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Sample records for 3-kinase negatively controls

  1. Phosphoinositide 3-kinase beta controls replication factor C assembly and function

    PubMed Central

    Redondo-Muñoz, Javier; Josefa Rodríguez, María; Silió, Virginia; Pérez-García, Vicente; María Valpuesta, José; Carrera, Ana C.

    2013-01-01

    Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1– and RFC–RAD17 complexes, but also RFC–CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes. PMID:23175608

  2. RAS signalling through PI3-Kinase controls cell migration via modulation of Reelin expression

    PubMed Central

    Castellano, Esther; Molina-Arcas, Miriam; Krygowska, Agata Adelajda; East, Philip; Warne, Patricia; Nicol, Alastair; Downward, Julian

    2016-01-01

    RAS signalling through phosphoinositide 3-kinase (PI3-Kinase) has been shown to have an essential role in tumour initiation and maintenance. RAS also regulates cell motility and tumour invasiveness, but the role of direct RAS binding to PI3-Kinase in this remains uncertain. Here, we provide evidence that disruption of RAS interaction with PI3-Kinase p110α decreases cell motility and prevents activation of Rac GTPase. Analysis of gene expression in cells lacking RAS interaction with p110α reveals increased levels of the extracellular matrix glycoprotein Reelin and activation of its downstream pathway resulting in upregulation of E-cadherin expression. Induction of the Reelin/E-cadherin axis is also observed in Kras mutant lung tumours that are regressing due to blockade of RAS interaction with PI3-Kinase. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis. PMID:27071537

  3. Phosphoinositide 3-Kinase Beta Protects Nuclear Envelope Integrity by Controlling RCC1 Localization and Ran Activity

    PubMed Central

    Redondo-Muñoz, Javier; Pérez-García, Vicente; Rodríguez, María J.; Valpuesta, José M.

    2014-01-01

    The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran. PMID:25348717

  4. Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms.

    PubMed

    Kaneko, Kazuma; Ueki, Kohjiro; Takahashi, Noriko; Hashimoto, Shinji; Okamoto, Masayuki; Awazawa, Motoharu; Okazaki, Yukiko; Ohsugi, Mitsuru; Inabe, Kazunori; Umehara, Toshihiro; Yoshida, Masashi; Kakei, Masafumi; Kitamura, Tadahiro; Luo, Ji; Kulkarni, Rohit N; Kahn, C Ronald; Kasai, Haruo; Cantley, Lewis C; Kadowaki, Takashi

    2010-12-01

    Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.

  5. Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase.

    PubMed

    Tornieri, Karine; Welshhans, Kristy; Geddis, Matthew S; Rehder, Vincent

    2006-04-01

    Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.

  6. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

    SciTech Connect

    Singh, Alok R.; Peirce, Susan K.; Joshi, Shweta; Durden, Donald L.

    2014-09-10

    -3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo. - Highlights: • First genetic evidence that PTEN controls LPS/TLR4 signaling in B lymphocytes. • Evidence that PTEN regulates LPS induced lymphoproliferation in vivo. • PI-3 kinase inhibitors block LPS induced lymphoproliferation in vivo.

  7. Oncogenic Kit controls neoplastic mast cell growth through a Stat5/PI3-kinase signaling cascade

    PubMed Central

    Harir, Noria; Boudot, Cédric; Friedbichler, Katrin; Sonneck, Karoline; Kondo, Rudin; Martin-Lannerée, Séverine; Kenner, Lukas; Kerenyi, Marc; Yahiaoui, Saliha; Gouilleux-Gruart, Valérie; Gondry, Jean; Bénit, Laurence; Dusanter-Fourt, Isabelle; Lassoued, Kaïss; Valent, Peter

    2008-01-01

    The D816V-mutated variant of Kit triggers multiple signaling pathways and is considered essential for malignant transformation in mast cell (MC) neoplasms. We here describe that constitutive activation of the Stat5-PI3K-Akt-cascade controls neoplastic MC development. Retrovirally transduced active Stat5 (cS5F) was found to trigger PI3K and Akt activation, and to transform murine bone marrow progenitors into tissue-infiltrating MCs. Primary neoplastic Kit D816V+ MCs in patients with mastocytosis also displayed activated Stat5, which was found to localize to the cytoplasm and to form a signaling complex with PI3K, with consecutive Akt activation. Finally, the knock-down of either Stat5 or Akt activity resulted in growth inhibition of neoplastic Kit D816V+ MCs. These data suggest that a downstream Stat5-PI3K-Akt signaling cascade is essential for Kit D816V-mediated growth and survival of neoplastic MCs. PMID:18579792

  8. A Receptor-associated Protein/Phosphatidylinositol 3-Kinase Pathway Controls Pseudopod Formation

    PubMed Central

    Kortholt, Arjan; Bolourani, Parvin; Rehmann, Holger; Keizer-Gunnink, Ineke; Weeks, Gerald; Wittinghofer, Alfred

    2010-01-01

    GbpD, a Dictyostelium discoideum guanine exchange factor specific for Rap1, has been implicated in adhesion, cell polarity, and chemotaxis. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. Phg2, a serine/threonine-specific kinase, mediates Rap1-regulated cell-substrate adhesion, but not cell polarity or chemotaxis. In this study we demonstrate that overexpression of GbpD in pi3k1/2-null cells does not induce the adhesion and cell morphology phenotype. Furthermore we show that Rap1 directly binds to the Ras binding domain of PI3K, and overexpression of GbpD leads to strongly enhanced PIP3 levels. Consistently, upon overexpression of the PIP3-degradating enzyme PTEN in GbpD-overexpressing cells, the strong adhesion and cell morphology phenotype is largely lost. These results indicate that a GbpD/Rap/PI3K pathway helps control pseudopod formation and cell polarity. As in Rap-regulated pseudopod formation in Dictyostelium, mammalian Rap and PI3K are essential for determining neuronal polarity, suggesting that the Rap/PI3K pathway is a conserved module regulating the establishment of cell polarity. PMID:20089846

  9. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment.

    PubMed

    Singh, Alok R; Peirce, Susan K; Joshi, Shweta; Durden, Donald L

    2014-09-10

    Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN(fl/fl) mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3

  10. Phosphoinositide 3-kinase targeting by the β galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death

    PubMed Central

    Wells, Valerie; Mallucci, Livio

    2009-01-01

    Introduction Phosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the β galactoside binding protein (βGBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, βGBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls. Methods Aggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by βGBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitro behaviour of the BT474 and SKBR3 cells. Results In aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor

  11. Signals controlling un-differentiated states in embryonic stem and cancer cells: role of the phosphatidylinositol 3' kinase pathway.

    PubMed

    Voskas, Daniel; Ling, Ling Sunny; Woodgett, James Robert

    2014-10-01

    The capacity of embryonic stem (ES) cells to differentiate into cell lineages comprising the three germ layers makes them powerful tools for studying mammalian early embryonic development in vitro. The human body consists of approximately 210 different somatic cell types, the majority of which have limited proliferative capacity. However, both stem cells and cancer cells bypass this replicative barrier and undergo symmetric division indefinitely when cultured under defined conditions. Several signal transduction pathways play important roles in regulating stem cell development, and aberrant expression of components of these pathways is linked to cancer. Among signaling systems, the critical role of leukemia inhibitory factor (LIF) coupled to the Jak/STAT3 (signal transduction and activation of transcription-3) pathway in maintaining stem cell self-renewal has been extensively reviewed. This pathway additionally plays multiple roles in tumorigenesis. Likewise, the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway has been determined to play an important role in both stem cell maintenance and tumor development. This pathway is often induced in cancer with frequent mutational activation of the catalytic subunit of PI3K or loss of a primary PI3K antagonist, phosphatase and tensin homolog deleted on chromosome ten (PTEN). This review focusses on roles of the PI3K signal transduction pathway components, with emphasis on functions in stem cell maintenance and cancer. Since the PI3K pathway impinges on and collaborates with other signaling pathways in regulating stem cell development and/or cancer, aspects of the canonical Wnt, Ras/mitogen-activated protein kinase (MAPK), and TGF-β signaling pathways are also discussed.

  12. An N-ethyl-N-nitrosourea (ENU)-induced dominant negative mutation in the JAK3 kinase protects against cerebral malaria.

    PubMed

    Bongfen, Silayuv E; Rodrigue-Gervais, Ian-Gael; Berghout, Joanne; Torre, Sabrina; Cingolani, Pablo; Wiltshire, Sean A; Leiva-Torres, Gabriel A; Letourneau, Louis; Sladek, Robert; Blanchette, Mathieu; Lathrop, Mark; Behr, Marcel A; Gruenheid, Samantha; Vidal, Silvia M; Saleh, Maya; Gros, Philippe

    2012-01-01

    Cerebral malaria (CM) is a lethal neurological complication of malaria. We implemented a genome-wide screen in mutagenized mice to identify host proteins involved in CM pathogenesis and whose inhibition may be of therapeutic value. One pedigree (P48) segregated a resistance trait whose CM-protective effect was fully penetrant, mapped to chromosome 8, and identified by genome sequencing as homozygosity for a mis-sense mutation (W81R) in the FERM domain of Janus-associated kinase 3 (Jak3). The causative effect of Jak3(W81R) was verified by complementation testing in Jak3(W81R/-) double heterozygotes that were fully protected against CM. Jak3(W81R) homozygotes showed defects in thymic development with depletion of CD8(+) T cell, B cell, and NK cell compartments, and defective T cell-dependent production of IFN-γ. Adoptive transfer of normal splenocytes abrogates CM resistance in Jak3(W81R) homozygotes, an effect attributed to the CD8(+) T cells. Jak3(W81R) behaves as a dominant negative variant, with significant CM resistance of Jak3(W81R/+) heterozygotes, compared to CM-susceptible Jak3(+/+) and Jak3(+/-) controls. CM resistance in Jak3(W81R/+) heterozygotes occurs in presence of normal T, B and NK cell numbers. These findings highlight the pathological role of CD8(+) T cells and Jak3-dependent IFN-γ-mediated Th1 responses in CM pathogenesis.

  13. Thyroid Hormone Controls Breast Cancer Cell Movement via Integrin αV/β3/SRC/FAK/PI3-Kinases.

    PubMed

    Flamini, Marina Inés; Uzair, Ivonne Denise; Pennacchio, Gisela Erika; Neira, Flavia Judith; Mondaca, Joselina Magali; Cuello-Carrión, Fernando Dario; Jahn, Graciela Alma; Simoncini, Tommaso; Sanchez, Angel Matías

    2017-02-01

    Thyroid hormones (TH) play a fundamental role in diverse processes, including cellular movement. Cell migration requires the integration of events that induce changes in cell structure towards the direction of migration. These actions are driven by actin remodeling and stabilized by the development of adhesion sites to extracellular matrix via transmembrane receptors linked to the actin cytoskeleton. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that promotes cell migration and invasion through the control of focal adhesion turnover. In this work, we demonstrate that the thyroid hormone triiodothyronine (T3) regulates actin remodeling and cell movement in breast cancer T-47D cells through the recruitment of FAK. T3 controls FAK phosphorylation and translocation at sites where focal adhesion complexes are assembled. This process is triggered via rapid signaling to integrin αV/β3, Src, phosphatidylinositol 3-OH kinase (PI3K), and FAK. In addition, we established a cellular model with different concentration of T3 levels: normal, absence, and excess in T-47D breast cancer cells. We found that the expression of Src, FAK, and PI3K remained at normal levels in the excess of T3 model, while it was significantly reduced in the absence model. In conclusion, these results suggest a novel role for T3 as an important modulator of cell migration, providing a starting point for the development of new therapeutic strategies for breast cancer treatment.

  14. The phosphoinositide 3-kinase signaling pathway is involved in the control of modified low-density lipoprotein uptake by human macrophages.

    PubMed

    Michael, Daryn R; Davies, Thomas S; Laubertová, Lucia; Gallagher, Hayley; Ramji, Dipak P

    2015-03-01

    The transformation of macrophages into lipid-loaded foam cells is a critical early event in the pathogenesis of atherosclerosis. Both receptor-mediated uptake of modified LDL, mediated primarily by scavenger receptors-A (SR-A) and CD36 along with other proteins such as lipoprotein lipase (LPL), and macropinocytosis contribute to macrophage foam cell formation. The signaling pathways that are involved in the control of foam cell formation are not fully understood. In this study, we have investigated the role of phosphoinositide 3-kinase (PI3K) in relation to foam cell formation in human macrophages. The pan PI3K inhibitor LY294002 attenuated the uptake of modified LDL and macropinocytosis, as measured by Lucifer Yellow uptake, by human macrophages. In addition, the expression of SR-A, CD36 and LPL was attenuated by LY294002. The use of isoform-selective PI3K inhibitors showed that PI3K-β, -γ and -δ were all required for the expression of SR-A and CD36 whereas only PI3K-γ was necessary in the case of LPL. These studies reveal a pivotal role of PI3K in the control of macrophage foam cell formation and provide further evidence for their potential as therapeutic target against atherosclerosis.

  15. The phosphoinositide 3-kinase pathway.

    PubMed

    Cantley, Lewis C

    2002-05-31

    Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

  16. Ceramide limits phosphatidylinositol-3-kinase C2β-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid

    PubMed Central

    Kitatani, Kazuyuki; Toshinori, Usui; Sriraman, Shravan Kumar; Toyoshima, Masafumi; Ishibashi, Masumi; Shigeta, Shogo; Nagase, Satoru; Sakamoto, Masahiro; Ogiso, Hideo; Okazaki, Toshiro; Hannun, Yusuf A.; Torchilin, Vladimir P.; Yaegashi, Nobuo

    2015-01-01

    Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. siRNA screening identified class II phosphatidylinositol 3-kinase C2β (PI3KC2β) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2β-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2β and affect its compartmentalization, thereby suppressing PI3KC2β activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2β knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2β in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2β-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for

  17. Transcriptional and post-transcriptional control of DNA methyltransferase 3B is regulated by phosphatidylinositol 3 kinase/Akt pathway in human hepatocellular carcinoma cell lines.

    PubMed

    Mei, Chuanzhong; Sun, Lidong; Liu, Yonglei; Yang, Yong; Cai, Xiumei; Liu, Mingzhu; Yao, Wantong; Wang, Can; Li, Xin; Wang, Liying; Li, Zengxia; Shi, Yinghong; Qiu, Shuangjian; Fan, Jia; Zha, Xiliang

    2010-09-01

    DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.

  18. Prolactin-Stimulated Activation of ERK1/2 Mitogen-Activated Protein Kinases is Controlled by PI3-Kinase/Rac/PAK Signaling Pathway in Breast Cancer Cells

    PubMed Central

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N.; Hoek, Jan B.; Kiyatkin, Anatoly

    2011-01-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  19. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

    PubMed

    Vicente-Manzanares, M; Rey, M; Jones, D R; Sancho, D; Mellado, M; Rodriguez-Frade, J M; del Pozo, M A; Yáñez-Mó, M; de Ana, A M; Martínez-A, C; Mérida, I; Sánchez-Madrid, F

    1999-10-01

    The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

  20. How do negative emotions impair self-control? A neural model of negative urgency.

    PubMed

    Chester, David S; Lynam, Donald R; Milich, Richard; Powell, David K; Andersen, Anders H; DeWall, C Nathan

    2016-05-15

    Self-control often fails when people experience negative emotions. Negative urgency represents the dispositional tendency to experience such self-control failure in response to negative affect. Neither the neural underpinnings of negative urgency nor the more general phenomenon of self-control failure in response to negative emotions are fully understood. Previous theorizing suggests that an insufficient, inhibitory response from the prefrontal cortex may be the culprit behind such self-control failure. However, we entertained an alternative hypothesis: negative emotions lead to self-control failure because they excessively tax inhibitory regions of the prefrontal cortex. Using fMRI, we compared the neural activity of people high in negative urgency with controls on an emotional, inhibitory Go/No-Go task. While experiencing negative (but not positive or neutral) emotions, participants high in negative urgency showed greater recruitment of inhibitory brain regions than controls. Suggesting a compensatory function, inhibitory accuracy among participants high in negative urgency was associated with greater prefrontal recruitment. Greater activity in the anterior insula on negatively-valenced, inhibitory trials predicted greater substance abuse one month and one year after the MRI scan among individuals high in negative urgency. These results suggest that, among people whose negative emotions often lead to self-control failure, excessive reactivity of the brain's regulatory resources may be the culprit.

  1. Controlling plasmon hybridization for negative refraction metamaterials

    NASA Astrophysics Data System (ADS)

    Kanté, B.; Burokur, S. N.; Sellier, A.; de Lustrac, A.; Lourtioz, J.-M.

    2009-02-01

    The hybridization scheme of plasmon modes in cut-wire-based left-handed metamaterials is shown to critically depend on the coupling between paired cut wires. We show that an inverted hybridization scheme obtained with an asymmetric alignment of paired cut wires is the most appropriate to negative refraction. This is validated (numerically and experimentally) by the first demonstration of negative refraction in the microwave domain using only periodic ensembles of cut wires.

  2. Phosphoinositide 3-kinase signaling in the vertebrate retina

    PubMed Central

    Rajala, Raju V. S.

    2010-01-01

    The phosphoinositide (PI) cycle, discovered over 50 years ago by Mabel and Lowell Hokin, describes a series of biochemical reactions that occur on the inner leaflet of the plasma membrane of cells in response to receptor activation by extracellular stimuli. Studies from our laboratory have shown that the retina and rod outer segments (ROSs) have active PI metabolism. Biochemical studies revealed that the ROSs contain the enzymes necessary for phosphorylation of phosphoinositides. We showed that light stimulates various components of the PI cycle in the vertebrate ROS, including diacylglycerol kinase, PI synthetase, phosphatidylinositol phosphate kinase, phospholipase C, and phosphoinositide 3-kinase (PI3K). This article describes recent studies on the PI3K-generated PI lipid second messengers in the control and regulation of PI-binding proteins in the vertebrate retina. PMID:19638643

  3. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed Central

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-01-01

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  4. No Negative Priming without Cognitive Control

    ERIC Educational Resources Information Center

    de Fockert, Jan W.; Mizon, Guy A.; D'Ubaldo, Mariangela

    2010-01-01

    There is evidence that the efficiency of selective attention depends on the availability of cognitive control mechanisms as distractor processing has been found to increase with high load on working memory or dual task coordination (Lavie, Hirst, de Fockert, & Viding, 2004). We tested the prediction that cognitive control load would also…

  5. The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels.

    PubMed

    Martin, Kayleigh J S; Muessel, Michelle J; Pullar, Christine E; Willars, Gary B; Wardlaw, Andrew J

    2015-01-01

    The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

  6. Class IA phosphoinositide 3-kinase regulates heart size and physiological cardiac hypertrophy.

    PubMed

    Luo, Ji; McMullen, Julie R; Sobkiw, Cassandra L; Zhang, Li; Dorfman, Adam L; Sherwood, Megan C; Logsdon, M Nicole; Horner, James W; DePinho, Ronald A; Izumo, Seigo; Cantley, Lewis C

    2005-11-01

    Class I(A) phosphoinositide 3-kinases (PI3Ks) are activated by growth factor receptors, and they regulate, among other processes, cell growth and organ size. Studies using transgenic mice overexpressing constitutively active and dominant negative forms of the p110alpha catalytic subunit of class I(A) PI3K have implicated the role of this enzyme in regulating heart size and physiological cardiac hypertrophy. To further understand the role of class I(A) PI3K in controlling heart growth and to circumvent potential complications from the overexpression of dominant negative and constitutively active proteins, we generated mice with muscle-specific deletion of the p85alpha regulatory subunit and germ line deletion of the p85beta regulatory subunit of class I(A) PI3K. Here we show that mice with cardiac deletion of both p85 subunits exhibit attenuated Akt signaling in the heart, reduced heart size, and altered cardiac gene expression. Furthermore, exercise-induced cardiac hypertrophy is also attenuated in the p85 knockout hearts. Despite such defects in postnatal developmental growth and physiological hypertrophy, the p85 knockout hearts exhibit normal contractility and myocardial histology. Our results therefore provide strong genetic evidence that class I(A) PI3Ks are critical regulators for the developmental growth and physiological hypertrophy of the heart.

  7. Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

    PubMed Central

    Juvekar, Ashish; Hu, Hai; Yadegarynia, Sina; Lyssiotis, Costas A.; Ullas, Soumya; Lien, Evan C.; Bellinger, Gary; Son, Jaekyoung; Hok, Rosanna C.; Seth, Pankaj; Daly, Michele B.; Kim, Baek; Scully, Ralph; Asara, John M.; Cantley, Lewis C.; Wulf, Gerburg M.

    2016-01-01

    We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1f/fp53f/f), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors. PMID:27402769

  8. Targeting phosphoinositide 3-kinase δ for allergic asthma.

    PubMed

    Rowan, Wendy C; Smith, Janet L; Affleck, Karen; Amour, Augustin

    2012-02-01

    Chronic inflammation in the lung has long been linked to the pathogenesis of asthma. Central to this airway inflammation is a T-cell response to allergens, with Th2 cytokines driving the differentiation, survival and function of the major inflammatory cells involved in the allergic cascade. PI3Kδ (phosphoinositide 3-kinase δ) is a lipid kinase, expressed predominantly in leucocytes, where it plays a critical role in immune receptor signalling. A selective PI3Kδ inhibitor is predicted to block T-cell activation in the lung, reducing the production of pro-inflammatory Th2 cytokines. PI3Kδ is also involved in B-cell and mast cell activation. Therefore the inhibition of PI3Kδ should dampen down the inflammatory cascade involved in the asthmatic response through a wide breadth of pharmacology. Current anti-inflammatory therapies, which are based on corticosteroids, are effective in controlling inflammation in mild asthmatics, but moderate/severe asthmatic patients remain poorly controlled, experiencing recurrent exacerbations. Corticosteroids have no effect on mast cell degranulation and do not act directly on B-cells, so, overall, a PI3Kδ inhibitor has the potential to deliver improvements in onset of action, efficacy and reduced exacerbations in moderate/severe asthmatics. Additionally, PI3Kδ inhibition is expected to block effects of Th17 cells, which are increasingly implicated in steroid-insensitive asthma.

  9. Improving the sensitivity of negative controls in ancient DNA extractions.

    PubMed

    Xu, Zhi; Zhang, Fan; Xu, Bosong; Tan, Jingze; Li, Shilin; Jin, Li

    2009-04-01

    Much attention has been paid on ancient DNA (aDNA) studies, and negative control was used as one of the stringent quality assurance criteria in order to detect potential contamination. However, the results of some aDNA studies showed the evidence of contamination despite their negative controls failed to do so. Using lambda DNA to mock extraneous contaminating DNA, our study showed that aDNA had a property of improving the efficiency of extraction including contaminating DNA, while negative controls had low sensitivity to detect contamination. To circumvent this problem, carrier DNA such as poly(dA) is suggested to be introduced into aDNA extraction.

  10. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

    PubMed Central

    Shepherd, P R; Withers, D J; Siddle, K

    1998-01-01

    Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. PMID:9677303

  11. Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers

    PubMed Central

    Geering, Barbara; Cutillas, Pedro R.; Nock, Gemma; Gharbi, Severine I.; Vanhaesebroeck, Bart

    2007-01-01

    Class IA phosphoinositide 3-kinases (PI3Ks) signal downstream of tyrosine kinases and Ras and control a wide variety of biological responses. In mammals, these heterodimeric PI3Ks consist of a p110 catalytic subunit (p110α, p110β, or p110δ) bound to any of five distinct regulatory subunits (p85α, p85β, p55γ, p55α, and p50α, collectively referred to as “p85s”). The relative expression levels of p85 and p110 have been invoked to explain key features of PI3K signaling. For example, free (i.e., non-p110-bound) p85α has been proposed to negatively regulate PI3K signaling by competition with p85/p110 for recruitment to phosphotyrosine docking sites. Using affinity and ion exchange chromatography and quantitative mass spectrometry, we demonstrate that the p85 and p110 subunits are present in equimolar amounts in mammalian cell lines and tissues. No evidence for free p85 or p110 subunits could be obtained. Cell lines contain 10,000–15,000 p85/p110 complexes per cell, with p110β and p110δ being the most prevalent catalytic subunits in nonleukocytes and leukocytes, respectively. These results argue against a role of free p85 in PI3K signaling and provide insights into the nonredundant functions of the different class IA PI3K isoforms. PMID:17470792

  12. Decreased inhibitory control of negative information in directed forgetting.

    PubMed

    Yang, Tianliang; Lei, Xu; Anderson, Michael

    2016-02-01

    A great deal of evidence suggests that emotion enhances memory. Thus, it may be harder to forget emotional information. By means of fMRI, this question was investigated in the item-method directed forgetting paradigm. Behavioral results demonstrated that although all kinds of material could be forgotten, negative words showed reduced directed forgetting effect. At the neural level, the initial viewing of negative words elicited increased activities in inferior frontal gyrus and superior parietal lobule when contrasted with neutral words, which reflected the capture of attention by negative content. Forgetting instructions for negative and neutral words led to enhanced activations in frontal and parietal cortex, consistent with the engagement of an active inhibitory process. Surprisingly, whereas successful directed forgetting of neutral words elicited stronger activations in right middle frontal gyrus compared with incidental forgetting, no such activation was observed for negative words. The lack of activation for negative words may be due to an attentional bias in processing negative words, which may briefly interfere with the deployment of inhibitory control. The present findings are consistent with the engagement of an active forgetting mechanism that contributes to the item-method directed forgetting. However, evidence of impeded inhibitory control suggests that forgetting negative words is harder.

  13. Two distinct functions for PI3-kinases in macropinocytosis

    PubMed Central

    Hoeller, Oliver; Bolourani, Parvin; Clark, Jonathan; Stephens, Len R.; Hawkins, Phillip T.; Weiner, Orion D.; Weeks, Gerald; Kay, Robert R.

    2013-01-01

    Summary Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins. PMID:23843627

  14. A biopsychosocial model based on negative feedback and control

    PubMed Central

    Carey, Timothy A.; Mansell, Warren; Tai, Sara J.

