applications when dealing with the specific activity determination of 14C in the sample.

  10. Production of high specific activity 27Mg by fast neutron irradiation and recoil-aided leaching

    International Nuclear Information System (INIS)

    Wierczinski, B.; Goeij, J.J.M. de; Volkers, K.J.

    2000-01-01

    High specific activity 27 Mg was produced via recoil-aided leaching from alumina in aqueous medium during irradiation with fast neutrons from a nuclear reactor. After irradiation the aqueous medium was passed through an IC-chelate column, the 24 Na formed during irradiation was removed by elution with 0.25 ml . l -1 sodium acetate and subsequently the 27 Mg was eluted with 2 mol . l -1 hydrochloric acid. Irradiation of alumina with a particle size of 3 μm and a specific surface area of 100 m 2 . g -1 in Milli-Q Plus Water yielded 90% of the total 27 Mg activity produced. Under standard conditions activities of about 8 . 10 5 Bq and specific activities of ca. 10 13 Bq . mol -1 were obtained at the end of irradiation. The standard working conditions involved irradiation of 200 mg alumina dispersed in 0.5 ml liquid in a fast neutron flux of 3 . 10 15 m -2 . s -1 for 15 min, a waiting time of 10 min, and a processing time of 15 minutes. Various alumina samples with different particle sizes and specific surfaces were tested, and the 27 Mg yields were fitted to a mathematical function. Since the high leaching yields cannot only be explained by recoil only, other phenomena such as diffusion and leaching aided by the high hydration energy of the Mg 2+ ion are probably involved. (orig.)

  11. Calculation of radiation production of high specific activity isotopes 192Ir and 60Co

    International Nuclear Information System (INIS)

    Zhou Quan; Zhong Wenfa; Xu Xiaolin

    1997-01-01

    The high specific activity isotopes: 192 Ir and 60 Co in the high neutron flux reactor are calculated with the method of reactor physics. The results of calculation are analyzed in two aspects: the production of isotopes and the influence to parameters of the reactor, and hence a better case is proposed as a reference to the production

  12. Transportation impact analysis for the shipment of low specific activity nitric acid. Revisison 1

    Energy Technology Data Exchange (ETDEWEB)

    Green, J.R.

    1995-05-16

    This is in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes potential toxicological and radiological risks associated with transportation of PUREX Facility LSA Nitric Acid from the Hanford Site to Portsmouth VA, Baltimore MD, and Port Elizabeth NJ.

  13. Rapid analysis method for the determination of 14C specific activity in irradiated graphite.

    Science.gov (United States)

    Remeikis, Vidmantas; Lagzdina, Elena; Garbaras, Andrius; Gudelis, Arūnas; Garankin, Jevgenij; Plukienė, Rita; Juodis, Laurynas; Duškesas, Grigorijus; Lingis, Danielius; Abdulajev, Vladimir; Plukis, Artūras

    2018-01-01

    14C is one of the limiting radionuclides used in the categorization of radioactive graphite waste; this categorization is crucial in selecting the appropriate graphite treatment/disposal method. We propose a rapid analysis method for 14C specific activity determination in small graphite samples in the 1-100 μg range. The method applies an oxidation procedure to the sample, which extracts 14C from the different carbonaceous matrices in a controlled manner. Because this method enables fast online measurement and 14C specific activity evaluation, it can be especially useful for characterizing 14C in irradiated graphite when dismantling graphite moderator and reflector parts, or when sorting radioactive graphite waste from decommissioned nuclear power plants. The proposed rapid method is based on graphite combustion and the subsequent measurement of both CO2 and 14C, using a commercial elemental analyser and the semiconductor detector, respectively. The method was verified using the liquid scintillation counting (LSC) technique. The uncertainty of this rapid method is within the acceptable range for radioactive waste characterization purposes. The 14C specific activity determination procedure proposed in this study takes approximately ten minutes, comparing favorably to the more complicated and time consuming LSC method. This method can be potentially used to radiologically characterize radioactive waste or used in biomedical applications when dealing with the specific activity determination of 14C in the sample.

  14. Designing Class Activities to Meet Specific Core Training Competencies: A Developmental Approach

    Science.gov (United States)

    Guth, Lorraine J.; McDonnell, Kelly A.

    2004-01-01

    This article presents a developmental model for designing and utilizing class activities to meet specific Association for Specialists in Group Work (ASGW) core training competencies for group workers. A review of the relevant literature about teaching group work and meeting core training standards is provided. The authors suggest a process by…

  15. Evaluation of Specific Activity in the Primary Coolant of PWRs by using SAEP

    International Nuclear Information System (INIS)

    Kim, Ha Yong; Song, Jae Seung; Kim, Keung Ku; Kim, Kyo Youn

    2008-07-01

    SAEP(Specific Activity Evaluation Program) to evaluate specific activities in the primary coolant of reactors due to fission products has been developed, which can be applied to the new concept nuclear reactor such as SMART as well as commercial PWRs in existence. Specific activities in the primary coolant were evaluated by using SAEP against reactor plants which are being operated currently in South Korea, respectively. We study the possibility of being applied to the developing commercial PWRs and the new concept reactors through the comparison the results by using SAEP with the results mentioned in the FSARs. We also verify SAEP itself through this evaluation. From the evaluation results, we know that the general trend is agreed with each other from the viewpoint of order of magnitude and that SAEP correctly executes the evaluation of specific activities in the primary coolant of reactor due to fission products for several reactor types, regardless of a reactor type. Therefore, SAEP can widely be applied to the new concept nuclear reactor development phase as well as already developed PWRs

  16. Rapid limb-specific modulation of vestibular contributions to ankle muscle activity during locomotion

    NARCIS (Netherlands)

    Forbes, P.A.; Vlutters, M; Dakin, CJ; van der Kooij, H.; Blouin, JS; Schouten, A.C.

    2017-01-01

    During walking, the vestibular influence on locomotor activity is phase-dependent and modulated in both limbs with changes in velocity. It is unclear, however, whether this bilateral modulation is due to a coordinated mechanism between both limbs or instead through limb-specific processes that

  17. Are There Gender-Specific Risk Factors for Suicidal Activity among Patients with Schizophrenia and Depression?

    Science.gov (United States)

    Kaplan, Kalman J.; Harrow, Martin; Faull, Robert N.

    2012-01-01

    Are there gender-specific risk factors for suicidal activity among patients with schizophrenia and depression? A total of 74 schizophrenia patients (51 men, 23 women) and 77 unipolar nonpsychotic depressed patients (26 men, 51 women) from the Chicago Follow-up Study were studied prospectively at 2 years posthospitalization and again at 7.5 years.…

  18. Transportation impact analysis for the shipment of low specific activity nitric acid. Revisison 1

    International Nuclear Information System (INIS)

    Green, J.R.

    1995-01-01

    This is in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes potential toxicological and radiological risks associated with transportation of PUREX Facility LSA Nitric Acid from the Hanford Site to Portsmouth VA, Baltimore MD, and Port Elizabeth NJ

  19. Transportation impact analysis for the shipment of Low Specific Activity Nitric Acid

    International Nuclear Information System (INIS)

    Green, J.R.

    1994-01-01

    This document was written in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes the potential toxicological and radiological risks associated with the transportation of PUREX Facility LSA Nitric Acid from the Hanford Site in Washington State to three Eastern ports

  20. Specific and Nonspecific Neural Activity during Selective Processing of Visual Representations in Working Memory

    Science.gov (United States)

    Oh, Hwamee; Leung, Hoi-Chung

    2010-01-01

    In this fMRI study, we investigated prefrontal cortex (PFC) and visual association regions during selective information processing. We recorded behavioral responses and neural activity during a delayed recognition task with a cue presented during the delay period. A specific cue ("Face" or "Scene") was used to indicate which one of the two…

  1. The effect of operational conditions on the sludge specific methanogenic activity and sludge biodegradability

    NARCIS (Netherlands)

    Leitao, R.; Santaellla, S.T.; Haandel, van A.C.; Zeeman, G.; Lettinga, G.

    2009-01-01

    The effects of hydraulic retention time (HRT) and influent COD concentration (CODInf) on Specific Methanogenic Activity (SMA) and the biodegradability of an anaerobic sludge need to be elucidated because of the discordant results available in literature. This information is important for the

  2. Context Fear Learning Specifically Activates Distinct Populations of Neurons in Amygdala and Hypothalamus

    Science.gov (United States)

    Trogrlic, Lidia; Wilson, Yvette M.; Newman, Andrew G.; Murphy, Mark

    2011-01-01

    The identity and distribution of neurons that are involved in any learning or memory event is not known. In previous studies, we identified a discrete population of neurons in the lateral amygdala that show learning-specific activation of a c-"fos"-regulated transgene following context fear conditioning. Here, we have extended these studies to…

  3. Consideration of statistical uncertainties for the determination of representative values of the specific activity of wastes

    International Nuclear Information System (INIS)

    Barthel, R.

    2008-01-01

    The German Radiation Protection Commission has recommended 'Principles and Methods for the Consideration of Statistical Uncertainties for the Determination of Representative Values of the Specific Activity of NORM wastes' concerning the proof of compliance with supervision limits or dose standards according to paragraph 97 and paragraph 98 of the Radiation Protection Ordinance, respectively. The recommendation comprises a method ensuring the representativeness of estimates for the specific activity of NORM wastes, which also assures the required evidence for conformity with respect to supervision limits or dose standards, respectively. On the basis of a sampling survey, confidence limits for expectation values of specific activities are determined, which will be used to show that the supervision limit or the dose standard is met or exceeded with certainty, or that the performed sampling is not sufficient for the intended assessment. The sampling effort depends on the type and the width of the distribution of specific activities and is determined by the position of the confidence interval with respect to the supervision limit or of the resulting doses with respect to the dose standard. The statistical uncertainties that are described by confidence limits may be reduced by an optimised extension of the sample number, as far as necessary. (orig.)

  4. Determination of specific activity of americium and plutonium in selected environmental samples

    International Nuclear Information System (INIS)

    Trebunova, T.

    1999-01-01

    The aim of this work was development of method for determination of americium and plutonium in environmental samples. Developed method was evaluated on soil samples and after they was applied on selected samples of fishes (smoked mackerel, herring and fillet from Alaska hake). The method for separation of americium is based on liquid separation with Aliquate-336, precipitation with oxalic acid and using of chromatographic material TRU-Spec TM .The intervals of radiochemical yields were from 13.0% to 80.9% for plutonium-236 and from 10.5% to 100% for americium-241. Determined specific activities of plutonium-239,240 were from (2.3 ± 1.4) mBq/kg to (82 ± 29) mBq/kg, the specific activities of plutonium-238 were from (14.2 ± 3.7) mBq/kg to (708 ± 86) mBq/kg. The specific activities of americium-241 were from (1.4 ± 0.9) mBq/kg to (3360 ± 210) mBq/kg. The fishes from Baltic Sea as well as from North Sea show highest specific activities then fresh-water fishes from Slovakia. Therefore the monitoring of alpha radionuclides in foods imported from territories with nuclear testing is recommended

  5. A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities.

    Science.gov (United States)

    Hitomi, Kiyotaka; Kitamura, Miyako; Alea, Mileidys Perez; Ceylan, Ismail; Thomas, Vincent; El Alaoui, Saïd

    2009-11-15

    Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.

  6. Human active X-specific DNA methylation events showing stability across time and tissues

    Science.gov (United States)

    Joo, Jihoon Eric; Novakovic, Boris; Cruickshank, Mark; Doyle, Lex W; Craig, Jeffrey M; Saffery, Richard

    2014-01-01

    The phenomenon of X chromosome inactivation in female mammals is well characterised and remains the archetypal example of dosage compensation via monoallelic expression. The temporal series of events that culminates in inactive X-specific gene silencing by DNA methylation has revealed a ‘patchwork' of gene inactivation along the chromosome, with approximately 15% of genes escaping. Such genes are therefore potentially subject to sex-specific imbalance between males and females. Aside from XIST, the non-coding RNA on the X chromosome destined to be inactivated, very little is known about the extent of loci that may be selectively silenced on the active X chromosome (Xa). Using longitudinal array-based DNA methylation profiling of two human tissues, we have identified specific and widespread active X-specific DNA methylation showing stability over time and across tissues of disparate origin. Our panel of X-chromosome loci subject to methylation on Xa reflects a potentially novel mechanism for controlling female-specific X inactivation and sex-specific dimorphisms in humans. Further work is needed to investigate these phenomena. PMID:24713664

  7. EFFECTS OF DYNAMIC AND STATIC STRETCHING WITHIN GENERAL AND ACTIVITY SPECIFIC WARM-UP PROTOCOLS

    Directory of Open Access Journals (Sweden)

    Michael Samson

    2012-06-01

    Full Text Available The purpose of the study was to determine the effects of static and dynamic stretching protocols within general and activity specific warm-ups. Nine male and ten female subjects were tested under four warm-up conditions including a 1 general aerobic warm-up with static stretching, 2 general aerobic warm-up with dynamic stretching, 3 general and specific warm-up with static stretching and 4 general and specific warm-up with dynamic stretching. Following all conditions, subjects were tested for movement time (kicking movement of leg over 0.5 m distance, countermovement jump height, sit and reach flexibility and 6 repetitions of 20 metre sprints. Results indicated that when a sport specific warm-up was included, there was an 0.94% improvement (p = 0.0013 in 20 meter sprint time with both the dynamic and static stretch groups. No such difference in sprint performance between dynamic and static stretch groups existed in the absence of the sport specific warm-up. The static stretch condition increased sit and reach range of motion (ROM by 2.8% more (p = 0.0083 than the dynamic condition. These results would support the use of static stretching within an activity specific warm-up to ensure maximal ROM along with an enhancement in sprint performance

  8. Effects of chronic Akt/mTOR inhibition by rapamycin on mechanical overload-induced hypertrophy and myosin heavy chain transition in masseter muscle.

    Science.gov (United States)

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2013-01-01

    To examine the effects of the Akt/mammalian target of rapamycin (mTOR) pathway on masseter muscle hypertrophy and myosin heavy chain (MHC) transition in response to mechanical overload, we analyzed the effects of bite-opening (BO) on the hypertrophy and MHC composition of masseter muscle of BO-rats treated or not treated with rapamycin (RAPA), a selective mTOR inhibitor. The masseter muscle weight in BO-rats was significantly greater than that in controls, and this increase was attenuated by RAPA treatment. Expression of slow-twitch MHC isoforms was significantly increased in BO-rats with/without RAPA treatment, compared with controls, but the magnitude of the increase was much smaller in RAPA-treated BO-rats. Phosphorylation of p44/42 MAPK (ERK1/2), which preserves fast-twitch MHC isoforms in skeletal muscle, was significantly decreased in BO-rats, but the decrease was abrogated by RAPA treatment. Calcineurin signaling is known to be important for masseter muscle hypertrophy and fast-to-slow MHC isoform transition, but expression of known calcineurin activity modulators was unaffected by RAPA treatment. Taken together, these results indicate that the Akt/mTOR pathway is involved in both development of masseter muscle hypertrophy and fast-to-slow MHC isoform transition in response to mechanical overload with inhibition of the ERK1/2 pathway and operates independently of the calcineurin pathway.

  9. Controlling nuclear JAKs and STATs for specific gene activation by IFNγ

    International Nuclear Information System (INIS)

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-01-01

    Highlights: → Gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interact with the promoter region of IFNγ-associated genes along with transcription factor STAT1α. → We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. → The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The

  10. Age-specific activation of cerebral areas in motor imagery - a fMRI study

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Li [Chongqing University, Key Laboratory of Biorheological Science and Technology of Ministry of Education, Bioengineering College, Chongqing (China); Third Military Medical University, Department of Medical Image, College of Biomedical Engineering, Chongqing (China); Qiu, Mingguo; Zhang, Jingna; Zhang, Ye; Sang, Linqiong [Third Military Medical University, Department of Medical Image, College of Biomedical Engineering, Chongqing (China); Liu, Chen; Yang, Jun [Third Military Medical University, Department of Radiology, Southwest Hospital, Chongqing (China); Yan, Rubing [Third Military Medical University, Department of Rehabilitation, Southwest Hospital, Chongqing (China); Zheng, Xiaolin [Chongqing University, Key Laboratory of Biorheological Science and Technology of Ministry of Education, Bioengineering College, Chongqing (China)

    2014-04-15

    The objectives of this study were to study the age-specific activation patterns of cerebral areas during motor execution (ME) and motor imaging (MI) of the upper extremities and to discuss the age-related neural mechanisms associated with ME or MI. The functional magnetic resonance imaging technique was used to monitor the pattern and intensity of brain activation during the ME and MI of the upper extremities in 20 elderly (>50 years) and 19 young healthy subjects (<25 years). No major differences were identified regarding the activated brain areas during ME or MI between the two groups; however, a minor difference was noted. The intensity of the activated brain area during ME was stronger in the older group than in the younger group, while the results with MI were the opposite. The posterior central gyrus and supplementary motor area during MI were more active in the younger group than in the older group. The putamen, lingual, and so on demonstrated stronger activation during dominant hand MI in the older group. The results of this study revealed that the brain structure was altered and that neuronal activity was attenuated with age, and the cerebral cortex and subcortical tissues were found to be over-activated to achieve the same level of ME and MI, indicating that the activating effects of the left hemisphere enhanced with age, whereas the inhibitory effects declined during ME, and activation of the right hemisphere became more difficult during MI. (orig.)

  11. Age-specific activation of cerebral areas in motor imagery - a fMRI study

    International Nuclear Information System (INIS)

    Wang, Li; Qiu, Mingguo; Zhang, Jingna; Zhang, Ye; Sang, Linqiong; Liu, Chen; Yang, Jun; Yan, Rubing; Zheng, Xiaolin

    2014-01-01

    The objectives of this study were to study the age-specific activation patterns of cerebral areas during motor execution (ME) and motor imaging (MI) of the upper extremities and to discuss the age-related neural mechanisms associated with ME or MI. The functional magnetic resonance imaging technique was used to monitor the pattern and intensity of brain activation during the ME and MI of the upper extremities in 20 elderly (>50 years) and 19 young healthy subjects (<25 years). No major differences were identified regarding the activated brain areas during ME or MI between the two groups; however, a minor difference was noted. The intensity of the activated brain area during ME was stronger in the older group than in the younger group, while the results with MI were the opposite. The posterior central gyrus and supplementary motor area during MI were more active in the younger group than in the older group. The putamen, lingual, and so on demonstrated stronger activation during dominant hand MI in the older group. The results of this study revealed that the brain structure was altered and that neuronal activity was attenuated with age, and the cerebral cortex and subcortical tissues were found to be over-activated to achieve the same level of ME and MI, indicating that the activating effects of the left hemisphere enhanced with age, whereas the inhibitory effects declined during ME, and activation of the right hemisphere became more difficult during MI. (orig.)

  12. Tramadol Pretreatment Enhances Ketamine-Induced Antidepressant Effects and Increases Mammalian Target of Rapamycin in Rat Hippocampus and Prefrontal Cortex

    Directory of Open Access Journals (Sweden)

    Chun Yang

    2012-01-01

    Full Text Available Several lines of evidence have demonstrated that acute administration of ketamine elicits fast-acting antidepressant effects. Moreover, tramadol also has potential antidepressant effects. The aim of this study was to investigate the effects of pretreatment with tramadol on ketamine-induced antidepressant activity and was to determine the expression of mammalian target of rapamycin (mTOR in rat hippocampus and prefrontal cortex. Rats were intraperitoneally administrated with ketamine at the dose of 10 mg/kg or saline 1 h before the second episode of the forced swimming test (FST. Tramadol or saline was intraperitoneally pretreated 30 min before the former administration of ketamine or saline. The locomotor activity and the immobility time of FST were both measured. After that, rats were sacrificed to determine the expression of mTOR in hippocampus and prefrontal cortex. Tramadol at the dose of 5 mg/kg administrated alone did not elicit the antidepressant effects. More importantly, pretreatment with tramadol enhanced the ketamine-induced antidepressant effects and upregulated the expression of mTOR in rat hippocampus and prefrontal cortex. Pretreatment with tramadol enhances the ketamine-induced antidepressant effects, which is associated with the increased expression of mTOR in rat hippocampus and prefrontal cortex.