    2014-01-01

    Although the biopsychosocial model has been a popular topic of discussion for over four decades it has not had the traction in fields of research that might be expected of such an intuitively appealing idea. One reason for this might be the absence of an identified mechanism or a functional architecture that is authentically biopsychosocial. What is needed is a robust mechanism that is equally important to biochemical processes as it is to psychological and social processes. Negative feedback may be the mechanism that is required. Negative feedback has been implicated in the regulation of neurotransmitters as well as important psychological and social processes such as emotional regulation and the relationship between a psychotherapist and a client. Moreover, negative feedback is purported to also govern the activity of all other organisms as well as humans. Perceptual Control Theory (PCT) describes the way in which negative feedback establishes control at increasing levels of perceptual complexity. Thus, PCT may be the first biopsychosocial model to be articulated in functional terms. In this paper we outline the working model of PCT and explain how PCT provides an embodied hierarchical neural architecture that utilizes negative feedback to control physiological, psychological, and social variables. PCT has major implications for both research and practice and, importantly, provides a guide by which fields of research that are currently separated may be integrated to bring about substantial progress in understanding the way in which the brain alters, and is altered by, its behavioral and environmental context. PMID:24616685

  15. Symmetry Switching of Negative Thermal Expansion by Chemical Control.

    PubMed

    Senn, Mark S; Murray, Claire A; Luo, Xuan; Wang, Lihai; Huang, Fei-Ting; Cheong, Sang-Wook; Bombardi, Alessandro; Ablitt, Chris; Mostofi, Arash A; Bristowe, Nicholas C

    2016-05-04

    The layered perovskite Ca3-xSrxMn2O7 is shown to exhibit a switching from a material exhibiting uniaxial negative to positive thermal expansion as a function of x. The switching is shown to be related to two closely competing phases with different symmetries. The negative thermal expansion (NTE) effect is maximized when the solid solution is tuned closest to this region of phase space but is switched off suddenly on passing though the transition. Our results show for the first time that, by understanding the symmetry of the competing phases alone, one may achieve unprecedented chemical control of this unusual property.

  16. Positive and Negative Control of the Lac Operon

    NASA Astrophysics Data System (ADS)

    Qaddour, Jihad S.; Werman, Steven D.; Misra, Prasanta K.

    1997-03-01

    We present a mathematical model for the positive and negative control of lac operon. We investigate a steady state solution for the coupled nonlinear differential equations representing the dynamic behaviors of the repressor-inducer components of negative control as well as the cyclic AMP receptor components of the positive control. A dimensionless derivation of the lac operon system is employed to produce singularly perturbed models. The first model represents the dynamical behavior of the operator while the slow model represents the dynamical behaviors of the inducer and the repressor. We use the singular perturbation theory to show that the behavior of the system can be described as a rapid on-off switch of structural gene transformation.

  17. Cesium control and diagnostics in surface plasma negative ion sources

    SciTech Connect

    Dudnikov, Vadim; Chapovsky, Pavel; Dudnikov, Andrei

    2010-02-15

    For efficient and reliable negative ion generation it is very important to improve a cesium control and diagnostics. Laser beam attenuation and resonance fluorescence can be used for measurement of cesium distribution and cesium control. Resonant laser excitation and two-photon excitation can be used for improved cesium ionization and cesium trapping in the discharge chamber. Simple and inexpensive diode lasers can be used for cesium diagnostics and control. Cesium migration along the surface is an important mechanism of cesium escaping. It is important to develop a suppression of cesium migration and cesium accumulation on the extraction system.

  18. Computer System for Unattended Control of Negative Ion Source

    SciTech Connect

    Zubarev, P. V.; Khilchenko, A. D.; Kvashnin, A. N.; Moiseev, D. V.; Puriga, E. A.; Sanin, A. L.; Savkin, V. Ya.

    2011-09-26

    The computer system for control of cw surface-plasma source of negative ions is described. The system provides an automatic handling of source parameters by the specified scenario. It includes the automatic source start and long-term operation with switching and control of the power supplies blocks, setting and reading of source parameters like hydrogen feed, cesium seed, electrodes' temperature, checking of the protection and blockings elements like vacuum degradation, absence of cooling water, etc. The semi-automatic mode of control is also available, where the order of steps and magnitude of parameters, included to scenario, is corrected in situ by the operator. Control system includes the main controller and a set of peripheral local controllers. Commands execution is carried out by the main controller. Each peripheral controller is driven by the stand-alone program, stored in its ROM. Control system is handled from PC via Ethernet. The PC and controllers are connected by fiber optic lines, which provide the high voltage insulation and the stable system operation in spite the breakdowns and electromagnetic noise of cross-field discharge. The PC program for data setting and information display is developed under the LabView.

  19. Brain and behavioral inhibitory control of kindergartners facing negative emotions.

    PubMed

    Farbiash, Tali; Berger, Andrea

    2016-09-01

    Inhibitory control (IC) - one of the most critical functions underlying a child's ability to self-regulate - develops significantly throughout the kindergarten years. Experiencing negative emotions imposes challenges on executive functioning and may specifically affect IC. In this study, we examined kindergartners' IC and its related brain activity during a negative emotional situation: 58 children (aged 5.5-6.5 years) performed an emotion-induction Go/NoGo task. During this task, we recorded children's performance and brain activity, focusing on the fronto-central N2 component in the event-related potential (ERP) and the power of its underlying theta frequency. Compared to Go trials, inhibition of NoGo trials was associated with larger N2 amplitudes and theta power. The negative emotional experience resulted in better IC performance and, at the brain level, in larger theta power. Source localization of this effect showed that the brain activity related to IC during the negative emotional experience was principally generated in the posterior frontal regions. Furthermore, the band power measure was found to be a more sensitive index for children's inhibitory processes than N2 amplitudes. This is the first study to focus on kindergartners' IC while manipulating their emotional experience to induce negative emotions. Our findings suggest that a kindergartner's experience of negative emotion can result in improved IC and increases in associated aspects of brain activity. Our results also suggest the utility of time-frequency analyses in the study of brain processes associated with response inhibition in young children.

  20. Negative regulation of the inflammasome: keeping inflammation under control.

    PubMed

    Pedraza-Alva, Gustavo; Pérez-Martínez, Leonor; Valdez-Hernández, Laura; Meza-Sosa, Karla F; Ando-Kuri, Masami

    2015-05-01

    In addition to its roles in controlling infection and tissue repair, inflammation plays a critical role in diverse and distinct chronic diseases, such as cancer, metabolic syndrome, and neurodegenerative disorders, underscoring the harmful effect of an uncontrolled inflammatory response. Regardless of the nature of the stimulus, initiation of the inflammatory response is mediated by assembly of a multimolecular protein complex called the inflammasome, which is responsible for the production of inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18. The different stimuli and mechanisms that control inflammasome activation are fairly well understood, but the mechanisms underlying the control of undesired inflammasome activation and its inactivation remain largely unknown. Here, we review recent advances in our understanding of the molecular mechanisms that negatively regulate inflammasome activation to prevent unwanted activation in the resting state, as well as those involved in terminating the inflammatory response after a specific insult to maintain homeostasis.

  1. Transduction of Growth or Mitogenic Signals into Translational Activation of TOP mRNAs Is Fully Reliant on the Phosphatidylinositol 3-Kinase-Mediated Pathway but Requires neither S6K1 nor rpS6 Phosphorylation

    PubMed Central

    Stolovich, Miri; Tang, Hua; Hornstein, Eran; Levy, Galit; Cohen, Ruth; Bae, Sun Sik; Birnbaum, Morris J.; Meyuhas, Oded

    2002-01-01

    Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Bα (PKBα). Likewise, overexpression of constitutively active PI3-kinase or PKBα can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway. PMID:12417714

  2. Perceived control increases the reward positivity and stimulus preceding negativity.

    PubMed

    Mühlberger, Christina; Angus, Douglas Jozef; Jonas, Eva; Harmon-Jones, Cindy; Harmon-Jones, Eddie

    2017-02-01

    The reward positivity (RewP) and the stimulus preceding negativity (SPN), two ERPs associated with reward delivery and reward anticipation, are modulated by motivational intensity. Motivational intensity is the effort organisms would make to exert behaviors, and it varies with the difficulty of exerting that behavior. If a task is perceived as impossible, which means that one does not have control over own outcomes, motivational intensity is low. In the current study, we tested the prediction that perceiving control over one's outcomes increases both the RewP to feedback and the SPN prior to feedback compared to perceiving no control. We also examined whether P300 and LPP amplitudes to reward and nonreward images were similarly modulated. Twenty-five female participants completed a gambling task in which correct choices were followed by pictures of attractive men and incorrect choices were followed by pictures of rocks. To manipulate perceived control, participants were told that, in one block of trials, they could learn a mouse-click rule in order to see only pictures of men (high perceived control condition), while in the other block, the pictures would appear randomly (low perceived control condition). However, in both conditions, feedback appeared randomly. Although the RewP was elicited in both blocks, the RewP and SPN were higher in the high perceived control condition (i.e., when participants thought that they could influence their outcomes). Perceived control did not modulate the P300 and LPP to pictures. The results suggest that approach motivation and its intensity modulate the processing of performance feedback.

  3. Realistic artificial DNA sequences as negative controls for computational genomics

    PubMed Central

    Caballero, Juan; Smit, Arian F. A.; Hood, Leroy; Glusman, Gustavo

    2014-01-01

    A common practice in computational genomic analysis is to use a set of ‘background’ sequences as negative controls for evaluating the false-positive rates of prediction tools, such as gene identification programs and algorithms for detection of cis-regulatory elements. Such ‘background’ sequences are generally taken from regions of the genome presumed to be intergenic, or generated synthetically by ‘shuffling’ real sequences. This last method can lead to underestimation of false-positive rates. We developed a new method for generating artificial sequences that are modeled after real intergenic sequences in terms of composition, complexity and interspersed repeat content. These artificial sequences can serve as an inexhaustible source of high-quality negative controls. We used artificial sequences to evaluate the false-positive rates of a set of programs for detecting interspersed repeats, ab initio prediction of coding genes, transcribed regions and non-coding genes. We found that RepeatMasker is more accurate than PClouds, Augustus has the lowest false-positive rate of the coding gene prediction programs tested, and Infernal has a low false-positive rate for non-coding gene detection. A web service, source code and the models for human and many other species are freely available at http://repeatmasker.org/garlic/. PMID:24803667

  4. Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics

    PubMed Central

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate diverse cellular processes including proliferation, adhesion, survival, and motility. Dysregulated PI3K pathway signaling occurs in one-third of human tumors. Aberrantly activated PI3K signaling also confers sensitivity and resistance to conventional therapies. PI3K has been recognized as an attractive molecular target for novel anti-cancer molecules. In the last few years, several classes of potent and selective small molecule PI3K inhibitors have been developed, and at least fifteen compounds have progressed into clinical trials as new anticancer drugs. Among these, idelalisib has advanced to phase III trials in patients with advanced indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. In this review, we summarized the major molecules of PI3K signaling pathway, and discussed the preclinical models and clinical trials of potent small-molecule PI3K inhibitors. PMID:24261963

  5. Dielectric Optical-Controllable Magnifying Lens by Nonlinear Negative Refraction

    PubMed Central

    Cao, Jianjun; Shang, Ce; Zheng, Yuanlin; Feng, Yaming; Chen, Xianfeng; Liang, Xiaogan; Wan, Wenjie

    2015-01-01

    A simple optical lens plays an important role for exploring the microscopic world in science and technology by refracting light with tailored spatially varying refractive indices. Recent advancements in nanotechnology enable novel lenses, such as, superlens and hyperlens, with sub-wavelength resolution capabilities by specially designed materials’ refractive indices with meta-materials and transformation optics. However, these artificially nano- or micro-engineered lenses usually suffer high losses from metals and are highly demanding in fabrication. Here, we experimentally demonstrate, for the first time, a nonlinear dielectric magnifying lens using negative refraction by degenerate four-wave mixing in a plano-concave glass slide, obtaining magnified images. Moreover, we transform a nonlinear flat lens into a magnifying lens by introducing transformation optics into the nonlinear regime, achieving an all-optical controllable lensing effect through nonlinear wave mixing, which may have many potential applications in microscopy and imaging science. PMID:26149952

  6. Dielectric Optical-Controllable Magnifying Lens by Nonlinear Negative Refraction

    NASA Astrophysics Data System (ADS)

    Cao, Jianjun; Shang, Ce; Zheng, Yuanlin; Feng, Yaming; Chen, Xianfeng; Liang, Xiaogan; Wan, Wenjie

    2015-07-01

    A simple optical lens plays an important role for exploring the microscopic world in science and technology by refracting light with tailored spatially varying refractive indices. Recent advancements in nanotechnology enable novel lenses, such as, superlens and hyperlens, with sub-wavelength resolution capabilities by specially designed materials’ refractive indices with meta-materials and transformation optics. However, these artificially nano- or micro-engineered lenses usually suffer high losses from metals and are highly demanding in fabrication. Here, we experimentally demonstrate, for the first time, a nonlinear dielectric magnifying lens using negative refraction by degenerate four-wave mixing in a plano-concave glass slide, obtaining magnified images. Moreover, we transform a nonlinear flat lens into a magnifying lens by introducing transformation optics into the nonlinear regime, achieving an all-optical controllable lensing effect through nonlinear wave mixing, which may have many potential applications in microscopy and imaging science.

  7. Negative Control Outcomes and the Analysis of Standardized Mortality Ratios

    PubMed Central

    Richardson, DB; Keil, A; Tchetgen, Tchetgen E; Cooper, GS

    2016-01-01

    In occupational cohort mortality studies, epidemiologists often compare the observed number of deaths in the cohort to the expected number obtained by multiplying person-time accrued in the study cohort by the mortality rate in an external reference population. Interpretation of the result may be difficult due to non-comparability of the occupational cohort and reference population. We describe an approach to estimate an adjusted standardized mortality ratio (aSMR) to control for bias due to unmeasured differences between the occupational cohort and the reference population. The approach draws on methods developed for the use of negative control outcomes. Conditions necessary for unbiased estimation are described, as well as looser conditions necessary for bias reduction. The approach is illustrated using data on bladder cancer mortality among male Oak Ridge National Laboratory workers. The SMR for bladder cancer was elevated among hourly-paid males (SMR=1.90; 1.27, 2.72) but not among monthly-paid males (SMR=0.96; 0.67, 1.33). After indirect adjustment using the proposed approach, the mortality ratios were similar in magnitude among hourly- and monthly-paid men (aSMR=2.22; 1.52, 3.24; and, aSMR=1.99; 1.43, 2.76, respectively). The proposed adjusted SMR offers a complement to typical standardized mortality ratio analyses. PMID:26172862

  8. Negative Control Outcomes and the Analysis of Standardized Mortality Ratios.

    PubMed

    Richardson, David B; Keil, Alexander P; Tchetgen Tchetgen, Eric; Cooper, Glinda

    2015-09-01

    In occupational cohort mortality studies, epidemiologists often compare the observed number of deaths in the cohort to the expected number obtained by multiplying person-time accrued in the study cohort by the mortality rate in an external reference population. Interpretation of the result may be difficult due to noncomparability of the occupational cohort and reference population with respect to unmeasured risk factors for the outcome of interest. We describe an approach to estimate an adjusted standardized mortality ratio (aSMR) to control for such bias. The approach draws on methods developed for the use of negative control outcomes. Conditions necessary for unbiased estimation are described, as well as looser conditions necessary for bias reduction. The approach is illustrated using data on bladder cancer mortality among male Oak Ridge National Laboratory workers. The SMR for bladder cancer was elevated among hourly-paid males (SMR = 1.9; 95% confidence interval [CI] = 1.3, 2.7) but not among monthly-paid males (SMR = 1.0; 95% CI = 0.67, 1.3). After indirect adjustment using the proposed approach, the mortality ratios were similar in magnitude among hourly- and monthly-paid men (aSMR = 2.2; 95% CI = 1.5, 3.2; and, aSMR = 2.0; 95% CI = 1.4, 2.8, respectively). The proposed adjusted SMR offers a complement to typical SMR analyses.

  9. The involvement of Gab1 and PI 3-kinase in {beta}1 integrin signaling in keratinocytes

    SciTech Connect

    Kuwano, Yoshihiro; Fujimoto, Manabu . E-mail: fujimoto-m@umin.ac.jp; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi

    2007-09-14

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. {beta}1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these {beta}1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of {beta}1 integrins, we established HaCaT cells with high {beta}1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing {beta}1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high {beta}1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the {beta}1 integrin-mediated regulation of the epidermal stem cell compartment.

  10. Neuroprotective Role of the PI3 Kinase/Akt Signaling Pathway in Zebrafish

    PubMed Central

    Chen, Shuang; Liu, Yunzhang; Rong, Xiaozhi; Li, Yun; Zhou, Jianfeng; Lu, Ling

    2017-01-01

    Neuronal survival and growth in the embryo is controlled partly by trophic factors. For most trophic factors (such as Insulin-like growth factor-1), the ability to regulate cell survival has been attributed to the phosphoinositide 3-kinase (PI3K)/Akt kinase cascade. This study presents data illustrating the role of PI3K/Akt in attainment of normal brain size during zebrafish embryogenesis. Blocking PI3K with inhibitor LY294002 caused a significant reduction in brain size (in addition to global growth retardation) during zebrafish embryogenesis. This PI3 Kinase inhibition-induced brain size decrease was recovered by the overexpression of myristoylated Akt (myr-Akt), a constitutive form of Akt. Further analysis reveals that expressing exogenous myr-Akt significantly augmented brain size. Whole mount in situ hybridization analysis of several marker genes showed that myr-Akt overexpression did not alter brain patterning. Furthermore, the expression of myr-Akt was found to protect neuronal cells from apoptosis induced by heat shock and UV light, suggesting that inhibition of neuronal cell death may be part of the underlying cause of the increased brain size. These data provide a foundation for addressing the role of PI3K/Akt in brain growth during zebrafish embryogenesis. PMID:28228749

  11. Pathophysiological roles of Pim-3 kinase in pancreatic cancer development and progression.

    PubMed

    Li, Ying-Yi; Mukaida, Naofumi

    2014-07-28

    Pim-3 is a member of the provirus integration site for Moloney murine leukemia virus (Pim) family proteins that exhibit serine/threonine kinase activity. Similar to the other Pim kinases (Pim-1 and Pim-2), Pim-3 is involved in many cellular processes, including cell proliferation, survival, and protein synthesis. Although Pim-3 is expressed in normal vital organs, it is overexpressed particularly in tumor tissues of endoderm-derived organs, including the liver, pancreas, and colon. Silencing of Pim-3 expression can retard in vitro cell proliferation of hepatocellular, pancreatic, and colon carcinoma cell lines by promoting cell apoptosis. Pim-3 lacks the regulatory domains similarly as Pim-1 and Pim-2 lack, and therefore, Pim-3 can exhibit its kinase activity once it is expressed. Pim-3 expression is regulated at transcriptional and post-transcriptional levels by transcription factors (e.g., Ets-1) and post-translational modifiers (e.g., translationally-controlled tumor protein), respectively. Pim-3 could promote growth and angiogenesis of human pancreatic cancer cells in vivo in an orthotopic nude mouse model. Furthermore, a Pim-3 kinase inhibitor inhibited cell proliferation when human pancreatic cancer cells were injected into nude mice, without inducing any major adverse effects. Thus, Pim-3 kinase may serve as a novel molecular target for developing targeting drugs against pancreatic and other types of cancer.

  12. Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy.

    PubMed

    Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja

    2011-11-11

    Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors.

  13. The Role of Antecedent Positive and Negative Comments in the Control of Children's Behavior

    ERIC Educational Resources Information Center

    Redd, William H.; Winston, Andrew S.

    1974-01-01

    The relative effectiveness of positive and negative adult preference statements in controlling children's behavior was studied. Results indicated that the adult's antecedent negative comments exerted greater control over the children than did the positive comments. (ST)

  14. Positive and negative peptide signals control stomatal density.

    PubMed

    Shimada, Tomoo; Sugano, Shigeo S; Hara-Nishimura, Ikuko

    2011-06-01

    The stoma is a micro valve found on aerial plant organs that promotes gas exchange between the atmosphere and the plant body. Each stoma is formed by a strict cell lineage during the early stages of leaf development. Molecular genetics research using the model plant Arabidopsis has revealed the genes involved in stomatal differentiation. Cysteine-rich secretory peptides of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family play crucial roles as extracellular signaling factors. Stomatal development is orchestrated by the positive factor STOMAGEN/EPFL9 and the negative factors EPF1, EPF2, and CHALLAH/EPFL6 in combination with multiple receptors. EPF1 and EPF2 are produced in the stomatal lineage cells of the epidermis, whereas STOMAGEN and CHALLAH are derived from the inner tissues. These findings highlight the complex cell-to-cell and intertissue communications that regulate stomatal development. To optimize gas exchange, particularly the balance between the uptake of carbon dioxide (CO(2)) and loss of water, plants control stomatal activity in response to environmental conditions. The CO(2) level and light intensity influence stomatal density. Plants sense environmental cues in mature leaves and adjust the stomatal density of newly forming leaves, indicating the involvement of long-distance systemic signaling. This review summarizes recent research progress in the peptide signaling of stomatal development and discusses the evolutionary model of the signaling machinery.

  15. Rubberband Effect in Temporal Control of Mismatch Negativity

    PubMed Central

    Wang, Lingyan; Lin, Xiaoxiong; Zhou, Bin; Pöppel, Ernst; Bao, Yan

    2016-01-01

    Mismatch negativity (MMN) is a difference event-related potential (ERP) wave reflecting the brain’s automatic reaction to deviant sensory stimuli, and it has been proven to be a useful tool in research on cognitive functions or clinical disorders. In most MMN studies, amplitude, peak latency, or the integral of the responses, in rare cases also the slopes of the responses, have been employed as parameters of the ERP responses for quantitative analyses. However, little is known about correlations between these parameters. To better understand the relations between different ERP parameters, we extracted and correlated several different parameters characterizing the MMN waves. We found an unexpected correlation which gives new insight into the temporal control of MMN: response amplitudes are positively correlated with downside slopes, whereas barely correlated with upside slopes. This result suggests an efficient feedback mechanism for the MMN to return to the baseline within a predefined time window, contradicting an exponential decay function as one might expect. As a metaphor we suggest a rubberband effect for the MMN responses, i.e., the larger the distance of the response from neural equilibrium, the stronger the return force to equilibrium. PMID:27642285

  16. Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

    PubMed Central

    Baskaran, Sulochanadevi; Carlson, Lars-Anders; Stjepanovic, Goran; Young, Lindsey N; Kim, Do Jin; Grob, Patricia; Stanley, Robin E; Nogales, Eva; Hurley, James H

    2014-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001 PMID:25490155

  17. Endosomal Phosphatidylinositol 3-Kinase Is Essential for Canonical GPCR Signaling.

    PubMed

    Uchida, Yasunori; Rutaganira, Florentine U; Jullié, Damien; Shokat, Kevan M; von Zastrow, Mark

    2017-01-01

    G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the β2-adrenoceptor (β2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by β2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.

  18. Phosphoinositide 3-kinase p85beta regulates invadopodium formation

    PubMed Central

    Cariaga-Martínez, Ariel E.; Cortés, Isabel; García, Esther; Pérez-García, Vicente; Pajares, María J.; Idoate, Miguel A.; Redondo-Muñóz, Javier; Antón, Inés M.; Carrera, Ana C.

    2014-01-01

    ABSTRACT The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85β, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85β might facilitate cell invasion. We show that p85β localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85β induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85β lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85β function in invadopodium-like formation, p85β levels increased in metastatic melanoma and p85β depletion reduced invadopodium formation and invasion. These results show that p85β enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85β levels. PMID:25217619

  19. PI3 kinase enzymology on fluid lipid bilayers.

    PubMed

    Dutta, Debjit; Pulsipher, Abigail; Luo, Wei; Yousaf, Muhammad N

    2014-10-21

    We report the use of fluid lipid bilayer membrane as a model platform to study the influence of the bilayer microenvironment and composition on the enzymology in membrane. As a model system we determined the enzyme kinetics on membranes for the transformation of bilayers containing phosphoinositol(4,5)-bisphosphate (PI(4,5)P2) to phosphoinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) by the enzyme phosphoinositol-3-kinase (PI3K) using radiolabeled ATP. The activity of the enzyme was monitored as a function of the radioactivity incorporated within the bilayer. The transformation of PI(4,5)P2 to PI(3,4,5)P3 was determined using a mass strip assay. The fluidity of the bilayer was confirmed by Fluorescence Recovery After Photobleaching (FRAP) experiments. Kinetic simulations were performed based on Langmuir adsorption and Michaelis-Menton kinetics equations to generate the rate constants for the enzymatic reaction. The effect of cholesterol on the enzyme kinetics was studied by doping the bilayer with 1% cholesterol. This leads to significant reduction in reaction rate due to change in membrane microenvironment. This strategy provides a method to study the enzymology of various kinases and phosphatases occurring at the membrane and also how these reactions are affected by the membrane composition and surface microenvironment.

  20. Clinical development of phosphatidylinositol 3-kinase inhibitors for cancer treatment

    PubMed Central

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer. In recent years, the results of the first phase I clinical trials with PI3K inhibitors have become available. In comparison to other targeted agents such v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors in melanoma or crizotinib in anaplastic lymphoma receptor tyrosine kinase (ALK) translocated tumors, the number of objective responses to PI3K inhibitors is less dramatic. In this review we propose possible strategies to optimize the clinical development of PI3K inhibitors: by exploring the potential role of PI3K isoform-specific inhibitors in improving the therapeutic index, molecular characterization as a basis for patient selection, and the relevance of performing serial tumor biopsies to understand the associated mechanisms of drug resistance. The main focus of this review will be on PI3K isoform-specific inhibitors by describing the functions of different PI3K isoforms, the preclinical activity of selective PI3K isoform-specific inhibitors and the early clinical data of these compounds. PMID:23232172

  1. Class (I) Phosphoinositide 3-Kinases in the Tumor Microenvironment

    PubMed Central

    Gyori, David; Chessa, Tamara; Hawkins, Phillip T.; Stephens, Len R.

    2017-01-01

    Phosphoinositide 3-kinases (PI3Ks) are a diverse family of enzymes which regulate various critical biological processes, such as cell proliferation and survival. Class (I) PI3Ks (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) mediate the phosphorylation of the inositol ring at position D3 leading to the generation of PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 can be dephosphorylated by several phosphatases, of which the best known is the 3-phosphatase PTEN (phosphatase and tensin homolog). The Class (I) PI3K pathway is frequently disrupted in human cancers where mutations are associated with increased PI3K-activity or loss of PTEN functionality within the tumor cells. However, the role of PI3Ks in the tumor stroma is less well understood. Recent evidence suggests that the white blood cell-selective PI3Kγ and PI3Kδ isoforms have an important role in regulating the immune-suppressive, tumor-associated myeloid cell and regulatory T cell subsets, respectively, and as a consequence are also critical for solid tumor growth. Moreover, PI3Kα is implicated in the direct regulation of tumor angiogenesis, and dysregulation of the PI3K pathway in stromal fibroblasts can also contribute to cancer progression. Therefore, pharmacological inhibition of the Class (I) PI3K family in the tumor microenvironment can be a highly attractive anti-cancer strategy and isoform-selective PI3K inhibitors may act as potent cancer immunotherapeutic and anti-angiogenic agents. PMID:28273837

  2. Does the control of negative emotions influence blood pressure control and its variability?

    PubMed

    Symonides, Bartosz; Holas, Paweł; Schram, Małgorzata; Śleszycka, Justyna; Bogaczewicz, Anna; Gaciong, Zbigniew

    2014-12-01

    The aim was to assess the control of negative emotions in treated patients with hypertension in comparison with normotensive individuals and to evaluate the association between suppression of negative emotions, control of blood pressure (BP) on ambulatory blood pressure monitoring (ABPM) and blood pressure variability (BPV). We studied 195 patients (women/men: 89/106); mean age 45.4 ± 15.9 years. All patients had ABPM and completed the Courtauld Emotional Control Scale (CECS). The total CECS score and scores for subscales for anger, depression and anxiety were analyzed together with mean BP values from ABPM, and their SD and coefficient of variation as BPV measures. The mean CECS score was 54 ± 12 in all subjects; highest in uncontrolled hypertension 56 ± 11, intermediate 53 ± 12 in controlled hypertension and lowest 48 ± 12 in normotensive subjects. The reference value for the Polish population is 50 ± 11. Significant differences of mean CECS scores among groups were observed (p = 0.0165) also in multivariate analysis. The difference between uncontrolled hypertension and normotension was significant (p = 0.0262). Few significant, weak correlations were observed between CECS score or its subscales and ABPM derivates in all subjects. Conclusion. Suppression of negative emotions may adversely affect BP control in treated hypertensive patients and it should be considered a cause of uncontrolled hypertension.

  3. Can Contextual Cues Control Consummatory Successive Negative Contrast?

    ERIC Educational Resources Information Center

    Daniel, Alan M.; Wood, Michael; Pellegrini, Santiago; Norris, Jacob N.; Papini, Mauricio R.

    2008-01-01

    Rats exposed to incentive downshift show behavioral deterioration. This phenomenon, called successive negative contrast (SNC), occurs in instrumental and consummatory responses (iSNC, cSNC). Whereas iSNC is related to the violation of reward expectancies retrieved in anticipation of the goal (cued-recall), cSNC involves reward rejection and may…

  4. Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma.

    PubMed

    Grugan, Katharine D; Ma, Chunguang; Singhal, Seema; Krett, Nancy L; Rosen, Steven T

    2008-06-01

    Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.

  5. Time course of the MAPK and PI3-kinase response within 24 h of skeletal muscle overload

    NASA Technical Reports Server (NTRS)

    Carlson, C. J.; Fan, Z.; Gordon, S. E.; Booth, F. W.

    2001-01-01

    Knowledge of the molecular mechanisms by which skeletal muscle hypertrophies in response to increased mechanical loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. To gain insight into potential early signaling mechanisms associated with skeletal muscle hypertrophy, the temporal pattern of mitogen-activated protein kinase (MAPK) phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activity during the first 24 h of muscle overload was determined in the rat slow-twitch soleus and fast-twitch plantaris muscles after ablation of the gastrocnemius muscle. p38alpha MAPK phosphorylation was elevated for the entire 24-h overload period in both muscles. In contrast, Erk 2 and p54 JNK phosphorylation were transiently increased by overload, returning to the levels of sham-operated controls by 24 h. PI3-kinase activity was increased by muscle overload only at 12 h of overload and only in the plantaris muscle. In summary, sustained elevation of p38alpha MAPK phosphorylation occurred early in response to muscle overload, identifying this pathway as a potential candidate for mediating early hypertrophic signals in response to skeletal muscle overload.

  6. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    PubMed

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

  7. Brief Report: Negative Controls to Detect Selection Bias and Measurement Bias in Epidemiologic Studies

    PubMed Central

    Ercumen, Ayse; Benjamin-Chung, Jade; Colford, John M.

    2016-01-01

    Biomedical laboratory experiments routinely use negative controls to identify possible sources of bias, but epidemiologic studies have infrequently used this type of control in their design or measurement approach. Recently, epidemiologists proposed the routine use of negative controls in observational studies and defined the structure of negative controls to detect bias due to unmeasured confounding. We extend this previous study and define the structure of negative controls to detect selection bias and measurement bias in both observational studies and randomized trials. We illustrate the strengths and limitations of negative controls in this context using examples from the epidemiologic literature. Given their demonstrated utility and broad generalizability, the routine use of prespecified negative controls will strengthen the evidence from epidemiologic studies. PMID:27182642

  8. Control and regulation of pathways via negative feedback

    PubMed Central

    2017-01-01

    The biochemical networks found in living organisms include a huge variety of control mechanisms at multiple levels of organization. While the mechanistic and molecular details of many of these control mechanisms are understood, their exact role in driving cellular behaviour is not. For example, yeast glycolysis has been studied for almost 80 years but it is only recently that we have come to understand the systemic role of the multitude of feedback and feed-forward controls that exist in this pathway. In this article, control theory is discussed as an approach to dissect the control logic of complex pathways. One of the key issues is distinguishing between the terms control and regulation and how these concepts are applied to regulated enzymes such as phosphofructokinase. In doing so, one of the paradoxes in metabolic regulation can be resolved where enzymes such as phosphofructokinase have little control but, nevertheless, possess significant regulatory influence. PMID:28202588

  9. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling.

    PubMed Central

    Kotani, K; Yonezawa, K; Hara, K; Ueda, H; Kitamura, Y; Sakaue, H; Ando, A; Chavanieu, A; Calas, B; Grigorescu, F

    1994-01-01

    Insulin, IGF-1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3-kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate-1 (IRS-1) with PI 3-kinase in a cell-free system and also inhibited insulin- or IGF-1-induced, but not EGF-induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild-type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS-1 with PI 3-kinase in a cell-free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3-kinase activity, inhibited insulin- or IGF-1-induced membrane ruffling. These results suggest that the association of IRS-1 with PI 3-kinase followed by the activation of PI 3-kinase are required for insulin- or IGF-1-induced, but not for EGF-induced, membrane ruffling. Images PMID:8194523

  10. Ultrasensitive Negative Feedback Control: A Natural Approach for the Design of Synthetic Controllers

    PubMed Central

    Montefusco, Francesco; Akman, Ozgur E.; Soyer, Orkun S.; Bates, Declan G.

    2016-01-01

    Many of the most important potential applications of Synthetic Biology will require the ability to design and implement high performance feedback control systems that can accurately regulate the dynamics of multiple molecular species within the cell. Here, we argue that the use of design strategies based on combining ultrasensitive response dynamics with negative feedback represents a natural approach to this problem that fully exploits the strongly nonlinear nature of cellular information processing. We propose that such feedback mechanisms can explain the adaptive responses observed in one of the most widely studied biomolecular feedback systems—the yeast osmoregulatory response network. Based on our analysis of such system, we identify strong links with a well-known branch of mathematical systems theory from the field of Control Engineering, known as Sliding Mode Control. These insights allow us to develop design guidelines that can inform the construction of feedback controllers for synthetic biological systems. PMID:27537373

  11. Context-specific control and the Stroop negative priming effect.

    PubMed

    Milliken, Bruce; Thomson, David R; Bleile, Karmen; MacLellan, Ellen; Giammarco, Maria

    2012-01-01

    The present study highlights the utility of context-specific learning for different probe types in accounting for the commonly observed dependence of negative priming on probe selection. Using a Stroop priming procedure, Experiments 1a and 1b offered a demonstration that Stroop priming effects can differ qualitatively for selection and no-selection probes when probe selection is manipulated between subjects, but not when it is manipulated randomly from trial to trial within subject (see also Moore, 1994). In Experiments 2 and 3, selection and no-selection probes served as two contexts that varied randomly from trial to trial, but for which proportion repeated was manipulated separately. A context-specific proportion repeated effect was observed in Experiment 2, characterized by modest quantitative shifts in the repetition effects as a function of the context-specific proportion repeated manipulation. However, with a longer intertrial interval in Experiment 3, a context-specific proportion repeated manipulation that focused on the no-selection probes changed the repetition effect qualitatively, from negative priming when the proportion repeated was .25 to positive priming when the proportion repeated was .75. The results are discussed with reference to the role of rapid, context-specific learning processes in the integration of prior experiences with current perception and action.

  12. Focusing on Plates: Controlling Guided Waves using Negative Refraction.

    PubMed

    Philippe, Franck D; Murray, Todd W; Prada, Claire

    2015-06-08

    Elastic waves are guided along finite structures such as cylinders, plates, or rods through reflection, refraction, and mode conversion at the interfaces. Such wave propagation is ubiquitous in the world around us, and studies of elastic waveguides first emerged in the later part of the 19(th) century. Early work on elastic waveguides revealed the presence of backward propagating waves, in which the phase velocity and group velocity are anti-parallel. While backward wave propagation exists naturally in very simple finite elastic media, there has been remarkably little attention paid to this phenomenon. Here we report the development of a tunable acoustic lens in an isotropic elastic plate showing negative refraction over a finite acoustic frequency bandwidth. As compared to engineered acoustic materials such as phononic crystals and metamaterials, the design of the acoustic lens is very simple, with negative refraction obtained through thickness changes rather than internal periodicity or sub-wavelength resonant structures. A new class of acoustic devices, including resonators, filters, lenses, and cloaks, may be possible through topography optimization of elastic waveguide structures to exploit the unique properties of backward waves.

  13. Standardization of Negative Controls in Diagnostic Immunohistochemistry: Recommendations From the International Ad Hoc Expert Panel

    PubMed Central

    Torlakovic, Emina E.; Francis, Glenn; Garratt, John; Gilks, Blake; Hyjek, Elizabeth; Ibrahim, Merdol; Miller, Rodney; Nielsen, Søren; Petcu, Eugen B.; Swanson, Paul E.; Taylor, Clive R.; Vyberg, Mogens

    2014-01-01

    Standardization of controls, both positive and negative controls, is needed for diagnostic immunohistochemistry (dIHC). The use of IHC-negative controls, irrespective of type, although well established, is not standardized. As such, the relevance and applicability of negative controls continues to challenge both pathologists and laboratory budgets. Despite the clear theoretical notion that appropriate controls serve to demonstrate the sensitivity and specificity of the dIHC test, it remains unclear which types of positive and negative controls are applicable and/or useful in day-to-day clinical practice. There is a perceived need to provide “best practice recommendations” for the use of negative controls. This perception is driven not only by logistics and cost issues, but also by increased pressure for accurate IHC testing, especially when IHC is performed for predictive markers, the number of which is rising as personalized medicine continues to develop. Herein, an international ad hoc expert panel reviews classification of negative controls relevant to clinical practice, proposes standard terminology for negative controls, considers the total evidence of IHC specificity that is available to pathologists, and develops a set of recommendations for the use of negative controls in dIHC based on “fit-for-use” principles. PMID:24714041

  14. Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert panel.

    PubMed

    Torlakovic, Emina E; Francis, Glenn; Garratt, John; Gilks, Blake; Hyjek, Elizabeth; Ibrahim, Merdol; Miller, Rodney; Nielsen, Søren; Petcu, Eugen B; Swanson, Paul E; Taylor, Clive R; Vyberg, Mogens

    2014-04-01

    Standardization of controls, both positive and negative controls, is needed for diagnostic immunohistochemistry (dIHC). The use of IHC-negative controls, irrespective of type, although well established, is not standardized. As such, the relevance and applicability of negative controls continues to challenge both pathologists and laboratory budgets. Despite the clear theoretical notion that appropriate controls serve to demonstrate the sensitivity and specificity of the dIHC test, it remains unclear which types of positive and negative controls are applicable and/or useful in day-to-day clinical practice. There is a perceived need to provide "best practice recommendations" for the use of negative controls. This perception is driven not only by logistics and cost issues, but also by increased pressure for accurate IHC testing, especially when IHC is performed for predictive markers, the number of which is rising as personalized medicine continues to develop. Herein, an international ad hoc expert panel reviews classification of negative controls relevant to clinical practice, proposes standard terminology for negative controls, considers the total evidence of IHC specificity that is available to pathologists, and develops a set of recommendations for the use of negative controls in dIHC based on "fit-for-use" principles.

  15. Low-bias negative differential conductance controlled by electrode separation

    NASA Astrophysics Data System (ADS)

    Yi, Xiao-Hua; Liu, Ran; Bi, Jun-Jie; Jiao, Yang; Wang, Chuan-Kui; Li, Zong-Liang

    2016-12-01

    The electronic transport properties of a single thiolated arylethynylene molecule with 9,10-dihydroanthracene core, denoted as TADHA, is studied by using non-equilibrium Green’s function formalism combined with ab initio calculations. The numerical results show that the TADHA molecule exhibits excellent negative differential conductance (NDC) behavior at lower bias regime as probed experimentally. The NDC behavior of TADHA molecule originates from the Stark effect of the applied bias voltage, by which the highest occupied molecular orbital (HOMO) and the HOMO-1 are pulled apart and become localized. The NDC behavior of TADHA molecular system is tunable by changing the electrode distance. Shortening the electrode separation can enhance the NDC effect which is attributed to the possible increase of coupling between the two branches of TADHA molecule. Project supported by the National Natural Science Foundation of China (Grant Nos. 11374195 and 11405098) and the Natural Science Foundation of Shandong Province, China (Grant No. ZR2013FM006).

  16. Control of absolute negative mobility via noise recycling procedure

    NASA Astrophysics Data System (ADS)

    Zeng, C. H.; Wang, H.; Qing, S.; Hu, J. H.; Li, K. Z.

    2012-10-01

    Absolute negative mobility (ANM) is investigated in a spatially-periodic symmetric system under the influence of noise consisting of the superposition of a white Gaussian noise with the same noise delayed by time τ. The effects of the noise intensity σ, the time delay τ and feedback intensity ɛ in the noise recycling are discussed. It is found that the noise intensity σ and time delay τ can induce the phenomenon of ANM, while the feedback intensity ɛ can not induce it. This phenomenon of ANM can be tested in the setup consisting of a resistively and capacitively shunted Josephson junction device by using a vertical cavity surface emitting laser to generate the noise recycling procedure.

  17. Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation.

    PubMed

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-21

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.

  18. Vav3 Modulates B Cell Receptor Responses by Regulating Phosphoinositide 3-Kinase Activation

    PubMed Central

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M.; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-01

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux. PMID:11805146

  19. Creatine inhibits adipogenesis by downregulating insulin-induced activation of the phosphatidylinositol 3-kinase signaling pathway.

    PubMed

    Lee, Nayeon; Kim, Inhee; Park, Soojeong; Han, Dasol; Ha, Soobong; Kwon, Mookwang; Kim, Juwan; Byun, Sung-Hyun; Oh, Wonil; Jeon, Hong Bae; Kweon, Dae-Hyuk; Cho, Jae Youl; Yoon, Keejung

    2015-04-15

    Creatine is a nitrogenous organic acid known to function in adenosine triphosphate (ATP) metabolism. Recent evidence indicates that creatine regulates the differentiation of mesenchymal stem cells (MSCs) in processes such as osteogenesis and myogenesis. In this study, we show that creatine also has a negative regulatory effect on fat cell formation. Creatine inhibits the accumulation of cytoplasmic triglycerides in a dose-dependent manner irrespective of the adipogenic cell models used, including a C3H10T1/2 MSC line, 3T3-L1 preadipocytes, and primary human MSCs. Consistently, a dramatic reduction in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), glucose transporters, 1 and 4 (Glut1, Glut4), and adipocyte markers, aP2 and adipsin, was observed in the presence of creatine. Creatine appears to exert its inhibitory effects on adipogenesis during early differentiation, but not late differentiation, or proliferation stages through inhibition of the PI3K-Akt-PPARγ signaling pathway. In an in vivo model, administration of creatine into mice resulted in body mass increase without fat accumulation. In summary, our results indicate that creatine downregulates adipogenesis through inhibition of phosphatidylinositol 3-kinase (PI3K) activation and imply the potent therapeutic value of creatine in treating obesity and obesity-related metabolic disorders.

  20. Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

    PubMed Central

    Young, Lindsey N.; Cho, Kelvin; Lawrence, Rosalie; Zoncu, Roberto; Hurley, James H.

    2016-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen–deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure. PMID:27385829

  1. Polycystin-1 Induces Resistance to Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Boca, Manila; Distefano, Gianfranco; Boletta, Alessandra; Qian, Feng; Bhunia, Anil K.; Germino, Gregory G.

    2006-01-01

    Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K β activity in PC-1- expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease. PMID:16452497

  2. Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries

    SciTech Connect

    Keating, Aileen F.; Mark, Connie J.; Sen, Nivedita; Sipes, I. Glenn; Hoyer, Patricia B.

    2009-12-01

    4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12 days in vehicle control, VCD (30 muM), or DMBA (1 muM), +- PI3 kinase inhibitor LY294002 (20 muM) or its inactive analog LY303511 (20 muM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P < 0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P < 0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P > 0.05) at any time, but did cause loss (P < 0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P < 0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P < 0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.

  3. Negative elongation factor controls energy homeostasis in cardiomyocytes.

    PubMed

    Pan, Haihui; Qin, Kunhua; Guo, Zhanyong; Ma, Yonggang; April, Craig; Gao, Xiaoli; Andrews, Thomas G; Bokov, Alex; Zhang, Jianhua; Chen, Yidong; Weintraub, Susan T; Fan, Jian-Bing; Wang, Degeng; Hu, Yanfen; Aune, Gregory J; Lindsey, Merry L; Li, Rong

    2014-04-10

    Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here, we show that whole-body conditional deletion of the B subunit of NELF (NELF-B) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptor α (PPARα), a master regulator of energy metabolism in the myocardium. Mechanistically, NELF helps stabilize the transcription initiation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARα-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes.

  4. Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk

    PubMed Central

    Stevens, K N; Garcia-Closas, M; Fredericksen, Z; Kosel, M; Pankratz, V S; Hopper, J L; Dite, G S; Apicella, C; Southey, M C; Schmidt, M K; Broeks, A; Van ‘t Veer, L J; Tollenaar, R A E M; Fasching, P A; Beckmann, M W; Hein, A; Ekici, A B; Johnson, N; Peto, J; dos Santos Silva, I; Gibson, L; Sawyer, E; Tomlinson, I; Kerin, M J; Chanock, S; Lissowska, J; Hunter, D J; Hoover, R N; Thomas, G D; Milne, R L; Pérez, JI Arias; González-Neira, A; Benítez, J; Burwinkel, B; Meindl, A; Schmutzler, R K; Bartrar, C R; Hamann, U; Ko, Y D; Brüning, T; Chang-Claude, J; Hein, R; Wang-Gohrke, S; Dörk, T; Schürmann, P; Bremer, M; Hillemanns, P; Bogdanova, N; Zalutsky, J V; Rogov, Y I; Antonenkova, N; Lindblom, A; Margolin, S; Mannermaa, A; Kataja, V; Kosma, V-M; Hartikainen, J; Chenevix-Trench, G; Chen, X; Peterlongo, P; Bonanni, B; Bernard, L; Manoukian, S; Wang, X; Cerhan, J; Vachon, C M; Olson, J; Giles, G G; Baglietto, L; McLean, C A; Severi, G; John, E M; Miron, A; Winqvist, R; Pylkäs, K; Jukkola-Vuorinen, A; Grip, M; Andrulis, I; Knight, J A; Glendon, G; Mulligan, A M; Cox, A; Brock, I W; Elliott, G; Cross, S S; Pharoah, P P; Dunning, A M; Pooley, K A; Humphreys, M K; Wang, J; Kang, D; Yoo, K-Y; Noh, D-Y; Sangrajrang, S; Gabrieau, V; Brennan, P; McKay, J; Anton-Culver, H; Ziogas, A; Couch, F J; Easton, D F

    2011-01-01

    Background: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. Methods: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC). Results: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95–0.99, P=4.6 × 10−3), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96–1.01, P=0.139). Conclusion: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer PMID:22033276

  5. Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

    PubMed Central

    Marty, Bérengère; Maire, Virginie; Gravier, Eléonore; Rigaill, Guillem; Vincent-Salomon, Anne; Kappler, Marion; Lebigot, Ingrid; Djelti, Fathia; Tourdès, Audrey; Gestraud, Pierre; Hupé, Philippe; Barillot, Emmanuel; Cruzalegui, Francisco; Tucker, Gordon C; Stern, Marc-Henri; Thiery, Jean-Paul; Hickman, John A; Dubois, Thierry

    2008-01-01

    Introduction Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs. Methods In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines. Results The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition. Conclusions These data provide insight into the molecular pathogenesis of BLCs and implicate the

  6. Functional genomics identifies negative regulatory nodes controlling phagocyte oxidative burst

    PubMed Central

    Graham, Daniel B.; Becker, Christine E.; Doan, Aivi; Goel, Gautam; Villablanca, Eduardo J.; Knights, Dan; Mok, Amanda; Ng, Aylwin C.Y.; Doench, John G.; Root, David E.; Clish, Clary B.; Xavier, Ramnik J.

    2015-01-01

    The phagocyte oxidative burst, mediated by Nox2 NADPH oxidase-derived reactive oxygen species, confers host defense against a broad spectrum of bacterial and fungal pathogens. Loss-of-function mutations that impair function of the Nox2 complex result in a life-threatening immunodeficiency, and genetic variants of Nox2 subunits have been implicated in pathogenesis of inflammatory bowel disease (IBD). Thus, alterations in the oxidative burst can profoundly impact host defense, yet little is known about regulatory mechanisms that fine-tune this response. Here we report the discovery of regulatory nodes controlling oxidative burst by functional screening of genes within loci linked to human inflammatory disease. Implementing a multi-omics approach, we define transcriptional, metabolic and ubiquitin-cycling nodes controlled by Rbpj, Pfkl and Rnf145, respectively. Furthermore, we implicate Rnf145 in proteostasis of the Nox2 complex by endoplasmic reticulum-associated degradation. Consequently, ablation of Rnf145 in murine macrophages enhances bacterial clearance, and rescues the oxidative burst defects associated with Ncf4 haploinsufficiency. PMID:26194095

  7. Nuclear but Not Cytosolic Phosphoinositide 3-Kinase Beta Has an Essential Function in Cell Survival ▿

    PubMed Central

    Kumar, Amit; Redondo-Muñoz, Javier; Perez-García, Vicente; Cortes, Isabel; Chagoyen, Monica; Carrera, Ana C.

    2011-01-01

    Class IA phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival. PMID:21383062

  8. Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance.

    PubMed Central

    Dib, K; Whitehead, J P; Humphreys, P J; Soos, M A; Baynes, K C; Kumar, S; Harvey, T; O'Rahilly, S

    1998-01-01

    Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor. PMID:9486982

  9. Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

    PubMed

    Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

    2015-10-01

    TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells.

  10. Endurance exercise training increases insulin responsiveness in isolated adipocytes through IRS/PI3-kinase/Akt pathway.

    PubMed

    Peres, Sidney B; de Moraes, Solange M Franzói; Costa, Cecilia E M; Brito, Luciana C; Takada, Julie; Andreotti, Sandra; Machado, Magaly A; Alonso-Vale, Maria Isabel C; Borges-Silva, Cristina N; Lima, Fabio B

    2005-03-01

    Endurance exercise training promotes important metabolic adaptations, and the adipose tissue is particularly affected. The aim of this study was to investigate how endurance exercise training modulates some aspects of insulin action in isolated adipocytes and in intact adipose tissue. Male Wistar rats were submitted to daily treadmill running (1 h/day) for 7 wk. Sedentary age-matched rats were used as controls. Final body weight, body weight gain, and epididymal fat pad weight did not show any statistical differences between groups. Adipocytes from trained rats were smaller than those from sedentary rats (205 +/- 16.8 vs. 286 +/- 26.4 pl; P < 0.05). Trained rats showed decreased plasma glucose (4.9 +/- 0.13 vs. 5.3 +/- 0.07 mM; P < 0.05) and insulin levels (0.24 +/- 0.012 vs. 0.41 +/- 0.049 mM; P < 0.05) and increased insulin-stimulated glucose uptake (23.1 +/- 3.1 vs. 12.1 +/- 2.9 pmol/cm(2); P < 0.05) compared with sedentary rats. The number of insulin receptors and the insulin-induced tyrosine phosphorylation of insulin receptor-beta subunit did not change between groups. Insulin-induced tyrosine phosphorylation insulin receptor substrates (IRS)-1 and -2 increased significantly (1.57- and 2.38-fold, respectively) in trained rats. Insulin-induced IRS-1/phosphatidylinositol 3 (PI3)-kinase (but not IRS-2/PI3-kinase) association and serine Akt phosphorylation also increased (2.06- and 3.15-fold, respectively) after training. The protein content of insulin receptor-beta subunit, IRS-1 and -2, did not differ between groups. Taken together, these data support the hypothesis that the increased adipocyte responsiveness to insulin observed after endurance exercise training is modulated by IRS/PI3-kinase/Akt pathway.

  11. Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

    PubMed Central

    Pérez-García, Vicente; Redondo-Muñoz, Javier; Kumar, Amit

    2014-01-01

    The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P3 levels. PMID:24958106

  12. Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways

    PubMed Central

    Zwang, N. A.; Zhang, R.; Germana, S.; Fan, M. Y.; Hastings, W. D.; Cao, A.; Turka, L. A.

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4+ and CD8+ lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform–specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4+ and CD8+ counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4+ and CD8+ lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity. PMID:27017850

  13. Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

    PubMed

    Yum, H K; Arcaroli, J; Kupfner, J; Shenkar, R; Penninger, J M; Sasaki, T; Yang, K Y; Park, J S; Abraham, E

    2001-12-01

    Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.

  14. Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

    PubMed

    Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

  15. Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase

    PubMed Central

    Williams, Karen D.; Busto, Macarena; Suster, Maximiliano L.; So, Anthony K.-C.; Ben-Shahar, Yehuda; Leevers, Sally J.; Sokolowski, Marla B.