  13. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

    International Nuclear Information System (INIS)

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-01-01

    Pestivirus N pro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N pro blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N pro' s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N pro -GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N pro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N pro' s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N pro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N pro' s autoproteolysis is studied using N pro -GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N pro prefers small amino acids with non-branched beta carbons at the P1 position

  14. Specificity of a prodrug-activating enzyme hVACVase: the leaving group effect.

    Science.gov (United States)

    Sun, Jing; Dahan, Arik; Walls, Zachary F; Lai, Longsheng; Lee, Kyung-Dall; Amidon, Gordon L

    2010-12-06

    Human valacyclovirase (hVACVase) is a prodrug-activating enzyme for amino acid prodrugs including the antiviral drugs valacyclovir and valganciclovir. In hVACVase-catalyzed reactions, the leaving group of the substrate corresponds to the drug moiety of the prodrug, making the leaving group effect essential for the rational design of new prodrugs targeting hVACVase activation. In this study, a series of valine esters, phenylalanine esters, and a valine amide were characterized for the effect of the leaving group on the efficiency of hVACVase-mediated prodrug activation. Except for phenylalanine methyl and ethyl esters, all of the ester substrates exhibited a relatively high specificity constant (k(cat)/K(m)), ranging from 850 to 9490 mM(-1)·s(-1). The valine amide Val-3-APG exhibited significantly higher K(m) and lower k(cat) values compared to the corresponding ester Val-3-HPG, indicating poor specificity for hVACVase. In conclusion, the substrate leaving group has been shown to affect both binding and specific activity of hVACVase-catalyzed activation. It is proposed that hVACVase is an ideal target for α-amino acid ester prodrugs with relatively labile leaving groups while it is relatively inactivate toward amide prodrugs.

  15. Purification of charybdotoxine, a specific inhibitor of the high-conductance Ca2+-activated K+ channel

    International Nuclear Information System (INIS)

    Smith, C.; Phillips, M.; Miller, C.

    1986-01-01

    Charybdotoxim is a high-affinity specific inhibitor of the high-conductance Ca 2+ -activated K + channel found in the plasma membranes of many vertebrate cell types. Using Ca 2+ -activated K + channels reconstituted into planar lipid bilayer membranes as an assay, the authors have purified the toxin from the venom of the scorpion Leiurus quinquestriatus by a two-step procedure involving chromatofocusing on SP-Sephadex, followed by reversed-phase high-performance liquid chromatography. Charybdotoxin is shown to be a highly basic protein with a mass of 10 kDa. Under the standard assay conditions, the purified toxin inhibits the Ca 2+ -activated K + channel with an apparent dissociation constant of 3.5 nM. The protein is unusually stable, with inhibitory potency being insensitive to boiling or exposure to organic solvents. The toxin's activity is sensitive to chymotrypsin treatment and to acylation of lysine groups. The protein may be radioiodinated without loss of activity

  16. Physical activity and gastric residuals as biomarkers for region-specific NEC lesions in preterm neonates

    DEFF Research Database (Denmark)

    Cao, Muqing; Andersen, Anders Daniel; Li, Yanqi

    2016-01-01

    onset of NEC can be predicted by decreased physical activity during the first few days after birth. Methods: Cesarean-delivered preterm pigs were fed parenteral nutrition and increasing amounts of formula for 5 days after birth (n = 120). Their physical activity was quantified by a continuous camera....... Results: Half of the pigs (48%) showed clear NEC-like lesions on day 5, and these individuals had more adverse clinical symptoms from day 3 but decreased physical activity already from day 2 relative to the unaffected pigs (both p ... physical activity on days 2 and 3, and the increased volume of gastric residuals was specifically related to colon lesions (both p physical activity precedes the clinical symptoms of NEC in the small intestine of preterm pigs, and increased gastric residuals predict NEC...

  17. 14C specific activity of farm products and marine products collected from the Rokkasho area in Aomori prefecture

    International Nuclear Information System (INIS)

    Muranaka, Takeshi; Honda, Kazuya

    1996-01-01

    We investigated 14 C specific activity of farm products and marine products which were collected from the Rokkasho area in Aomori prefecture from 1988 to 1990. The measured 14 C specific activity of farm products was almost equal to one another with the averaged 14 C specific activity of 0.261 [Bq/g·C]. On the other hand, 14 C specific activity of marine products was slightly lower than those of farm products. Especially that of squid was the lowest among studied marine products. This may be due to the low 14 C specific activity of the sea water surrounding squid. (author)

  18. Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD.

    Science.gov (United States)

    Lattante, Serena; de Calbiac, Hortense; Le Ber, Isabelle; Brice, Alexis; Ciura, Sorana; Kabashi, Edor

    2015-03-15

    Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK

    Directory of Open Access Journals (Sweden)

    Sung-Jun Park

    2017-04-01

    Full Text Available The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK, an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

  20. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  1. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  2. [Validity of a standard questionnaire to assess physical activity for specific medical checkups and health guidance].

    Science.gov (United States)

    Kawakami, Ryoko; Miyachi, Motohiko

    2010-10-01

    This study aimed to determine the validity of a standard questionnaire to assess amount of physical activity (PA) and cardiorespiratory fitness (VO2peak). A total of 483 men and women, aged 20 to 69 years, participated. The standard questionnaire included 3 items about exercise, PA, and walking speed. All questions were designed to require an answer of Yes or No. Subjects were classified into one of four groups regarding the number of Yes answers to the three questions, giving activity levels of 0 to 3. The amount of PA was measured objectively with a tn-axial accelerometer which could also calculate daily step counts, and the amounts of PA under 3 metabolic equivalents (METs) and at 3 METs or more. VO2peak. was measured by incremental cycle exercise tests with indirect calorimetry. The daily step counts, the amount of PA at 3 METs or more, and the VO2peak. were significantly higher in subjects who answered Yes to each question than in those who answered No. Sensitivity and specificity of each question were 62-73% and 45-71% for the amount of PA established with the "Exercise and Physical Activity Reference for Health Promotion 2006 (EPAR2006)". The sum of sensitivity and specificity was the highest when the cutoff value was activity level 2 (sensitivity 73%, specificity 68%). Sensitivity and specificity for VO2max established by EPAR2006 were lower than those for the amount of PA. These results suggest that only answering simple questions with a standard questionnaire is sufficient for estimation of PA levels for specific medical checkups and health guidance, even though the accuracy is somewhat limited.

  3. Preparation and quantification of 3'-phosphoadenosine 5'-phospho[35S]sulfate with high specific activity

    International Nuclear Information System (INIS)

    Vargas, F.

    1988-01-01

    The synthesis and quantitation of the sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAP35S), prepared from inorganic [35S]sulfate and ATP, were studied. An enzymatic transfer method based upon the quantitative transfer of [35S]sulfate from PAP35S to 2-naphthol and 4-methylumbelliferone by the action of phenolsulfotransferase activity from rat brain cytosol was also developed. The 2-naphthyl[35S]sulfate or 35S-methylumbelliferone sulfate formed was isolated by polystyrene bead chromatography. This method allows the detection of between 0.1 pmol and 1 nmol/ml of PAP35S. PAP35S of high specific activity (75 Ci/mmol) was prepared by incubating ATP and carrier-free Na2 35SO4 with a 100,000g supernatant fraction from rat spleen. The product was purified by ion-exchange chromatography. The specific activity and purity of PAP35S were estimated by examining the ratios of Km values for PAP35S of the tyrosyl protein sulfotransferase present in microsomes from rat cerebral cortex. The advantage and applications of these methods for the detection of femtomole amounts, and the synthesis of large scale quantities of PAP35S with high specific activity are discussed

  4. Experimental study on an active specific immunotherapy modified with irradiation, 1

    International Nuclear Information System (INIS)

    Ogawa, Yasuhiro; Takashima, Hitoshi; Imanaka, Kazufumi

    1982-01-01

    Active specific immunotherapy using low-dose-irradiated tumor cells and activated mononuclear cells was studied on the transplanted MM 46 tumor of female C3H/He mice aged 12 weeks, after radiotherapy was performed. Local irradiation with the dose of 3,000 rads by high energy electron beam was performed on the fifth day after 5 x 10 6 tumor cells were inoculated in the right hind paws. Tumor cells and mononuclear cells were separated from the tumor tissue which was irradiated with the dose of 2,000 rads on the fifth day and were injected subcutaneously into the left hind paws of the tumor burden mice on the eleventh day. Anti-tumor effect was evaluated by the changes of the tumor volume and survival curves. Tumor growth was significantly inhibited in the group which received irradiation combined with active specific immunotherapy compared with the control group (p < 0.01). Survival rate was significantly higher in the group which received irradiation combined with active specific immunotherapy than the control group (p < 0.025). (author)

  5. Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

    Science.gov (United States)

    Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

    2008-01-01

    The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

  6. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    KAUST Repository

    Walkiewicz, Katarzyna Wiktoria

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  7. Subnanomole detection and quantitation of high specific activity 32P-nucleotides

    International Nuclear Information System (INIS)

    Coniglio, C.; Pappas, G.; Gill, W.J.; Kashdan, M.; Maniscalco, M.

    1991-01-01

    Microbore liquid chromatography utilizes conventional HPLC and ultraviolet detection principles to determine subnanomole mass quantities of biologically significant molecules. This system takes advantage of specifically designed microflow equipment to analyze ultraviolet absorbing species at the picomole range. 32P-labeled nucleotides are examples of compounds routinely used at picomole quantities that are extremely difficult to accurately quantify using standard mass measurement techniques. The procedure described in this paper has the capability of measuring nucleotides down to 10 pmol using commercially available microbore ultraviolet detection equipment. The technique can be used to accurately measure the specific activity of as little as 10 microCi of an aqueous 32P-nucleotide solution

  8. StrigoQuant: A genetically encoded biosensor for quantifying strigolactone activity and specificity

    KAUST Repository

    Samodelov, S. L.

    2016-11-05

    Strigolactones are key regulators of plant development and interaction with symbiotic fungi; however, quantitative tools for strigolactone signaling analysis are lacking. We introduce a genetically encoded hormone biosensor used to analyze strigolactone-mediated processes, including the study of the components involved in the hormone perception/signaling complex and the structural specificity and sensitivity of natural and synthetic strigolactones in Arabidopsis, providing quantitative insights into the stereoselectivity of strigolactone perception. Given the high specificity, sensitivity, dynamic range of activity, modular construction, ease of implementation, and wide applicability, the biosensor StrigoQuant will be useful in unraveling multiple levels of strigolactone metabolic and signaling networks.

  9. Synthesis of high specific activity carbon-11 labeled tracers for neuroreceptor studies

    International Nuclear Information System (INIS)

    Dannals, R.F.; Ravert, H.T.; Wilson, A.A.; Wagner, H.N. Jr; Johns Hopkins Medical Institutions, Baltimore, MD

    1989-01-01

    The use of short-lived positron-emitting radiotracers together with positron emission tomography (PET) has allowed scientists to acquire previously inaccessible information regarding problems in physiology, biochemistry, and pharmacology in the living human body. In the past five years, successes in the application of PET to the non-invasive determination of the spatial distribution and regional concentration of a variety of neurotransmitter binding sites within the living brain often followed the successful selections and syntheses of appropriately radiolabeled ligands. This presentation concentrates on the synthesis of these high specific activity radiotracers for neuroreceptor PET studies labeled specifically with carbon-11. (author). 15 refs.; 1 fig

  10. Luciferase-Specific Coelenterazine Analogues for Optical Contamination-Free Bioassays.

    Science.gov (United States)

    Nishihara, Ryo; Abe, Masahiro; Nishiyama, Shigeru; Citterio, Daniel; Suzuki, Koji; Kim, Sung Bae

    2017-04-19

    Spectral overlaps among the multiple optical readouts commonly cause optical contamination in fluorescence and bioluminescence. To tackle this issue, we created five-different lineages of coelenterazine (CTZ) analogues designed to selectively illuminate a specific luciferase with unique luciferase selectivity. In the attempt, we found that CTZ analogues with ethynyl or styryl groups display dramatically biased bioluminescence to specific luciferases and pHs by modifying the functional groups at the C-2 and C-6 positions of the imidazopyradinone backbone of CTZ. The optical contamination-free feature was exemplified with the luciferase-specific CTZ analogues, which illuminated anti-estrogenic and rapamycin activities in a mixture of optical probes. This unique bioluminescence platform has great potential for specific and high throughput imaging of multiple optical readouts in bioassays without optical contamination.

  11. Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

    Science.gov (United States)

    Zhao, Hua

    2015-01-01

    There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

  12. Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity.

    Directory of Open Access Journals (Sweden)

    Laura M J Ylinen

    2010-08-01

    Full Text Available TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5alpha but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations.

  13. Context-Specific Associations of Physical Activity and Sedentary Behavior With Cognition in Children.

    Science.gov (United States)

    Aggio, Daniel; Smith, Lee; Fisher, Abigail; Hamer, Mark

    2016-06-15

    In the present study, we investigated how overall and specific domains of physical activity and sedentary behavior at the age of 7 years were associated with cognition at the age of 11 years in 8,462 children from the Millennium Cohort Study. Data were collected from 2001 to 2013. Participation in domains of physical activity and sedentary behavior at 7 years of age were reported. Activity levels were also measured objectively. Cognition was assessed using the British Ability Scales. General linear models were used to assess longitudinal associations of physical activity and sedentary behavior, measured both objectively and via self-report, with cognition. Analyses were adjusted for prespecified covariates. Sports/physical activity club attendance (B = 0.6, 95% confidence interval (CI): 0.2, 1.1), doing homework (B = 0.5, 95% CI: 0.0, 0.9), and objectively measured sedentary time (B = 0.8, 95% CI: 0.1, 1.4) at age 7 years were positively associated with cognition at age 11 years in final the models. Television viewing was negatively associated with cognition (B = -1.7, 95% CI: -2.4, -1.0), although the association was attenuated to the null after adjustments for baseline cognition. Objectively measured light physical activity was inversely associated with cognition (B = -0.7, 95% CI: -1.3, -0.1). Moderate-to-vigorous physical activity was also inversely associated with cognition in girls only (B = -1.1, 95% CI: -2.0, -0.3). Associations of physical activity and sedentary behavior with cognition appear to be context-specific in young people. © The Author 2016. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health.

  14. Toxicidade pulmonar induzida pela rapamicina Lung toxicity induced by rapamycin

    Directory of Open Access Journals (Sweden)

    C Damas

    2006-11-01

    complication associated to this medication. Interstitial pneumonitis and more rarely alveolar haemorrhage have been described. The authors describe 4 cases (3 men and 1 woman between 46-71 years, transplanted three years ago (1 patient and 7 years ago (3 patients. All of them were medicated with micofenolato mofetil, prednisone and sirolimus. All patients had fever at admission, 3 patients had dyspnoea and 2 productive cough. Diffuse pulmonary infiltrates with basal predominance in HRCT scan were present in the four patients. BAL showed lymphocytic alveolitis in 3 cases, however with a different CD4/CD8 ratio. In additio to lymphocytosis, neutrophilia was observed in 2 patients. One patient showed serious alveolar haemorrhage in BAL. Pulmonary infections were ruled out by specific BAL staining and cultures. After drug suspension, all patients showed a clear improvement. These case studies show some diversity in clinical presentation and in the features of some exams, namely in BAL. This may suggest different underlying pathophysiology entities induced by sirolimus.

  15. Carrageenan activates monocytes via type-specific binding with interleukin-8: an implication for design of immuno-active biomaterials.

    Science.gov (United States)

    Chan, Weng-I; Zhang, Guangpan; Li, Xin; Leung, Chung-Hang; Ma, Dik-Lung; Dong, Lei; Wang, Chunming

    2017-02-28

    Polymers that can activate the immune system may become useful biomaterials tools, given that the mechanisms underlying their actions are well understood. Herein, we report a novel type of interaction between polymers and immune cells - in studying the influence of the three major types of carrageenan (CGN) polysaccharides on monocyte behaviour in vitro, we found only the λ-type induced monocyte adhesion and this action requires the presence of an adequate amount of serum. Further analyses indicated λ-CGN bound interleukin-8 (IL-8) in the serum and activated the cultured monocytes through an IL-8-dependent pathway. This is the first demonstration that a polymer, with a renowned immunostimulatory effect, activates the immune system via binding and harnessing the function of a specific cytokine in the microenvironment. This is a new mechanism underlying polymer-immunity interactions that may shed light on future design and application of biomaterials tools targeting the immune system for a wide variety of therapeutic applications.

  16. PEGylated DX-1000: Pharmacokinetics and Antineoplastic Activity of a Specific Plasmin Inhibitor

    Directory of Open Access Journals (Sweden)

    Laetitia Devy

    2007-11-01

    Full Text Available Novel inhibitors of the urokinase-mediated plasminogen (plg activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pin (Ki = 99 pM. When tested in vitro, DX-1000 blocks plasminmediated pro-matrix metal loproteinase-9 (proMMP-9 activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor (~ 7 kDa exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogenactivated protein kinase (MAPK in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

  17. Gender-specific activity demands experienced during semiprofessional basketball game play.

    Science.gov (United States)

    Scanlan, Aaron T; Dascombe, Ben J; Kidcaff, Andrew P; Peucker, Jessica L; Dalbo, Vincent J

    2015-07-01

    To compare game activity demands between female and male semiprofessional basketball players. Female (n=12) and male (n=12) semiprofessional basketball players were monitored across 3 competitive games. Time-motion-analysis procedures quantified player activity into predefined movement categories across backcourt (BC) and frontcourt (FC) positions. Activity frequencies, durations, and distances were calculated relative to live playing time (min). Work:rest ratios were also calculated using the video data. Game activity was compared between genders for each playing position and all players. Female players performed at greater running work-rates than male players (45.7±1.4 vs. 42.1±1.7 m/min, P=.05), while male players performed more dribbling than female players (2.5±0.3 vs. 3.0±0.2 s/min; 8.4±0.3 vs. 9.7±0.7 m/min, P=.05). Positional analyses revealed that female BC players performed more low-intensity shuffling (P=.04) and jumping (P=.05), as well as longer (P=.04) jogging durations, than male BC players. Female FC players executed more upper-body activity (P=.03) and larger work:rest ratios (Pbasketball. Furthermore, position-specific variations between female and male basketball players should be considered. These data may prove useful in the development of gender-specific conditioning plans relative to playing position in basketball.

  18. Radioimmunoassay for platelet activation specific protein GMP-140 on the platelet surface and in plasma

    International Nuclear Information System (INIS)

    Wu Guoxin; Li Jianyong; Ruan Changgeng

    1991-08-01

    Using monoclonal antibody (McAb) SZ-51 which is specific for an alpha-granule membrane protein (GMP-140) on the surface of human activated platelets, the platelet GMP-140 expression in fixed whole blood was measured by direct radioimmunoassay and GMP-140 microparticles in plasma was measured by sandwich method. The GMP-140 molecules per platelet or milliliter (mL) were calculated for the following subjects; acute myocardial infarction; cerebro thrombosis; diabetic mellitus; asthma attack; epidemic hemorrhagic fever etc.. By comparing with the concentration of thromboxane B 2 (TXB 2 ) and von Willebrand factor (vWF) in plasma, it is confirmed that the measurement of GMP-140 molecules is better than that of TXB 2 and vWF. It is a sensitive and specific method for evaluating the platelet activation degree in vivo. The establishment of this method will be useful to diagnosing the thrombotic disorders and studying the pathogenesis of some other diseases

  19. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity

    Directory of Open Access Journals (Sweden)

    Nicolas Huguenin-Dezot

    2016-07-01

    Full Text Available Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.