    2006-01-01

    This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses. PMID:17043223

  16. Improved emotional conflict control triggered by the processing priority of negative emotion

    PubMed Central

    Yang, Qian; Wang, Xiangpeng; Yin, Shouhang; Zhao, Xiaoyue; Tan, Jinfeng; Chen, Antao

    2016-01-01

    The prefrontal cortex is responsible for emotional conflict resolution, and this control mechanism is affected by the emotional valence of distracting stimuli. In the present study, we investigated effects of negative and positive stimuli on emotional conflict control using a face-word Stroop task in combination with functional brain imaging. Emotional conflict was absent in the negative face context, in accordance with the null activation observed in areas regarding emotional face processing (fusiform face area, middle temporal/occipital gyrus). Importantly, these visual areas negatively coupled with the dorsolateral prefrontal cortex (DLPFC). However, the significant emotional conflict was observed in the positive face context, this effect was accompanied by activation in areas associated with emotional face processing, and the default mode network (DMN), here, DLPFC mainly negatively coupled with DMN, rather than visual areas. These results suggested that the conflict control mechanism exerted differently between negative faces and positive faces, it implemented more efficiently in the negative face condition, whereas it is more devoted to inhibiting internal interference in the positive face condition. This study thus provides a plausible mechanism of emotional conflict resolution that the rapid pathway for negative emotion processing efficiently triggers control mechanisms to preventively resolve emotional conflict. PMID:27086908

  17. Psychological control in daily parent-child interactions increases children's negative emotions.

    PubMed

    Aunola, Kaisa; Tolvanen, Asko; Viljaranta, Jaana; Nurmi, Jari-Erik

    2013-06-01

    The aim of the present study was to investigate the temporal dynamics between parental behaviors in daily interactions with their offspring, that is, affection and psychological control, and children's negative emotions. The participants were 152 Finnish families with a 6- to 7-year-old child. Children's negative emotions and parental affection and psychological control in interactions with their child were measured daily using diary questionnaires filled in by the mothers and fathers over 7 successive days. The results of multilevel modeling showed that psychological control applied by mothers and fathers in daily interactions with their child leads to an increase in negative emotions in the child. Parental affection in daily interactions with their child was not associated with the child's negative emotions.

  18. Inhibitory control and negative emotional processing in psychopathy and antisocial personality disorder.

    PubMed

    Verona, Edelyn; Sprague, Jenessa; Sadeh, Naomi

    2012-05-01

    The field of personality disorders has had a long-standing interest in understanding interactions between emotion and inhibitory control, as well as neurophysiological indices of these processes. More work in particular is needed to clarify differential deficits in offenders with antisocial personality disorder (APD) who differ on psychopathic traits, as APD and psychopathy are considered separate, albeit related, syndromes. Evidence of distinct neurobiological processing in these disorders would have implications for etiology-based personality disorder taxonomies in future psychiatric classification systems. To inform this area of research, we recorded event-related brain potentials during an emotional-linguistic Go/No-Go task to examine modulation of negative emotional processing by inhibitory control in three groups: psychopathy (n = 14), APD (n = 16), and control (n = 15). In control offenders, inhibitory control demands (No-Go vs. Go) modulated frontal-P3 amplitude to negative emotional words, indicating appropriate prioritization of inhibition over emotional processing. In contrast, the psychopathic group showed blunted processing of negative emotional words regardless of inhibitory control demands, consistent with research on emotional deficits in psychopathy. Finally, the APD group demonstrated enhanced processing of negative emotion words in both Go and No-Go trials, suggesting a failure to modulate negative emotional processing when inhibitory control is required. Implications for emotion-cognition interactions and putative etiological processes in these personality disorders are discussed.

  19. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells.

    PubMed Central

    Scott, P H; Belham, C M; al-Hafidh, J; Chilvers, E R; Peacock, A J; Gould, G W; Plevin, R

    1996-01-01

    In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation. PMID:8836145

  20. Phosphoinositide 3-kinase p110δ mediates estrogen- and FSH-stimulated ovarian follicle growth.

    PubMed

    Li, Qian; He, Hui; Zhang, Yin-Li; Li, Xiao-Meng; Guo, Xuejiang; Huo, Ran; Bi, Ye; Li, Jing; Fan, Heng-Yu; Sha, Jiahao

    2013-09-01

    In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development.

  1. Positive Emotion, Negative Emotion, and Emotion Control in the Externalizing Problems of School-Aged Children

    ERIC Educational Resources Information Center

    Kim, Geunyoung; Walden, Tedra; Harris, Vicki; Karrass, Jan; Catron, Thomas

    2007-01-01

    The present study examined the role of emotion and emotion control in children's externalizing problems. Third- to sixth-grade children were administered a self-report measure of positive emotion, negative emotion, and emotion control. Peer- and teacher-reported adjustment problems were assessed. Structural equations modeling revealed that…

  2. Role of class III phosphatidylinositol 3-kinase during programmed nuclear death of Tetrahymena thermophila.

    PubMed

    Akematsu, Takahiko; Fukuda, Yasuhiro; Attiq, Rizwan; Pearlman, Ronald E

    2014-02-01

    Programmed nuclear death (PND) in the ciliate protozoan Tetrahymena thermophila is a novel type of autophagy that occurs during conjugation, in which only the parental somatic macronucleus is destined to die and is then eliminated from the progeny cytoplasm. Other coexisting nuclei, however, such as new micro- and macronuclei are unaffected. PND starts with condensation in the nucleus followed by apoptotic DNA fragmentation, lysosomal acidification, and final resorption. Because of the peculiarity in the process and the absence of some ATG genes in this organism, the mechanism of PND has remained unclear. In this study, we focus on the role of class III phosphatidylinositol 3-kinase (PtdIns3K, corresponding to yeast Vps34) in order to identify central regulators of PND. We identified the sole Tetrahymena thermophila ortholog (TtVPS34) to yeast Vps34 and human PIK3C3 (the catalytic subunit of PtdIns3K), through phylogenetic analysis, and generated the gene knockdown mutant for functional analysis. Loss of TtVPS34 activity prevents autophagosome formation on the parental macronucleus, and this nucleus escapes from the lysosomal pathway. In turn, DNA fragmentation and final resorption of the nucleus are drastically impaired. These phenotypes are similar to the situation in the ATG8Δ mutants of Tetrahymena, implying an inextricable link between TtVPS34 and TtATG8s in controlling PND as well as general macroautophagy. On the other hand, TtVPS34 does not appear responsible for the nuclear condensation and does not affect the progeny nuclear development. These results demonstrate that TtVPS34 is critically involved in the nuclear degradation events of PND in autophagosome formation rather than with an involvement in commitment to the death program.

  3. PHOSPHOINOSITIDE 3-KINASE REGULATES THE ROLE OF RETROMER IN TRANSCYTOSIS OF THE POLYMERIC IMMUNOGLOBULIN RECEPTOR

    PubMed Central

    Vergés, Marcel; Sebastián, Isabel; Mostov, Keith E.

    2007-01-01

    Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer’s role in pIgR-pIgA transcytosis. PMID:17184770

  4. Negative Affectivity and Effortful Control in Mothers With Borderline Personality Disorder and in Their Young Children.

    PubMed

    Mena, Christina G; Macfie, Jenny; Strimpfel, Jennifer M

    2016-07-07

    Research has examined temperament in individuals with borderline personality disorder (BPD) but not in their offspring, despite offspring's risk of developing BPD and the importance of temperament in the etiology of BPD. We recruited a low-socioeconomic sample of 36 mothers with BPD and their children ages 4 through 7, and 34 normative comparisons. Replicating prior studies, mothers with BPD reported themselves as having more negative affectivity (frustration, fear) and less effortful control (inhibitory control, attentional control, activation control) than did comparisons. Mothers with BPD also reported that their offspring had more negative affectivity (anger/frustration, fear) and less effortful control (inhibitory control, attentional focusing) than did comparisons. We were concerned about potential bias and shared method variance. We therefore provided validity support for mothers' ratings of their children with teacher ratings of child behavior and child self-report via their story-stem completion narratives. We discuss children's temperamental vulnerability versus differential susceptibility to the environment.

  5. A negative association between video game experience and proactive cognitive control.

    PubMed

    Bailey, Kira; West, Robert; Anderson, Craig A

    2010-01-01

    Some evidence demonstrates that video game experience has a beneficial effect on visuospatial cognition. In contrast, other evidence indicates that video game experience may be negatively related to cognitive control. In this study we examined the specificity of the influence of video game experience on cognitive control. Participants with high and low video game experience performed the Stroop task while event-related brain potentials were recorded. The behavioral data revealed no difference between high and low gamers for the Stroop interference effect and a reduction in the conflict adaptation effect in high gamers. The amplitude of the medial frontal negativity and a frontal slow wave was attenuated in high gamers, and there was no effect of gaming status on the conflict slow potential. These data lead to the suggestion that video game experience has a negative influence on proactive, but not reactive, cognitive control.

  6. Negative thermal expansion materials: technological key for control of thermal expansion

    PubMed Central

    Takenaka, Koshi

    2012-01-01

    Most materials expand upon heating. However, although rare, some materials contract upon heating. Such negative thermal expansion (NTE) materials have enormous industrial merit because they can control the thermal expansion of materials. Recent progress in materials research enables us to obtain materials exhibiting negative coefficients of linear thermal expansion over −30 ppm K−1. Such giant NTE is opening a new phase of control of thermal expansion in composites. Specifically examining practical aspects, this review briefly summarizes materials and mechanisms of NTE as well as composites containing NTE materials, based mainly on activities of the last decade. PMID:27877465

  7. Negative thermal expansion materials: technological key for control of thermal expansion.

    PubMed

    Takenaka, Koshi

    2012-02-01

    Most materials expand upon heating. However, although rare, some materials contract upon heating. Such negative thermal expansion (NTE) materials have enormous industrial merit because they can control the thermal expansion of materials. Recent progress in materials research enables us to obtain materials exhibiting negative coefficients of linear thermal expansion over -30 ppm K(-1). Such giant NTE is opening a new phase of control of thermal expansion in composites. Specifically examining practical aspects, this review briefly summarizes materials and mechanisms of NTE as well as composites containing NTE materials, based mainly on activities of the last decade.

  8. Negative stereotype activation alters interaction between neural correlates of arousal, inhibition and cognitive control

    PubMed Central

    Cox, Christine L.; Schmader, Toni; Ryan, Lee

    2012-01-01

    Priming negative stereotypes of African Americans can bias perceptions toward novel Black targets, but less is known about how these perceptions ultimately arise. Examining how neural regions involved in arousal, inhibition and control covary when negative stereotypes are activated can provide insight into whether individuals attempt to downregulate biases. Using fMRI, White egalitarian-motivated participants were shown Black and White faces at fast (32 ms) or slow (525 ms) presentation speeds. To create a racially negative stereotypic context, participants listened to violent and misogynistic rap (VMR) in the background. No music (NM) and death metal (DM) were used as control conditions in separate blocks. Fast exposure of Black faces elicited amygdala activation in the NM and VMR conditions (but not DM), that also negatively covaried with activation in prefrontal regions. Only in VMR, however, did amygdala activation for Black faces persist during slow exposure and positively covary with activation in dorsolateral prefrontal cortex while negatively covarying with activation in orbitofrontal cortex. Findings suggest that contexts that prime negative racial stereotypes seem to hinder the downregulation of amygdala activation that typically occurs when egalitarian perceivers are exposed to Black faces. PMID:21954239

  9. Negative stereotype activation alters interaction between neural correlates of arousal, inhibition and cognitive control.

    PubMed

    Forbes, Chad E; Cox, Christine L; Schmader, Toni; Ryan, Lee

    2012-10-01

    Priming negative stereotypes of African Americans can bias perceptions toward novel Black targets, but less is known about how these perceptions ultimately arise. Examining how neural regions involved in arousal, inhibition and control covary when negative stereotypes are activated can provide insight into whether individuals attempt to downregulate biases. Using fMRI, White egalitarian-motivated participants were shown Black and White faces at fast (32 ms) or slow (525 ms) presentation speeds. To create a racially negative stereotypic context, participants listened to violent and misogynistic rap (VMR) in the background. No music (NM) and death metal (DM) were used as control conditions in separate blocks. Fast exposure of Black faces elicited amygdala activation in the NM and VMR conditions (but not DM), that also negatively covaried with activation in prefrontal regions. Only in VMR, however, did amygdala activation for Black faces persist during slow exposure and positively covary with activation in dorsolateral prefrontal cortex while negatively covarying with activation in orbitofrontal cortex. Findings suggest that contexts that prime negative racial stereotypes seem to hinder the downregulation of amygdala activation that typically occurs when egalitarian perceivers are exposed to Black faces.

  10. Multiple roles for PI 3-kinase in the regulation of PLCgamma activity and Ca2+ mobilization in antigen-stimulated mast cells.

    PubMed

    Barker, S A; Lujan, D; Wilson, B S

    1999-03-01

    Cross-linking the IgE-bound FcepsilonRI with polyvalent antigen leads to Ca2+-dependent degranulation from mast cells and basophils, initiating the allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of FcepsilonRI-mediated tyrosine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLCgamma activity and is demonstrated by a dramatic inhibition of FcepsilonRI-induced Ins(1,4,5)P3 production after treatment of RBL-2H3 cells with wortmannin, a PI 3-kinase inhibitor. We show that PI 3-kinase lipid products support Ins(1,4,5)P3 production in at least two ways: by promoting translocation and phosphorylation of PLCgamma1 and by direct stimulation of both PLCgamma isoforms. In vitro stimulation of PLCgamma activity by PtdIns(3,4,5)P3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca2+ influx. Compared with control cells, Ca2+ responses are markedly diminished in antigen-stimulated cells after wortmannin pretreatment. Differences include both a longer lag time to the initial elevation in Ca2+ after antigen and an inhibition of the sustained Ca2+ influx phase. However, thapsigargin challenge during the sustained phase demonstrates no difference in the state of the Ca2+ stores in antigen-stimulated cells in the presence or absence of wortmannin. These data suggest that sufficient Ins(1,4,5)P3 is synthesized in wortmannin-treated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca2+ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca2+ influx in antigen-stimulated RBL-2H3 cells, one that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase.

  11. On negative outcome control of unobserved confounding as a generalization of difference-in-differences

    PubMed Central

    Sofer, Tamar; Richardson, David B.; Colicino, Elena; Schwartz, Joel; Tchetgen Tchetgen, Eric J.

    2016-01-01

    The difference-in-differences (DID) approach is a well known strategy for estimating the effect of an exposure in the presence of unobserved confounding. The approach is most commonly used when pre-and post-exposure outcome measurements are available, and one can assume that the association of the unobserved confounder with the outcome is equal in the two exposure groups, and constant over time. Then, one recovers the treatment effect by regressing the change in outcome over time on the exposure. In this paper, we interpret the difference-in-differences as a negative outcome control (NOC) approach. We show that the pre-exposure outcome is a negative control outcome, as it cannot be influenced by the subsequent exposure, and it is affected by both observed and unobserved confounders of the exposure-outcome association of interest. The relation between DID and NOC provides simple conditions under which negative control outcomes can be used to detect and correct for confounding bias. However, for general negative control outcomes, the DID-like assumption may be overly restrictive and rarely credible, because it requires that both the outcome of interest and the control outcome are measured on the same scale. Thus, we present a scale-invariant generalization of the DID that may be used in broader NOC contexts. The proposed approach is demonstrated in simulations and on a Normative Aging Study data set, in which Body Mass Index is used for NOC of the relationship between air pollution and inflammatory outcomes. PMID:28239233

  12. Sound shielding by a piezoelectric membrane and a negative capacitor with feedback control.

    PubMed

    Sluka, Tomás; Kodama, Hidekazu; Fukada, Eiichi; Mokrý, Pavel

    2008-08-01

    The design and realization of an adaptive sound-shielding system based on a method to control the effective elastic stiffness of piezoelectric materials are presented in this paper. In this system, the sound-shielding effect is achieved by a sound reflection from the piezoelectric curved membrane fixed in rigid frame and connected to an active analog circuit that behaves as a negative capacitor. The acoustic transmission loss through the curved membrane was measured for the incident sound of frequency 1.6 kHz and of acoustic pressure level 80 dB. When the negative capacitor in the system was properly adjusted, the acoustic pressure level of the transmitted sound was reduced from the initial 60 dB to 15 dB by the action of the negative capacitor. Then the system was exposed to naturally changing operational conditions, and their effect on sound-shielding efficiency was studied. It is shown that the acoustic transmission loss of the system dropped by 35 dB within 30 min from the moment of negative capacitor adjustment. Therefore, a self-adjustment of the system has been implemented by appending an additional digital control circuit to the negative capacitor. It is shown that the aforementioned deteriorating effect has been eliminated by the adjusting action of the control circuit. The long-time sustainable value of 60 dB in the acoustic transmission loss of the adaptive sound shielding system has been achieved.

  13. Effects of eicosapentaenoic acid on synaptic plasticity, fatty acid profile and phosphoinositide 3-kinase signaling in rat hippocampus and differentiated PC12 cells.

    PubMed

    Kawashima, Akiko; Harada, Tsuyoshi; Kami, Hideaki; Yano, Takashi; Imada, Kazunori; Mizuguchi, Kiyoshi

    2010-04-01

    Placebo-controlled clinical studies suggest that intake of n-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimer's disease, Huntington's disease and schizophrenia. To evaluate the impact of eicosapentaenoic acid (EPA), we orally administered highly purified ethyl EPA (EPA-E) to rats at a dose of 1.0 mg/g per day and measured long-term potentiation of the CA1 hippocampal region, a physiological correlate of synaptic plasticity that is thought to underlie learning and memory. The mean field excitatory postsynaptic potential slope of the EPA-E group was significantly greater than that of the control group in the CA1 region. Gene expression of hippocampal p85alpha, one of the regulatory subunits of phosphatidylinositol 3-kinase (PI3-kinase), was increased with EPA-E administration. Investigation of fatty acid profiles of neuronal and glia-enriched fractions demonstrated that a single administration of EPA-E significantly increased neuronal and glial EPA content and glial docosahexaenoic acid content, clearly suggesting that EPA was indeed taken up by both neurons and glial cells. In addition, we investigated the direct effects of EPA on the PI3-kinase/Akt pathway in differentiated PC12 cells. Phosphorylated-Akt expression was significantly increased in EPA-treated cells, and nerve growth factor withdrawal-induced increases in cell death and caspase-3 activity were suppressed by EPA treatment. These findings suggest that EPA protects against neurodegeneration by modulating synaptic plasticity and activating the PI3-kinase/Akt pathway, possibly by its own functional effects in neurons and glial cells and by its capacity to increase brain docosahexaenoic acid.

  14. Automatic control of negative emotions: evidence that structured practice increases the efficiency of emotion regulation.

    PubMed

    Christou-Champi, Spyros; Farrow, Tom F D; Webb, Thomas L

    2015-01-01

    Emotion regulation (ER) is vital to everyday functioning. However, the effortful nature of many forms of ER may lead to regulation being inefficient and potentially ineffective. The present research examined whether structured practice could increase the efficiency of ER. During three training sessions, comprising a total of 150 training trials, participants were presented with negatively valenced images and asked either to "attend" (control condition) or "reappraise" (ER condition). A further group of participants did not participate in training but only completed follow-up measures. Practice increased the efficiency of ER as indexed by decreased time required to regulate emotions and increased heart rate variability (HRV). Furthermore, participants in the ER condition spontaneously regulated their negative emotions two weeks later and reported being more habitual in their use of ER. These findings indicate that structured practice can facilitate the automatic control of negative emotions and that these effects persist beyond training.

  15. On the control of single-prime negative priming: the effects of practice and time course.

    PubMed

    Chao, Hsuan-Fu

    2009-09-01

    Single-prime negative priming refers to the phenomenon wherein repetition of a prime as the probe target results in delayed response. Sometimes this effect has been found to be contingent on participants' unawareness of the primes, and sometimes it has not. Further, sometimes this effect has been found to be eliminated when the prime could predict the following probe target, and sometimes it has not. An integrative account is postulated to account for these findings. Three experiments supported this account by demonstrating that (a) regardless of the proportion of prime repetition, negative priming was the default effect; (b) the control mechanism was triggered to activate the prime after there was enough practice for the detection of the contingency between the prime and probe; and (c) it took time for the control mechanism to overcome the negative-priming effect and produce a positive-priming effect.

  16. Parental Perceptions of Aggressive Behavior in Preschoolers: Inhibitory Control Moderates the Association with Negative Emotionality

    ERIC Educational Resources Information Center

    Suurland, Jill; van der Heijden, Kristiaan B.; Huijbregts, Stephan C. J.; Smaling, Hanneke J. A.; de Sonneville, Leo M. J.; Van Goozen, Stephanie H. M.; Swaab, Hanna

    2016-01-01

    Inhibitory control (IC) and negative emotionality (NE) are both linked to aggressive behavior, but their interplay has not yet been clarified. This study examines different NE × IC interaction models in relation to aggressive behavior in 855 preschoolers (aged 2-5 years) using parental questionnaires. Hierarchical regression analyses revealed that…

  17. 100 s extraction of negative ion beams by using actively temperature-controlled plasma grid

    SciTech Connect

    Kojima, A. Hanada, M.; Yoshida, M.; Tobari, H.; Kashiwagi, M.; Umeda, N.; Watanabe, K.; Grisham, L. R.

    2014-02-15

    Long pulse beam extraction with a current density of 120 A/m{sup 2} for 100 s has been achieved with a newly developed plasma grid (PG) for the JT-60SA negative ion source which is designed to produce high power and long pulse beams with a negative ion current of 130 A/m{sup 2} (22 A) and a pulse length of 100 s. The PG temperature is regulated by fluorinated fluids in order to keep the high PG temperature for the cesium-seeded negative ion production. The time constant for temperature controllability of the PG was measured to be below 10 s, which was mainly determined by the heat transfer coefficient of the fluorinated fluid. The measured decay time of the negative ion current extracted from the actively temperature-controlled PG was 430 s which was sufficient for the JT-60SA requirement, and much longer than that by inertial-cooling PG of 60 s. Obtained results of the long pulse capability are utilized to design the full size PG for the JT-60SA negative ion source.

  18. Utilization of Positive and Negative Controls to Examine Comorbid Associations in Observational Database Studies

    PubMed Central

    Hyde, Craig L.; Kabadi, Shaum; St Louis, Matthew; Bonato, Vinicius; Katrina Loomis, A.; Galaznik, Aaron; Berger, Marc L.

    2017-01-01

    Background: Opportunities to leverage observational data for precision medicine research are hampered by underlying sources of bias and paucity of methods to handle resulting uncertainty. We outline an approach to account for bias in identifying comorbid associations between 2 rare genetic disorders and type 2 diabetes (T2D) by applying a positive and negative control disease paradigm. Research Design: Association between 10 common and 2 rare genetic disorders [Hereditary Fructose Intolerance (HFI) and α-1 antitrypsin deficiency] and T2D was compared with the association between T2D and 7 negative control diseases with no established relationship with T2D in 4 observational databases. Negative controls were used to estimate how much bias and variance existed in datasets when no effect should be observed. Results: Unadjusted association for common and rare genetic disorders and T2D was positive and variable in magnitude and distribution in all 4 databases. However, association between negative controls and T2D was 200% greater than expected indicating the magnitude and confidence intervals for comorbid associations are sensitive to systematic bias. A meta-analysis using this method demonstrated a significant association between HFI and T2D but not for α-1 antitrypsin deficiency. Conclusions: For observational studies, when covariate data are limited or ambiguous, positive and negative controls provide a method to account for the broadest level of systematic bias, heterogeneity, and uncertainty. This provides greater confidence in assessing associations between diseases and comorbidities. Using this approach we were able to demonstrate an association between HFI and T2D. Leveraging real-world databases is a promising approach to identify and corroborate potential targets for precision medicine therapies. PMID:27787351

  19. Treatments of Negative Symptoms in Schizophrenia: Meta-Analysis of 168 Randomized Placebo-Controlled Trials

    PubMed Central

    Fusar-Poli, Paolo; Papanastasiou, Evangelos; Stahl, Daniel; Rocchetti, Matteo; Carpenter, William; Shergill, Sukhwinder; McGuire, Philip

    2015-01-01

    Objectives: Existing treatments for schizophrenia can improve positive symptoms, but it is unclear if they have any impact on negative symptoms. This meta-analysis was conducted to assess the efficacy of available treatments for negative symptoms in schizophrenia. Methods: All randomized-controlled trials of interventions for negative symptoms in schizophrenia until December 2013 were retrieved; 168 unique and independent placebo-controlled trials were used. Negative symptom scores at baseline and follow-up, duration of illness, doses of medication, type of interventions, and sample demographics were extracted. Heterogeneity was addressed with the I 2 and Q statistic. Standardized mean difference in values of the Negative Symptom Rating Scale used in each study was calculated as the main outcome measure. Results: 6503 patients in the treatment arm and 5815 patients in the placebo arm were included. No evidence of publication biases found. Most treatments reduced negative symptoms at follow-up relative to placebo: second-generation antipsychotics: −0.579 (−0.755 to −0.404); antidepressants: −0.349 (−0.551 to −0.146); combinations of pharmacological agents: −0.518 (−0.757 to −0.279); glutamatergic medications: −0.289 (−0.478 to −0.1); psychological interventions: −0.396 (−0.563 to −0.229). No significant effect was found for first-generation antipsychotics: −0.531 (−1.104 to 0.041) and brain stimulation: −0.228 (−0.775 to 0.319). Effects of most treatments were not clinically meaningful as measured on Clinical Global Impression Severity Scale. Conclusions and Relevance: Although some statistically significant effects on negative symptoms were evident, none reached the threshold for clinically significant improvement. PMID:25528757

  20. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  1. Phosphoinositide lipid phosphatases: natural regulators of phosphoinositide 3-kinase signaling in T lymphocytes.

    PubMed

    Harris, Stephanie J; Parry, Richard V; Westwick, John; Ward, Stephen G

    2008-02-01

    The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.

  2. The association of phosphoinositide 3-kinase enhancer A with hepatic insulin receptor enhances its kinase activity.

    PubMed

    Chan, Chi Bun; Liu, Xia; He, Kunyan; Qi, Qi; Jung, Dae Y; Kim, Jason K; Ye, Keqiang

    2011-07-01

    Dysfunction of hepatic insulin receptor tyrosine kinase (IRTK) causes the development of type 2 diabetes. However, the molecular mechanism regulating IRTK activity in the liver remains poorly understood. Here, we show that phosphoinositide 3-kinase enhancer A (PIKE-A) is a new insulin-dependent enhancer of hepatic IRTK. Liver-specific Pike-knockout (LPKO) mice display glucose intolerance with impaired hepatic insulin sensitivity. Specifically, insulin-provoked phosphoinositide 3-kinase/Akt signalling is diminished in the liver of LPKO mice, leading to the failure of insulin-suppressed gluconeogenesis and hyperglycaemia. Thus, hepatic PIKE-A has a key role in mediating insulin signal transduction and regulating glucose homeostasis in the liver.

  3. Experimental Comparison of two Active Vibration Control Approaches: Velocity Feedback and Negative Capacitance Shunt Damping

    NASA Technical Reports Server (NTRS)

    Beck, Benjamin; Schiller, Noah

    2013-01-01

    This paper outlines a direct, experimental comparison between two established active vibration control techniques. Active vibration control methods, many of which rely upon piezoelectric patches as actuators and/or sensors, have been widely studied, showing many advantages over passive techniques. However, few direct comparisons between different active vibration control methods have been made to determine the performance benefit of one method over another. For the comparison here, the first control method, velocity feedback, is implemented using four accelerometers that act as sensors along with an analog control circuit which drives a piezoelectric actuator. The second method, negative capacitance shunt damping, consists of a basic analog circuit which utilizes a single piezoelectric patch as both a sensor and actuator. Both of these control methods are implemented individually using the same piezoelectric actuator attached to a clamped Plexiglas window. To assess the performance of each control method, the spatially averaged velocity of the window is compared to an uncontrolled response.