  20. Specific-activity and concentration model applied to cesium movement in an oligotrophic lake

    International Nuclear Information System (INIS)

    Vanderploeg, H.A.; Booth, R.S.; Clark, F.H.

    1975-01-01

    A linear systems-analysis model was derived to simulate the time-dependent dynamics of specific activity and concentration of radionuclides in aquatic systems. Transfer coefficients were determined for movement of 137 Cs in the components of an oligotrophic lake. These coefficients were defined in terms of basic environmental and ecological data so that the model can be applied to a wide variety of sites. Simulations with a model that ignored sediment--water interactions predicted much higher 137 Cs specific activities in the lake water and biota than did those with the complete model. Comparing 137 Cs concentrations predicted by the model with concentrations reported for the biota of an experimentally contaminated oligotrophic lake indicated that the transfer coefficients derived for the biota are adequate

  1. Graded territories: Towards the design, specification and simulation of materially graded bending active structures

    DEFF Research Database (Denmark)

    Nicholas, Paul; Tamke, Martin; Ramsgaard Thomsen, Mette

    2012-01-01

    these structures, the property of bending is activated and varied through bespoke material means so as to match a desired form. Within the architectural design process, formal control depends upon design approaches for material specification and simulation that consider behavior at the level of the material...... element as well as the structure. We describe an evolving approach to material specification and simulation, and highlight the digital and material considerations that frame the process.......The ability to make materials with bespoke behavior affords new perspectives on incorporating material properties within the design process not available through natural materials. This paper reports the design and assembly of two bending-active, fibre-reinforced composite structures. Within...

  2. A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs.

    Science.gov (United States)

    Urfer, Silvan R; Kaeberlein, Tammi L; Mailheau, Susan; Bergman, Philip J; Creevy, Kate E; Promislow, Daniel E L; Kaeberlein, Matt

    2017-04-01

    Age is the single greatest risk factor for most causes of morbidity and mortality in humans and their companion animals. As opposed to other model organisms used to study aging, dogs share the human environment, are subject to similar risk factors, receive comparable medical care, and develop many of the same age-related diseases humans do. In this study, 24 middle-aged healthy dogs received either placebo or a non-immunosuppressive dose of rapamycin for 10 weeks. All dogs received clinical and hematological exams before, during, and after the trial and echocardiography before and after the trial. Our results showed no clinical side effects in the rapamycin-treated group compared to dogs receiving the placebo. Echocardiography suggested improvement in both diastolic and systolic age-related measures of heart function (E/A ratio, fractional shortening, and ejection fraction) in the rapamycin-treated dogs. Hematological values remained within the normal range for all parameters studied; however, the mean corpuscular volume (MCV) was decreased in rapamycin-treated dogs. Based on these results, we will test rapamycin on a larger dog cohort for a longer period of time in order to validate its effects on cardiac function and to determine whether it can significantly improve healthspan and reduce mortality in companion dogs.

  3. Prolonged activation of virus-specific CD8+T cells after acute B19 infection

    DEFF Research Database (Denmark)

    Isa, Adiba; Kasprowicz, Victoria; Norbeck, Oscar

    2005-01-01

    BACKGROUND: Human parvovirus B19 (B19) is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. METHODS AND FINDINGS......: The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA-peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia......, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function...

  4. Synthesis of high specific activity tritium labelled [2-3H]-adenosine-5'-triphosphate

    International Nuclear Information System (INIS)

    Jaiswal, D.K.; Morimoto, H.; Trump, E.L.; Williams, P.G.; Wemmer, D.E.

    1996-01-01

    A procedure for high level tritium labelling at the C2-H position of adenosine 5'-triphosphate ([2- 3 H]-ATP, 1), based on the tritiodehalogenation reaction of 2-bromoadenosine 5'-triphosphate (2) has been elaborated. This precursor was prepared in a six-step synthesis from guanosine. The tritiodehalogenation of (2) for three hours over palladium oxide in phosphate buffer yielded tritium labelled ATP with high specific activity, in good chemical yield. (author)

  5. Preparation and characterization of high-specific activity radiolabeled 50 S measles virus RNA

    International Nuclear Information System (INIS)

    Spruance, S.L.; Ashton, B.N.; Smith, C.B.

    1980-01-01

    A method is described to radiolabeled measles virus RNA for hybridization studies. Tritiated nucleosides were added to the media of measles virus infected Vero cells and negative-strand (genome) RNA with a specific activity of 6X10 5 c.p.m./μg was purified from viral nucleocapsids. 50 S RNA was the sole RNA present in nucleocapsids and self-annealed to 50% due to the presence of 25% 50 S plus-strands (anti-genomes). (Auth.)

  6. The Evolution of Lineage-Specific Regulatory Activities in the Human Embryonic Limb

    OpenAIRE

    Cotney, Justin; Leng, Jing; Yin, Jun; Reilly, Steven K.; DeMare, Laura E.; Emera, Deena; Ayoub, Albert E.; Rakic, Pasko; Noonan, James P.

    2013-01-01

    The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find...

  7. Estimation of the specific activity of radioiodinated gonadotrophins: comparison of three methods

    Energy Technology Data Exchange (ETDEWEB)

    Englebienne, P [Centre for Research and Diagnosis in Endocrinology, Kain (Belgium); Slegers, G [Akademisch Ziekenhuis, Ghent (Belgium). Lab. voor Analytische Chemie

    1983-01-14

    The authors compared 3 methods for estimating the specific activity of radioiodinated gonadotrophins. Two of the methods (column recovery and isotopic dilution) gave similar results, while the third (autodisplacement) gave significantly higher estimations. In the autodisplacement method, B/T ratios, obtained when either labelled hormone alone, or labelled and unlabelled hormone, are added to the antibody, were compared as estimates of the mass of hormone iodinated. It is likely that immunologically unreactive impurities present in the labelled hormone solution invalidate such comparison.

  8. Multiple containment for LSA [low specific activity] and SCO [surface contaminated objects] wastes

    International Nuclear Information System (INIS)

    Burgess, M.H.

    1993-09-01

    Radioactive wastes are generally transported in the form of Low Specific Activity (LSA) materials or Surface Contaminated Objects (SCO). This report proposes that a method of acknowledging the beneficial effects of multiple containment for such wastes should be written into the 1996 Edition of the IAEA Transport Regulations. Experience used to assess risks from on-site movements of radioactive material in the UK can be applied to develop safety arguments justifying the alleviation of off-site transport risks. (UK)

  9. Method of preparing tritium-labelled thymidine-5'-monophosphates of high specific activity

    International Nuclear Information System (INIS)

    Filip, J.; Vesely, J.; Cihak, A.

    1976-01-01

    A method is described of preparing thymidine-5'-monophosphates labelled with tritium of high specific activity based on enzyme synthesis in vitro. Phosphorylation was carried out using the catalytic effect of an enzyme contained in the supernatant fraction prepared from Yoshida ascites carcinoma in rats. The course of the enzyme reaction can be controlled by the concentration of the individual reaction mixture components. The method described allows obtaining thymidine-5'-monophosphate of radiochemical purity better than 95%. (J.B.)

  10. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

    Directory of Open Access Journals (Sweden)

    Raj Kumar

    2008-08-01

    Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

  11. A benefit-cost framework of motivation for a specific activity.

    Science.gov (United States)

    Studer, B; Knecht, S

    2016-01-01

    How can an individual be motivated to perform a target exercise or activity? This question arises in training, therapeutic, and education settings alike, yet despite-or even because of-the large range of extant motivation theories, finding a clear answer to this question can be challenging. Here we propose an application-friendly framework of motivation for a specific activity or exercise that incorporates core concepts from several well-regarded psychological and economic theories of motivation. The key assumption of this framework is that motivation for performing a given activity is determined by the expected benefits and the expected costs of (performance of) the activity. Benefits comprise positive feelings, gains, and rewards experienced during performance of the activity (intrinsic benefits) or achieved through the activity (extrinsic benefits). Costs entail effort requirements, time demands, and other expenditure (intrinsic costs) as well as unwanted associated outcomes and missing out on alternative activities (extrinsic costs). The expected benefits and costs of a given exercise are subjective and state dependent. We discuss convergence of the proposed framework with a selection of extant motivation theories and briefly outline neurobiological correlates of its main components and assumptions. One particular strength of our framework is that it allows to specify five pathways to increasing motivation for a target exercise, which we illustrate and discuss with reference to previous empirical data. © 2016 Elsevier B.V. All rights reserved.

  12. The length of a lantibiotic hinge region has profound influence on antimicrobial activity and host specificity

    Directory of Open Access Journals (Sweden)

    Liang eZhou

    2015-01-01

    Full Text Available Lantibiotics are ribosomally synthesized (methyllanthionine containing peptides which can efficiently inhibit the growth of Gram-positive bacteria. As lantibiotics kill bacteria efficiently and resistance to them is difficult to be obtained, they have the potential to be used in many applications, e.g. in pharmaceutical industry or food industry. Nisin can inhibit the growth of Gram-positive bacteria by binding to lipid II and by making pores in their membrane. The C-terminal part of nisin is known to play an important role during translocation over the membrane and forming pore complexes. However, as the thickness of bacterial membranes varies between different species and environmental conditions, this property could have an influence on the pore forming activity of nisin. To investigate this, the so-called hinge region of nisin (residues NMK was engineered to vary from one to six amino acid residues and specific activity against different indicators was compared. Antimicrobial activity in liquid culture assays showed that wild type nisin is most active, while truncation of the hinge region dramatically reduced the activity of the peptide. However, one or two amino acids extensions showed only slightly reduced activity against most indicator strains. Notably, some variants (+2, +1, -1, -2 exhibited higher antimicrobial activity than nisin in agar well diffusion assays against Lactococcus lactis MG1363, Listeria monocytogenes, Enterococcus faecalis VE14089, Bacillus sporothermodurans IC4 and Bacillus cereus 4153 at certain temperatures.

  13. Levels for the specific activity at disposing low-level contaminated municipal wastes

    International Nuclear Information System (INIS)

    Poschner, J.; Schaller, G.

    1993-12-01

    Using radioecological models, nuclide-specific ''basic values'' were established for the specific activity of radioactive contaminated waste. These basic values were determined in order to avoid dose equivalents of more than 10 μSv/a (de minimis concept) when disposing the waste in conventional waste deposits or burning it in incineration plants. The basic values vary within 16 orders of magnitude, ranging between 4.3E-02 Bq/g for Cm-250 and 2.2E+14 Bq/g for Po-212. About 82% of the basic values are between 1 Bq/g und 1000 Bq/g. The critical exposure for most of the radionuclides is that from direct radiation or inhalation at the slag-deposit. For 54% of the radionuclides the basic value for the specific activity of waste is lower at least by a factor of 10 than the 10 -4 -fold value per gram of the release limit (German Radiation Protection Ordinance). Appropriate nuclide-specific and dose-related limits were derived from these basic values. The respective limits are ranging between 1E-01 Bq/g and 1E+07 Bq/g. About 95% of the limits are between 1 Bq/g and 1000 Bq/g. (orig./HP) [de

  14. Guidance and methods for satisfying low specific activity material and surface contaminated object regulatory requirements

    International Nuclear Information System (INIS)

    Pope, R.B.; Shappert, L.B.; Michelhaugh, R.D.; Boyle, R.W.; Easton, E.P.; Coodk, J.R.

    1998-01-01

    The U.S. Department of Transportation (DOT) and the U.S. Nuclear Regulatory Commission (NRC) have prepared a comprehensive set of draft guidance for shippers and inspectors to use when applying the newly imposed regulatory requirements for low specific activity (LSA) material and surface contaminated objects (SCOs). These requirements represent significant departures in some areas from the manner in which these materials and objects were regulated by the earlier versions of the regulations. The proper interpretation and application of the regulatory criteria can require a fairly complex set of decisions be made. To assist those trying these regulatory requirements, a detailed set of logic-flow diagrams representing decisions related to multiple factors were prepared and included in the draft report for comment on Categorizing and Transporting Low Specific Activity Materials and Surface Contaminated Objects, (DOT/NRC, 1997). These logic-flow diagrams, as developed, are specific to the U.S. regulations, but were readily adaptable to the IAEA regulations. The diagrams have been modified accordingly and tied directly to specific paragraphs in IAEA Safety Series No. 6. This paper provides the logic-flow diagrams adapted in the IAEA regulations, and demonstrated how these diagrams can be used to assist consignors and inspectors in assessing compliance of shipments with the LSA material and SCO regulatory requirements. (authors)

  15. Selective radiolabeling of cell surface proteins to a high specific activity

    International Nuclear Information System (INIS)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-01-01

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125 I-(hydroxyphenyl)propionyl groups via 125 I-sulfosuccinimidyl (hydroxyphenyl)propionate ( 125 II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[ 125 I]iodide labeling technique revealed that 125 I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125 I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane

  16. Determination of specific activity of phosphorus-32 labelled o-phosphoric acid

    International Nuclear Information System (INIS)

    Sane, S.U.

    2015-01-01

    Phosphorus-32 is one of the important radioisotopes used in therapeutic nuclear medicine. This work was aimed at developing a fast and sensitive procedure to determine trace amounts of 32 P which is present in various acidic chemical form thereby enabling to determine its specific activity. The method utilizes ammonium molybdate and metol for complexing with phosphorus in presence of sulphuric acid which was measured using UV-VIS spectrophotometer. The phosphate and molybdate ions form a stable complex which turns blue (molybdenum blue) by reduction with sulphuric acid. The absorbance of the complex thus formed was measured at 700 nm. Five batches of 32 P produced were analyzed using the procedure and specific activity was determined. It was found that radioactivity of 32 P did not interfere in absorbance measurements and the method could be successfully adopted for the determination of specific activity of 32 P. A scope of the method is to find the chemical purity of radioactive phosphorus ( 32 P) in quality control analysis. (author)

  17. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  18. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro.

    Science.gov (United States)

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H

    2014-03-01

    Pestivirus N(pro) is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N(pro) blocks the host׳s interferon response by inducing degradation of interferon regulatory factor-3. N(pro׳)s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N(pro)-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N(pro) proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N(pro׳)s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N(pro) does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila.

    Directory of Open Access Journals (Sweden)

    Yuri G Strukov

    2011-01-01

    Full Text Available The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding

  20. The interaction between early life epilepsy and autistic-like behavioral consequences: a role for the mammalian target of rapamycin (mTOR pathway.

    Directory of Open Access Journals (Sweden)

    Delia M Talos

    Full Text Available Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1 signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46, phospho-p70S6K (Thr389 and phospho-S6 (Ser235/236, as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308 and phospho-ERK (Thr202/Tyr204. Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.

  1. Branched-chain amino acids enhance premature senescence through mammalian target of rapamycin complex I-mediated upregulation of p21 protein.

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    Masayuki Nakano

    Full Text Available Branched-chain amino acids (BCAAs have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer's ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.

  2. Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

    Directory of Open Access Journals (Sweden)

    Jeffrey W Koehler

    2007-02-01

    Full Text Available We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

  3. The maximum specific hydrogen-producing activity of anaerobic mixed cultures: definition and determination.

    Science.gov (United States)

    Mu, Yang; Yang, Hou-Yun; Wang, Ya-Zhou; He, Chuan-Shu; Zhao, Quan-Bao; Wang, Yi; Yu, Han-Qing

    2014-06-10

    Fermentative hydrogen production from wastes has many advantages compared to various chemical methods. Methodology for characterizing the hydrogen-producing activity of anaerobic mixed cultures is essential for monitoring reactor operation in fermentative hydrogen production, however there is lack of such kind of standardized methodologies. In the present study, a new index, i.e., the maximum specific hydrogen-producing activity (SHAm) of anaerobic mixed cultures, was proposed, and consequently a reliable and simple method, named SHAm test, was developed to determine it. Furthermore, the influences of various parameters on the SHAm value determination of anaerobic mixed cultures were evaluated. Additionally, this SHAm assay was tested for different types of substrates and bacterial inocula. Our results demonstrate that this novel SHAm assay was a rapid, accurate and simple methodology for determining the hydrogen-producing activity of anaerobic mixed cultures. Thus, application of this approach is beneficial to establishing a stable anaerobic hydrogen-producing system.

  4. Antiviral activity and specific modes of action of bacterial prodigiosin against Bombyx mori nucleopolyhedrovirus in vitro.

    Science.gov (United States)

    Zhou, Wei; Zeng, Cheng; Liu, RenHua; Chen, Jie; Li, Ru; Wang, XinYan; Bai, WenWen; Liu, XiaoYuan; Xiang, TingTing; Zhang, Lin; Wan, YongJi

    2016-05-01

    Prodigiosin, the tripyrrole red pigment, is a bacterial secondary metabolite with multiple bioactivities; however, the antiviral activity has not been reported yet. In the present study, we found the antiviral activity of bacterial prodigiosin on Bombyx mori nucleopolyhedrovirus (BmNPV)-infected cells in vitro, with specific modes of action. Prodigiosin at nontoxic concentrations selectively killed virus-infected cells, inhibited viral gene transcription, especially viral early gene ie-1, and prevented virus-mediated membrane fusion. Under prodigiosin treatment, both progeny virus production and viral DNA replication were significantly inhibited. Fluorescent assays showed that prodigiosin predominantly located in cytoplasm which suggested it might interact with cytoplasm factors to inhibit virus replication. In conclusion, the present study clearly indicates that prodigiosin possesses significant antiviral activity against BmNPV.

  5. The maximum specific hydrogen-producing activity of anaerobic mixed cultures: definition and determination

    Science.gov (United States)

    Mu, Yang; Yang, Hou-Yun; Wang, Ya-Zhou; He, Chuan-Shu; Zhao, Quan-Bao; Wang, Yi; Yu, Han-Qing

    2014-06-01

    Fermentative hydrogen production from wastes has many advantages compared to various chemical methods. Methodology for characterizing the hydrogen-producing activity of anaerobic mixed cultures is essential for monitoring reactor operation in fermentative hydrogen production, however there is lack of such kind of standardized methodologies. In the present study, a new index, i.e., the maximum specific hydrogen-producing activity (SHAm) of anaerobic mixed cultures, was proposed, and consequently a reliable and simple method, named SHAm test, was developed to determine it. Furthermore, the influences of various parameters on the SHAm value determination of anaerobic mixed cultures were evaluated. Additionally, this SHAm assay was tested for different types of substrates and bacterial inocula. Our results demonstrate that this novel SHAm assay was a rapid, accurate and simple methodology for determining the hydrogen-producing activity of anaerobic mixed cultures. Thus, application of this approach is beneficial to establishing a stable anaerobic hydrogen-producing system.

  6. Phosphatidylinositol-specific phospholipase C activity in Lactobacillus rhamnosus with capacity to translocate.

    Science.gov (United States)

    Rodriguez, A V; Baigorí, M D; Alvarez, S; Castro, G R; Oliver, G

    2001-10-16

    Phosphatidylinositol-specific phospholipase C (PI-PLC) activity was investigated in 25 different lactic acid bacteria (LAB) strains belonging to the genera Lactobacillus, Weisella, and Enterococcus. PI-PLC activity was detected in 44% of the strains studied in culture medium without carbon source. From the PI-PLC positive strains, Lactobacillus rhamnosus ATCC 7469 was selected for translocation studies. Healthy mice were orally administered with a daily dose of 2.0 x 10(9) of viable L. rhamnosus suspension. Viable bacteria were detected in liver and spleen of mice fed with LAB for 7 days. Bacterial colonies isolated from liver were biochemically characterized, and further subjected to randomly amplified polymorphic DNA. Amplification patterns of five strains displayed identical profiles to L. rhamnosus. PI-PLC activity was determined in the strains recovered from liver.

  7. Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

    Science.gov (United States)

    Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

    2014-04-10

    Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. White blood cell counts mediate the effects of physical activity on prostate-specific antigen levels.

    Science.gov (United States)

    Loprinzi, Paul D; Richart, Sarah M

    2014-09-01

    The purpose of this study was to examine whether white blood cell (WBC) level mediated the relationship between physical activity and prostate-specific antigen (PSA) levels. Data from the 2003-2006 National Health and Nutrition Examination Survey were used; 1,726 U.S. adult men (aged 40 years or older) provided complete data on the study variables. Participants wore an ActiGraph 7164 accelerometer for a 7-day period to measure their physical activity behavior, and PSA and WBC levels were obtained from a blood sample. After adjustments, results showed that moderate-to-vigorous physical activity (MVPA) was inversely associated with WBC count (b = - .03; 95% CI [ - 0.04, - 0.006; p = .01), and WBC count (b = .10; 95% CI [0.009, 0.18; p = .04) was positively associated with PSA. Both the Sobel (coef. = - .004, SE = .002; z = - 2.0; p = .03) and the Aroian (coef. = - .004, SE = .002; z = - 1.9; p = .03) tests demonstrated that WBC mediated the relationship between physical activity and PSA. Additionally, among 107 participants with prostate cancer, survivors engaging in more MVPA had lower levels of WBC (b = - .04; 95% CI [ - 0.09, - 0.0009; p = .04). Conclusion Physical activity may influence PSA levels through WBC modulation; however, future research is needed to determine the direction of causality. Additionally, prostate cancer survivors engaging in higher levels of MVPA had lower levels of WBC, underscoring the importance of promoting physical activity among prostate cancer survivors.

  9. Active training and driving-specific feedback improve older drivers' visual search prior to lane changes

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    Lavallière Martin

    2012-03-01

    Full Text Available Abstract Background Driving retraining classes may offer an opportunity to attenuate some effects of aging that may alter driving skills. Unfortunately, there is evidence that classroom programs (driving refresher courses do not improve the driving performance of older drivers. The aim of the current study was to evaluate if simulator training sessions with video-based feedback can modify visual search behaviors of older drivers while changing lanes in urban driving. Methods In order to evaluate the effectiveness of the video-based feedback training, 10 older drivers who received a driving refresher course and feedback about their driving performance were tested with an on-road standardized evaluation before and after participating to a simulator training program (Feedback group. Their results were compared to a Control group (12 older drivers who received the same refresher course and in-simulator active practice as the Feedback group without receiving driving-specific feedback. Results After attending the training program, the Control group showed no increase in the frequency of the visual inspection of three regions of interests (rear view and left side mirrors, and blind spot. In contrast, for the Feedback group, combining active training and driving-specific feedbacks increased the frequency of blind spot inspection by 100% (32.3 to 64.9% of verification before changing lanes. Conclusions These results suggest that simulator training combined with driving-specific feedbacks helped older drivers to improve their visual inspection strategies, and that in-simulator training transferred positively to on-road driving. In order to be effective, it is claimed that driving programs should include active practice sessions with driving-specific feedbacks. Simulators offer a unique environment for developing such programs adapted to older drivers' needs.

  10. Active training and driving-specific feedback improve older drivers' visual search prior to lane changes.

    Science.gov (United States)

    Lavallière, Martin; Simoneau, Martin; Tremblay, Mathieu; Laurendeau, Denis; Teasdale, Normand

    2012-03-02

    Driving retraining classes may offer an opportunity to attenuate some effects of aging that may alter driving skills. Unfortunately, there is evidence that classroom programs (driving refresher courses) do not improve the driving performance of older drivers. The aim of the current study was to evaluate if simulator training sessions with video-based feedback can modify visual search behaviors of older drivers while changing lanes in urban driving. In order to evaluate the effectiveness of the video-based feedback training, 10 older drivers who received a driving refresher course and feedback about their driving performance were tested with an on-road standardized evaluation before and after participating to a simulator training program (Feedback group). Their results were compared to a Control group (12 older drivers) who received the same refresher course and in-simulator active practice as the Feedback group without receiving driving-specific feedback. After attending the training program, the Control group showed no increase in the frequency of the visual inspection of three regions of interests (rear view and left side mirrors, and blind spot). In contrast, for the Feedback group, combining active training and driving-specific feedbacks increased the frequency of blind spot inspection by 100% (32.3 to 64.9% of verification before changing lanes). These results suggest that simulator training combined with driving-specific feedbacks helped older drivers to improve their visual inspection strategies, and that in-simulator training transferred positively to on-road driving. In order to be effective, it is claimed that driving programs should include active practice sessions with driving-specific feedbacks. Simulators offer a unique environment for developing such programs adapted to older drivers' needs.

  11. Preclinical evaluation of somatostatin analogs bearing two macrocyclic chelators for high specific activity labeling with radiometals

    International Nuclear Information System (INIS)

    Storch, D.; Schmitt, J.S.; Waldherr, C.; Maecke, H.R.; Waser, B.; Reubi, J.C.

    2007-01-01

    Radiometallated analogues of the regulatory peptide somatostatin are of interest in the in vivo localization and targeted radiotherapy of somatostatin receptor-overexpressing tumors. An important aspect of their use in vivo is a fast and efficient labeling (complexation) protocol for radiometals along with a high specific activity. We describe in this manuscript synthetic methods for the coupling of two chelators (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid = DOTA) to the bioactive peptide [Tyr 3 ,Thr 8 ]-octreotide (TATE) in order to increase the specific activity (radioactivity in Bq per mole peptide). The full chelator-linker-peptide conjugate was assembled on solid support using standard Fmoc chemistry. Two DOTA-chelators were linked to the peptide using lysine or N,N'-bis(3-aminopropyl)-glycine (Apg); in addition, pentasarcosine (Sar 5 ) was used as a spacer between the chelators and the peptide to probe its influence on biology and pharmacology. Complexation rates with In 3+ and Y 3+ salts and the corresponding radiometals were high, the bis-DOTA-derivatives showed higher complexation rates and gave higher specific activity than DOTA-TATE. Pharmacological and biological data of the complexed molecules did not show significant differences if compared to the parent peptide [ 111/nat In-DOTA]-TATE except for [( 111/nat In-DOTA) 2 -Apg]-TATE which showed a lower binding affinity and rate of internalization into tumor cells. The biodistribution of [( 111/nat In-DOTA)-Lys( 111/nat In-DOTA)]-TATE in the rat tumor model (AR4-2J) showed a high and specific (as shown by a blocking experiment) tracer uptake in somatostatin receptor-positive tissue but a lower tumor uptake compared to [ 111/nat In-DOTA]-TATE. (orig.)

  12. Patterns of muscle activity underlying object-specific grasp by the macaque monkey.

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    Brochier, T; Spinks, R L; Umilta, M A; Lemon, R N

    2004-09-01

    During object grasp, a coordinated activation of distal muscles is required to shape the hand in relation to the physical properties of the object. Despite the fundamental importance of the grasping action, little is known of the muscular activation patterns that allow objects of different sizes and shapes to be grasped. In a study of two adult macaque monkeys, we investigated whether we could distinguish between EMG activation patterns associated with grasp of 12 differently shaped objects, chosen to evoke a wide range of grasping postures. Each object was mounted on a horizontal shuttle held by a weak spring (load force 1-2 N). Objects were located in separate sectors of a "carousel," and inter-trial rotation of the carousel allowed sequential presentation of the objects in pseudorandom order. EMG activity from 10 to 12 digit, hand, and arm muscles was recorded using chronically implanted electrodes. We show that the grasp of different objects was characterized by complex but distinctive patterns of EMG activation. Cluster analysis shows that these object-related EMG patterns were specific and consistent enough to identify the object unequivocally from the EMG recordings alone. EMG-based object identification required a minimum of six EMGs from simultaneously recorded muscles. EMG patterns were consistent across recording sessions in a given monkey but showed some differences between animals. These results identify the specific patterns of activity required to achieve distinct hand postures for grasping, and they open the way to our understanding of how these patterns are generated by the central motor network.

  13. Experimental study on active specific immunotherapy utilizing the immune reaction of low-dose irradiated tumor tissue, 4

    International Nuclear Information System (INIS)

    Imanaka, Kazufumi; Ogawa, Yasuhiro; Gose, Kyuhei; Imajo, Yoshinari; Kimura, Shuji

    1982-01-01

    We have already reported that the effectiveness of active specific immunotherapy using low-dose irradiated tumor cells and infiltrating mononuclear cells. The present study was designed to investigate the effect of non-specific immunopotentiator combined with the active specific immunotherapy. Twelve-week-aged female C3H/He mice transplanted with MM46 tumor were received local radiotherapy with the dose of 3,000 rads by high energy electron beam on the sixth day after inoculation. Active specific immunotherapy was performed on the 13th day, and daily dose of 1.0 KE of OK-432 was injected intraperitoneally from the 13th day to the 17th day. The inhibition of the tumor growth and the elongation of survival period were noted in the group which received active specific immunotherapy combined with non-specific immunopotentiator, OK-432 compared with that active specific immunotherapy alone. (author)

  14. Sex-specific genetic effects in physical activity: results from a quantitative genetic analysis.

    Science.gov (United States)

    Diego, Vincent P; de Chaves, Raquel Nichele; Blangero, John; de Souza, Michele Caroline; Santos, Daniel; Gomes, Thayse Natacha; dos Santos, Fernanda Karina; Garganta, Rui; Katzmarzyk, Peter T; Maia, José A R

    2015-08-01

    The objective of this study is to present a model to estimate sex-specific genetic effects on physical activity (PA) levels and sedentary behaviour (SB) using three generation families. The sample consisted of 100 families covering three generations from Portugal. PA and SB were assessed via the International Physical Activity Questionnaire short form (IPAQ-SF). Sex-specific effects were assessed by genotype-by-sex interaction (GSI) models and sex-specific heritabilities. GSI effects and heterogeneity were tested in the residual environmental variance. SPSS 17 and SOLAR v. 4.1 were used in all computations. The genetic component for PA and SB domains varied from low to moderate (11% to 46%), when analyzing both genders combined. We found GSI effects for vigorous PA (p = 0.02) and time spent watching television (WT) (p < 0.001) that showed significantly higher additive genetic variance estimates in males. The heterogeneity in the residual environmental variance was significant for moderate PA (p = 0.02), vigorous PA (p = 0.006) and total PA (p = 0.001). Sex-specific heritability estimates were significantly higher in males only for WT, with a male-to-female difference in heritability of 42.5 (95% confidence interval: 6.4, 70.4). Low to moderate genetic effects on PA and SB traits were found. Results from the GSI model show that there are sex-specific effects in two phenotypes, VPA and WT with a stronger genetic influence in males.

  15. Performance Effects of Repetition Specific Gluteal Activation Protocols on Acceleration in Male Rugby Union Players

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    Barry Lorna

    2016-12-01

    Full Text Available Warm-up protocols have the potential to cause an acute enhancement of dynamic sprinting performance. The purpose of this study was to evaluate the effects of three repetition specific gluteal activation warm-up protocols on acceleration performance in male rugby union players. Forty male academy rugby union players were randomly assigned to one of 4 groups (control, 5, 10 or 15 repetition gluteal activation group and performed 10 m sprints at baseline and 30 s, 2, 4, 6 and 8 min after their specific intervention protocol. Five and ten meter sprint times were the dependent variable and dual-beam timing gates were used to record all sprint times. Repeated measures analysis of variance found no significant improvement in 5 and 10 m sprint times between baseline and post warm-up scores (p ≥ 0.05 for all groups. There were no reported significant differences between groups at any of the rest interval time points (p ≥ 0.05. However, when individual responses to the warm-up protocols were analyzed, the 15 repetition gluteal activation group had faster 10 m times post-intervention and this improvement was significant (p = 0.021. These results would indicate that there is no specific rest interval for any of the gluteal interventions that results in a potentiation effect on acceleration performance. However, the individual response analysis would seem to indicate that a 15 repetition gluteal activation warm-up protocol has a potentiating effect on acceleration performance provided that the rest interval is adequately and individually determined.

  16. Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts.

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    Linda Petrone

    2015-11-01

    Full Text Available Cystic echinococcosis (CE is a complex disease caused by Echinococcus granulosus (E.granulosus, and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS, the presence of CE specific immune signatures.We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25 and "inactive stages" (17. The ability of CD4+ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4+ T-cells producing IL-2+TNF-α+Th2+ or TNF-α+Th2+ were significantly increased in the "active stages" group compared to the "inactive stages" group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE.We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2+TNF-α+Th2+ triple-positive and TNF-α+Th2+ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and

  17. CD4~+Foxp3~+ regulatory T cells converted by rapamycin from peripheral CD4~+ CD25~-naive T cells display more potent regulatory ability in vitro

    Institute of Scientific and Technical Information of China (English)

    CHEN Jian-fei; GAO Jie; ZHANG Dong; WANG Zi-han; ZHU Ji-ye

    2010-01-01

    Background Rapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells. Methods Peripheral CD4~+CD25~- naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4~+CD25~- T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4~+CD25~+CD127~- subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4~+CD25~+ T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1×10~5 carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4~+CD25~- T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25×10~5/well) in 96-well round-bottom plates for 6 days. Then 1×10~5 or 0.25×10~5 converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4~+CD25~- T cells using flow cytometry. Data were analyzed using CellQuest software.Results We found that RAPA can convert peripheral CD4~+CD25~- naive T Cells to CD4~+Foxp3~+ Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity. Conclusion Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and

  18. Levels for the specific activity at disposing low-level contaminated municipal wastes

    International Nuclear Information System (INIS)

    Poschner, J.; Schaller, G.

    1995-01-01

    Using radioecological models, nuclide specific values were calculated for the specific activity of low contaminated radioactive waste, which is disposed in conventional waste deposits or burned in incineration plants. The calculation of these values is based on a limit of 10 μSv effective dose in one year, i.e. effective dose possibly resulting from waste disposal or burning should not exceed a 'de-minimis'-value of some 10 μSv per year. The applied radioecological models describe exposure of the public by direct radiation, inhalation and ingestion for the operational period of a deposit or an incineration plant, but also cover post-operational scenarios, collecting and sorting of waste and road accidents of the waste-truck. Referring to the dose limit of 10 μSv/a, a value for the specific activity of waste was calculated for each scenario and each radionuclide considered. The smallest of these values for a radionuclide, the 'basic value' was rounded to a 'reference value'. For about 600 radionuclides reference values were derived. About 90% of the reference values are ranging between 1 and 1 000 Bq/g. For about 90% of the radionuclides direct radiation or inhalation at the deposit proved to be the critical path of exposure. (orig.) [de

  19. Biocatalytically Oligomerized Epicatechin with Potent and Specific Anti-proliferative Activity for Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ramaswamy Nagarajan

    2008-11-01

    Full Text Available Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP as a catalyst yielding water-soluble oligo(epicatechins. The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.

  20. Evaluation of the specificity and effectiveness of selected oral hygiene actives in salivary biofilm microcosms.

    Science.gov (United States)

    Ledder, Ruth G; Sreenivasan, Prem K; DeVizio, William; McBain, Andrew J

    2010-12-01

    The microbiological effects of biocidal products used for the enhancement of oral hygiene relate to the active compound(s) as well as other formulation components. Here, we test the specificities of selected actives in the absence of multiple excipients. Salivary ecosystems were maintained in tissue culture plate-based hydroxyapatite disc models (HDMs) and modified drip-flow biofilm reactors (MDFRs). Test compounds stannous fluoride (SF), SDS, triclosan (TCS), zinc lactate (ZL) and ZL with SF in combination (ZLSF) were delivered to the HDMs once and four times daily for 6 days to MDFRs. Plaques were characterized by differential viable counting and PCR-denaturing gradient gel electrophoresis (DGGE). TCS and SDS were the most effective compounds against HDM plaques, significantly reducing total viable counts (P<0.05), whilst SF, ZL and ZLSF were comparatively ineffective. TCS exhibited specificity for streptococci (P<0.01) and Gram-negative anaerobes (P<0.01) following a single dosing and also on repeated dosing in MDFRs. In contrast to single exposures, multiple dosing with ZLSF also significantly reduced all bacterial groups, whilst SF and ZL caused significant but transient reductions. According to PCR-DGGE analyses, significant (P<0.05) reductions in eubacterial diversity occurred following 6 day dosing with both TCS and ZLSF. Concordance of MDFR eubacterial profiles with salivary inocula ranged between 58 and 97%. TCS and ZL(SF) exhibited similar specificities to those reported for formulations. TCS was the most potent antibacterial, after single and multiple dosage regimens.

  1. Rapamycin combined with anti-CD45RB mAb and IL-10 or with G-CSF induces tolerance in a stringent mouse model of islet transplantation.

    Directory of Open Access Journals (Sweden)

    Nicola Gagliani

    Full Text Available BACKGROUND: A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg cells. Among the various Treg-cell types, Foxp3(+Treg and IL-10-producing T regulatory type 1 (Tr1 cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell-associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model. We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent. METHODOLOGY/PRINCIPAL FINDINGS: Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3(+Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4(+IL-10(+IL-4(- T (i.e., Tr1 cells localized in the spleen. In long-term tolerant mice, only CD4(+IL-10(+IL-4(- T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF; this drug combination promoted tolerance associated with CD4(+IL-10(+IL-4(- T cells. CONCLUSIONS/SIGNIFICANCE: The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces

  2. Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002.