  4. Recent development of ATP-competitive small molecule phosphatidylinostitol-3-kinase inhibitors as anticancer agents

    PubMed Central

    Liu, Yu; Wan, Wen-zhu; Li, Yan; Zhou, Guan-lian; Liu, Xin-guang

    2017-01-01

    Phosphatidylinostitol-3-kinase (PI3K) is the potential anticancer target in the PI3K/Akt/ mTOR pathway. Here we reviewed the ATP-competitive small molecule PI3K inhibitors in the past few years, including the pan Class I PI3K inhibitors, the isoform-specific PI3K inhibitors and/or the PI3K/mTOR dual inhibitors. PMID:27769061

  5. Short-term low-protein diet during pregnancy alters islet area and protein content of phosphatidylinositol 3-kinase pathway in rats.

    PubMed

    Salvatierra, Cristiana S B; Reis, Sílvia R L; Pessoa, Ana F M; De Souza, Letícia M I; Stoppiglia, Luiz F; Veloso, Roberto V; Reis, Marise A B; Carneiro, Everardo M; Boschero, Antonio C; Colodel, Edson M; Arantes, Vanessa C; Latorraca, Márcia Q

    2015-01-01

    The phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways mediate β cell growth, proliferation, survival and death. We investigated whether protein restriction during pregnancy alters islet morphometry or the expression and phosphorylation of several proteins involved in the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. As controls, adult pregnant and non-pregnant rats were fed a normal-protein diet (17%). Pregnant and non-pregnant rats in the experimental groups were fed a low-protein diet (6%) for 15 days. Low protein diet during pregnancy increased serum prolactin level, reduced serum corticosterone concentration and the expression of both protein kinase B/AKT1 (AKT1) and p70 ribosomal protein S6 kinase (p70S6K), as well as the islets area, but did not alter the insulin content of pancreatic islets. Pregnancy increased the expression of the Src homology/collagen (SHC) protein and the extracellular signal-regulated kinases 1/2 (ERK1/2) independent of diet. ERK1/2 phosphorylation (pERK1/2) was similar in islets from pregnant and non-pregnant rats fed a low-protein diet, and was higher in islets from pregnant rats than in islets from non-pregnant rats fed a normal-protein diet. Thus, a short-term, low-protein diet during pregnancy was sufficient to reduce the levels of proteins in the phosphatidylinositol 3-kinase pathway and affect islet morphometry.

  6. Risk for Depression and Anxiety in Youth: The Interaction between Negative Affectivity, Effortful Control, and Stressors.

    PubMed

    Gulley, Lauren D; Hankin, Benjamin L; Young, Jami F

    2016-02-01

    Theories of temperament suggest that individual differences in affective reactivity (e.g., negative affectivity) may confer risk for internalizing psychopathology in youth and that self-regulatory aspects of temperament (e.g., effortful control) may protect against the deleterious effects of high negative affective reactivity. However, no study to date has examined how the relationship between temperament and youth internalizing psychopathology may be moderated by stress. The current study used a prospective longitudinal design to test the interaction of temperament (e.g., negative affectivity and effortful control) and stressors as a predictor of youth (ages 7-16; 56 % female; N = 576) depressive and anxious symptoms over a 3-month period. Findings show that at low levels of stress, high levels of effortful control protect against the development of depressive and anxious symptoms among youth with high levels of negative affectivity. However, at high levels of stress, this buffering effect is not observed. Gender and grade did not moderate this relationship. Overall, findings extend current understanding of how the interaction of individual psychosocial vulnerabilities and environmental factors may confer increased or decreased risk for depressive and anxious symptoms.

  7. Risk for Depression and Anxiety in Youth: The Interaction between Negative Affectivity, Effortful Control, and Stressors

    PubMed Central

    Gulley, Lauren D.; Hankin, Benjamin L.; Young, Jami F.

    2015-01-01

    Theories of temperament suggest that individual differences in affective reactivity (e.g., negative affectivity) may confer risk for internalizing psychopathology in youth and that self-regulatory aspects of temperament (e.g., effortful control) may protect against the deleterious effects of high negative affective reactivity. However, no study to date has examined how the relationship between temperament and youth internalizing psychopathology may be moderated by stress. The current study used a prospective longitudinal design to test the interaction of temperament (e.g., negative affectivity and effortful control) and stressors as a predictor of youth (ages 7–16; 56% female; N = 576) depressive and anxious symptoms over a 3-month period. Findings show that at low levels of stress, high levels of effortful control protect against the development of depressive and anxious symptoms among youth with high levels of negative affectivity. However, at high levels of stress, this buffering effect is not observed. Gender and grade did not moderate this relationship. Overall, findings extend current understanding of how the interaction of individual psychosocial vulnerabilities and environmental factors may confer increased or decreased risk for depressive and anxious symptoms. PMID:25870113

  8. Ribonuclease 5 facilitates corneal endothelial wound healing via activation of PI3-kinase/Akt pathway

    PubMed Central

    Kim, Kyoung Woo; Park, Soo Hyun; Lee, Soo Jin; Kim, Jae Chan

    2016-01-01

    To maintain corneal transparency, corneal endothelial cells (CECs) exert a pump function against aqueous inflow. However, human CECs are arrested in the G1-phase and non-proliferative in vivo. Thus, treatment of corneal endothelial decompensation is limited to corneal transplantation, and grafts are vulnerable to immune rejection. Here, we show that ribonuclease (RNase) 5 is more highly expressed in normal human CECs compared to decompensated tissues. Furthermore, RNase 5 up-regulated survival of CECs and accelerated corneal endothelial wound healing in an in vitro wound of human CECs and an in vivo cryo-damaged rabbit model. RNase 5 treatment rapidly induced accumulation of cytoplasmic RNase 5 into the nucleus, and activated PI3-kinase/Akt pathway in human CECs. Moreover, inhibition of nuclear translocation of RNase 5 using neomycin reversed RNase 5-induced Akt activation. As a potential strategy for proliferation enhancement, RNase 5 increased the population of 5-bromo-2′-deoxyuridine (BrdU)-incorporated proliferating CECs with concomitant PI3-kinase/Akt activation, especially in CECs deprived of contact-inhibition. Specifically, RNase 5 suppressed p27 and up-regulated cyclin D1, D3, and E by activating PI3-kinase/Akt in CECs to initiate cell cycle progression. Together, our data indicate that RNase 5 facilitates corneal endothelial wound healing, and identify RNase 5 as a novel target for therapeutic exploitation. PMID:27526633

  9. Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.

    PubMed

    Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C

    2003-07-01

    Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

  10. Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.

    PubMed

    Matsunaga, Kohichi; Morita, Eiji; Saitoh, Tatsuya; Akira, Shizuo; Ktistakis, Nicholas T; Izumi, Tetsuro; Noda, Takeshi; Yoshimori, Tamotsu

    2010-08-23

    Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

  11. DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Ishizuka, T; Kajita, K; Miura, A; Ishizawa, M; Kanoh, Y; Itaya, S; Kimura, M; Muto, N; Mune, T; Morita, H; Yasuda, K

    1999-01-01

    We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

  12. Pooled Analysis of Phosphatidylinositol 3-kinase Pathway Variants and Risk of Prostate Cancer

    PubMed Central

    Koutros, Stella; Schumacher, Fredrick R.; Hayes, Richard B.; Ma, Jing; Huang, Wen-Yi; Albanes, Demetrius; Canzian, Federico; Chanock, Stephen J.; Crawford, E. David; Diver, W. Ryan; Feigelson, Heather Spencer; Giovanucci, Edward; Haiman, Christopher A.; Henderson, Brian E.; Hunter, David J.; Kaaks, Rudolf; Kolonel, Laurence N.; Kraft, Peter; Le Marchand, Loïc; Riboli, Elio; Siddiq, Afshan; Stampfer, Mier J.; Stram, Daniel O.; Thomas, Gilles; Travis, Ruth C.; Thun, Michael J.; Yeager, Meredith; Berndt, Sonja I.

    2010-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway regulates various cellular processes, including cellular proliferation and intracellular trafficking and may impact prostate carcinogenesis. Thus, we explored the association between single nucleotide polymorphisms (SNPs) in PI3K genes and prostate cancer. Pooled data from the National Cancer Institute Breast and Prostate Cancer Cohort Consortium were examined for associations between 89 SNPs in PI3K genes (PIK3C2B, PIK3AP1, PIK3C2A, PIK3CD, and PIK3R3) and prostate cancer risk in 8,309 cases and 9,286 controls. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression. SNP rs7556371 in PIK3C2B was significantly associated with prostate cancer risk (ORper allele=1.08 (95% CI: 1.03, 1.14), p-trend = 0.0017) after adjustment for multiple testing (Padj=0.024). Simultaneous adjustment of rs7556371 for nearby SNPs strengthened the association (ORper allele=1.21 (95% CI: 1.09, 1.34); p-trend =0.0003). The adjusted association was stronger for men who were diagnosed before 65 years (ORper allele= 1.47 (95% CI: 1.20, 1.79), p-trend = 0.0001) or had a family history (ORper allele= 1.57 (95% CI: 1.11, 2.23), p-trend = 0.0114), and was strongest in those with both characteristics (ORper allele= 2.31 (95% CI: 1.07, 5.07), p-interaction = 0.005). Increased risks were observed among men in the top tertile of circulating insulin like growth factor-1 (IGF-1) levels (ORper allele= 1.46 (95% CI: 1.04, 2.06), p-trend=0.075). No differences were observed with disease aggressiveness (≥8/stage T3/T4/fatal). In conclusion, we observed a significant association between PIK3C2B and prostate cancer risk, especially for familial, early onset disease, which may be attributable to IGF-dependent PI3K signaling. PMID:20197460

  13. Negative control in two-component signal transduction by transmitter phosphatase activity.

    PubMed

    Huynh, TuAnh Ngoc; Stewart, Valley

    2011-10-01

    Bifunctional sensor transmitter modules of two-component systems exert both positive and negative control on the receiver domain of the cognate response regulator. In negative control, the transmitter module accelerates the rate of phospho-receiver dephosphorylation. This transmitter phosphatase reaction serves the important physiological functions of resetting response regulator phosphorylation level and suppressing cross-talk. Although the biochemical reactions underlying positive control are reasonably well understood, the mechanism for transmitter phosphatase activity has been unknown. A recent hypothesis is that the transmitter phosphatase reaction is catalysed by a conserved Gln, Asn or Thr residue, via a hydrogen bond between the amide or hydroxyl group and the nucleophilic water molecule in acyl-phosphate hydrolysis. This hypothetical mechanism closely resembles the established mechanisms of auxiliary phosphatases such as CheZ and CheX, and may be widely conserved in two-component signal transduction. In addition to the proposed catalytic residues, transmitter phosphatase activity also requires the correct transmitter conformation and appropriate interactions with the receiver. Evidence suggests that the phosphatase-competent and autokinase-competent states are mutually exclusive, and the corresponding negative and positive activities are likely to be reciprocally regulated through dynamic control of transmitter conformations.

  14. Cell Shape and Negative Links in Regulatory Motifs Together Control Spatial Information Flow in Signaling Networks

    PubMed Central

    Neves, Susana R.; Tsokas, Panayiotis; Sarkar, Anamika; Grace, Elizabeth A.; Rangamani, Padmini; Taubenfeld, Stephen M.; Alberini, Cristina M.; Schaff, James C.; Blitzer, Robert D.; Moraru, Ion I.; Iyengar, Ravi

    2009-01-01

    Summary The role of cell size and shape in controlling local intracellular signaling reactions, and how this spatial information originates and is propagated, is not well understood. We have used partial differential equations to model the flow of spatial information from the β-adrenergic receptor to MAPK1,2 through the cAMP/PKA/B-Raf/MAPK1,2 network in neurons using real geometries. The numerical simulations indicated that cell shape controls the dynamics of local biochemical activity of signal-modulated negative regulators, such as phosphodiesterases and protein phosphatases within regulatory loops to determine the size of microdomains of activated signaling components. The model prediction that negative regulators control the flow of spatial information to downstream components was verified experimentally in rat hippocampal slices. These results suggest a mechanism by which cellular geometry, the presence of regulatory loops with negative regulators, and key reaction rates all together control spatial information transfer and microdomain characteristics within cells. PMID:18485874

  15. Effectiveness of group body psychotherapy for negative symptoms of schizophrenia: multicentre randomised controlled trial†

    PubMed Central

    Priebe, S.; Savill, M.; Wykes, T.; Bentall, R. P.; Reininghaus, U.; Lauber, C.; Bremner, S.; Eldridge, S.; Röhricht, F.

    2016-01-01

    Background Negative symptoms of schizophrenia have a severe impact on functional outcomes and treatment options are limited. Arts therapies are currently recommended but more evidence is required. Aims To assess body psychotherapy as a treatment for negative symptoms compared with an active control (trial registration: ISRCTN84216587). Method Schizophrenia out-patients were randomised into a 20-session body psychotherapy or Pilates group. The primary outcome was negative symptoms at end of treatment. Secondary outcomes included psychopathology, functional, social and treatment satisfaction outcomes at treatment end and 6-months later. Results In total, 275 participants were randomised. The adjusted difference in negative symptoms was 0.03 (95% CI −1.11 to 1.17), indicating no benefit from body psychotherapy. Small improvements in expressive deficits and movement disorder symptoms were detected in favour of body psychotherapy. No other outcomes were significantly different. Conclusions Body psychotherapy does not have a clinically relevant beneficial effect in the treatment of patients with negative symptoms of schizophrenia. PMID:27151073

  16. A causal role for the anterior mid-cingulate cortex in negative affect and cognitive control.

    PubMed

    Tolomeo, Serenella; Christmas, David; Jentzsch, Ines; Johnston, Blair; Sprengelmeyer, Reiner; Matthews, Keith; Douglas Steele, J

    2016-06-01

    Converging evidence has linked the anterior mid-cingulate cortex to negative affect, pain and cognitive control. It has previously been proposed that this region uses information about punishment to control aversively motivated actions. Studies on the effects of lesions allow causal inferences about brain function; however, naturally occurring lesions in the anterior mid-cingulate cortex are rare. In two studies we therefore recruited 94 volunteers, comprising 15 patients with treatment-resistant depression who had received bilateral anterior cingulotomy, which consists of lesions made within the anterior mid-cingulate cortex, 20 patients with treatment-resistant depression who had not received surgery and 59 healthy control subjects. Using the Ekman 60 faces paradigm and two Stroop paradigms, we tested the hypothesis that patients who received anterior cingulotomy were impaired in recognizing negative facial affect expressions but not positive or neutral facial expressions, and impaired in Stroop cognitive control, with larger lesions being associated with more impairment. Consistent with this hypothesis, we found that larger volume lesions predicted more impairment in recognizing fear, disgust and anger, and no impairment in recognizing facial expressions of surprise or happiness. However, we found no impairment in recognizing expressions of sadness. Also consistent with the hypothesis, we found that larger volume lesions predicted impaired Stroop cognitive control. Notably, this relationship was only present when anterior mid-cingulate cortex lesion volume was defined as the overlap between cingulotomy lesion volume and Shackman's meta-analysis-derived binary masks for negative affect and cognitive control. Given substantial evidence from healthy subjects that the anterior mid-cingulate cortex is part of a network associated with the experience of negative affect and pain, engaging cognitive control processes for optimizing behaviour in the presence of such

  17. Sensitivity control through attenuation of signal transfer efficiency by negative regulation of cellular signalling.

    PubMed

    Toyoshima, Yu; Kakuda, Hiroaki; Fujita, Kazuhiro A; Uda, Shinsuke; Kuroda, Shinya

    2012-03-13

    Sensitivity is one of the hallmarks of biological and pharmacological responses. However, the principle of controlling sensitivity remains unclear. Here we theoretically analyse a simple biochemical reaction and find that the signal transfer efficiency of the transient peak amplitude attenuates depending on the strength of negative regulation. We experimentally find that many signalling pathways in various cell lines, including the Akt and ERK pathways, can be approximated by simple biochemical reactions and that the same property of the attenuation of signal transfer efficiency was observed for such pathways. Because of this property, a downstream molecule should show higher sensitivity to an activator and lower sensitivity to an inhibitor than an upstream molecule. Indeed, we experimentally verify that S6, which lies downstream of Akt, shows lower sensitivity to an epidermal growth factor receptor inhibitor than Akt. Thus, cells can control downstream sensitivity through the attenuation of signal transfer efficiency by changing the expression level of negative regulators.

  18. Long-pulse production of high current negative ion beam by using actively temperature controlled plasma grid for JT-60SA negative ion source

    SciTech Connect

    Kojima, A.; Hanada, M.; Yoshida, M.; Umeda, N.; Hiratsuka, J.; Kashiwagi, M.; Tobari, H.; Watanabe, K.; Grisham, L. R.

    2015-04-08

    The temperature control system of the large-size plasma grid has been developed to realize the long pulse production of high-current negative ions for JT-60SA. By using this prototype system for the JT-60SA ion source, 15 A negative ions has been sustained for 100 s for the first time, which is three times longer than that obtained in JT-60U. In this system, a high-temperature fluorinated fluid with a high boiling point of 270 degree Celsius is circulated in the cooling channels of the plasma grids (PG) where a cesium (Cs) coverage is formed to enhance the negative ion production. Because the PG temperature control had been applied to only 10% of the extraction area previously, the prototype PG with the full extraction area (110 cm × 45 cm) was developed to increase the negative ion current in this time. In the preliminary results of long pulse productions of high-current negative ions at a Cs conditioning phase, the negative ion production was gradually degraded in the last half of 100 s pulse where the temperature of an arc chamber wall was not saturated. From the spectroscopic measurements, it was found that the Cs flux released from the wall might affect to the negative ion production, which implied the wall temperature should be kept low to control the Cs flux to the PG for the long-pulse high-current production. The obtained results of long-pulse production and the PG temperature control method contributes the design of the ITER ion source.

  19. Hunger and negative alliesthesia to aspartame and sucrose in patients treated with antipsychotic drugs and controls.

    PubMed

    Khazaal, Y; Chatton, A; Claeys, F; Ribordy, F; Khan, R; Zullino, D

    2009-12-01

    The present study explores sweet stimuli effects on hunger and negative alliesthesia in patients treated with antipsychotic drugs and controls. Those phenomena were examined in relation to previous weight gain, eating and weight-related cognitions and type of sweet stimuli: aspartame or sucrose. Alliesthesia is delayed in participants who gained weight regardless of cross group differences. A similar reduction of hunger was observed after the intake of two kinds of sweet stimuli (aspartame or sucrose) whereas alliesthesia measures were not affected. Whereas atypical antipsychotic drug-induced weight gain is linked to delayed satiety, the phenomenon is similar in magnitude in non-psychiatric controls who gained weight.

  20. Control algorithm of lighting color for LED considering human's negative feeling

    NASA Astrophysics Data System (ADS)

    Yamagishi, Yoko; Mita, Akira

    2014-03-01

    This research looks for appropriate colors of lighting depending on several kinds of feelings, covering not only colors which are on trajectory of a complete radiator but also other various colors, and aims to propose a control algorithm of lighting color of LED considering human's negative feelings. This research consists of three experiments. The first experiment aims to seek an appropriate color of lighting when we feel anger, sadness, happy and joy. The second experiment is to seek for reasons of results of the first experiment and to look for what kind of impression of lighting color leads to comfortable lighting environment. The third experiment aims to look for an applicable control of lighting colors when considering people's feelings. From these experiments, it was found that comfortable lighting color differ depending on feelings and the possibility of an effective use of colors which are not on the trajectory of a complete radiator was shown. Moreover, many elements which lead to comfortable lighting control were found, and as the controlling method which consider those elements was more preferred than the method which does not change colors at all, the usability of controlling lighting colors considering people's negative feelings was proved.

  1. Adolescent Neurodevelopment of Cognitive Control and Risk-taking in Negative Family Contexts

    PubMed Central

    McCormick, Ethan M.; Qu, Yang; Telzer, Eva H.

    2015-01-01

    Adolescents have an increased need to regulate their behavior as they gain access to opportunities for risky behavior; however, cognitive control systems necessary for this regulation remain relatively immature. Parents can impact their adolescent child's abilities to regulate their behavior and engagement in risk taking. Since adolescents undergo significant neural change, negative parent-child relationship quality may impede or alter development in prefrontal regions subserving cognitive control. To test this hypothesis, 20 adolescents completed a go/nogo task during two fMRI scans occurring 1 year apart. Adolescents reporting greater family conflict and lower family cohesion showed longitudinal increases in risk-taking behavior, which was mediated by longitudinal increases in left VLPFC activation during cognitive control. These results underscore the importance of parent-child relationships during early adolescence, and the neural processes by which cognitive control may be derailed and lead to increased risk taking. PMID:26434803

  2. The Interaction between Negative Emotionality and Effortful Control in Early Social-emotional Development

    PubMed Central

    Moran, Lyndsey R.; Lengua, Liliana J.; Zalewski, Maureen

    2014-01-01

    Interactions between reactive and regulatory dimensions of temperament may be particularly relevant to children’s adjustment but are examined infrequently. This study investigated these interactions by examining effortful control as a moderator of the relations of fear and frustration reactivity to children’s social competence, internalizing, and externalizing problems. Participants included 306 three-year-old children and their mothers. Children’s effortful control was measured using observational measures, and reactivity was assessed with both observational and mother-reported measures. Mothers reported on children’s adjustment. Significant interactions indicated that children with higher mother-reported fear or higher observed frustration and lower executive control showed higher externalizing problems whereas children with higher observed fear and higher delay ability demonstrated lower externalizing problems. These results highlight effortful control as a moderator of the relation between reactivity and adjustment, and may inform the development of interventions geared toward the management of specific negative affects. PMID:25429192

  3. The Interaction between Negative Emotionality and Effortful Control in Early Social-emotional Development.

    PubMed

    Moran, Lyndsey R; Lengua, Liliana J; Zalewski, Maureen

    2013-05-01

    Interactions between reactive and regulatory dimensions of temperament may be particularly relevant to children's adjustment but are examined infrequently. This study investigated these interactions by examining effortful control as a moderator of the relations of fear and frustration reactivity to children's social competence, internalizing, and externalizing problems. Participants included 306 three-year-old children and their mothers. Children's effortful control was measured using observational measures, and reactivity was assessed with both observational and mother-reported measures. Mothers reported on children's adjustment. Significant interactions indicated that children with higher mother-reported fear or higher observed frustration and lower executive control showed higher externalizing problems whereas children with higher observed fear and higher delay ability demonstrated lower externalizing problems. These results highlight effortful control as a moderator of the relation between reactivity and adjustment, and may inform the development of interventions geared toward the management of specific negative affects.

  4. Adolescent neurodevelopment of cognitive control and risk-taking in negative family contexts.

    PubMed

    McCormick, Ethan M; Qu, Yang; Telzer, Eva H

    2016-01-01

    Adolescents have an increased need to regulate their behavior as they gain access to opportunities for risky behavior; however, cognitive control systems necessary for this regulation remain relatively immature. Parents can impact their adolescent child's abilities to regulate their behavior and engagement in risk taking. Since adolescents undergo significant neural change, negative parent-child relationship quality may impede or alter development in prefrontal regions subserving cognitive control. To test this hypothesis, 20 adolescents completed a Go/NoGo task during two fMRI scans occurring 1year apart. Adolescents reporting greater family conflict and lower family cohesion showed longitudinal increases in risk-taking behavior, which was mediated by longitudinal increases in left VLPFC activation during cognitive control. These results underscore the importance of parent-child relationships during early adolescence, and the neural processes by which cognitive control may be derailed and may lead to increased risk taking.

  5. Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.

    PubMed

    Martelli, A M; Evangelisti, C; Follo, M Y; Ramazzotti, G; Fini, M; Giardino, R; Manzoli, L; McCubrey, J A; Cocco, L

    2011-01-01

    Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.

  6. Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras-Induced Lung Cancer

    PubMed Central

    Yang, Yanan; Iwanaga, Kentaro; Raso, Maria Gabriela; Wislez, Marie; Hanna, Amy E.; Wieder, Eric D.; Molldrem, Jeffrey J.; Wistuba, Ignacio I.; Powis, Garth; Demayo, Francesco J.; Kim, Carla F.; Kurie, Jonathan M.

    2008-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined. Methodology/Principal Findings We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K. Conclusions/Significance We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients. PMID:18493606

  7. Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis.

    PubMed

    Mishima, Elina; Sharma, Ashu

    2011-08-01

    Tannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of 'lipid rafts'), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process.

  8. Enhancement of morphological plasticity in hippocampal neurons by a physically modified saline via phosphatidylinositol-3 kinase.

    PubMed

    Roy, Avik; Modi, Khushbu K; Khasnavis, Saurabh; Ghosh, Supurna; Watson, Richard; Pahan, Kalipada

    2014-01-01

    Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer's disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias.

  9. PP6 controls T cell development and homeostasis by negatively regulating distal TCR signaling.

    PubMed

    Ye, Jian; Shi, Hao; Shen, Ye; Peng, Chao; Liu, Yan; Li, Chenyu; Deng, Kejing; Geng, Jianguo; Xu, Tian; Zhuang, Yuan; Zheng, Biao; Tao, Wufan

    2015-02-15

    T cell development and homeostasis are both regulated by TCR signals. Protein phosphorylation and dephosphorylation, which are catalyzed by protein kinases and phosphatases, respectively, serve as important switches controlling multiple downstream pathways triggered by TCR recognition of Ags. It has been well documented that protein tyrosine phosphatases are involved in negative regulation of proximal TCR signaling. However, how TCR signals are terminated or attenuated in the distal TCR signaling pathways is largely unknown. We investigated the function of Ser/Thr protein phosphatase (PP) 6 in TCR signaling. T cell lineage-specific ablation of PP6 in mice resulted in enhanced thymic positive and negative selection, and preferential expansion of fetal-derived, IL-17-producing Vγ6Vδ1(+) T cells. Both PP6-deficient peripheral CD4(+) helper and CD8(+) cytolytic cells could not maintain a naive state and became fast-proliferating and short-lived effector cells. PP6 deficiency led to profound hyperactivation of multiple distal TCR signaling molecules, including MAPKs, AKT, and NF-κB. Our studies demonstrate that PP6 acts as a critical negative regulator, not only controlling both αβ and γδ lineage development, but also maintaining naive T cell homeostasis by preventing their premature activation before Ag stimulation.

  10. Structural basis for isoform selectivity in a class of benzothiazole inhibitors of phosphoinositide 3-kinase γ.

    PubMed

    Collier, Philip N; Martinez-Botella, Gabriel; Cornebise, Mark; Cottrell, Kevin M; Doran, John D; Griffith, James P; Mahajan, Sudipta; Maltais, François; Moody, Cameron S; Huck, Emilie Porter; Wang, Tiansheng; Aronov, Alex M

    2015-01-08

    Phosphoinositide 3-kinase γ (PI3Kγ) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3Kγ, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region.

  11. Synthesis and Pharmacological Evaluation of 4-Iminothiazolidinones for Inhibition of PI3 Kinase

    PubMed Central

    Pinson, Jo-Anne; Schmidt-Kittler, Oleg; Frazzetto, Mark; Zheng, Zhaohua; Jennings, Ian G.; Kinzler, Kenneth W.; Vogelstein, Bert; Chalmers, David K.; Thompson, Philip E.

    2012-01-01

    The thiazolidinedione, compound 1, has previously shown pan-inhibition of the phosphoinositide 3-kinase (PI3K) class I isoforms. We hypothesized the derivatization of the thiazolidinedione core of compound 1 could introduce isoform selectivity. We report the synthesis, characterization, and inhibitory activity of a novel series of 4-iminothiazolidin-2-ones for inhibition of the class I PI3K isoforms. Their synthesis was successfully achieved by multiple pathways described in this paper. Initial in vitro data of 28 analogues demonstrated poor inhibition of all class I PI3K isoforms. However, we identified an alternate target, the phosphodiesterases, and present preliminary screening results showing improved inhibitory activity. PMID:23997244

  12. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  13. Constitutively activated phosphatidylinositol 3-kinase primes platelets from patients with chronic myelogenous leukemia for thrombopoietin-induced aggregation.

    PubMed

    Kubota, Y; Tanaka, T; Ohnishi, H; Kitanaka, A; Okutani, Y; Taminato, T; Ishida, T; Kamano, H

    2004-06-01

    In this study, we examined the effect of thrombopoietin (TPO) on the aggregation of platelets from 40 patients with myeloproliferative disorders (MPDs), including 17 patients with chronic myelogenous leukemia in the chronic phase (CML-CP), 10 with polycythemia vera, 10 with essential thrombocythemia, and three with myelofibrosis. TPO by itself dose-dependently induced the aggregation of platelets from patients with CML-CP but not from those with other MPDs or with CML-CP in cytogenetical complete remission. The expression of CD63 in CML-CP platelets was induced by TPO treatment. Phosphatidylinositol 3-kinase (PI3-kinase) was constitutively activated in CML-CP platelets. Pretreatment with PI3-kinase inhibitors (wortmannin and LY294002) dose-dependently inhibited TPO-induced aggregation of CML-CP platelets. The Abl kinase inhibitor imatinib mesylate and the Jak inhibitor AG490 suppressed TPO-induced aggregation of CML-CP platelets. Pretreatment with imatinib mesylate, but not with AG490, inhibited the activity of PI3-kinase in CML-CP platelets. In addition, tyrosine phosphorylation of Jak2 was undetected in CML-CP platelets before TPO treatment. These findings indicate that the constitutive activation of PI3-kinase primes CML-CP platelets for the aggregation induced by TPO, and that Bcr-Abl, but not Jak family protein tyrosine kinases, are involved in the constitutive activation of PI3-kinase in CML-CP platelets.

  14. CUSUM control charts to monitor series of Negative Binomial count data.

    PubMed

    Alencar, Airlane Pereira; Lee Ho, Linda; Albarracin, Orlando Yesid Esparza

    2015-06-26

    To detect outbreaks of diseases in public health, several control charts have been proposed in the literature. In this context, the usual generalized linear model may be fitted for counts under a Negative Binomial distribution with a logarithm link function and the population size included as offset to model hospitalization rates. Different statistics are used to build CUSUM control charts to monitor daily hospitalizations and their performances are compared in simulation studies. The main contribution of the current paper is to consider different statistics based on transformations and the deviance residual to build control charts to monitor counts with seasonality effects and evaluate all the assumptions of the monitored statistics. The monitoring of daily number of hospital admissions due to respiratory diseases for people aged over 65 years in the city São Paulo-Brazil is considered as an illustration of the current proposal.

  15. The Phosphatidylinositol 3-Kinase/Akt Pathway Negatively Regulates Nod2-Mediated NF-kB Pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways including NF-kB pathway. Here, we show that Nod2 agonist MDP induces Ak...

  16. Idelalisib: Targeting the PI3 Kinase Pathway in Non-Hodgkin Lymphoma.

    PubMed

    Sujobert, Pierre; Rioufol, Catherine; Salles, Gilles A

    2016-01-01

    Based on substantial preclinical rationale, the restricted hematopoietic expression of the δ isoform of the phosphatidylinositol 3-kinase represents an attractive therapeutic target in B-cell malignancies. Its inhibition results in a direct antiproliferative effect on tumor cells as well as several modifications of their cellular microenvironment, all accounting for the potential therapeutic interest. Idelalisib, the first-in-class phosphatidylinositol 3-kinase δ-specific inhibitor, was developed in patients with B-cell lymphomas and chronic lymphocytic leukemia. Early clinical results demonstrated a potent antitumor effect across different subtypes of indolent and mantle cell lymphomas (where response duration was short). Adverse events, including transaminitis, neutropenia, pneumonitis, and diarrhea, were observed. A pivotal phase II study in patients with double refractory disease showed a 57% response rate, with response lasting for about 1 year, leading to market approval of the drug in the United States and Europe. Further developments of idelalisib combinations will contribute to delineate the position of this drug in the therapeutic strategy of indolent lymphomas.

  17. Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy.

    PubMed

    Yu, Xinlei; Long, Yun Chau; Shen, Han-Ming

    2015-01-01

    Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases.

  18. LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

    SciTech Connect

    Tong, W.-G.; Ding, X.-Z.; Talamonti, Mark S.; Bell, Richard H.; Adrian, Thomas E. . E-mail: tadrian@northwestern.edu

    2005-09-30

    We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.

  19. Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects.

    PubMed Central

    Goodyear, L J; Giorgino, F; Sherman, L A; Carey, J; Smith, R J; Dohm, G L

    1995-01-01

    To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9 +/- 3.6 vs 25.7 +/- 0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with 10(-7) M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), measured by immunoblotting with anti-phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10- vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulin-stimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action. Images PMID:7537758

  20. Phosphoinositide-3-kinases p110α and p110β mediate S phase entry in astroglial cells in the marginal zone of rat neocortex.

    PubMed

    Müller, Rabea; Fischer, Catharina; Wilmes, Thomas; Heimrich, Bernd; Distel, Vanessa; Klugbauer, Norbert; Meyer, Dieter K

    2013-01-01

    In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative (dn) isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine (BrdU). Only in astroglial cells harvested from the marginal zone (MZ) of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with BrdU, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110α and p110β were essential for S phase entry. p110α diminished the level of the p27(Kip1) which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110β phosphorylated and inhibited glycogen synthase kinase-3β which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

  1. Irradiation effect of carbon negative-ion implantation on polytetrafluoroethylene for controlling cell-adhesion property

    NASA Astrophysics Data System (ADS)

    Sommani, Piyanuch; Tsuji, Hiroshi; Kojima, Hiroyuki; Sato, Hiroko; Gotoh, Yasuhito; Ishikawa, Junzo; Takaoka, Gikan H.

    2010-10-01

    We have investigated the irradiation effect of negative-ion implantation on the changes of physical surface property of polytetrafluoroethylene (PTFE) for controlling the adhesion property of stem cells. Carbon negative ions were implanted into PTFE sheets at fluences of 1 × 10 14-1 × 10 16 ions/cm 2 and energies of 5-20 keV. Wettability and atomic bonding state including the ion-induced functional groups on the modified surfaces were investigated by water contact angle measurement and XPS analysis, respectively. An initial value of water contact angles on PTFE decreased from 104° to 88° with an increase in ion influence to 1 × 10 16 ions/cm 2, corresponding to the peak shifting of XPS C1s spectra from 292.5 eV to 285 eV with long tail on the left peak-side. The change of peak position was due to decrease of C-F 2 bonds and increase of C-C bonds with the formation of hydrophilic oxygen functional groups of OH and C dbnd O bonds after the ion implantation. After culturing rat mesenchymal stem cells (MSC) for 4 days, the cell-adhesion properties on the C --patterned PTFE were observed by fluorescent microscopy with staining the cell nuclei and their actin filament (F-actin). The clear adhesion patterning of MSCs on the PTFE was obtained at energies of 5-10 keV and a fluence of 1 × 10 15 ions/cm 2. While the sparse patterns and the uncontrollable patterns were found at a low fluence of 3 × 10 14 ions/cm 2 and a high fluence of 3 × 10 15 ions/cm 2, respectively. As a result, we could improve the surface wettability of PTFE to control the cell-adhesion property by carbon negative-ion implantation.

  2. Control of adolescents' arbitrary matching-to-sample by positive and negative stimulus relations.

    PubMed Central

    Stromer, R; Osborne, J G

    1982-01-01

    In Experiment 1, four developmentally delayed adolescents were taught an A-B matching-to-sample task with nonidentical stimuli: given Sample A1, select Comparison B1; given A2, select B2. During nonreinforced test trials, appropriate matching occurred when B stimuli appeared as samples and A stimuli as comparisons, i.e., the sample and comparison functions were symmetrical (B-A matching). During A-B or B-A matching test trials in which familiar samples and correct comparisons were presented along with novel comparisons, the subjects selected the correct comparisons. In tests with familiar samples and both incorrect and novel comparisons, subjects selected the novel comparisons, demonstrating control by both positive ("matching") and negative ("nonmatching") stimulus relations in A-B and B-A arrays. In Experiment 2, 12 developmentally delayed subjects were taught a two-stage arbitrary-matching task (e.g., A-B, C-B matching). Test sessions showed sample-comparison symmetry (e.g., B-A, B-C matching) and derived sample-comparison relations (e.g., A-C, C-A matching) for 11 subjects. These subjects also demonstrated control by positive and negative stimulus relations in the derived relations. PMID:6178786

  3. Negative control of BAK1 by protein phosphatase 2A during plant innate immunity.

    PubMed

    Segonzac, Cécile; Macho, Alberto P; Sanmartín, Maite; Ntoukakis, Vardis; Sánchez-Serrano, José Juan; Zipfel, Cyril

    2014-09-17

    Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors (PRRs) activates plant innate immunity, mainly through activation of numerous protein kinases. Appropriate induction of immune responses must be tightly regulated, as many of the kinases involved have an intrinsic high activity and are also regulated by other external and endogenous stimuli. Previous evidences suggest that PAMP-triggered immunity (PTI) is under constant negative regulation by protein phosphatases but the underlying molecular mechanisms remain unknown. Here, we show that protein Ser/Thr phosphatase type 2A (PP2A) controls the activation of PRR complexes by modulating the phosphostatus of the co-receptor and positive regulator BAK1. A potential PP2A holoenzyme composed of the subunits A1, C4, and B'η/ζ inhibits immune responses triggered by several PAMPs and anti-bacterial immunity. PP2A constitutively associates with BAK1 in planta. Impairment in this PP2A-based regulation leads to increased steady-state BAK1 phosphorylation, which can poise enhanced immune responses. This work identifies PP2A as an important negative regulator of plant innate immunity that controls BAK1 activation in surface-localized immune receptor complexes.

  4. Negative control of BAK1 by protein phosphatase 2A during plant innate immunity

    PubMed Central

    Segonzac, Cécile; Macho, Alberto P; Sanmartín, Maite; Ntoukakis, Vardis; Sánchez-Serrano, José Juan; Zipfel, Cyril

    2014-01-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors (PRRs) activates plant innate immunity, mainly through activation of numerous protein kinases. Appropriate induction of immune responses must be tightly regulated, as many of the kinases involved have an intrinsic high activity and are also regulated by other external and endogenous stimuli. Previous evidences suggest that PAMP-triggered immunity (PTI) is under constant negative regulation by protein phosphatases but the underlying molecular mechanisms remain unknown. Here, we show that protein Ser/Thr phosphatase type 2A (PP2A) controls the activation of PRR complexes by modulating the phosphostatus of the co-receptor and positive regulator BAK1. A potential PP2A holoenzyme composed of the subunits A1, C4, and B’η/ζ inhibits immune responses triggered by several PAMPs and anti-bacterial immunity. PP2A constitutively associates with BAK1 in planta. Impairment in this PP2A-based regulation leads to increased steady-state BAK1 phosphorylation, which can poise enhanced immune responses. This work identifies PP2A as an important negative regulator of plant innate immunity that controls BAK1 activation in surface-localized immune receptor complexes. PMID:25085430

  5. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  6. SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis

    PubMed Central

    Yoshida, Makoto M.; Ting, Lily; Gygi, Steven P.

    2016-01-01

    DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. PMID:27325792

  7. Expression of beta-catenin is regulated by PI-3 kinase and sodium butyrate in colorectal cancer cells.

    PubMed

    Turecková, Jolana; Kucerová, Dana; Vojtechová, Martina; Sloncová, Eva; Tuhácková, Zdena

    2006-01-01

    beta-catenin has a dual function; it is implicated in intercellular junctions and transcriptional co-activation. Here we examined the regulation of the expression and localization of beta-catenin in HT29 colorectal adenocarcinoma cells. Our results showed that inhibition of PI-3 kinase with wortmannin was accompanied by a considerably reduced expression of beta-catenin. This effect was overcome by butyrate and occurred at the protein level, not at the level of mRNA. Moreover, NaBT significantly increased the phosphorylation of the ribosomal protein, S6, known to participate in the translational control of gene expression. This was accompanied by the increased phosphorylation of p70 S6K and MAPKs, the effector proteins that are upstream of protein S6 in the distinct signaling pathways. These facts indicate that different signaling pathways may be involved in the regulation of beta-catenin synthesis. Modulation of beta-catenin expression induced by NaBT appeared to occur at the level of protein translation, suggesting that NaBT may act as a translational regulator.

  8. Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci*

    PubMed Central

    Popkie, Anthony P.; Zeidner, Leigh C.; Albrecht, Ashley M.; D'Ippolito, Anthony; Eckardt, Sigrid; Newsom, David E.; Groden, Joanna; Doble, Bradley W.; Aronow, Bruce; McLaughlin, K. John; White, Peter; Phiel, Christopher J.

    2010-01-01

    Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. PMID:21047779

  9. Active vibration control of structure by Active Mass Damper and Multi-Modal Negative Acceleration Feedback control algorithm

    NASA Astrophysics Data System (ADS)

    Yang, Don-Ho; Shin, Ji-Hwan; Lee, HyunWook; Kim, Seoug-Ki; Kwak, Moon K.

    2017-03-01

    In this study, an Active Mass Damper (AMD) consisting of an AC servo motor, a movable mass connected to the AC servo motor by a ball-screw mechanism, and an accelerometer as a sensor for vibration measurement were considered. Considering the capability of the AC servo motor which can follow the desired displacement accurately, the Negative Acceleration Feedback (NAF) control algorithm which uses the acceleration signal directly and produces the desired displacement for the active mass was proposed. The effectiveness of the NAF control was proved theoretically using a single-degree-of-freedom (SDOF) system. It was found that the stability condition for the NAF control is static and it can effectively increase the damping of the target natural mode without causing instability in the low frequency region. Based on the theoretical results of the SDOF system, the Multi-Modal NAF (MMNAF) control is proposed to suppress the many natural modes of multi-degree-of-freedom (MDOF) systems using a single AMD. It was proved both theoretically and experimentally that the MMNAF control can suppress vibrations of the MDOF system.

  10. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  11. Inhibition of PI-3 kinase for treating respiratory disease: good idea or bad idea?

    PubMed

    Thomas, Matt; Owen, Charles

    2008-06-01

    Inhibition of one or more members of the phosphoinositide 3-kinase (PI3K) family for the treatment of respiratory diseases remains the goal of many pharmaceutical companies over the past 20 years. Here we briefly review the PI3K family, then focus on the assessment of each isoform as a drug discovery target. The rationale for PI3Kalpha inhibition in the treatment of lung cancer, and PI3Kbeta inhibitors in pulmonary thrombotic processes, are balanced with a potential side effect profile affecting metabolism and/or foetal development. Roles for PI3Kdelta in inflammatory lung diseases and PI3Kgamma in asthma are weighed against the consequences of manipulating key immune cell populations. We also discuss the current status and future potential of PI3K inhibitors in respiratory disease.

  12. Targeting the Phosphatidylinositol-3-kinase Pathway in Gastric Cancer: Can Omics Improve Outcomes?

    PubMed Central

    Tran, Phu; Nguyen, Cham

    2016-01-01

    Phosphatidylinositol-3-kinase (PI3K) pathway signaling is an established oncogenic signal transduction pathway implicated in multiple malignancies. Therapeutic targeting of PI3K pathway components has improved outcomes in chronic lymphocytic leukemia, kidney cancer, breast cancer, and neuroendocrine tumors. Gastric cancers harbor some of the highest rates of oncogenic alterations in PI3K but attempts to translate this genomic observation have met with limited clinical success and novel approaches are needed. In the following review we discuss PI3K signaling, previous preclinical and clinical investigations in gastric cancer, and discuss future strategies aimed at overcoming resistance and improving efficacy. Identification and refinement of molecular tumor subtypes, development of predictive biomarkers along, and rational drug combination strategies are key to capitalizing on the therapeutic potential of PI3K pathway directed therapies in gastric cancers. PMID:27915478

  13. Measurement of phosphoinositide 3-kinase products in cultured Mammalian cells by HPLC.

    PubMed

    Cooke, Frank T

    2010-01-01

    The phosphoinositide 3-kinase (PI3K) family catalyses the addition of a phosphate group to the D-3 position of polyphosphoinositides (PPIn). Since the discovery in the late 80s that phosphatidylinositol is phosphorylated in the D-3 position in eukaryotic cells, there has been an explosion of interest in these PPIn. Although the four D-3 PPIn (phosphatidylinositol 3-phophate (PtdIns3P), PtdIns(3,4)P(2), PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3)) represent only a small proportion of PPIn, production of D-3 PPIn is required for an ever-increasing number of processes. Measurement of the PPIn levels in intact cells cultured cells has been vital to our understanding of the metabolism and function of these important signalling molecules; methods are described herein that allow measurement of PPIn levels in cultured cells, with emphasis on the 3-OH PPIn.

  14. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  15. Negative oncologic impact of poor postoperative pain control in left-sided pancreatic cancer

    PubMed Central

    Min, Eun-Ki; Chong, Jae Uk; Hwang, Ho Kyoung; Pae, Sang Joon; Kang, Chang Moo; Lee, Woo Jung

    2017-01-01

    AIM To investigate the association between postoperative pain control and oncologic outcomes in resected pancreatic ductal adenocarcinoma (PDAC). METHODS From January 2009 to December 2014, 221 patients were diagnosed with PDAC and underwent resection with curative intent. Retrospective review of the patients was performed based on electronic medical records system. One patient without records of numerical rating scale (NRS) pain intensity scores was excluded and eight patients who underwent total pancreatectomy were also excluded. NRS scores during 7 postoperative days following resection of PDAC were reviewed along with clinicopathologic characteristics. Patients were stratified into a good pain control group and a poor pain control group according to the difference in average pain intensity between the early (POD 1, 2, 3) and late (POD 5, 7) postoperative periods. Cox-proportional hazards multivariate analysis was performed to determine association between postoperative pain control and oncologic outcomes. RESULTS A total of 212 patients were dichotomized into good pain control group (n = 162) and poor pain control group (n = 66). Median follow-up period was 17 mo. A negative impact of poor postoperative pain control on overall survival (OS) was observed in the group of patients receiving distal pancreatectomy (DP group; 42.0 mo vs 5.0 mo, P = 0.001). Poor postoperative pain control was also associated with poor disease-free survival (DFS) in the DP group (18.0 mo vs 8.0 mo, P = 0.001). Patients undergoing pancreaticoduodenectomy or pylorus-preserving pancreaticoduodenectomy (PD group) did not show associations between postoperative pain control and oncologic outcomes. Poor patients’ perceived pain control was revealed as an independent risk factor of both DFS (HR = 4.157; 95%CI: 1.938-8.915; P < 0.001) and OS (HR = 4.741; 95%CI: 2.214-10.153; P < 0.001) in resected left-sided pancreatic cancer. CONCLUSION Adequate postoperative pain relief during the early

  16. Cognitive reappraisal and secondary control coping: associations with working memory, positive and negative affect, and symptoms of anxiety/depression.

    PubMed

    Andreotti, Charissa; Thigpen, Jennifer E; Dunn, Madeleine J; Watson, Kelly; Potts, Jennifer; Reising, Michelle M; Robinson, Kristen E; Rodriguez, Erin M; Roubinov, Danielle; Luecken, Linda; Compas, Bruce E

    2013-01-01

    The current study examined the relations of measures of cognitive reappraisal and secondary control coping with working memory abilities, positive and negative affect, and symptoms of anxiety and depression in young adults (N=124). Results indicate significant relations between working memory abilities and reports of secondary control coping and between reports of secondary control coping and cognitive reappraisal. Associations were also found between measures of secondary control coping and cognitive reappraisal and positive and negative affect and symptoms of depression and anxiety. Further, the findings suggest that reports of cognitive reappraisal may be more strongly predictive of positive affect whereas secondary control coping may be more strongly predictive of negative affect and symptoms of depression and anxiety. Overall, the results suggest that current measures of secondary control coping and cognitive reappraisal capture related but distinct constructs and suggest that the assessment of working memory may be more strongly related to secondary control coping in predicting individual differences in distress.

  17. LINGO-1 receptor promotes neuronal apoptosis by inhibiting WNK3 kinase activity.

    PubMed

    Zhang, Zhaohuan; Xu, Xiaohui; Xiang, Zhenghua; Yu, Zhongwang; Feng, Jifeng; He, Cheng

    2013-04-26

    LINGO-1 is a functional component of the Nogo receptor 1 · p75(NTR) · LINGO-1 and Nogo receptor 1 · TAJ (TNFRSF19/TROY)·LINGO-1 signaling complexes. It has recently been shown that LINGO-1 antagonists significantly improve neuronal survival after neural injury. However, the mechanism by which LINGO-1 signaling influences susceptibility to apoptosis remains unknown. In an effort to better understand how LINGO-1 regulates these signaling pathways, we used an established model of serum deprivation (SD) to induce neuronal apoptosis. We demonstrate that treatment either with a construct containing the intracellular domain of LINGO-1 or with Nogo66, a LINGO-1 receptor complex agonist, resulted in an enhanced rate of apoptosis in primary cultured cortical neurons under SD. Reducing the expression levels of the serine/threonine kinase WNK3 using shRNA or inhibiting its kinase activity had similar effects on the survival of serum-deprived neurons. Consistent with these observations, we found that LINGO-1 and WNK3 co-localized and co-precipitated in cultured cortical neurons and brain tissue. Significantly, this co-association was enhanced by Nogo66 treatment. Binding of WNK3 to the intracellular domain of LINGO-1 led to a reduction in WNK3 kinase activity, as did Nogo66 stimulation. Moreover, in vitro and in vivo evidence indicates that endogenous WNK3 suppresses SD-induced neuronal apoptosis in a kinase-dependent manner, as the expression of either a WNK3 RNAi construct or a kinase-dead N-terminal fragment of WNK3 led to increased apoptosis. Taken together, our results show that LINGO-1 potentiates neuronal apoptosis, likely by inhibiting WNK3 kinase activity.

  18. PAQR3 modulates insulin signaling by shunting phosphoinositide 3-kinase p110α to the Golgi apparatus.

    PubMed

    Wang, Xiao; Wang, Lingdi; Zhu, Lu; Pan, Yi; Xiao, Fei; Liu, Weizhong; Wang, Zhenzhen; Guo, Feifan; Liu, Yong; Thomas, Walter G; Chen, Yan

    2013-02-01

    Phosphoinositide 3-kinase (PI3K) mediates insulin actions by relaying signals from insulin receptors (IRs) to downstream targets. The p110α catalytic subunit of class IA PI3K is the primary insulin-responsive PI3K implicated in insulin signaling. We demonstrate here a new mode of spatial regulation for the p110α subunit of PI3K by PAQR3 that is exclusively localized in the Golgi apparatus. PAQR3 interacts with p110α, and the intracellular targeting of p110α to the Golgi apparatus is reduced by PAQR3 downregulation and increased by PAQR3 overexpression. Insulin-stimulated PI3K activity and phosphoinositide (3,4,5)-triphosphate production are enhanced by Paqr3 deletion and reduced by PAQR3 overexpression in hepatocytes. Deletion of Paqr3 enhances insulin-stimulated phosphorylation of AKT and glycogen synthase kinase 3β, but not phosphorylation of IR and IR substrate-1 (IRS-1), in hepatocytes, mouse liver, and skeletal muscle. Insulin-stimulated GLUT4 translocation to the plasma membrane and glucose uptake are enhanced by Paqr3 ablation. Furthermore, PAQR3 interacts with the domain of p110α involved in its binding with p85, the regulatory subunit of PI3K. Overexpression of PAQR3 dose-dependently reduces the interaction of p85α with p110α. Thus, PAQR3 negatively regulates insulin signaling by shunting cytosolic p110α to the Golgi apparatus while competing with p85 subunit in forming a PI3K complex with p110α.