    Science.gov (United States)

    Goltsov, Alexey; Tashkandi, Ghassan; Langdon, Simon P; Harrison, David J; Bown, James L

    2017-01-15

    The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC 50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC 50 on ATP concentration that allows prediction of the IC 50 at different ATP concentrations in enzyme and cellular assays. Comparison of drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K

  3. Endurance exercise training induces fat depot-specific differences in basal autophagic activity

    International Nuclear Information System (INIS)

    Tanaka, Goki; Kato, Hisashi; Izawa, Tetsuya

    2015-01-01

    The purpose of this study was to uncover the effect of exercise training on the expression of autophagy marker proteins in epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), and the stromal vascular fraction (SVF) collected from eWAT. Male Wistar rats aged 4–5 weeks were randomly divided into two groups, sedentary control (n = 7) and exercise-trained (n = 7). Rats in the exercise-trained group were exercised on a treadmill set at a 5° incline 5 days/week for 9 weeks. We determined that the expression levels of an autophagosome-associating form of microtubule-associated protein 1 light chain 3 (LC3)-II and of p62 were significantly higher in eWAT from exercise-trained than from control rats, while those of adipose-specific deletion of autophagy-related protein (ATG7) and lysosomal-associated membrane protein type 2A (LAMP2a) showed no difference between groups. However, in iWAT, the expression levels of LC3-II and ATG7 were significantly higher in exercise-trained than in control rats. The expression of p62 was highly correlated with that of peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and lipid metabolism, in both WAT types (eWAT, r = 0.856, P < 0.05; iWAT, r = 0.762, P < 0.05), whereas LC3-II and PPARγ levels were highly correlated in eWAT (r = 0.765, P < 0.05) but not in iWAT (r = −0.306, ns). In SVF, the expression levels of LC3II, ATG7, and LAMP2a were significantly higher in exercise-trained than in control rats. These results suggest that exercise training suppresses basal autophagy activity in eWAT, but that this activity is enhanced in iWAT and SVF collected from eWAT. Thus, the adaptation of basal autophagic activity following exercise training exhibits fat depot-specific differences. - Highlights: • Autophagy has been associated with obesity and associated diseases. • We examined exercise-associated rat white adipose tissue (WAT) autophagy markers. • Exercise increased

  4. Endurance exercise training induces fat depot-specific differences in basal autophagic activity

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    Tanaka, Goki; Kato, Hisashi; Izawa, Tetsuya, E-mail: tizawa@mail.doshisha.ac.jp

    2015-10-23

    The purpose of this study was to uncover the effect of exercise training on the expression of autophagy marker proteins in epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), and the stromal vascular fraction (SVF) collected from eWAT. Male Wistar rats aged 4–5 weeks were randomly divided into two groups, sedentary control (n = 7) and exercise-trained (n = 7). Rats in the exercise-trained group were exercised on a treadmill set at a 5° incline 5 days/week for 9 weeks. We determined that the expression levels of an autophagosome-associating form of microtubule-associated protein 1 light chain 3 (LC3)-II and of p62 were significantly higher in eWAT from exercise-trained than from control rats, while those of adipose-specific deletion of autophagy-related protein (ATG7) and lysosomal-associated membrane protein type 2A (LAMP2a) showed no difference between groups. However, in iWAT, the expression levels of LC3-II and ATG7 were significantly higher in exercise-trained than in control rats. The expression of p62 was highly correlated with that of peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and lipid metabolism, in both WAT types (eWAT, r = 0.856, P < 0.05; iWAT, r = 0.762, P < 0.05), whereas LC3-II and PPARγ levels were highly correlated in eWAT (r = 0.765, P < 0.05) but not in iWAT (r = −0.306, ns). In SVF, the expression levels of LC3II, ATG7, and LAMP2a were significantly higher in exercise-trained than in control rats. These results suggest that exercise training suppresses basal autophagy activity in eWAT, but that this activity is enhanced in iWAT and SVF collected from eWAT. Thus, the adaptation of basal autophagic activity following exercise training exhibits fat depot-specific differences. - Highlights: • Autophagy has been associated with obesity and associated diseases. • We examined exercise-associated rat white adipose tissue (WAT) autophagy markers. • Exercise increased

  5. Targeted radiotherapy of multicell neuroblastoma spheroids with high specific activity [125I]meta-iodobenzylguanidine

    International Nuclear Information System (INIS)

    Roa, Wilson H.Y.; Miller, Gerald G.; McEwan, Alexander J.B.; McQuarrie, Steve A.; Tse, Jeanie; Wu, Jonn; Wiebe, Leonard I.

    1998-01-01

    Purpose: Iodine-125 induces cell death by a mechanism similar to that of high linear energy transfer (high-LET) radiation. This study investigates the cytotoxicity of high-specific-activity [ 125 I]meta-iodobenzylguanidine ( 125 I-mIBG) in human SK-N-MC neuroblastoma cells grown as three-dimensional multicellular spheroids. Materials and Methods: Spheroids were incubated with high-specific-activity 125 I-mIBG (6 mCi/μg, 1000 times that of the conventional specific activity used for autoradiography). Cytotoxicity was assessed by fluorescence viability markers and confocal microscopy for intact spheroids, fluorescence-activated cell sorting and clonogenic assay, and clonogenic assays for dispersed whole spheroids. Distribution of radioactive mIBG was determined by quantitative light-microscope autoradiography of spheroid cryostat sections. Dose estimation was based on temporal knowledge of the retained radioactivity inside spheroids, and of the radiolabel's emission characteristics. Findings were compared with those of spheroids treated under the same conditions with 131 I-mIBG, cold mIBG, and free iodine-125. Results: 125 I-mIBG exerted significant cell killing. Complete spheroids were eradicated when they were treated with 500 μCi of 125 I-mIBG, while those treated with 500 μCi or 1000 μCi of 131 I-mIBG were not. The observed difference in cytotoxicity between treatments with 125 I- and 131 I-mIBG could not be accounted for by the absorbed dose of spheroid alone. The peripheral, proliferating cell layer of the spheroids remained viable at the moderate radioactivity of 100 μCi for both isotopes. Cytotoxicity induced by 125 I-mIBG was quantitatively comparable by the peripheral rim thickness to that of 131 I-mIBG at the dose of 100 μCi. The peripheral rim thickness decreased most significantly in the first 17 hours after initial treatment. There was no statistical decrease in the rim thickness identified afterwards for the second, third, and fourth days of

  6. Cell-type-specific activation of mitogen-activated protein kinases in PAN-induced progressive renal disease in rats

    International Nuclear Information System (INIS)

    Park, Sang-Joon; Jeong, Kyu-Shik

    2004-01-01

    We examined the time-course activation and the cell-type specific role of MAP kinases in puromycin aminonucleoside (PAN)-induced renal disease. The maximal activation of c-Jun-NH 2 -terminal kinase (JNK), extracellular signal regulated kinase (ERK), and p38 MAP kinase was detected on Days 52, 38, and 38 after PAN-treatment, respectively. p-JNK was localized in mesangial and proximal tubular cells at the early renal injury. It was expressed, therefore, in the inflammatory cells of tubulointerstitial lesions. While, p-ERK was markedly increased in the glomerular regions and macrophages p-p38 was observed in glomerular endothelial cells, tubular cells, and some inflammatory cells. The results show that the activation of MAP kinases in the early renal injury by PAN-treatment involves cellular changes such as cell proliferation or apoptosis in renal native cells. The activation of MAP kinases in infiltrated inflammatory cells and fibrotic cells plays an important role in destructive events such as glomerulosclerosis and tubulointerstitial fibrosis

  7. A central role for the mammalian target of rapamycin in LPS-induced anorexia in mice.

    Science.gov (United States)

    Yue, Yunshuang; Wang, Yi; Li, Dan; Song, Zhigang; Jiao, Hongchao; Lin, Hai

    2015-01-01

    Bacterial lipopolysaccharide (LPS), also known as endotoxin, induces profound anorexia. However, the LPS-provoked pro-inflammatory signaling cascades and the neural mechanisms underlying the development of anorexia are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. This study aimed to determine whether the mTOR pathway is involved in LPS-induced anorexia. Effects of LPS on hypothalamic gene/protein expression in mice were measured by RT-PCR or western blotting analysis. To determine whether inhibition of mTOR signaling could attenuate LPS-induced anorexia, we administered an i.c.v. injection of rapamycin, an mTOR inhibitor, on LPS-treated male mice. In this study, we showed that LPS stimulates the mTOR signaling pathway through the enhanced phosphorylation of mTOR(Ser2448) and p70S6K(Thr389). We also showed that LPS administration increased the phosphorylation of FOXO1(Ser256), the p65 subunit of nuclear factor kappa B (Panorexia by decreasing the phosphorylation of p70S6K(Thr389), FOXO1(Ser256), and FOXO1/3a(Thr) (24) (/) (32). These results suggest promising approaches for the prevention and treatment of LPS-induced anorexia. © 2015 Society for Endocrinology.

  8. Frequency specific activity in subthalamic nucleus correlates with hand bradykinesia in Parkinson's disease.

    Science.gov (United States)

    Tan, Huiling; Pogosyan, Alek; Anzak, Anam; Foltynie, Thomas; Limousin, Patricia; Zrinzo, Ludvic; Ashkan, Keyoumars; Bogdanovic, Marko; Green, Alexander L; Aziz, Tipu; Brown, Peter

    2013-02-01

    Local field potential recordings made from the basal ganglia of patients undergoing deep brain stimulation have suggested that frequency specific activity is involved in determining the rate of force development and the peak force at the outset of a movement. However, the extent to which the basal ganglia might be involved in motor performance later on in a sustained contraction is less clear. We therefore recorded from the subthalamic nucleus region (STNr) in patients with Parkinson's disease (PD) as they made maximal voluntary grips. Relative to age-matched controls they had more rapid force decrement when contraction was meant to be sustained and prolonged release reaction time and slower rate of force offset when they were supposed to release the grip. These impairments were independent from medication status. Increased STNr power over 5-12 Hz (in the theta/alpha band) independently predicted better performance-reduced force decrement, shortened release reaction time and faster rate of force offset. In contrast, lower mean levels and progressive reduction of STNr power over 55-375 Hz (high gamma/high frequency) over the period when contraction was meant to be sustained were both strongly associated with greater force decrement over time. Higher power over 13-23 Hz (low beta) was associated with more rapid force decrement during the period when grip should have been sustained, and with a paradoxical shortening of the release reaction time. These observations suggest that STNr activities at 5-12 Hz and 55-375 Hz are necessary for optimal grip performance and that deficiencies of such activities lead to motor impairments. In contrast, increased levels of 13-25 Hz activity both promote force decrement and shorten the release reaction time, consistent with a role in antagonising (and terminating) voluntary movement. Frequency specific oscillatory activities in the STNr impact on motor performance from the beginning to the end of a voluntary grip. Copyright © 2012

  9. Application of the quantitative autoradiography for determination of specific activity of labelled non-metallic inclusions

    International Nuclear Information System (INIS)

    Kowalczyk, J.T.; Wilczynski, A.W.

    1983-01-01

    The knowledge of specific activity of labelled non-metallic inclusions, i.e. the knowledge of the content of the radiotracer in a single inclusion, allows to obtain new information about the mechanism and the kinetics of steel deoxidation. In order to determine this specific activity quantitative autoradiography was used. Fo; this purpose, various standards of aluminium oxides with different amounts of cerium oxide Ce 2 O 3 and an aluminium-cerium alloy were prepared. The standards and the alloy were activated with thermal neutrons. Then several autoradiographs were made for these standards (ORWO AF-3 films were used). The autoradiographs served as the basis for evaluation of the standardization curves: optical density versus dimension of particles for a constant cerium concentration; optical density versus concentration of cerium for a constant dimension of particle. The samples of liquid steel were deoxidated with Al-Ce alloy. After labelled non-metallic inclusions had been isolated, the autoradiographs were made under the same conditions as for the standards. The standardization curves were used to determine the cerium content in the single inclusions. (author)

  10. Production of high-specific activity radionuclides using SM high-flux reactor

    International Nuclear Information System (INIS)

    Karelin, Ye.A.; Toporov, Yu.G.; Filimonov, V.T.; Vakhetov, F.Z.; Tarasov, V.A.; Kuznetsov, R.A.; Lebedev, V.M.; Andreev, O.I.; Melnik, M.I.; Gavrilov, V.D.

    1997-01-01

    The development of High Specific Activity (HSA) radionuclides production technologies is one of the directions of RIAR activity, and the high flux research reactor SM, having neutron flux density up to 2.10 15 cm -2 s 1 in a wide range of neutron spectra hardness, plays the principal role in this development. The use of a high-flux reactor for radionuclide production provides the following advantages: production of radionuclides with extremely high specific activity, decreasing of impurities content in irradiated targets (both radioactive and non-radioactive), cost-effective use of expensive isotopically enriched target materials. The production technologies of P-33, Gd-153, W-188, Ni-63, Fe-55,59, Sn-113,117m,119m, Sr- 89, applied in industry, nuclear medicine, research, etc, were developed by RIAR during the last 5-10 years. The research work included the development of calculation procedures for radionuclide reactor accumulation forecast, experimental determination of neutron cross-sections, the development of irradiated materials reprocessing procedures, isolation and purification of radionuclides. The principal results are reviewed in the paper. (authors)

  11. Using ion-selective electrode for determining iodine-131 preparation specific activity

    International Nuclear Information System (INIS)

    Melnik, M.I.; Nazirova, T.E.

    2002-01-01

    A pilot facility was developed in 2000 for the production of iodine-131. The parameters of the preparation are as follows: chemical form: sodium iodide solution (NaI-131) in a carbonate-bicarbonate buffer (or in 0.001M NaOH); specific activity: carrier free (> 5 Ci/mg); solution pH: 7-10; radionuclide purity: > 99.9%; radiochemical purity: > 97%; bulk activity: 0.15 Ci/ml. The experimental results of investigation aimed at the determination of the specific activity of the I-131 preparation using a iodine-selective electrode are described. The method enables the analytical concentration of iodide ions in the carbonate-bicarbonate buffer (pH = 8-11) and NaOH solution (0.01 mol/l, pH = 8-11) to be determined. A micro-cell has been developed for the analysis of the I-131 solution allowing the sample volume to be reduced to below 0.3 ml. The relative error of determination of the analytical concentration of iodide (10 -6 to 10 -1 mol/l) does not exceed 1%

  12. Calibration of context-specific survey items to assess youth physical activity behaviour.

    Science.gov (United States)

    Saint-Maurice, Pedro F; Welk, Gregory J; Bartee, R Todd; Heelan, Kate

    2017-05-01

    This study tests calibration models to re-scale context-specific physical activity (PA) items to accelerometer-derived PA. A total of 195 4th-12th grades children wore an Actigraph monitor and completed the Physical Activity Questionnaire (PAQ) one week later. The relative time spent in moderate-to-vigorous PA (MVPA % ) obtained from the Actigraph at recess, PE, lunch, after-school, evening and weekend was matched with a respective item score obtained from the PAQ's. Item scores from 145 participants were calibrated against objective MVPA % using multiple linear regression with age, and sex as additional predictors. Predicted minutes of MVPA for school, out-of-school and total week were tested in the remaining sample (n = 50) using equivalence testing. The results showed that PAQ β-weights ranged from 0.06 (lunch) to 4.94 (PE) MVPA % (P PAQ and accelerometer MVPA at school and out-of-school ranged from -15.6 to +3.8 min and the PAQ was within 10-15% of accelerometer measured activity. This study demonstrated that context-specific items can be calibrated to predict minutes of MVPA in groups of youth during in- and out-of-school periods.

  13. High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

    Science.gov (United States)

    Kretz, Colin A; Tomberg, Kärt; Van Esbroeck, Alexander; Yee, Andrew; Ginsburg, David

    2018-02-12

    We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10 7 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

  14. Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Kevin V. Solomon

    2016-12-01

    Full Text Available Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV, and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1 were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae. We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production. Keywords: Pseudomonas, Isobutanol, Dehydrogenase, Mitochondria, Saccharomyces cerevisiae, Metabolic engineering

  15. Gender specific changes in cortical activation patterns during exposure to artificial gravity

    Science.gov (United States)

    Schneider, Stefan; Robinson, Ryan; Smith, Craig; von der Wiesche, Melanie; Goswami, Nandu

    2014-11-01

    Keeping astronauts healthy during long duration spaceflight remains a challenge. Artificial gravity (AG) generated by a short arm human centrifuges (SAHC) is proposed as the next generation of integrated countermeasure devices that will allow human beings to safely spend extended durations in space, although comparatively little is known about any psychological side effects of AG on brain function. 16 participants (8 male and 8 female, GENDER) were exposed to 10 min at a baseline gravitational load (G-Load) of +.03 Gz, then 10 min at +.6 Gz for females and +.8 Gz for males, before being exposed to increasing levels of AG in a stepped manner by increasing the acceleration by +.1 Gz every 3 min until showing signs of pre-syncope. EEG recordings were taken of brain activity during 2 min time periods at each AG level. Analysing the results of the mixed total population of participants by two way ANOVA, a significant effect of centrifugation on alpha and beta activity was found (p<.01). Furthermore results revealed a significant interaction between G-LOAD and GENDER alpha-activity (p<.01), but not for beta-activity. Although the increase in alpha and beta activity with G-LOAD does not reflect a general model of cortical arousal and therefore cannot support previous findings reporting that AG may be a cognitively arousing environment, the gender specific responses identified in this study may have wider implications for EEG and AG research.

  16. Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity.

    Science.gov (United States)

    Fezza, Filomena; Gasperi, Valeria; Mazzei, Cinzia; Maccarrone, Mauro

    2005-04-01

    A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.

  17. Structural and biochemical analysis of atypically low dephosphorylating activity of human dual-specificity phosphatase 28.

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    Bonsu Ku

    Full Text Available Dual-specificity phosphatases (DUSPs constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central β-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.

  18. Immunohistochemical analysis of the mechanistic target of rapamycin and hypoxia signalling pathways in basal cell carcinoma and trichoepithelioma.

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    Tjinta Brinkhuizen

    Full Text Available BACKGROUND: Basal cell carcinoma (BCC is the most common cancer in Caucasians. Trichoepithelioma (TE is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1 and mechanistic/mammalian target of rapamycin (mTOR are key players in these pathways. OBJECTIVES: To determine whether HIF1/mTOR signalling is involved in BCC and TE. METHODS: We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45 and TE (n = 35 samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, 80%. RESULTS: Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3, 73% and 75% (CAIX, 79% and 86% (GLUT1, 50% and 19% (HIF1α, 89% and 88% (pAKT, 55% and 61% (pS6, 15% and 25% (pMTOR, 44% and 63% (PHD2 and 44% and 49% (VEGF-A. CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included. CONCLUSIONS: HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE.

  19. Expression and mechanism of mammalian target of rapamycin in age-related renal cell senescence and organ aging.

    Science.gov (United States)

    Zhuo, Li; Cai, Guangyan; Liu, Fuyou; Fu, Bo; Liu, Weiping; Hong, Quan; Ma, Qiang; Peng, Youming; Wang, Jianzhong; Chen, Xiangmei

    2009-10-01

    The mammalian target of rapamycin (mTOR) is relevant to cell senescence and organismal aging. This study firstly showed that the level of mTOR expression increased with aging in rat kidneys, rat mesangial cells and WI-38 cells (P aging-related phenotypes were all reduced in cells treated with rapamycin (an inhibitor of mTOR) than in control cells (P aging, and that mTOR may promote cellular senescence by regulating the cell cycle through p21(WAF1/CIP1/SDI1), which might provide a new target for preventing renal aging.

  20. CDX2 Stimulates the Proliferation of Porcine Intestinal Epithelial Cells by Activating the mTORC1 and Wnt/β-Catenin Signaling Pathways.

    Science.gov (United States)

    Fan, Hong-Bo; Zhai, Zhen-Ya; Li, Xiang-Guang; Gao, Chun-Qi; Yan, Hui-Chao; Chen, Zhe-Sheng; Wang, Xiu-Qi

    2017-11-18

    Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/β-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/β-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/β-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/β-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H -thiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/β-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/β-catenin signaling pathways.

  1. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions.

    Science.gov (United States)

    Walkiewicz, Katarzyna W; Girault, Jean-Antoine; Arold, Stefan T

    2015-10-01

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein 'nanomachines' to become activated in a site-specific manner. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Tissue-specific metabolic activation and mutagenicity of 3-nitrobenzanthrone in MutaMouse.