  19. Control of the negative IRES trans-acting factor KHSRP by ubiquitination

    PubMed Central

    Kung, Yu-An; Hung, Chuan-Tien; Chien, Kun-Yi; Shih, Shin-Ru

    2017-01-01

    Cells and viruses can utilize internal ribosome entry sites (IRES) to drive translation when cap-dependent translation is inhibited by stress or viral factors. IRES trans-acting factors (ITAFs) are known to participate in such cap-independent translation, but there are gaps in the understanding as to how ITAFs, particularly negative ITAFs, regulate IRES-driven translation. This study found that Lys109, Lys121 and Lys122 represent critical ubiquitination sites for far upstream element-binding protein 2 (KHSRP, also known as KH-type splicing regulatory protein or FBP2), a negative ITAF. Mutations at these sites subsequently reduced KHSRP ubiquitination and abolished its inhibitory effect on IRES-driven translation. We further found that interaction between the Kelch domain of Kelch-like protein 12 (KLHL12) and the C-terminal domain of KHSRP contributed to KHSRP ubiquitination, leading to downregulation of enterovirus IRES-mediated translation in infected cells and increased competition against other positive ITAFs. Together, these results show that ubiquitination can exert control over IRES-driven translation via modification of ITAFs, and to the best of our knowledge, this is the first description of such a regulatory mechanism for IRES-dependent translation. PMID:27899653

  20. Positive and negative regulation of a SNARE protein by control of intracellular localization.

    PubMed

    Nakanishi, Hideki; de los Santos, Pablo; Neiman, Aaron M

    2004-04-01

    In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

  1. ITIM-dependent negative signaling pathways for the control of cell-mediated xenogeneic immune responses.

    PubMed

    del Rio, Maria-Luisa; Seebach, Jörg D; Fernández-Renedo, Carlos; Rodriguez-Barbosa, Jose-Ignacio

    2013-01-01

    Xenotransplantation is an innovative field of research with the potential to provide us with an alternative source of organs to face the severe shortage of human organ donors. For several reasons, pigs have been chosen as the most suitable source of organs and tissues for transplantation in humans. However, porcine xenografts undergo cellular immune responses representing a major barrier to their acceptance and normal functioning. Innate and adaptive xenogeneic immunity is mediated by both the recognition of xenogeneic tissue antigens and the lack of inhibition due to molecular cross-species incompatibilities of regulatory pathways. Therefore, the delivery of immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent and related negative signals to control innate (NK cells, macrophages) and adaptive T and B cells might overcome cell-mediated xenogeneic immunity. The proof of this concept has already been achieved in vitro by the transgenic overexpression of human ligands of several inhibitory receptors in porcine cells resulting in their resistance against xenoreactivity. Consequently, several transgenic pigs expressing tissue-specific human ligands of inhibitory coreceptors (HLA-E, CD47) or soluble competitors of costimulation (belatacept) have already been generated. The development of these robust and innovative approaches to modulate human anti-pig cellular immune responses, complementary to conventional immunosuppression, will help to achieve long-term xenograft survival. In this review, we will focus on the current strategies to enhance negative signaling pathways for the regulation of undesirable cell-mediated xenoreactive immune responses.

  2. RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis

    PubMed Central

    Yonekawa, Tohru; Gamez, Graciela; Kim, Jihye; Tan, Aik Choon; Thorburn, Jackie; Gump, Jacob; Thorburn, Andrew; Morgan, Michael J

    2015-01-01

    In a synthetic lethality/viability screen, we identified the serine–threonine kinase RIP1 (RIPK1) as a gene whose knockdown is highly selected against during growth in normal media, in which autophagy is not critical, but selected for in conditions that increase reliance on basal autophagy. RIP1 represses basal autophagy in part due to its ability to regulate the TFEB transcription factor, which controls the expression of autophagy-related and lysosomal genes. RIP1 activates ERK, which negatively regulates TFEB though phosphorylation of serine 142. Thus, in addition to other pro-death functions, RIP1 regulates cellular sensitivity to pro-death stimuli by modulating basal autophagy. PMID:25908842

  3. Hindbrain DPP-IV inhibition improves glycemic control and promotes negative energy balance.

    PubMed

    Mietlicki-Baase, Elizabeth G; McGrath, Lauren E; Koch-Laskowski, Kieran; Krawczyk, Joanna; Pham, Tram; Lhamo, Rinzin; Reiner, David J; Hayes, Matthew R

    2017-05-01

    The beneficial glycemic and food intake-suppressive effects of glucagon-like peptide-1 (GLP-1) have made this neuroendocrine system a leading target for pharmacological approaches to the treatment of diabetes and obesity. One strategy to increase the activity of endogenous GLP-1 is to prevent the rapid degradation of the hormone by the enzyme dipeptidyl peptidase-IV (DPP-IV). However, despite the expression of both DPP-IV and GLP-1 in the brain, and the clear importance of central GLP-1 receptor (GLP-1R) signaling for glycemic and energy balance control, the metabolic effects of central inhibition of DPP-IV activity are unclear. To test whether hindbrain DPP-IV inhibition suppresses blood glucose, feeding, and body weight gain, the effects of 4th intracerebroventricular (ICV) administration of the FDA-approved DPP-IV inhibitor sitagliptin were evaluated. Results indicate that hindbrain delivery of sitagliptin improves glycemic control in a GLP-1R-dependent manner, suggesting that this effect is due at least in part to increased endogenous brainstem GLP-1 activity after sitagliptin administration. Furthermore, 4th ICV injection of sitagliptin reduced 24h body weight gain and energy intake, with a selective suppression of high-fat diet, but not chow, intake. These data reveal a novel role for hindbrain GLP-1R activation in glycemic control and also demonstrate that DPP-IV inhibition in the caudal brainstem promotes negative energy balance.

  4. The restorative effects of smoking upon self-control resources: a negative reinforcement pathway.

    PubMed

    Heckman, Bryan W; Ditre, Joseph W; Brandon, Thomas H

    2012-02-01

    Based on a model in which self-control (SC) is considered to be a limited resource, research suggests that diminished SC resources may increase the likelihood of tobacco smoking. Yet, the inverse--how smoking may influence SC resources--has not been tested. The authors of this study utilized a randomized, 2 × 2 crossed-factorial (SC Depletion Manipulation × Smoking Manipulation), between-subjects design to test the hypothesis that smoking restores depleted SC resources. To manipulate SC depletion, experimenters instructed half of 132 nicotine dependent smokers to suppress their emotional reaction to a brief video depicting environmental damage (i.e., depletion), whereas the other half were instructed to "act natural" (i.e., no depletion) during viewing. Half of the participants in each condition then smoked a cigarette, whereas the other half sat patiently without smoking (i.e., smoke vs. no smoke). All participants then completed behavioral measures of SC. As hypothesized, an interaction occurred between the depletion and smoking manipulations for duration of time spent on a frustrating mirror-tracing task. That is, depletion reduced persistence on the task, unless depletion was followed by smoking. This effect was mediated by positive affect (PA). Thus, smoking appeared to restore depleted SC resources via modulation of PA, but independent of negative affect or smoking urges. These findings suggest that restoration of SC resources may represent another means by which smoking is negatively reinforced. The application of the self-control strength model to the study of nicotine dependence may inform the development of novel treatment modalities.

  5. Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.

    PubMed

    Chandrasekar, Bysani; Melby, Peter C; Sarau, Henry M; Raveendran, Muthuswamy; Perla, Rao P; Marelli-Berg, Federica M; Dulin, Nickolai O; Singh, Ishwar S

    2003-02-14

    It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.

  6. Incisional Negative Pressure Wound Therapy in High Risk Patients Undergoing Panniculectomy: A Prospective Randomized Controlled Trial

    ClinicalTrials.gov

    2017-01-03

    Complications Wounds; Negative Pressure Wound Therapy; Wound Healing Delayed; Incisional; Panniculectomy; Incisional Negative Pressure Wound Therapy; Incisional Vac; Wound Vac; Obese; Renal Failure; Kidney Transplant; Complications; Wound Healing Complication

  7. Longitudinal investigation on learned helplessness tested under negative and positive reinforcement involving stimulus control.

    PubMed

    Oliveira, Emileane C; Hunziker, Maria Helena

    2014-07-01

    In this study, we investigated whether (a) animals demonstrating the learned helplessness effect during an escape contingency also show learning deficits under positive reinforcement contingencies involving stimulus control and (b) the exposure to positive reinforcement contingencies eliminates the learned helplessness effect under an escape contingency. Rats were initially exposed to controllable (C), uncontrollable (U) or no (N) shocks. After 24h, they were exposed to 60 escapable shocks delivered in a shuttlebox. In the following phase, we selected from each group the four subjects that presented the most typical group pattern: no escape learning (learned helplessness effect) in Group U and escape learning in Groups C and N. All subjects were then exposed to two phases, the (1) positive reinforcement for lever pressing under a multiple FR/Extinction schedule and (2) a re-test under negative reinforcement (escape). A fourth group (n=4) was exposed only to the positive reinforcement sessions. All subjects showed discrimination learning under multiple schedule. In the escape re-test, the learned helplessness effect was maintained for three of the animals in Group U. These results suggest that the learned helplessness effect did not extend to discriminative behavior that is positively reinforced and that the learned helplessness effect did not revert for most subjects after exposure to positive reinforcement. We discuss some theoretical implications as related to learned helplessness as an effect restricted to aversive contingencies and to the absence of reversion after positive reinforcement. This article is part of a Special Issue entitled: insert SI title.

  8. Negative thermal expansion in functional materials: controllable thermal expansion by chemical modifications.

    PubMed

    Chen, Jun; Hu, Lei; Deng, Jinxia; Xing, Xianran

    2015-06-07

    Negative thermal expansion (NTE) is an intriguing physical property of solids, which is a consequence of a complex interplay among the lattice, phonons, and electrons. Interestingly, a large number of NTE materials have been found in various types of functional materials. In the last two decades good progress has been achieved to discover new phenomena and mechanisms of NTE. In the present review article, NTE is reviewed in functional materials of ferroelectrics, magnetics, multiferroics, superconductors, temperature-induced electron configuration change and so on. Zero thermal expansion (ZTE) of functional materials is emphasized due to the importance for practical applications. The NTE functional materials present a general physical picture to reveal a strong coupling role between physical properties and NTE. There is a general nature of NTE for both ferroelectrics and magnetics, in which NTE is determined by either ferroelectric order or magnetic one. In NTE functional materials, a multi-way to control thermal expansion can be established through the coupling roles of ferroelectricity-NTE, magnetism-NTE, change of electron configuration-NTE, open-framework-NTE, and so on. Chemical modification has been proved to be an effective method to control thermal expansion. Finally, challenges and questions are discussed for the development of NTE materials. There remains a challenge to discover a "perfect" NTE material for each specific application for chemists. The future studies on NTE functional materials will definitely promote the development of NTE materials.

  9. A comparison of controlled negative pressure and aerosol quantitative respirator fit test systems by using fixed leaks.

    PubMed

    Crutchfield, C D; Murphy, R W; Van Ert, M D

    1991-06-01

    An automated version of a new method for quantitative respirator fit testing by controlled negative pressure was compared with a computerized aerosol fit test system. The controlled negative pressure technique eliminates many of the problems associated with aerosol and pressure decay fit test methods. A series of fixed leaks was used to compare the leak measurement capabilities of the controlled negative pressure system against a standard computerized aerosol fit test system. Negative pressure and aerosol fit factors determined for a series of fixed leaks through hypodermic needles were highly correlated with each other (r = 0.998) and with the cross-sectional areas of the leak needles (r greater than 0.995).

  10. Dehydroepiandrosterone Stimulates Phosphorylation of FoxO1 in Vascular Endothelial Cells via Phosphatidylinositol 3-Kinase- and Protein Kinase A-dependent Signaling Pathways to Regulate ET-1 Synthesis and Secretion*

    PubMed Central

    Chen, Hui; Lin, Alice Seraphina; Li, Yunhua; Reiter, Chad E. N.; Ver, Maria R.; Quon, Michael J.

    2008-01-01

    Dehydroepiandrosterone (DHEA) is an endogenous adrenal steroid hormone with controversial actions in humans. We previously reported that DHEA has opposing actions in endothelial cells to stimulate phosphatidylinositol (PI) 3-kinase/Akt/endothelial nitric-oxide synthase leading to increased production of nitric oxide while simultaneously stimulating MAPK-dependent secretion of the vasoconstrictor ET-1. In the present study we hypothesized that DHEA may stimulate PI 3-kinase-dependent phosphorylation of FoxO1 in endothelial cells to help regulate endothelial function. In bovine or human aortic endothelial cells (BAEC and HAEC), treatment with DHEA (100 nm) acutely enhanced phosphorylation of FoxO1. DHEA-stimulated phosphorylation of FoxO1 was inhibited by pretreatment of cells with wortmannin (PI 3-kinase inhibitor) or H89 (protein kinase A (PKA) inhibitor) but not ICI182780 (estrogen receptor blocker), or PD98059 (MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor). Small interfering RNA knockdown of PKA inhibited DHEA-stimulated phosphorylation of FoxO1. DHEA promoted nuclear exclusion of FoxO1 that was blocked by pretreatment of cells with wortmannin, H89, or by small interfering RNA knockdown of PKA. DHEA treatment of endothelial cells increased PKA activity and intracellular cAMP concentrations. Transfection of BAEC with a constitutively nuclear FoxO1 mutant transactivated a co-transfected ET-1 promoter luciferase reporter. Treatment of BAEC with DHEA inhibited transactivation of the ET-1 promoter reporter in cells overexpressing FoxO1. ET-1 promoter activity and secretion in response to DHEA treatment was augmented by PI 3-kinase blockade and inhibited by MAPK blockade. We conclude that DHEA stimulates phosphorylation of FoxO1 via PI 3-kinase- and PKA-dependent pathways in endothelial cells that negatively regulates ET-1 promoter activity and secretion. Balance between PI 3-kinase-dependent inhibition and MAPK-dependent stimulation of ET-1

  11. Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus.

    PubMed Central

    Folli, F; Saad, M J; Backer, J M; Kahn, C R

    1993-01-01

    Insulin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which in turn binds to and activates phosphatidylinositol 3-kinase (PI 3-kinase). In the present study, we have examined these processes in animal models of insulin-resistant and insulin-deficient diabetes mellitus. After in vivo insulin stimulation, there was a 60-80% decrease in IRS-1 phosphorylation in liver and muscle of the ob/ob mouse. There was no insulin stimulation of PI 3-kinase (85 kD subunit) association with IRS-1, and IRS-1-associated PI 3-kinase activity was reduced 90%. Insulin-stimulated total PI 3-kinase activity was also absent in both tissues of the ob/ob mouse. By contrast, in the streptozotocin diabetic rat, IRS-1 phosphorylation increased 50% in muscle, IRS-1-associated PI 3-kinase activity was increased two- to threefold in liver and muscle, and there was a 50% increase in the p85 associated with IRS-1 after insulin stimulation in muscle. In conclusion, (a) IRS-1-associated PI 3-kinase activity is differentially regulated in hyperinsulinemic and hypoinsulinemic diabetic states; (b) PI 3-kinase activation closely correlates with IRS-1 phosphorylation; and (c) reduced PI 3-kinase activity may play a role in the pathophysiology of insulin resistant diabetic states, such as that seen in the ob/ob mouse. Images PMID:7691886

  12. Class A scavenger receptor-mediated cell adhesion requires the sequential activation of Lyn and PI3-kinase.

    PubMed

    Nikolic, Dejan M; Cholewa, Jill; Gass, Cecelia; Gong, Ming C; Post, Steven R

    2007-04-01

    Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A-/- macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting G(i/o) activation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading.

  13. Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product.

    PubMed

    Kim, H H; Sierke, S L; Koland, J G

    1994-10-07

    The ErbB3 protein is a member of the ErbB subfamily of receptor protein tyrosine kinases. In the present study, the mechanism by which the ErbB3 protein is phosphorylated and the signal-transducing functions of this phosphorylated protein were investigated. When phosphorylated by the epidermal growth factor receptor in vitro, the ErbB3 protein strongly associated with the regulatory p85 subunit and the catalytic activity of phosphatidylinositol (PI) 3-kinase. The association of PI 3-kinase with ErbB3 in human breast cancer cells was found to be correlated with the constitutive phosphorylation of ErbB3 on tyrosine residues. In MDA-MB-468 breast cancer cells in which the ErbB3 protein is not constitutively phosphorylated, stimulation with epidermal growth factor led to the phosphorylation of ErbB3 on tyrosine residues and the formation of a functional signal transduction complex involving the ErbB3 protein and PI 3-kinase. These results suggest that the ErbB3 protein can be phosphorylated on tyrosine residues by a cross-phosphorylation mechanism and that the phosphorylated ErbB3 protein can couple other growth factor receptor protein tyrosine kinases to the PI 3-kinase pathway in a manner similar to the insulin receptor substrate 1 protein.

  14. Kinetic analysis of platelet-derived growth factor receptor/phosphoinositide 3-kinase/Akt signaling in fibroblasts.

    PubMed

    Park, Chang Shin; Schneider, Ian C; Haugh, Jason M

    2003-09-26

    Isoforms of the serine-threonine kinase Akt coordinate multiple cell survival pathways in response to stimuli such as platelet-derived growth factor (PDGF). Activation of Akt is a multistep process, which relies on the production of 3'-phosphorylated phosphoinositide (PI) lipids by PI 3-kinases. To quantitatively assess the kinetics of PDGF receptor/PI 3-kinase/Akt signaling in fibroblasts, a systematic study of this pathway was performed, and a mechanistic mathematical model that describes its operation was formulated. We find that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, and its kinetics are quantitatively consistent with a mechanism in which receptor dimerization is initially mediated by the association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high concentrations of PDGF, consistent with the loss of activated receptors upon endocytosis. By comparison, Akt activation responds to lower PDGF concentrations and exhibits more sustained kinetics. Further analysis and modeling suggest that the pathway is saturated at the level of PI 3-kinase activation, and that the p110alpha catalytic subunit of PI 3-kinase contributes most to PDGF-stimulated 3'-PI production. Thus, at high concentrations of PDGF the kinetics of 3'-PI production are limited by the turnover rate of these lipids, while the Akt response is additionally influenced by the rate of Akt deactivation.

  15. Phosphatidylinositol 3-kinase mediates the ability of retinol to decrease colorectal cancer cell invasion.

    PubMed

    Lengyel, Jennifer N Griffin; Park, Eun Young; Brunson, Anna R; Pinali, Daniel; Lane, Michelle A

    2014-01-01

    Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor-independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 μM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 μM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P = 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P = 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis.

  16. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  17. Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage

    PubMed Central

    Angulo, Ivan; Vadas, Oscar; Garçon, Fabien; Banham-Hall, Edward; Plagnol, Vincent; Leahy, Timothy R.; Baxendale, Helen; Coulter, Tanya; Curtis, James; Wu, Changxin; Blake-Palmer, Katherine; Perisic, Olga; Smyth, Deborah; Maes, Mailis; Fiddler, Christine; Juss, Jatinder; Cilliers, Deirdre; Markelj, Gašper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debré, Marianne; Picard, Capucine; Pellier, Isabelle; Jabado, Nada; Morris, James A.; Barcenas-Morales, Gabriela; Fischer, Alain; Stephens, Len; Hawkins, Phillip; Barrett, Jeffrey C.; Abinun, Mario; Clatworthy, Menna; Durandy, Anne; Doffinger, Rainer; Chilvers, Edwin; Cant, Andrew J.; Kumararatne, Dinakantha; Okkenhaug, Klaus; Williams, Roger L.; Condliffe, Alison; Nejentsev, Sergey

    2014-01-01

    Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-of-function mutation E1021K in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3,346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased IgM and reduced IgG2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, suggesting a therapeutic approach for patients with APDS. PMID:24136356

  18. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling

    PubMed Central

    Cinnamon, Einat; Sawala, Annick; Tittiger, Claus; Paroush, Ze'ev

    2016-01-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  19. Diosgenin inhibits melanogenesis through the activation of phosphatidylinositol-3-kinase pathway (PI3K) signaling.

    PubMed

    Lee, Jongsung; Jung, Kwangseon; Kim, Yeong Shik; Park, Deokhoon

    2007-06-27

    An increased level of melanin is characteristic of a large number of skin diseases, including acquired hyperpigmentation conditions such as melasma, post inflammatory melanoderma, and solar lentigo. Thus, there is an increasing need for the development of depigmenting agents. In order to evaluate the depigmenting capacity of diosgenin and elucidate its mechanism of action, several experiments were performed in B16 melanoma cells. Melanin content and Western blots for proteins that are involved in melanogenesis were assessed in this study. The melanin content was significantly inhibited by diosgenin. To clarify the mechanism of the depigmenting property of diosgenin, we examined the involvement of diosgenin in the phosphatidylinositol-3-kinase (PI3K) pathway. In this study, diosgenin inhibited the reduction of Akt and GSK 3beta phosphorylation induced by LY294,002, a PI3K inhibitor. In accordance with this result, production levels of MITF (microphthalmia-associated transcription factor) and tyrosinase were increased by diosgenin. These data suggest that diosgenin inhibits melanogenesis through the activation of the PI3K pathway. This suggestion was further confirmed by the fact that the increased production level of melanin by LY294,002 was reduced by diosgenin in B16 melanoma cells. Our study shows that diosgenin inhibits melanogenesis by activating the PI3K pathway, and also suggests that diosgenin may be an effective inhibitor of hyperpigmentation.

  20. IPD-196, a novel phosphatidylinositol 3-kinase inhibitor with potent anticancer activity against hepatocellular carcinoma.

    PubMed

    Lee, Ju-Hee; Lee, Hyunseung; Yun, Sun-Mi; Jung, Kyung Hee; Jeong, Yujeong; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

    2013-02-01

    As the activation of phosphatidylinositol 3-kinase (PI3K) is associated with a wide variety of human malignancies, it is emerging as an attractive target for cancer treatment. In this study we synthesized a novel PI3Kα inhibitor, IPD-196 [ethyl 6-(5-(2,4-difluorophenylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate], and evaluated its anticancer effects on human hepatocellular carcinoma (HCC) cells. IPD-196 effectively inhibited the phosphorylation of downstream PI3K effectors such as Akt, mTOR, p70S6K, and 4E-BP1, and its antiproliferative effect was more potent than that of sorafenib or LY294002. It also induced cell cycle arrest at the G0/G1 phase as well as apoptosis by increasing the proportion of sub-G1 apoptotic cells, and the levels of cleaved PARP, caspase-3, and caspase-9. Furthermore, it decreased the expression of HIF-1α and VEGF in Huh-7 cells, and inhibited tube formation and migration of human umbilical vein endothelial cells, which was confirmed by a Matrigel plug assay in mice. Taken together, IPD-196 exhibited its anticancer activity through disruption of the PI3K/Akt pathway that caused cell cycle arrest, apoptosis induction, and inhibition of angiogenesis in human HCC cells. We therefore suggest that IPD-196 may be a potential candidate drug for targeted HCC therapy.

  1. Phosphoinositide-3-kinases as the novel therapeutic targets for the inflammatory diseases: Current and future perspectives.

    PubMed

    Vyas, Preeti; Vohora, Divya

    2016-10-13

    Recent findings have publicized phosphoinositide-3-kinases (PI3Ks) as novel therapeutic targets, which are also purported to be involved in the complex pathophysiology of inflammatory and various other diseases. They are recognized to participate in the inflammatory cellular responses by modulating the growth, development and proliferation of various immune cells and hence, affect the release of various cytokines and other inflammatory mediators involved in these manifestations. The review presents a brief synopsis of the PI3K/AKT/mTOR signalling pathway along with the current and future prospects of targeting PI3Ks for various diseases, like malignant, autoimmune, inflammatory, cardiovascular, neurological disorders etc., laying special emphasis on the inflammatory diseases and associated cellular responses. The recent literature relating this pathway with these diseases is highlighted, with a hope, which remains for the progression of PI3K inhibitors in the market as a treatment option. With Idelalisib entering the market for cancer, PI3K/AKT signalling has also gained significance as an investigational target for other diseases, particularly for inflammation. Further exploration of this pathway may also uncover its involvement in these disorders, which may further contribute to developing the new treatments and can turn out to be an innovative brainwave in the field of experimental and clinical pharmacology in future.

  2. Biliverdin Reductase Mediates Hypoxia-Induced EMT via PI3-Kinase and Akt

    PubMed Central

    Zeng, Rui; Yao, Ying; Han, Min; Zhao, Xiaoqin; Liu, Xiao-Cheng; Wei, Juncheng; Luo, Yun; Zhang, Juan; Zhou, Jianfeng; Wang, Shixuan; Ma, Ding; Xu, Gang

    2008-01-01

    Chronic hypoxia in the renal parenchyma is thought to induce epithelial-to-mesenchymal transition (EMT), leading to fibrogenesis and ultimately end-stage renal failure. Biliverdin reductase, recently identified as a serine/threonine/tyrosine kinase that may activate phosphatidylinositol 3-kinase (PI3K) and Akt, is upregulated in response to reactive oxygen species that may accompany hypoxia. We investigated this potential role of biliverdin reductase in hypoxia-induced renal tubular EMT. Expression of biliverdin reductase was upregulated in a human proximal tubule cell line (HK-2) cultured in hypoxic conditions (1% O2), and this was accompanied by reduced expression of E-cadherin and increased expression of the mesenchymal marker vimentin. Inhibiting PI3K reversed these changes, consistent with EMT. In normoxic conditions, overexpression of biliverdin reductase promoted similar characteristics of EMT, which were also reversed by inhibiting PI3K. Furthermore, using small interfering RNA (siRNA) to knockdown biliverdin reductase, we demonstrated that the enzyme associates with phosphorylated Akt and mediates the hypoxia-induced EMT phenotype. In vivo, expression of biliverdin reductase increased in the tubular epithelia of 5/6-nephrectomized rats, and immunohistochemistry of serial sections demonstrated similar localization of phosphorylated Akt and biliverdin reductase. In conclusion, biliverdin reductase mediates hypoxia-induced EMT through a PI3K/Akt-dependent pathway. PMID:18184861

  3. Tyrosol Suppresses Allergic Inflammation by Inhibiting the Activation of Phosphoinositide 3-Kinase in Mast Cells.

    PubMed

    Je, In-Gyu; Kim, Duk-Sil; Kim, Sung-Wan; Lee, Soyoung; Lee, Hyun-Shik; Park, Eui Kyun; Khang, Dongwoo; Kim, Sang-Hyun

    2015-01-01

    Allergic diseases such as atopic dermatitis, rhinitis, asthma, and anaphylaxis are attractive research areas. Tyrosol (2-(4-hydroxyphenyl)ethanol) is a polyphenolic compound with diverse biological activities. In this study, we investigated whether tyrosol has anti-allergic inflammatory effects. Ovalbumin-induced active systemic anaphylaxis and immunoglobulin E-mediated passive cutaneous anaphylaxis models were used for the immediate-type allergic responses. Oral administration of tyrosol reduced the allergic symptoms of hypothermia and pigmentation in both animal models. Mast cells that secrete allergic mediators are key regulators on allergic inflammation. Tyrosol dose-dependently decreased mast cell degranulation and expression of inflammatory cytokines. Intracellular calcium levels and activation of inhibitor of κB kinase (IKK) regulate cytokine expression and degranulation. Tyrosol blocked calcium influx and phosphorylation of the IKK complex. To define the molecular target for tyrosol, various signaling proteins involved in mast cell activation such as Lyn, Syk, phosphoinositide 3-kinase (PI3K), and Akt were examined. Our results showed that PI3K could be a molecular target for tyrosol in mast cells. Taken together, these findings indicated that tyrosol has anti-allergic inflammatory effects by inhibiting the degranulation of mast cells and expression of inflammatory cytokines; these effects are mediated via PI3K. Therefore, we expect tyrosol become a potential therapeutic candidate for allergic inflammatory disorders.