    Science.gov (United States)

    Chen, Guosheng; Gingerich, John; Soper, Lynda; Douglas, George R; White, Paul A

    2008-10-01

    3-Nitrobenzanthrone (3-NBA) is a mutagen and suspected human carcinogen detected in diesel exhaust, airborne particulate matter, and urban soil. We investigated the tissue specific mutagenicity of 3-NBA at the lacZ locus of transgenic MutaMouse following acute single dose or 28-day repeated-dose oral administration. In the acute high dose (50 mg/kg) exposure, increased lacZ mutant frequency was observed in bone marrow and colonic epithelium, but not in liver and bladder. In the repeated-dose study, a dose-dependent increase in lacZ mutant frequency was observed in bone marrow and liver (2- and 4-fold increase above control), but not in lung or intestinal epithelium. In addition, a concentration-dependent increase in mutant frequency (8.5-fold above control) was observed for MutaMouse FE1 lung epithelial cells exposed in vitro. 1-Nitropyrene reductase, 3-NBA reductase, and acetyltransferase activities were measured in a variety of MutaMouse specimens in an effort to link metabolic activation and mutagenicity. High 3-NBA nitroreductase activities were observed in lung, liver, colon and bladder, and detectable N-acetyltransferase activities were found in all tissues except bone marrow. The relatively high 3-NBA nitroreductase activity in MutaMouse tissues, as compared with those in Salmonella TA98 and TA100, suggests that 3-NBA is readily reduced and activated in vivo. High 3-NBA nitroreductase levels in liver and colon are consistent with the elevated lacZ mutant frequency values, and previously noted inductions of hepatic DNA adducts. Despite an absence of induced lacZ mutations, the highest 3-NBA reductase activity was detected in lung. Further studies are warranted, especially following inhalation or intratracheal exposures. Published 2008 Wiley-Liss, Inc.

  3. MicroRNA-99a inhibits insulin-induced proliferation, migration, dedifferentiation, and rapamycin resistance of vascular smooth muscle cells by inhibiting insulin-like growth factor-1 receptor and mammalian target of rapamycin

    International Nuclear Information System (INIS)

    Zhang, Zi-wei; Guo, Rui-wei; Lv, Jin-lin; Wang, Xian-mei; Ye, Jin-shan; Lu, Ni-hong; Liang, Xing; Yang, Li-xia

    2017-01-01

    Patients with type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and are subsequently at high risk for atherosclerosis. Hyperinsulinemia has been associated with proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) during the pathogenesis of atherosclerosis. Moreover, insulin-like growth factor-1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) have been demonstrated to be the underlying signaling pathways. Recently, microRNA-99a (miR-99a) has been suggested to regulate the phenotypic changes of VSMCs in cancer cells. However, whether it is involved in insulin-induced changes of VSCMs has not been determined. In this study, we found that insulin induced proliferation, migration, and dedifferentiation of mouse VSMCs in a dose-dependent manner. Furthermore, the stimulating effects of high-dose insulin on proliferation, migration, and dedifferentiation of mouse VSMCs were found to be associated with the attenuation of the inhibitory effects of miR-99a on IGF-1R and mTOR signaling activities. Finally, we found that the inducing effect of high-dose insulin on proliferation, migration, and dedifferentiation of VSMCs was partially inhibited by an active mimic of miR-99a. Taken together, these results suggest that miR-99a plays a key regulatory role in the pathogenesis of insulin-induced proliferation, migration, and phenotype conversion of VSMCs at least partly via inhibition of IGF-1R and mTOR signaling. Our results provide evidence that miR-99a may be a novel target for the treatment of hyperinsulinemia-induced atherosclerosis. - Highlights: • Suggesting a new mechanism of insulin-triggered VSMC functions. • Providing a new therapeutic strategies that target atherosclerosis in T2DM patients. • Providing a new strategies that target in-stent restenosis in T2DM patients.

  4. Prolonged activation of virus-specific CD8+T cells after acute B19 infection.

    Directory of Open Access Journals (Sweden)

    Adiba Isa

    2005-12-01

    Full Text Available Human parvovirus B19 (B19 is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously.The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA-peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%-0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low.This is the first example to our knowledge of an "acute" human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation--analogous to that for cytomegalovirus infection--is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development.

  5. Prolonged activation of virus-specific CD8+T cells after acute B19 infection.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available BACKGROUND: Human parvovirus B19 (B19 is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. METHODS AND FINDINGS: The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA-peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%-0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low. CONCLUSION: This is the first example to our knowledge of an "acute" human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation--analogous to that for cytomegalovirus infection--is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development.

  6. Interpretation of biological-rate coefficients derived from radionuclide content, radionuclide concentration and specific activity experiments

    International Nuclear Information System (INIS)

    Vanderploeg, H.A.; Booth, R.S.

    1976-01-01

    Rigorous expressions are derived for the biological-rate coefficients (BRCs) determined from time-dependent measurements of three different dependent variables of radionuclide tracer experiments. These variables, which apply to a single organism, are radionuclide content, radionuclide concentration and specific activity. The BRCs derived from these variables have different mathematical expressions and, for high growth rates, their numerical values can be quite different. The precise mathematical expressions for the BRCs are presented here to aid modelers in selecting the correct parameters for their models and to aid experiments in interpreting their results. The usefulness of these three variables in quantifying elemental uptakes and losses by organisms is discussed. (U.K.)

  7. Optimization of the synthesis of a high specific activity 125I-labelled hapten for radioimmunoassays

    International Nuclear Information System (INIS)

    Suraez, C.; Paz, D.; Simon, M. A.; Romero del Hombrebueno, B.

    1994-01-01

    In this first report it is described the synthesis, separation and purification of the 2-radioiodinated histamine- I-labelled histamine by a mixed anhydride reaction. About 75% incorporation of I - 125, from Na 1 25I, was achieved with a molecular ratio of 1:1 mixed anhydride:histamine. The radiochemical purity of the conjugate by TLC was > 99% and its theoretical specific activity, 3850 μCi/μg. Dissolved in ethanol and held at -20 degree centigree under darkness decomposition on storage didn't exceed 1% per month. (Author) 13 refs

  8. Optimization of the synthesis of a high specific activity 125 I-labelled hapten for radioimmunoassays

    International Nuclear Information System (INIS)

    Suarez, C.; Simon, M.A.; Paz, D.; Romero del Hombrebueno, B.

    1994-01-01

    In this first report it is described the synthesis, separation and purification of the 2-radioiodinated histamine ''125 I-labelled histamine by a mixed anhydride reaction. About 75% incorporation of I''1125, from Na''125, I, was achieved with a molecular ratio of 1:1 mixed anhydride:histamine. The radiochemical purity of the conjugate by TLC was >99% and its theoretical specific activity, 3850 mu Ci/mug. Dissolved in ethanol and held at -20 degree centigree under darkness decomposition on storage did not exceed 1% per month

  9. Application of modeling methods for an estimation of a specific activity 137Cs geologic environment

    International Nuclear Information System (INIS)

    Kalinovskij, A.K.; Batij, V.G.; Pravdivyj, A.A.; Krasnov, V.A.

    2004-01-01

    The manual application of methods of mathematical and physical modeling for an estimation of the specific activity 137 Cs in soils composing a geological profile of site of object 'Ukryttya' is demonstrated. The calculations are executed by the software packages of Micro Shield, CYCLON, MCNP5. The experimental measurements are carried out by logging radiometers of different type on the borehole models. The value of a conversion coefficient of infinite environment for quantitative interpretation of gamma-ray logging data is determined over calculations outcomes and experimental measurements. The calculated and experimental values have agreement among themselves. the error estimation of the obtained outcomes is executed. 26 refs., 3 tab., 10 figs

  10. The effect of operational conditions on the sludge specific methanogenic activity and sludge biodegradability

    Energy Technology Data Exchange (ETDEWEB)

    Leitao, R. C.; Santaella, S. T.; Haandel, A. C. van; Zeeman, G.; Lettinga, G.

    2009-07-01

    The Specific Methanogenic Activity (SMA) and sludge biodegradability of an anaerobic sludge depends on various operational and environmental conditions imposed to the anaerobic reactor. However, the effects of hydraulic retention time (HRT), influent COD concentration (COD{sub i}nf) and sludge retention time (SRT) on those two parameters need to be elucidated. This knowledge about SMA can provide insights about the capacity of the UASB reactors to withstand organic and hydraulic shock loads, whereas the biodegradability gives information necessary for final disposal of the sludge. (Author)

  11. The effect of operational conditions on the sludge specific methanogenic activity and sludge biodegradability

    International Nuclear Information System (INIS)

    Leitao, R. C.; Santaella, S. T.; Haandel, A. C. van; Zeeman, G.; Lettinga, G.

    2009-01-01

    The Specific Methanogenic Activity (SMA) and sludge biodegradability of an anaerobic sludge depends on various operational and environmental conditions imposed to the anaerobic reactor. However, the effects of hydraulic retention time (HRT), influent COD concentration (COD i nf) and sludge retention time (SRT) on those two parameters need to be elucidated. This knowledge about SMA can provide insights about the capacity of the UASB reactors to withstand organic and hydraulic shock loads, whereas the biodegradability gives information necessary for final disposal of the sludge. (Author)

  12. Soil Moisture Active Passive (SMAP) Mission Level 4 Carbon (L4_C) Product Specification Document

    Science.gov (United States)

    Glassy, Joe; Kimball, John S.; Jones, Lucas; Reichle, Rolf H.; Ardizzone, Joseph V.; Kim, Gi-Kong; Lucchesi, Robert A.; Smith, Edmond B.; Weiss, Barry H.

    2015-01-01

    This is the Product Specification Document (PSD) for Level 4 Surface and Root Zone Soil Moisture (L4_SM) data for the Science Data System (SDS) of the Soil Moisture Active Passive (SMAP) project. The L4_SM data product provides estimates of land surface conditions based on the assimilation of SMAP observations into a customized version of the NASA Goddard Earth Observing System, Version 5 (GEOS-5) land data assimilation system (LDAS). This document applies to any standard L4_SM data product generated by the SMAP Project.

  13. The Hyperpolarization-Activated Current Determines Synaptic Excitability, Calcium Activity and Specific Viability of Substantia Nigra Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Carmen Carbone

    2017-06-01

    Full Text Available Differential vulnerability between Substantia Nigra pars compacta (SNpc and Ventral Tegmental Area (VTA dopaminergic (DAergic neurons is a hallmark of Parkinson’s disease (PD. Understanding the molecular bases of this key histopathological aspect would foster the development of much-needed disease-modifying therapies. Non-heterogeneous DAergic degeneration is present in both toxin-based and genetic animal models, suggesting that cellular specificity, rather than causing factors, constitutes the background for differential vulnerability. In this regard, we previously demonstrated that MPP+, a neurotoxin able to cause selective nigrostriatal degeneration in animal rodents and primates, inhibits the Hyperpolarization-activated current (Ih in SNpc DAergic neurons and that pharmacological Ih antagonism causes potentiation of evoked Excitatory post-synaptic potentials (EPSPs. Of note, the magnitude of such potentiation is greater in the SNpc subfield, consistent with higher Ih density. In the present work, we show that Ih block-induced synaptic potentiation leads to the amplification of somatic calcium responses (SCRs in vitro. This effect is specific for the SNpc subfield and largely mediated by L-Type calcium channels, as indicated by sensitivity to the CaV 1 blocker isradipine. Furthermore, Ih is downregulated by low intracellular ATP and determines the efficacy of GABAergic inhibition in SNpc DAergic neurons. Finally, we show that stereotaxic administration of Ih blockers causes SNpc-specific neurodegeneration and hemiparkinsonian motor phenotype in rats. During PD progression, Ih downregulation may result from mitochondrial dysfunction and, in concert with PD-related disinhibition of excitatory inputs, determine a SNpc-specific disease pathway.

  14. Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab.

    Science.gov (United States)

    Galli, Valentina; Sebastian, Rafael; Moutel, Sandrine; Ecard, Jason; Perez, Franck; Roux, Aurélien

    2017-10-12

    Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.

  15. Specific methanogenic activity (SMA of industrial sludge from the aerobic and anaerobic biological treatment

    Directory of Open Access Journals (Sweden)

    Danieli Schneiders

    2013-08-01

    Full Text Available In this study, specific methanogenic activity (SMA tests were performed on textile sludge and food industry sludge. The textile sludge from an activated sludge was collected at the entrance of the secondary biologic clarifier and the food sludge was collected in a UASB reactor. Once collected, the sludges were characterized and tested for SMA. It was found that the microrganisms present in the food sludge had SMA of 0.17 gCOD-CH4 gSSV.d-1 and 337.05 mL of methane production, while the microrganisms of the textile sludge presented 0.10 gCOD-CH4 gSSV.d-1 of SMA and 3.04 mL of methane production. Therefore, the food sludge was more suitable to be used as a starting inoculum in UASB.

  16. Effects of oxytetracycline, tylosin, and amoxicillin antibiotics on specific methanogenic activity of anaerobic biomass

    Directory of Open Access Journals (Sweden)

    Mohammad Mehdi Amin

    2012-01-01

    Materials and Methods: To evaluate the inhibitory antibiotics amoxicillin, tetracycline, and tylosin on biomass activity, specific methanogenic activity (SMA using anerobic biomass batch; into 120 ml vials: 30 ml biomass and 70 ml substrate including volatile fatty acids, mainly acetic acid and various concentrations of antibiotics were added. Methane gas production replacement through solution of KOH (2 N as an absorber of CO 2 and bromine thymol blue as indicator was measured. Each batch was tested for 10 days. Results: Based on the findings, inhibitory concentration of oxytetracycline, amoxicillin, and tylosin were 8000, 9000, and 9000 mg/L, respectively. Conclusions: This study showed that with increasing concentrations of antibiotics, the produced biogas volume from biomass per unit weight is decreased. COD removal was 42-82 % due to long retention time and adsorption to flocks.

  17. Specific activity of uranium and thorium in marketable rock phosphate as a function of particle size

    Energy Technology Data Exchange (ETDEWEB)

    Metzger, R; McKlveen, J W [Arizona State Univ., Tempe (USA); Jenkins, R [Phillip Morris Research Center, Richmond, VA (USA); McDowell, W J [Oak Ridge National Lab., TN (USA)

    1980-07-01

    Marketable rock phosphate fertilizer from Florida was classified into seven particle size fractions ranging from 149 ..mu..m to less than 0.5 ..mu..m using a Bahco Microparticle Classifier and air elutriation. The resulting size fractions were assayed for U and /sup 230/Th by solvent extraction and liquid scintillation ..cap alpha..-spectroscopy. Results indicated that the specific activity of U and /sup 230/Th increased with decreasing particle size. Maximum activities of 110 pCi/g U and 50 pCi/g /sup 230/Th were found in particles less than 1.0 ..mu..m in aerodynamic diameter. Qualitative emission spectrographic analysis of the fractions revealed that the concentrations of Al, Cu, Mg, Na, Ti and Zn also increased with decreasing particle size.

  18. Nodal-dependent mesendoderm specification requires the combinatorial activities of FoxH1 and Eomesodermin.

    Directory of Open Access Journals (Sweden)

    Christopher E Slagle

    2011-05-01

    Full Text Available Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA-binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals.

  19. Oxytocin Reduces Cocaine Cued Fos Activation in a Regionally Specific Manner

    Science.gov (United States)

    Leong, Kah-Chung; Freeman, Linnea R; Berini, Carole R; Ghee, Shannon M; See, Ronald E

    2017-01-01

    Abstract Background Oxytocin may be a possible treatment for multiple neuropsychiatric disorders, including cocaine addiction. Little is known about the site-specific effects of oxytocin on various drug addiction-related brain regions. Furthermore, sexually dimorphic effects of oxytocin on neural function in the addiction circuit have not been established. Here, we studied Fos expression following cocaine-cued reinstatement in both male and female rats. Methods Male and female rats underwent self-administration, extinction, and reinstatement tests. On test days, rats were given oxytocin or vehicle, and lever pressing was measured in response to conditioned cocaine cues. Rats were perfused and Fos staining measured in the central amygdala, medial prefrontal cortex, nucleus accumbens core, and subthalamic nucleus. Fos/oxytocin double labeling occurred in the paraventricular nucleus of the hypothalamus. Results Rats reinstated to cocaine cues relative to extinction responding and oxytocin reduced cocaine seeking. Oxytocin combined with contingent cue presentations increased Fos+ oxytocin cell bodies within the paraventricular nucleus of the hypothalamus relative to vehicle. Fos expression robustly increased in the central amygdala following oxytocin administration. Oxytocin reversed cue-induced Fos expression in the medial prefrontal cortex, nucleus accumbens core, and subthalamic nucleus. Central oxytocin infusion also attenuated reinstated cocaine seeking. Conclusions Oxytocin decreased reinstated cocaine seeking, increased Fos activation in the paraventricular nucleus of the hypothalamus and central amygdala, but normalized cue-induced Fos activation in the medial prefrontal cortex, nucleus accumbens core, and subthalamic nucleus, thereby demonstrating regionally specific activation patterns. No sex differences were seen for the effects of oxytocin on cocaine seeking and Fos activation, indicating that oxytocin acts on similar central neural circuits critical to

  20. Continuous High Frequency Activity: A peculiar SEEG pattern related to specific brain regions

    Science.gov (United States)

    Melani, Federico; Zelmann, Rina; Mari, Francesco; Gotman, Jean

    2015-01-01

    Objective While visually marking the high frequency oscillations in the stereo-EEG of epileptic patients, we observed a continuous/semicontinuous activity in the ripple band (80–250 Hz), which we defined continuous High Frequency Activity (HFA). We aim to analyze in all brain regions the occurrence and significance of this particular pattern. Methods Twenty patients implanted in mesial temporal and neocortical areas were studied. One minute of slow-wave sleep was reviewed. The background was classified as continuous/semicontinuous, irregular, or sporadic based on the duration of the fast oscillations. Each channel was classified as inside/outside the seizure onset zone (SOZ) or a lesion. Results The continuous/semicontinuous HFA occurred in 54 of the 790 channels analyzed, with a clearly higher prevalence in hippocampus and occipital lobe. No correlation was found with the SOZ or lesions. In the occipital lobe the continuous/semicontinuous HFA was present independently of whether eyes were open or closed. Conclusions We describe what appears to be a new physiological High Frequency Activity, independent of epileptogenicity, present almost exclusively in the hippocampus and occipital cortex but independent of the alpha rhythm. Significance The continuous HFA may be an intrinsic characteristic of specific brain regions, reflecting a particular type of physiological neuronal activity. PMID:23768436

  1. Passive Stretch Versus Active Stretch on Intervertebral Movement in Non - Specific Neck Pain

    International Nuclear Information System (INIS)

    Abd El - Aziz, A.H.; Amin, D.I.; Moustafa, I.

    2016-01-01

    Neck pain is one of the most common and painful musculoskeletal conditions. Point prevalence ranges from 6% to 22% and up to 38% of the elderly population, while lifetime prevalence ranges from 14,2% to 71%. Up till now no randomized study showed the effect between controversy of active and passive stretch on intervertebral movement. The purpose: the current study was to investigate the effect of the passive and active stretch on intervertebral movement in non - specific neck pain. Material and methods: Forty five subjects from both sexes with age range between 18 and 30 years and assigned in three groups, group I (15) received active stretch, ultrasound and TENS. Group II (15) received passive stretch, ultrasound and TENS. Group III (15) received ultrasound and TENS. The radiological assessment was used to measure rotational and translational movement of intervertebral movement before and after treatment. Results: MANOVA test was used for radiological assessment before and after treatment there was significant increase in intervertebral movement in group I as p value =0.0001. Conclusion: active stretch had a effect in increasing the intervertebral movement compared to the passive stretch

  2. Domain-Specific Self-Reported and Objectively Measured Physical Activity in Children

    Directory of Open Access Journals (Sweden)

    Ole Sprengeler

    2017-03-01

    Full Text Available Little is known about the extent that different domains contribute to total sedentary (SED, light (LPA and moderate-to-vigorous physical activity (MVPA. We aimed to identify domain-specific physical activity (PA patterns in school-aged children who were assessed by questionnaire and accelerometry. For the study, 298 German school children and adolescents aged 6–17 years wore an accelerometer for one week and completed a PA recall-questionnaire for the same period. Spearman coefficients (r were used to evaluate the agreement between self-reported and objectively measured PA in five domains (transport, school hours, physical education, leisure-time, organized sports activities. School hours mainly contributed to the total objectively measured SED, LPA and MVPA (55%, 53% and 46%, respectively, whilst sports activities contributed only 24% to total MVPA. Compared to accelerometry, the proportion of self-reported LPA and MVPA during school hours was substantially underestimated but overestimated during leisure-time. The agreement of self-reported and objectively measured PA was low for total LPA (r = 0.09, 95% CI (confidence interval: −0.03–0.20 and total MVPA (r = 0.21, 95% CI: 0.10–0.32, while moderate agreement was only found for total SED (r = 0.44, 95% CI: 0.34–0.53, LPA during transport (r = 0.59; 95% CI: 0.49–0.67 and MVPA during organized sports activities (r = 0.54; 95% CI: 0.38–0.67. Since school hours mainly contribute to total SED, LPA and MVPA and self-reported LPA and MVPA during school were importantly underestimated compared to objectively measured LPA and MVPA, the application of objective measurements is compulsory to characterize the entire activity pattern of school-aged children.

  3. Development of a GAL4-VP16/UAS trans-activation system for tissue specific expression in Medicago truncatula.

    Directory of Open Access Journals (Sweden)

    Amélie Sevin-Pujol

    Full Text Available Promoters with tissue-specific activity are very useful to address cell-autonomous and non cell autonomous functions of candidate genes. Although this strategy is widely used in Arabidopsis thaliana, its use to study tissue-specific regulation of root symbiotic interactions in legumes has only started recently. Moreover, using tissue specific promoter activity to drive a GAL4-VP16 chimeric transcription factor that can bind short upstream activation sequences (UAS is an efficient way to target and enhance the expression of any gene of interest. Here, we developed a collection of promoters with different root cell layers specific activities in Medicago truncatula and tested their abilities to drive the expression of a chimeric GAL4-VP16 transcription factor in a trans-activation UAS: β-Glucuronidase (GUS reporter gene system. By developing a binary vector devoted to modular Golden Gate cloning together with a collection of adapted tissue specific promoters and coding sequences we could test the activity of four of these promoters in trans-activation GAL4/UAS systems and compare them to "classical" promoter GUS fusions. Roots showing high levels of tissue specific expression of the GUS activity could be obtained with this trans-activation system. We therefore provide the legume community with new tools for efficient modular Golden Gate cloning, tissue specific expression and a trans-activation system. This study provides the ground work for future development of stable transgenic lines in Medicago truncatula.

  4. Activity, specificity, and titer of naturally occurring canine anti-DEA 7 antibodies.

    Science.gov (United States)

    Spada, Eva; Proverbio, Daniela; Baggiani, Luciana; Canzi, Ilaria; Perego, Roberta

    2016-11-01

    The reported prevalence of naturally occurring anti-dog erythrocyte antigen (DEA) 7 antibodies in DEA 7-negative dogs is as high as 50%. Characterization of these antibodies may better define their importance in canine transfusion medicine. We determined in vitro activity, specificity, and titer of anti-DEA 7 antibodies in DEA 7-negative dogs. Plasma samples from 317 DEA 7-negative dogs were cross-matched with DEA 7-positive red blood cells (RBCs) using gel column technology. Agglutination occurred with DEA 7-positive RBCs but not with DEA 7-negative RBCs in 73 samples (23%), which were hence classified as containing anti-DEA 7 antibodies. These samples were evaluated for hemolytic and agglutinating activity, strength of agglutination, and antibody specificity and titers. All samples showed agglutination but none showed hemolysis. Gel agglutination was graded as 1+ for 20 samples (27%), 2+ for 49 samples (67%), 3+ for 4 samples (6%); no samples were graded 4+. The agglutination titer was DEA 7 antibodies were found in 23% of DEA 7-negative dogs. The presence of naturally occurring anti-DEA 7 antibodies suggests that cross-matching of canine blood recipients is advisable, even at first transfusion, to minimize delayed transfusion reactions. © 2016 The Author(s).

  5. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    Science.gov (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  6. Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting.

    Science.gov (United States)

    Wu, Shwu-Yuan; Lee, A-Young; Lai, Hsien-Tsung; Zhang, Hong; Chiang, Cheng-Ming

    2013-03-07

    Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Preparation of a high specific activity I-125 labeled styryl dye for leukocyte membrane labeling

    International Nuclear Information System (INIS)

    Lambert, C.; Mease, R.C.; Le, T.; Sabet, H.; Avren, L.I.; McAfee, J.G.

    1994-01-01

    The purpose of this work was to develop a high specific activity radioiodinated cell membrane probe for tracking lymphocytes in-vivo to replace the nucleus localizing, cytotoxic lipophilic chelates (In-111 oxine and Tc-99m HMPAO) currently used. Alkylation of parent dye 4-[2-[-N,N-didecylamino]phenyl]ethenyl pyridine with E-1-tributylstannyl-3-tosylpropene (prepared form E-1-tributylstannyl-1-propene-3-ol), gave a tributyltin precursor 1. Radiolabeled 3-[4-[2-[4-(N,N-didecylamino)phenyl]ethenyl]pyridino] E-[I-125]-1-iodopropene (2), was prepared from 1 using peracetic acid in acetonitrile/water. Labeling yields and specific activities achieved were 26% (∼2170 Ci/mmol), 40% (1220 Ci/mmol), and 55% (200 Ci/mmol) for nca, 0.4, and 2 nanomole carrier iodide runs respectively. Canine mixed leukocytes (0.5-1.0 x 10 8 cells) were labeled with 2 (67% and 42% yields for 200 Ci/mol and 1220 Ci/mmol preparations) and showed blood clearance similar to In 111 oxine. Radioiodinated styryl dye 2 appears to be a promising leukocyte labeling agent. Imaging studies with I-131 labeled 2 are in progress

  8. Region-specific proteolysis differentially regulates type 1 inositol 1,4,5-trisphosphate receptor activity.

    Science.gov (United States)

    Wang, Liwei; Wagner, Larry E; Alzayady, Kamil J; Yule, David I

    2017-07-14

    The inositol 1,4,5 trisphosphate receptor (IP 3 R) is an intracellular Ca 2+ release channel expressed predominately on the membranes of the endoplasmic reticulum. IP 3 R1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood. Our previous work has demonstrated that IP 3 R1 channels, formed following either enzymatic fragmentation or expression of the corresponding complementary polypeptide chains, retain tetrameric architecture and are still activated by IP 3 binding despite the loss of peptide continuity. In this study, we demonstrate that region-specific receptor fragmentation modifies channel regulation. Specifically, the agonist-evoked temporal Ca 2+ release profile and protein kinase A modulation of Ca 2+ release are markedly altered. Moreover, we also demonstrate that activation of fragmented IP 3 R1 can result in a distinct functional outcome. Our work suggests that proteolysis of IP 3 R1 may represent a novel form of modulation of IP 3 R1 channel function and increases the repertoire of Ca 2+ signals achievable through this channel. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Specific activity and concentration model applied to 137Cs movement in a eutrophic lake

    International Nuclear Information System (INIS)

    Vanderploeg, H.A.; Booth, R.S.; Clark, F.H.

    1976-01-01

    A linear systems-analysis model which simulates time-dependent dynamics of specific activity and concentration of radiocesium in lake ecosystems was applied to a shallow, eutrophic lake that had received a pulse input of 137 Cs. Best estimates of transfer coefficients for abiotic compartments (sediment, interstitial water and lake water) and the macrophyte compartment which controlled the mass balance of cesium in water were determined by ''tuning'' our initial estimates of the transfer coefficients to observed data on 137 Cs concentrations and contents of these compartments. In most cases, the optimized transfer coefficients for the abiotic compartments were not greatly different from our independently derived initial estimates, and the simulations for optimized coefficients were close to those based on initial estimates. The 137 Cs concentrations in water as predicted by the optimized transfer coefficients were then used to calculate 137 Cs kinetics in biota other than macrophytes. In general, model simulations were close to concentrations observed in the biota. The agreement between 137 Cs concentrations and simulations in bottom invertebrates supported our assumption that bottom sediments are not a major source of Cs to the biota. Our specific activity and concentration model was compared to the radionuclide content model, the model used in terrestrial ecosystems. For biotic components of aquatic ecosystems, values of α/sub ij/, the transfer coefficients of our model, are easily estimated from turnover rates of radiocesium in individual organisms in the laboratory

  10. Domain-specific physical activity and health-related quality of life in university students.

    Science.gov (United States)

    Pedišić, Zeljko; Rakovac, Marija; Titze, Sylvia; Jurakić, Danijel; Oja, Pekka

    2014-01-01

    Information on the relationship between domain-specific physical activity (PA) and health-related quality of life (HRQoL) in the general population and specific groups is still scarce. The aim of this study was to determine the relationship between PA in work, transport, domestic and leisure-time domains and HRQoL among university students. PA and HRQoL were assessed in a random stratified sample of 1750 university students using the International Physical Activity Questionnaire - long form and 12-item Short Form Health Survey, respectively. The Spearman's rank correlations, adjusted for age, community size, personal monthly budget, body mass index, smoking habits and alcohol intake ranged from -0.11 to 0.18 in female students and -0.29 to 0.19 in male students. Leisure-time, domestic, transport-related PA and total PA were positively related to HRQoL. Inverse correlations with HRQoL were only found for work-related PA in male students. Multiple linear regression analysis showed that only leisure-time PA was related to the Physical Summary Component score (β = 0.08 for females and β = 0.10 for males, P leisure-time, transport and domestic PA with HRQoL can potentially be used to support evidence-based promotion of PA in a university setting, and as a hypothesis for future longitudinal studies on such potential causal relationships.

  11. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    Directory of Open Access Journals (Sweden)

    Damián ePérez-Mazliah

    2012-09-01

    Full Text Available Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

  12. Layer- and Cell Type-Specific Modulation of Excitatory Neuronal Activity in the Neocortex

    Directory of Open Access Journals (Sweden)

    Gabriele Radnikow

    2018-01-01

    Full Text Available From an anatomical point of view the neocortex is subdivided into up to six layers depending on the cortical area. This subdivision has been described already by Meynert and Brodmann in the late 19/early 20. century and is mainly based on cytoarchitectonic features such as the size and location of the pyramidal cell bodies. Hence, cortical lamination is originally an anatomical concept based on the distribution of excitatory neuron. However, it has become apparent in recent years that apart from the layer-specific differences in morphological features, many functional properties of neurons are also dependent on cortical layer or cell type. Such functional differences include changes in neuronal excitability and synaptic activity by neuromodulatory transmitters. Many of these neuromodulators are released from axonal afferents from subcortical brain regions while others are released intrinsically. In this review we aim to describe layer- and cell-type specific differences in the effects of neuromodulator receptors in excitatory neurons in layers 2–6 of different cortical areas. We will focus on the neuromodulator systems using adenosine, acetylcholine, dopamine, and orexin/hypocretin as examples because these neuromodulator systems show important differences in receptor type and distribution, mode of release and functional mechanisms and effects. We try to summarize how layer- and cell type-specific neuromodulation may affect synaptic signaling in cortical microcircuits.

  13. Plasticity during Sleep Is Linked to Specific Regulation of Cortical Circuit Activity

    Directory of Open Access Journals (Sweden)

    Niels Niethard

    2017-09-01

    Full Text Available Sleep is thought to be involved in the regulation of synaptic plasticity in two ways: by enhancing local plastic processes underlying the consolidation of specific memories and by supporting global synaptic homeostasis. Here, we briefly summarize recent structural and functional studies examining sleep-associated changes in synaptic morphology and neural excitability. These studies point to a global down-scaling of synaptic strength across sleep while a subset of synapses increases in strength. Similarly, neuronal excitability on average decreases across sleep, whereas subsets of neurons increase firing rates across sleep. Whether synapse formation and excitability is down or upregulated across sleep appears to partly depend on the cell’s activity level during wakefulness. Processes of memory-specific upregulation of synapse formation and excitability are observed during slow wave sleep (SWS, whereas global downregulation resulting in elimination of synapses and decreased neural firing is linked to rapid eye movement sleep (REM sleep. Studies of the excitation/inhibition balance in cortical circuits suggest that both processes are connected to a specific inhibitory regulation of cortical principal neurons, characterized by an enhanced perisomatic inhibition via parvalbumin positive (PV+ cells, together with a release from dendritic inhibition by somatostatin positive (SOM+ cells. Such shift towards increased perisomatic inhibition of principal cells appears to be a general motif which underlies the plastic synaptic changes observed during sleep, regardless of whether towards up or downregulation.

  14. Antitumor activity of sequence-specific alkylating agents: pyrolle-imidazole CBI conjugates with indole linker.

    Science.gov (United States)

    Shinohara, Ken-ichi; Bando, Toshikazu; Sasaki, Shunta; Sakakibara, Yogo; Minoshima, Masafumi; Sugiyama, Hiroshi

    2006-03-01

    DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (Ralkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.

  15. miR-30c is specifically repressed in patients with active pulmonary tuberculosis.

    Science.gov (United States)

    Spinelli, Silvana V; Fernández, Rocío Del V; Zoff, Luciana; Bongiovanni, Bettina; Díaz, Ariana; D'Attilio, Luciano; Santucci, Natalia; Alvarez, Tomás; Marchesini, Marcela M; Bogue, Cristina; Bay, Maria L; Bottasso, Oscar A

    2017-07-01

    Tuberculous pleurisy (PLTB) is a common form of extrapulmonary tuberculosis. It often resolves without chemotherapy being hence considered a rather benign manifestation of the disease. Patients with PLTB mount an effective anti-mycobacterial response, unlike those with active pulmonary TB (pTB) that were shown to present an imbalance in plasma immune and endocrine mediators. In this work, we explored whether expression of the active isoform of the glucocorticoid receptor (hGRα) in the context of the inflammatory-anti-inflammatory responses of TB patients may be associated to microRNA levels. As expected, the inflammatory response triggered in patients coexists with increased circulating cortisol and altered hGRα levels in the peripheral blood mononuclear cells. However, while hGRα expression is significantly downregulated in PLTB, its levels in pTB patients are higher within the control values. These results point out to the existence of an additional mechanism tending to preserve hGRα levels probably to deal with the chronic inflammation observed in pTB. In this regard, we found that miR-30c is strongly downregulated in mononuclear cells of pTB patients compared to PLTB cases, showing an expression profile opposite to that seen with hGRα. Interestingly, low levels of miR-30c are specific for this active form of TB, as its expression is not altered in mononuclear cells from either healthy controls or patients with tuberculous or non-tuberculous pleurisy. Moreover, miR-30c and hGRα also showed an inverse expression pattern in M. tuberculosis-stimulated THP-1 macrophage cultures. In sum, our studies identify miR-30c as a specific correlate of pulmonary manifestations of TB, potentially involved in the altered glucocorticoid sensitivity observed in these patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Specific marker of feigned memory impairment: The activation of left superior frontal gyrus.

    Science.gov (United States)

    Chen, Zi-Xiang; Xue, Li; Liang, Chun-Yu; Wang, Li-Li; Mei, Wei; Zhang, Qiang; Zhao, Hu

    2015-11-01

    Faking memory impairment means normal people complain lots of memory problems without organic damage in forensic assessments. Using alternative forced-choice paradigm, containing digital or autobiographical information, previous neuroimaging studies have indicated that faking memory impairment could cause the activation in the prefrontal and parietal regions, and might involve a fronto-parietal-subcortical circuit. However, it is still unclear whether different memory types have influence on faking or not. Since different memory types, such as long-term memory (LTM) and short-term memory (STM), were found supported by different brain areas, we hypothesized that feigned STM or LTM impairment had distinct neural activation mapping. Besides that, some common neural correlates may act as the general characteristic of feigned memory impairment. To verify this hypothesis, the functional magnetic resonance imaging (fMRI) combined with an alternative word forced-choice paradigm were used in this study. A total of 10 right-handed participants, in this study, had to perform both STW and LTM tasks respectively under answering correctly, answering randomly and feigned memory impairment conditions. Our results indicated that the activation of the left superior frontal gyrus and the left medial frontal gyrus was associated with feigned LTM impairment, whereas the left superior frontal gyrus, the left precuneus and the right anterior cingulate cortex (ACC) were highly activated while feigning STM impairment. Furthermore, an overlapping was found in the left superior frontal gyrus, and it suggested that the activity of the left superior frontal gyrus might be acting as a specific marker of feigned memory impairment. Copyright © 2015. Published by Elsevier Ltd.

  17. Experimental study on active specific immunotherapy utilizing the immune reaction of low-dose irradiated tumor tissue, 7

    International Nuclear Information System (INIS)

    Ogawa, Yasuhiro; Maeda, Tomoho; Yoshida, Shoji; Yamamoto, Yoichi; Morita, Masaru

    1983-01-01

    We have already reported the remarkable effect of the active specific immunotherapy utilizing cryopreserved tumor cells and infiltrating mononuclear cells prepared from a lowdose irradiated tumor tissue after cytoreductive radiotherapy. In the present study, the effect of a biological response modifier, PSK combined with this active specific immunotherapy was investigated. Twelve-week-aged female C3H/He mice transplanted with MM46 tumor cells were received local radiotherapy with the dose of 3,000 rads by high energy electron beam on the fifth day after tumor inoculation. This active specific immunotherapy was performed on the twelveth day, and daily dose of 200 mg/kg of PSK was injected intraperitoneally from the sixth day to the tenth day. The more inhibition of the tumor growth was observed in the group which received this active specific immunotherapy combined with a biological response modifier, PSK compared with that received this active specific immunotherapy alone. (author)

  18. Experimental study on active specific immunotherapy utilizing the immune reaction of low-dose irradiated tumor tissue, 5

    International Nuclear Information System (INIS)

    Ogawa, Yasuhiro; Imanaka, Kazufumi; Gose, Kyuhei; Imajo, Yoshinari; Kimura, Shuji

    1982-01-01

    We have already reported the remarkable effect of the active specific immunotherapy utilizing cryopreserved tumor cells and infiltrating mononuclear cells prepared from a low-dose irradiated tumor tissue after cytoreductive radiotherapy. In the present study, the effect of a biological response modifier, OK-432 combined with this active specific immunotherapy was investigated. Twelve-week-aged female C3H/He mice transplanted with MM46 tumor cells were received local radiotherapy with the dose of 3,000 rads by high energy electron beam on the sixth day after inoculation. This active specific immunotherapy was performed on the thirteenth day, and daily dose of 1.0 KE of OK-432 was injected intraperitoneally from the thirteenth day to the seventeenth day. The inhibition of the tumor growth was observed in the group which received this active specific immunotherapy combined with a biological response modifier, OK-432 compared with that received this active specific immunotherapy alone. (author)

  19. Target of rapamycin signalling mediates the lifespan-extending effects of dietary restriction by essential amino acid alteration

    NARCIS (Netherlands)

    Emran, S.; Yang, M.Y.; He, X.L.; Zandveld, J.; Piper, M.D.W.

    2014-01-01

    Dietary restriction (DR), defined as a moderate reduction in food intake short of malnutrition, has been shown to extend healthy lifespan in a diverse range of organisms, from yeast to primates. Reduced signalling through the insulin/IGF-like (IIS) and Target of Rapamycin (TOR) signalling pathways

  20. Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats

    NARCIS (Netherlands)

    Drion, Cato M.; Borm, Lars E.; Kooijman, Lieneke; Aronica, Eleonora; Wadman, Wytse J.; Hartog, Aloysius F.; van Vliet, Erwin A.; Gorter, Jan A.

    2016-01-01

    Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the

  1. Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats.

    NARCIS (Netherlands)

    Drion, C.M.; Borm, L.E.; Kooijman, L.; Aronica, E.; Wadman, W.J.; Hartog, A.F.; van Vliet, E.A.; Gorter, J.A.

    2016-01-01

    OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment

  2. Mechanistic target of rapamycin (MTOR) protein expression in the tumor and its microenvironment correlates with more aggressive pathology at cystectomy

    NARCIS (Netherlands)

    Winters, B.R. (Brian R.); Vakar-Lopez, F. (Funda); Brown, L. (Lisha); Montgomery, B. (Bruce); Seiler, R. (Roland); P.C. Black (Peter C.); J.L. Boormans (Joost); Dall′Era, M. (Marc); Davincioni, E. (Elai); Douglas, J. (James); Gibb, E.A. (Ewan A.); B.W. van Rhijn (Bas); M.S. van der Heijden (Michiel); Hsieh, A.C. (Andrew C.); Wright, J.L. (Jonathan L.); Lam, H.-M. (Hung-Ming)

    2018-01-01

    textabstractBackground: The mechanistic target of rapamycin (mTOR) has been implicated in driving tumor biology in multiple malignancies, including urothelial carcinoma (UC). We investigate how mTOR and phosphorylated mTOR (pmTOR) protein expression correlate with chemoresponsiveness in the tumor

  3. Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53.

    Directory of Open Access Journals (Sweden)

    Kristina Kirschner

    2015-03-01

    Full Text Available The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage and chronically activated (in senescent or pro-apoptotic conditions p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.

  4. Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

    Science.gov (United States)

    Gamba, Elia; Mori, Mattia; Kovalenko, Lesia; Giannini, Alessia; Sosic, Alice; Saladini, Francesco; Fabris, Dan; Mély, Yves; Gatto, Barbara; Botta, Maurizio

    2018-02-10

    In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2

    Science.gov (United States)

    Solli, Ann Iren; Fadnes, Bodil; Winberg, Jan-Olof; Uhlin-Hansen, Lars

    2013-01-01

    Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions. PMID:23482328

  6. Species-specific calls evoke asymmetric activity in the monkey's temporal poles.

    Science.gov (United States)

    Poremba, Amy; Malloy, Megan; Saunders, Richard C; Carson, Richard E; Herscovitch, Peter; Mishkin, Mortimer

    2004-01-29

    It has often been proposed that the vocal calls of monkeys are precursors of human speech, in part because they provide critical information to other members of the species who rely on them for survival and social interactions. Both behavioural and lesion studies suggest that monkeys, like humans, use the auditory system of the left hemisphere preferentially to process vocalizations. To investigate the pattern of neural activity that might underlie this particular form of functional asymmetry in monkeys, we measured local cerebral metabolic activity while the animals listened passively to species-specific calls compared with a variety of other classes of sound. Within the superior temporal gyrus, significantly greater metabolic activity occurred on the left side than on the right, only in the region of the temporal pole and only in response to monkey calls. This functional asymmetry was absent when these regions were separated by forebrain commissurotomy, suggesting that the perception of vocalizations elicits concurrent interhemispheric interactions that focus the auditory processing within a specialized area of one hemisphere.

  7. ChIP-seq Accurately Predicts Tissue-Specific Activity of Enhancers

    Energy Technology Data Exchange (ETDEWEB)

    Visel, Axel; Blow, Matthew J.; Li, Zirong; Zhang, Tao; Akiyama, Jennifer A.; Holt, Amy; Plajzer-Frick, Ingrid; Shoukry, Malak; Wright, Crystal; Chen, Feng; Afzal, Veena; Ren, Bing; Rubin, Edward M.; Pennacchio, Len A.

    2009-02-01

    A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover since they are scattered amongst the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here, we performed chromatin immunoprecipitation with the enhancer-associated protein p300, followed by massively-parallel sequencing, to map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain, and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases revealed reproducible enhancer activity in those tissues predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities and suggest that such datasets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

  8. Alternative method to determine Specific Activity of (177)Lu by HPLC.

    Science.gov (United States)

    Breeman, Wouter A P; de Zanger, Rory M S; Chan, Ho Sze; de Blois, Erik

    2015-01-01

    Peptide Receptor Radionuclide Therapy (PRRT) with (177)Lu-DOTA-peptides requires (177)Lu with high specific activity (SA) and values >740 GBq (177)Lu per mg Lu to maximise the atom% of (177)Lu over total Lu. Vendors provide SA values which are based on activity and mass of the target, whereas due to "burn-up" of target, these SA values are not accurate. For a radiochemist the SA of (177)Lu is of interest prior to radiolabeling. An alternative method to determine SA was developed by HPLC, which includes a metal titration of a known amount of DOTA-peptide with a known amount of activity ((177)Lu), and a unknown amount of metal ((177+nat)Lu). Based on an HPLC separation of radiometal-DOTA-peptide and DOTA-peptide, and the concordant ratio of these components the metal content ((177+nat)Lu) can be calculated, and eventually the SA of (177)Lu can be accurately determined. These experimentally determined SA values exceeded the estimated values provided by vendors by 27 ± 16%, (range 6-73 %). The deviation of SA values for samples from the same Lu batch was <2% (n ≥ 10). the SA of (177)Lu is apparently often higher as stated by vendors in comparison to the experimentally determined actual values. For this reason, the SA of (177)Lu-DOTA-TATE and other Lu-DOTA-peptides could be increased accordingly.

  9. A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.

    Science.gov (United States)

    Maccarrone, M; Bari, M; Agrò, A F

    1999-02-15

    A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low. Copyright 1999 Academic Press.

  10. Inhibitory Effect of Chinese Propolis on Phosphatidylcholine-Specific Phospholipase C Activity in Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hongzhuan Xuan

    2011-01-01

    Full Text Available To understand the mechanisms underlying the anti-inflammatory action of Chinese propolis, we investigated its effect on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC that plays critical roles in control of vascular endothelial cell (VEC function and inflammatory responses. Furthermore, p53 and reactive oxygen species (ROS levels and mitochondrial membrane potential (Δψm were investigated. Our data indicated that treatment of Chinese propolis 6.25 and 12.5 μg/ml for 12 hours increased VEC viability obviously. Exposure to Chinese propolis 6.25, 12.5, and 25 μg/ml for 6 and 12 hours significantly decreased PC-PLC activity and p53 level, and ROS levels were depressed by Chinese propolis 12.5 μg/ml and 25 μg/ml dramatically. The Δψm of VECs was not affected by Chinese propolis at low concentration but disrupted by the propolis at 25 μg/ml significantly, which indicated that Chinese propolis depressed PC-PLC activity and the levels of p53 and ROS in VECs but disrupted Δψm at a high concentration.

  11. Activity and specificity of TRV-mediated gene editing in plants

    KAUST Repository

    Ali, Zahir

    2015-06-03

    © 2015 Taylor and Francis Group, LLC. Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRVmediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.

  12. The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

    Science.gov (United States)

    Lloyd, Chelsea R; Park, Seongjin; Fei, Jingyi; Vanderpool, Carin K

    2017-06-01

    The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria. IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic

  13. Active hydrocarbon (methane) seepage at the Alboran Sea mud volcanoes indicated by specific lipid biomarkers.

    Science.gov (United States)

    Lopez-Rodriguez, C.; Stadnitskaia, A.; De Lange, G. J.; Martínez-Ruiz, F.; Comas, M.; Sinninghe Damsté, J. S.

    2012-04-01

    Mud volcanoes (MVs) and pockmark fields are known to occur in the Alboran Basin (Westernmost Mediterranean). These MVs occur above a major sedimentary depocenter that includes up to 7 km thick early Miocene to Holocene sequences. MVs located on the top of diapiric structures that originated from undercompacted Miocene clays and olistostromes. Here we provide results from geochemical data-analyses of four gravity cores acquired in the Northern Mud Volcano Field (north of the 36°N): i.e. Perejil, Kalinin and Schneiderś Heart mud expulsion structures. Extruded materials include different types of mud breccias. Specific lipid biomarkers (n-alkanes, hopanes, irregular isoprenoid hydrocarbons and Dialkyl Glycerol Diethers (DGDs) were analysed by gas chromatography (GC) and gas chromatography mass spectrometry (GC-MS). Determination of Glycerol Dialkyl Glycerol Tetraethers (GDGTs) by high performance liquid chromatography-spectrometry (HPLC-MS), and analysis of biomarker δ13C values were performed in selected samples. Lipid biomarker analysis from the three MVs revealed similar n-alkane distributions in all mud breccia intervals, showing significant hydrocarbon-derived signals and the presence of thermally immature organic-matter admixture. This suggests that similar strata fed these MVs. The hemipelagic drapes reveal comparable n-alkane distributions, suggesting that significant upward diffusion of fluids occurs. Distributions of GDGTs are generally accepted as usefull biomarkers to locate the anaerobic oxidation of methane (AOM) in marine sediments. However, our GDGT profiles only reflect the marine thaumarchaeotal signature. There seems to be no archaea producing specific GDGTs involved in AOM in the recovered interval. Evidence of recent activity (i.e., methane gas-bubbling and chemosynthetic fauna at the Perejil MV) and the presence of specific lipid biomarker related with methanotropic archaea (Irregular Isoprenoids and DGDs), however, suggest the existence of

  14. Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Pei-Jie Shi

    2016-02-01

    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL is an aggressive malignant disorder of lymphoid progenitor cells in both children and adults. Although improvements in contemporary therapy and development of new treatment strategies have led to dramatic increases in the cure rate in children with ALL, the relapse rate remains high and the prognosis of relapsed childhood ALL is poor. Molecularly targeted therapies have emerged as the leading treatments in cancer therapy. Multi-cytotoxic drug regimens have achieved success, yet many studies addressing targeted therapies have focused on only one single agent. In this study, we attempted to investigate whether the effect of the mammalian target of rapamycin (mTOR inhibitor rapamycin is synergistic with the effect of focal adhesion kinase (FAK down-regulation in the treatment of ALL. Methods The effect of rapamycin combined with FAK down-regulation on cell proliferation, the cell cycle, and apoptosis was investigated in the human precursor B acute lymphoblastic leukemia cells REH and on survival time and leukemia progression in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID mouse model. Results When combined with FAK down-regulation, rapamycin-induced suppression of cell proliferation, G0/G1 cell cycle arrest, and apoptosis were significantly enhanced. In addition, REH cell-injected NOD/SCID mice treated with rapamycin and a short-hairpin RNA (shRNA to down-regulate FAK had significantly longer survival times and slower leukemia progression compared with mice injected with REH-empty vector cells and treated with rapamycin. Moreover, the B-cell CLL/lymphoma-2 (BCL-2 gene family was shown to be involved in the enhancement, by combined treatment, of REH cell apoptosis. Conclusions FAK down-regulation enhanced the in vitro and in vivo inhibitory effects of rapamycin on REH cell growth, indicating that the simultaneous targeting of mTOR- and FAK-related pathways might offer a novel

  15. Development of the radiosynthesis of high-specific-activity {sup 123}I-NKJ64

    Energy Technology Data Exchange (ETDEWEB)

    Tavares, Adriana Alexandre S., E-mail: a.tavares.1@research.gla.ac.u [Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, G12 8QQ Glasgow (United Kingdom); Jobson, Nicola K. [WestCHEM, School of Chemistry, The Joseph Black Building, University of Glasgow, G12 8QQ Glasgow (United Kingdom); Dewar, Deborah [Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, G12 8QQ Glasgow (United Kingdom); Sutherland, Andrew [WestCHEM, School of Chemistry, The Joseph Black Building, University of Glasgow, G12 8QQ Glasgow (United Kingdom); Pimlott, Sally L. [West of Scotland Radionuclide Dispensary, University of Glasgow and North Glasgow University Hospital NHS Trust, G11 6NT Glasgow (United Kingdom)

    2011-05-15

    Introduction: {sup 123}I-NKJ64, a reboxetine analogue, is currently under development as a potential novel single photon emission computed tomography radiotracer for imaging the noradrenaline transporter in brain. This study describes the development of the radiosynthesis of {sup 123}I-NKJ64, highlighting the advantages and disadvantages, pitfalls and solutions encountered while developing the final radiolabelling methodology. Methods: The synthesis of {sup 123}I-NKJ64 was evaluated using an electrophilic iododestannylation method, where a Boc-protected trimethylstannyl precursor was radioiodinated using peracetic acid as an oxidant and deprotection was investigated using either trifluoroacetic acid (TFA) or 2 M hydrochloric acid (HCl). Results: Radioiodination of the Boc-protected trimethylstannyl precursor was achieved with an incorporation yield of 92{+-}6%. Deprotection with 2 M HCl produced {sup 123}I-NKJ64 with the highest radiochemical yield of 98.05{+-}1.63% compared with 83.95{+-}13.24% with TFA. However, the specific activity of the obtained {sup 123}I-NKJ64 was lower when measured after using 2 M HCl (0.15{+-}0.23 Ci/{mu}mol) as the deprotecting agent in comparison to TFA (1.76{+-}0.60 Ci/{mu}mol). Further investigation of the 2 M HCl methodology found a by-product, identified as the deprotected proto-destannylated precursor, which co-eluted with {sup 123}I-NKJ64 during the high-performance liquid chromatography (HPLC) purification. Conclusions: The radiosynthesis of {sup 123}I-NKJ64 was achieved with good isolated radiochemical yield of 68% and a high specific activity of 1.8 Ci/{mu}mol. TFA was found to be the most suitable deprotecting agent, since 2 M HCl generated a by-product that could not be fully separated from {sup 123}I-NKJ64 using the HPLC methodology investigated. This study highlights the importance of HPLC purification and accurate measurement of specific activity while developing new radiosynthesis methodologies.

  16. Development of the radiosynthesis of high-specific-activity 123I-NKJ64

    International Nuclear Information System (INIS)

    Tavares, Adriana Alexandre S.; Jobson, Nicola K.; Dewar, Deborah; Sutherland, Andrew; Pimlott, Sally L.

    2011-01-01

    Introduction: 123 I-NKJ64, a reboxetine analogue, is currently under development as a potential novel single photon emission computed tomography radiotracer for imaging the noradrenaline transporter in brain. This study describes the development of the radiosynthesis of 123 I-NKJ64, highlighting the advantages and disadvantages, pitfalls and solutions encountered while developing the final radiolabelling methodology. Methods: The synthesis of 123 I-NKJ64 was evaluated using an electrophilic iododestannylation method, where a Boc-protected trimethylstannyl precursor was radioiodinated using peracetic acid as an oxidant and deprotection was investigated using either trifluoroacetic acid (TFA) or 2 M hydrochloric acid (HCl). Results: Radioiodination of the Boc-protected trimethylstannyl precursor was achieved with an incorporation yield of 92±6%. Deprotection with 2 M HCl produced 123 I-NKJ64 with the highest radiochemical yield of 98.05±1.63% compared with 83.95±13.24% with TFA. However, the specific activity of the obtained 123 I-NKJ64 was lower when measured after using 2 M HCl (0.15±0.23 Ci/μmol) as the deprotecting agent in comparison to TFA (1.76±0.60 Ci/μmol). Further investigation of the 2 M HCl methodology found a by-product, identified as the deprotected proto-destannylated precursor, which co-eluted with 123 I-NKJ64 during the high-performance liquid chromatography (HPLC) purification. Conclusions: The radiosynthesis of 123 I-NKJ64 was achieved with good isolated radiochemical yield of 68% and a high specific activity of 1.8 Ci/μmol. TFA was found to be the most suitable deprotecting agent, since 2 M HCl generated a by-product that could not be fully separated from 123 I-NKJ64 using the HPLC methodology investigated. This study highlights the importance of HPLC purification and accurate measurement of specific activity while developing new radiosynthesis methodologies.

  17. Convergence of the mammalian target of rapamycin complex 1- and glycogen synthase kinase 3-β-signaling pathways regulates the innate inflammatory response.

    Science.gov (United States)

    Wang, Huizhi; Brown, Jonathan; Gu, Zhen; Garcia, Carlos A; Liang, Ruqiang; Alard, Pascale; Beurel, Eléonore; Jope, Richard S; Greenway, Terrance; Martin, Michael

    2011-05-01

    The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-β converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-β. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-β associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-β. Inhibition of S6K1 abrogated the phosphorylation of GSK3-β while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin's ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3.

  18. Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

    Science.gov (United States)

    Inobe, Tomonao; Nukina, Nobuyuki

    2016-07-01

    Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Is generic physical activity or specific exercise associated with motor abilities?

    Science.gov (United States)

    Rinne, Marjo; Pasanen, Matti; Miilunpalo, Seppo; Mälkiä, Esko

    2010-09-01

    Evidence of the effect of leisure time physical activity (LTPA) modes on the motor abilities of a mature population is scarce. The purpose of this study was to compare the motor abilities of physically active and inactive men and women and to examine the associations of different exercise modes and former and recent LTPA (R-LTPA) with motor ability and various physical tests. The LTPA of the participants (men n = 69, women n = 79; aged 41-47 yr) was ascertained by a modified Physical Activity Readiness Questionnaire, including questions on the frequency, duration, and intensity of R-LTPA and former LTPA and on exercise modes. Motor abilities in terms of balance, agility, and coordination were assessed with a battery of nine tests supplemented with five physical fitness tests. Multiple statistical methods were used in analyses that were conducted separately for men and women. The MET-hours per week of R-LTPA correlated statistically significantly with the tests of agility and static balance (rs = -0.28, P = 0.022; rs = -0.25, P = 0.043, respectively) among men and with the static balance (rs = 0.41), 2-km walking (rs = 0.36), step squat (rs = 0.36) (P women. In the stepwise regression among men, the most frequent statistically significant predictor was the playing of several games. For women, a history of LTPA for more than 3 yr was the strongest predictor for good results in almost all tests. Participants with long-term and regular LTPA had better motor performance, and especially a variety of games improve components of motor ability. Diverse, regular, and long-term exercise including both specific training and general activity develops both motor abilities and physical fitness.

  20. Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer

    Directory of Open Access Journals (Sweden)

    Tai Phillip WL

    2011-07-01

    Full Text Available Abstract Background Hundreds of genes, including muscle creatine kinase (MCK, are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood. Results Modulatory region 1 (MR1 is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE containing a paired E-box/myocyte enhancer factor 2 (MEF2 regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively, but is not required for expression in fast-twitch muscle fibers (types IIb and IId. Conclusions In this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.

  1. Critical analysis of the potential for therapeutic targeting of mammalian target of rapamycin (mTOR in gastric cancer

    Directory of Open Access Journals (Sweden)

    Inokuchi M

    2014-04-01

    Full Text Available Mikito Inokuchi,1 Keiji Kato,1 Kazuyuki Kojima,2 Kenichi Sugihara1 1Department of Surgical Oncology, 2Department of Minimally Invasive Surgery, Tokyo Medical and Dental University, Tokyo, Japan Abstract: Multidisciplinary treatment including chemotherapy has become the global standard of care for patients with metastatic gastric cancer (mGC; nonetheless, survival remains poor. Although many molecular-targeted therapies have been developed for various cancers, only anti-HER2 treatment has produced promising results in patients with mGC. Mammalian target of rapamycin (mTOR plays a key role in cell proliferation, antiapoptosis, and metastasis in signaling pathways from the tyrosine kinase receptor, and its activation has been demonstrated in gastric cancer (GC cells. This review discusses the clinical relevance of mTOR in GC and examines its potential as a therapeutic target in patients with mGC. Preclinical studies in animal models suggest that suppression of the mTOR pathway inhibits the proliferation of GC cells and delays tumor progression. The mTOR inhibitor everolimus has been evaluated as second- or third-line treatment in clinical trials. Adverse events were well tolerated although the effectiveness of everolimus alone was limited. Everolimus is now being evaluated in combi