  4. Targeting phosphoinositide 3-kinase δ for the treatment of respiratory diseases.

    PubMed

    Sriskantharajah, Srividya; Hamblin, Nicole; Worsley, Sally; Calver, Andrew R; Hessel, Edith M; Amour, Augustin

    2013-03-01

    Asthma and chronic obstructive pulmonary disease (COPD) are characterized in their pathogenesis by chronic inflammation in the airways. Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase expressed predominantly in leukocytes, is thought to hold much promise as a therapeutic target for such inflammatory conditions. Of particular interest for the treatment of severe respiratory disease is the observation that inhibition of PI3Kδ may restore steroid effectiveness under conditions of oxidative stress. PI3Kδ inhibition may also prevent recruitment of inflammatory cells, including T lymphocytes and neutrophils, as well as the release of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species, and proteolytic enzymes. In addition, targeting the PI3Kδ pathway could reduce the incidence of pathogen-induced exacerbations by improving macrophage-mediated bacterial clearance. In this review, we discuss the potential and highlight the unknowns of targeting PI3Kδ for the treatment of respiratory disease, focusing on recent developments in the role of the PI3Kδ pathway in inflammatory cell types believed to be critical to the pathogenesis of COPD.

  5. Phosphatidylinositol 3-Kinase γ is required for the development of experimental cerebral malaria.

    PubMed

    Lacerda-Queiroz, Norinne; Brant, Fatima; Rodrigues, David Henrique; Vago, Juliana Priscila; Rachid, Milene Alvarenga; Sousa, Lirlândia Pires; Teixeira, Mauro Martins; Teixeira, Antonio Lucio

    2015-01-01

    Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3+CD8+ T cells were significantly reduced in the brain of infected PI3Kγ-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.

  6. Phosphoinositide 3-kinase: a new kid on the block in vascular anomalies.

    PubMed

    Castillo, Sandra D; Vanhaesebroeck, Bart; Sebire, Neil J

    2016-12-01

    Vascular anomalies are broadly divided into vascular tumours and malformations. These lesions are composed of abnormal vascular elements of various types, and mainly affect infants, children, and young adults. Vascular anomalies may be painful, may be complicated by bleeding, infection, or organ dysfunction, and can have secondary effects on other tissues. Current treatment strategies include surgical excision, pulsed laser, and sclerotherapy, which are invasive, with risks of recurrence. There are growing pharmacological options for these vascular anomalies, but, to date, no specific targeted therapies have been developed. Phosphoinositide 3-kinases (PI3Ks) constitute a family of lipid kinases that are involved in signal transduction and vesicular traffic, and that modulate important cellular processes such as proliferation, growth, and migration. Recent findings have indicated that the PI3K signalling pathway is important in the pathogenesis of vascular anomalies. This provides an opportunity to use PI3K inhibitors, which are in clinical trials for cancer treatment, for such lesions. Here, we provide an update on the classification of vascular anomalies, with their major features, and discuss the role of the PI3K signalling pathway in the pathogenesis of vascular anomalies, and their clinical implications and therapeutic opportunities. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  7. ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor identified using the JFCR39 drug discovery system.

    PubMed

    Kong, De-xin; Yamori, Takao

    2010-09-01

    JFCR39 is an informatic anticancer drug discovery system that utilizes a panel of 39 human cancer cells coupled with a drug-activity database. This system not only provides disease-oriented information but can also predict the mechanism of action of a given antitumor agent. Development of a phosphatidylinositol 3-kinase (PI3K) inhibitor as an anticancer drug candidate has attracted a great deal of attention from both academia and industry because PI3K is known to be closely involved in carcinogenesis. ZSTK474 was identified as a PI3K inhibitor using JFCR39 system in combination with COMPARE analysis program. These findings were based on the similar fingerprint (growth inhibition profiles for JFCR39 human cancer cell line panel) with that of a classical PI3K inhibitor LY294002. Biochemical experiments confirmed ZSTK474 to be a potent pan-class I PI3K inhibitor, with high selectivity over other classes of PI3K and protein kinases. We previously reported the in vitro and in vivo antitumor efficacy of ZSTK474, together with the G(0)/G(1) arrest and antiangiogenic activity. Here, we review the JFCR39 system and summarize recent studies on PI3K biology and the development of PI3K inhibitors before discussing ZSTK474 in some detail.

  8. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    SciTech Connect

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori . E-mail: hirokato@pharm.kyoto-u.ac.jp

    2007-08-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.

  9. Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase.

    PubMed

    Linassier, C; MacDougall, L K; Domin, J; Waterfield, M D

    1997-02-01

    Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. We have identified a representative member of each class of PI3Ks in Drosophila using a PCR-based approach. In the present paper we describe the molecular cloning of a PI3K from Drosophila, P13K_59F, that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologues, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes.

  10. G-CSF maintains controlled neutrophil mobilization during acute inflammation by negatively regulating CXCR2 signaling.

    PubMed

    Bajrami, Besnik; Zhu, Haiyan; Kwak, Hyun-Jeong; Mondal, Subhanjan; Hou, Qingming; Geng, Guangfeng; Karatepe, Kutay; Zhang, Yu C; Nombela-Arrieta, César; Park, Shin-Young; Loison, Fabien; Sakai, Jiro; Xu, Yuanfu; Silberstein, Leslie E; Luo, Hongbo R

    2016-09-19

    Cytokine-induced neutrophil mobilization from the bone marrow to circulation is a critical event in acute inflammation, but how it is accurately controlled remains poorly understood. In this study, we report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation. Nevertheless, although serum CXCR2 ligand concentrations increased during inflammation, neutrophil mobilization slowed after an initial acute fast phase, suggesting a suppression of neutrophil response to CXCR2 ligands after the acute phase. We demonstrate that granulocyte colony-stimulating factor (G-CSF), usually considered a prototypical neutrophil-mobilizing cytokine, was expressed later in the acute inflammatory response and unexpectedly impeded CXCR2-induced neutrophil mobilization by negatively regulating CXCR2-mediated intracellular signaling. Blocking G-CSF in vivo paradoxically elevated peripheral blood neutrophil counts in mice injected intraperitoneally with Escherichia coli and sequestered large numbers of neutrophils in the lungs, leading to sterile pulmonary inflammation. In a lipopolysaccharide-induced acute lung injury model, the homeostatic imbalance caused by G-CSF blockade enhanced neutrophil accumulation, edema, and inflammation in the lungs and ultimately led to significant lung damage. Thus, physiologically produced G-CSF not only acts as a neutrophil mobilizer at the relatively late stage of acute inflammation, but also prevents exaggerated neutrophil mobilization and the associated inflammation-induced tissue damage during early-phase infection and inflammation.

  11. Source Control and Graft Preservation Using Negative Pressure Wound Therapy with Antibiotic Instillation: A Case Report

    PubMed Central

    Kelly, Timothy; Grossi, Robert

    2016-01-01

    Negative pressure wound therapy (NPWT) is widely used to treat many types of complex wounds, and the advent of the instillation and dwell time (NPWTi-d) technique has enhanced this system with the addition of automated treatment with topical solutions. In the field of vascular surgery, NPWT is utilized to help close wounds over underlying grafts; however keeping these wounds free of infection and avoiding large reoperation when infection occurs remains a challenge. In this case report we present a patient who required acute intervention for limb ischemia, with a large wound created in the groin for anastomosis of a prosthetic graft bypass. Postoperatively, the wound became infected, and the challenge became balancing infection control and graft preservation with the patient’s multiple comorbidities including postoperative non-ST segment elevation myocardial infarction (NSTEMI). To avoid a large reoperation, we chose NPWTi-d with automated instillation of an antibiotic solution. There was no reinfection or return to the operating room (OR), the patient was discharged after four weeks and the wound closed on its own shortly thereafter. This case demonstrates that for high-risk surgical patients with known wound infections in the proximity of a bypass graft, NPWTi-d with antibiotic instillation may be an effective augmentation to current treatment strategies and may be considered as a stand-alone technique for wound closure in select cases. PMID:27909643

  12. Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

    PubMed

    Andreozzi, Francesco; Melillo, Rosa Marina; Carlomagno, Francesca; Oriente, Francesco; Miele, Claudia; Fiory, Francesca; Santopietro, Stefania; Castellone, Maria Domenica; Beguinot, Francesco; Santoro, Massimo; Formisano, Pietro

    2003-05-15

    We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

  13. Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

    PubMed Central

    Watkins, Herschel M.; Simon, Anna J.; Sosnick, Tobin R.; Lipman, Everett A.; Hjelm, Rex P.; Plaxco, Kevin W.

    2015-01-01

    Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant. PMID:25964362

  14. JNK1 negatively controls antifungal innate immunity by suppressing CD23 expression.

    PubMed

    Zhao, Xueqiang; Guo, Yahui; Jiang, Changying; Chang, Qing; Zhang, Shilei; Luo, Tianming; Zhang, Bin; Jia, Xinming; Hung, Mien-Chie; Dong, Chen; Lin, Xin

    2017-03-01

    Opportunistic fungal infections are a leading cause of death among immune-compromised patients, and there is a pressing need to develop new antifungal therapeutic agents because of toxicity and resistance to the antifungal drugs currently in use. Although C-type lectin receptor- and Toll-like receptor-induced signaling pathways are key activators of host antifungal immunity, little is known about the mechanisms that negatively regulate host immune responses to a fungal infection. Here we found that JNK1 activation suppresses antifungal immunity in mice. We showed that JNK1-deficient mice had a significantly higher survival rate than wild-type control mice in response to Candida albicans infection, and the expression of JNK1 in hematopoietic innate immune cells was critical for this effect. JNK1 deficiency leads to significantly higher induction of CD23, a novel C-type lectin receptor, through NFATc1-mediated regulation of the CD23 gene promoter. Blocking either CD23 upregulation or CD23-dependent nitric oxide production eliminated the enhanced antifungal response found in JNK1-deficient mice. Notably, JNK inhibitors exerted potent antifungal therapeutic effects in both mouse and human cells infected with C. albicans, indicating that JNK1 may be a therapeutic target for treating fungal infection.

  15. Mutant DnaAs of Escherichia coli that are refractory to negative control

    PubMed Central

    Chodavarapu, Sundari; Felczak, Magdalena M.; Simmons, Lyle A.; Murillo, Alec; Kaguni, Jon M.

    2013-01-01

    DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli. PMID:23990329

  16. Urocortin3 mediates somatostatin-dependent negative feedback control of insulin secretion

    PubMed Central

    van der Meulen, Talitha; Donaldson, Cynthia J.; Cáceres, Elena; Hunter, Anna E.; Cowing–Zitron, Christopher; Pound, Lynley D.; Adams, Michael W.; Zembrzycki, Andreas; Grove, Kevin L.; Huising, Mark O.

    2015-01-01

    The peptide hormone Urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Here we demonstrate that Ucn3 is stored and co–released with insulin and potentiates glucose–stimulated somatostatin secretion via cognate receptor on delta cells. Further, we found that islets lacking endogenous Ucn3 demonstrate fewer delta cells, reduced somatostatin content, impaired somatostatin secretion and exaggerated insulin release, and that these defects are rectified by synthetic Ucn3 in vitro. Our observations indicate that the paracrine actions of Ucn3 activate a negative feedback loop that promotes somatostatin release to ensure the timely reduction of insulin secretion upon normalization of plasma glucose. Moreover, Ucn3 is markedly depleted from beta cells in mouse and macaque diabetes models and in human diabetic islets. This suggests that Ucn3 is a key contributor to stable glycemic control whose reduction during diabetes aggravates glycemic volatility and contributes to the pathophysiology of this disease. PMID:26076035

  17. Cyclin A expression is under negative transcriptional control during the cell cycle.

    PubMed Central

    Huet, X; Rech, J; Plet, A; Vié, A; Blanchard, J M

    1996-01-01

    Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression. PMID:8668196

  18. G-CSF maintains controlled neutrophil mobilization during acute inflammation by negatively regulating CXCR2 signaling

    PubMed Central

    Bajrami, Besnik; Zhu, Haiyan; Zhang, Yu C.

    2016-01-01

    Cytokine-induced neutrophil mobilization from the bone marrow to circulation is a critical event in acute inflammation, but how it is accurately controlled remains poorly understood. In this study, we report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation. Nevertheless, although serum CXCR2 ligand concentrations increased during inflammation, neutrophil mobilization slowed after an initial acute fast phase, suggesting a suppression of neutrophil response to CXCR2 ligands after the acute phase. We demonstrate that granulocyte colony-stimulating factor (G-CSF), usually considered a prototypical neutrophil-mobilizing cytokine, was expressed later in the acute inflammatory response and unexpectedly impeded CXCR2-induced neutrophil mobilization by negatively regulating CXCR2-mediated intracellular signaling. Blocking G-CSF in vivo paradoxically elevated peripheral blood neutrophil counts in mice injected intraperitoneally with Escherichia coli and sequestered large numbers of neutrophils in the lungs, leading to sterile pulmonary inflammation. In a lipopolysaccharide-induced acute lung injury model, the homeostatic imbalance caused by G-CSF blockade enhanced neutrophil accumulation, edema, and inflammation in the lungs and ultimately led to significant lung damage. Thus, physiologically produced G-CSF not only acts as a neutrophil mobilizer at the relatively late stage of acute inflammation, but also prevents exaggerated neutrophil mobilization and the associated inflammation-induced tissue damage during early-phase infection and inflammation. PMID:27551153

  19. Regulation of PI-3-Kinase and Akt Signaling in T Lymphocytes and Other Cells by TNFR Family Molecules

    PubMed Central

    So, Takanori; Croft, Michael

    2013-01-01

    Activation of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) is a common response triggered by a range of membrane-bound receptors on many cell types. In T lymphocytes, the PI3K-Akt pathway promotes clonal expansion, differentiation, and survival of effector cells and suppresses the generation of regulatory T cells. PI3K activation is tightly controlled by signals through the T cell receptor (TCR) and the co-stimulatory receptor CD28, however sustained and periodic signals from additional co-receptors are now being recognized as critical contributors to the activation of this pathway. Accumulating evidence suggests that many members of the Tumor Necrosis Factor receptor (TNFR) superfamily, TNFR2 (TNFRSF1B), OX40 (TNFRSF4), 4-1BB (TNFRSF9), HVEM (TNFRSF14), and DR3 (TNFRSF25), that are constitutive or inducible on T cells, can directly or indirectly promote activity in the PI3K-Akt pathway. We discuss recent data which suggests that ligation of one TNFR family molecule organizes a signalosome, via TNFR-associated factor (TRAF) adapter proteins in T cell membrane lipid microdomains, that results in the subsequent accumulation of highly concentrated depots of PI3K and Akt in close proximity to TCR signaling units. We propose this may be a generalizable mechanism applicable to other TNFR family molecules that will result in a quantitative contribution of these signalosomes to enhancing and sustaining PI3K and Akt activation triggered by the TCR. We also review data that other TNFR molecules, such as CD40 (TNFRSF5), RANK (TNFRSF11A), FN14 (TNFRSF12A), TACI (TNFRSF13B), BAFFR (TNFRSF13C), and NGFR (TNFRSF16), contribute to the activation of this pathway in diverse cell types through a similar ability to recruit PI3K or Akt into their signaling complexes. PMID:23760533

  20. Pulmonary administration of phosphoinositide 3-kinase inhibitor is a curative treatment for chronic obstructive pulmonary disease by alveolar regeneration.

    PubMed

    Horiguchi, Michiko; Oiso, Yuki; Sakai, Hitomi; Motomura, Tomoki; Yamashita, Chikamasa

    2015-09-10

    Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, causing widespread and irreversible alveoli collapse. The discovery of a low-molecular-weight compound that induces regeneration of pulmonary alveoli is of utmost urgency to cure intractable pulmonary diseases such as COPD. However, a practically useful compound for regenerating pulmonary alveoli is yet to be reported. Previously, we have elucidated that Akt phosphorylation is involved in a differentiation-inducing molecular mechanism of human alveolar epithelial stem cells, which play a role in regenerating pulmonary alveoli. In the present study, we directed our attention to phosphoinositide 3-kinase (PI3K)-Akt signaling and examined whether PI3K inhibitors display the pulmonary alveolus regeneration. Three PI3K inhibitors with different PI3K subtype specificities (Wortmannin, AS605240, PIK-75 hydrochloride) were tested for the differentiation-inducing effect on human alveolar epithelial stem cells, and Wortmannin demonstrated the most potent differentiation-inducing activity. We evaluated Akt phosphorylation in pulmonary tissues of an elastase-induced murine COPD model and found that Akt phosphorylation in the pulmonary tissue was enhanced in the murine COPD model compared with normal mice. Then, the alveolus-repairing effect of pulmonary administration of Wortmannin to murine COPD model was evaluated using X-ray CT analysis and hematoxylin-eosin staining. As a result, alveolar damages were repaired in the Wortmannin-administered group to a similar level of normal mice. Furthermore, pulmonary administration of Wortmannin induced a significant recovery of the respiratory function, compared to the control group. These results indicate that Wortmannin is capable of inducing differentiation of human alveolar epithelial stem cells and represents a promising drug candidate for curative treatment of pulmonary alveolar destruction in COPD.

  1. Impact of negative affectively charged stimuli and response style on cognitive-control-related neural activation: an ERP study.

    PubMed

    Lamm, C; Pine, D S; Fox, N A

    2013-11-01

    The canonical AX-CPT task measures two forms of cognitive control: sustained goal-oriented control ("proactive" control) and transient changes in cognitive control following unexpected events ("reactive" control). We modified this task by adding negative and neutral International Affective Picture System (IAPS) pictures to assess the effects of negative emotion on these two forms of cognitive control. Proactive and reactive control styles were assessed based on measures of behavior and electrophysiology, including the N2 event-related potential component and source space activation (Low Resolution Tomography [LORETA]). We found slower reaction-times and greater DLPFC activation for negative relative to neutral stimuli. Additionally, we found that a proactive style of responding was related to less prefrontal activation (interpreted to reflect increased efficiency of processing) during actively maintained previously cued information and that a reactive style of responding was related to less prefrontal activation (interpreted to reflect increased efficiency of processing) during just-in-time environmentally triggered information. This pattern of results was evident in relatively neutral contexts, but in the face of negative emotion, these associations were not found, suggesting potential response style-by-emotion interaction effects on prefrontal neural activation.

  2. A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells

    SciTech Connect

    Iqbal, Mohd S.; Tsuyama, Naohiro; Obata, Masanori; Ishikawa, Hideaki

    2010-02-12

    Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21{sup Cip1} proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.

  3. PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

    PubMed

    Desai, S; Pillai, P; Win-Piazza, H; Acevedo-Duncan, M

    2011-06-01

    The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

  4. Moderating Effect of Negative Peer Group Climate on the Relation Between Men's Locus of Control and Aggression Toward Intimate Partners.

    PubMed

    Schmidt, Megan R; Lisco, Claire G; Parrott, Dominic J; Tharp, Andra T

    2016-03-01

    The present study sought to examine the interactive effects of an external locus of control and interaction in a negative peer group climate on men's perpetration of physical aggression and infliction of injury toward their female intimate partners. Participants were 206 heterosexual males recruited from the metro-Atlanta community who completed self-report measures of external locus of control, involvement in a negative peer group climate, and physical aggression and infliction of injury against intimate partners during the past 12 months. Negative peer group climate was conceptualized as a peer group that displays behavior which may instigate aggressive norms, attitudes, and behaviors. Results indicated that men with an external locus of control were more likely to perpetrate physical aggression toward and inflict injury on their intimate partners if they reported high, but not low, involvement in a negative peer group climate. These results extend current research suggesting external locus of control as a risk factor for intimate partner aggression by highlighting the impact of negative peer groups. Implications and future intervention research are discussed.

  5. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

    PubMed

    Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

    1995-10-24

    Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

  6. Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae.

    PubMed

    Barrett, LaKisha; Orlova, Marianna; Maziarz, Marcin; Kuchin, Sergei

    2012-02-01

    Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.

  7. Cellular response to low dose radiation: Role of phosphatidylinositol-3 kinase like kinases

    SciTech Connect

    Balajee, A.S.; Meador, J.A.; Su, Y.

    2011-03-24

    It is increasingly realized that human exposure either to an acute low dose or multiple chronic low doses of low LET radiation has the potential to cause different types of cancer. Therefore, the central theme of research for DOE and NASA is focused on understanding the molecular mechanisms and pathways responsible for the cellular response to low dose radiation which would not only improve the accuracy of estimating health risks but also help in the development of predictive assays for low dose radiation risks associated with tissue degeneration and cancer. The working hypothesis for this proposal is that the cellular mechanisms in terms of DNA damage signaling, repair and cell cycle checkpoint regulation are different for low and high doses of low LET radiation and that the mode of action of phosphatidylinositol-3 kinase like kinases (PIKK: ATM, ATR and DNA-PK) determines the dose dependent cellular responses. The hypothesis will be tested at two levels: (I) Evaluation of the role of ATM, ATR and DNA-PK in cellular response to low and high doses of low LET radiation in simple in vitro human cell systems and (II) Determination of radiation responses in complex cell microenvironments such as human EpiDerm tissue constructs. Cellular responses to low and high doses of low LET radiation will be assessed from the view points of DNA damage signaling, DNA double strand break repair and cell cycle checkpoint regulation by analyzing the activities (i.e. post-translational modifications and kinetics of protein-protein interactions) of the key target proteins for PI-3 kinase like kinases both at the intra-cellular and molecular levels. The proteins chosen for this proposal are placed under three categories: (I) sensors/initiators include ATM ser1981, ATR, 53BP1, gamma-H2AX, MDC1, MRE11, Rad50 and Nbs1; (II) signal transducers include Chk1, Chk2, FANCD2 and SMC1; and (III) effectors include p53, CDC25A and CDC25C. The primary goal of this proposal is to elucidate the

  8. Phosphoinositide 3-Kinase Gamma Contributes to Neuroinflammation in a Rat Model of Surgical Brain Injury

    PubMed Central

    Huang, Lei; Sherchan, Prativa; Wang, Yuechun; Reis, Cesar; Applegate, Richard L.; Tang, Jiping

    2015-01-01

    Neuroinflammation plays an important role in the pathophysiology of surgical brain injury (SBI). Phosphoinositide 3-kinase gamma (PI3Kγ), predominately expressed in immune and endothelial cells, activates multiple inflammatory responses. In the present study, we investigated the role of PI3Kγ and PI3Kγ-activated phosphodiesterase 3B (PDE3B) in neuroinflammation in a rat model of SBI. One hundred and fifty-two male Sprague Dawley rats (weight 280–350 g) were subjected to a partial right frontal lobe corticotomy model of SBI. A PI3Kγ pharmacological inhibitor (AS252424 or AS605240) was administered intraperitoneally. PI3Kγ siRNA, human recombinant active-PI3Kγ protein, or human recombinant active-PDE3B protein were administered intracerebroventricularly. Post-SBI assessments included neurobehavioral tests, brain water content, Western blot, and immunohistochemistry. Endogenous PI3Kγ levels were increased within peri-resection brain tissues after SBI, accompanied by increased brain water content and neurological functional deficits. There was a trend toward increased endogenous PDE3B phosphorylation after SBI. The selective PI3Kγ inhibitors AS252424 and AS605240 reduced brain water content surrounding corticotomy and improved neurological function after SBI. SBI increased and PI3Kγ inhibitor decreased levels of myeloperoxidase, cluster of differentiation 3, mast cell degranulation, E-selectin, and IL-1 in peri-resection brain tissues. Direct administration of human recombinant active-PI3Kγ protein and active-PDE3B protein countered the protective effect of AS252424. PI3Kγ siRNA reduced PI3Kγ levels, decreased brain water content within peri-resection brain tissues, and improved neurological function after SBI. Collectively, our findings suggest that PI3Kγ contributed to neuroinflammation after SBI. The use of selective PI3Kγ inhibitors may be a novel approach to ameliorating SBI via their anti-inflammation effects. SIGNIFICANCE STATEMENT Life-saving or

  9. A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation.

    PubMed

    Franco-Echevarría, Elsa; Baños-Sanz, Jose I; Monterroso, Begoña; Round, Adam; Sanz-Aparicio, Julia; González, Beatriz

    2014-11-01

    IP3-3K [Ins(1,4,5)P3 3-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3 and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3 is a key point in Ca2+ metabolism that promotes Ca2+ release from intracellular stores and together with Ins(1,3,4,5)P4 regulates Ca2+ homoeostasis. In addition, Ins(1,3,4,5)P4 is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.

  10. Rapamycin regulates connective tissue growth factor expression of lung epithelial cells via phosphoinositide 3-kinase.

    PubMed

    Xu, Xuefeng; Wan, Xuan; Geng, Jing; Li, Fei; Yang, Ting; Dai, Huaping

    2013-09-01

    The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains largely unknown. It is believed that IPF is mainly driven by activated alveolar epithelial cells that have a compromised migration capacity, and that also produce substances (such as connective tissue growth factor, CTGF) that contribute to fibroblast activation and matrix protein accumulation. Because the mechanisms regulating these processes are unclear, the aim of this study was to determine the role of rapamycin in regulating epithelial cell migration and CTGF expression. Transformed epithelial cell line A549 and normal human pulmonary alveolar or bronchial epithelial cells were cultured in regular medium or medium containing rapamycin. Real time reverse transcriptase polymerase chain reaction was employed to determine CTGF mRNA expression. Western blotting and an enzyme-linked immunosorbent assay were used for detecting CTGF protein. Wound healing and migration assays were used to determine the cell migration potential. Transforming growth factor (TGF)-β type I receptor (TβRI) inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. It was found that treatment of A549 and normal human alveolar or bronchial epithelial cells with rapamycin significantly promoted basal or TGF-β1 induced CTGF expression. LY294002, not SB431542 attenuated the promotional effect of rapamycin on CTGF expression. Cell mobility was not affected by rapamycin in wound healing and migration assays. These data suggest rapamycin has a profibrotic effect in vitro and underscore the potential of combined therapeutic approach with PI3K and mammalian target of rapamycin inhibitors for the treatment of animal or human lung fibrosis.

  11. Supramolecular nanoparticles that target phosphatidylinositol-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy

    PubMed Central

    Kulkarni, Ashish A.; Roy, Bhaskar; Rao, Poornima S.; Wyant, Gregory A.; Mahmoud, Ayaat; Ramachandran, Madhumitha; Sengupta, Poulomi; Goldman, Aaron; Kotamraju, Venkata Ramana; Basu, Sudipta; Mashelkar, Raghunath A; Ruoslahti, Erkki; Dinulescu, Daniela M.; Sengupta, Shiladitya

    2013-01-01

    The centrality of phosphatidylinositol-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intra-tumoral concentration and an insulin resistance ‘class effect’. The current study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG. The supramolecular nanoparticles that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-RasLSL/+/Ptenfl/fl ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the supramolecular nanoparticles highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the supramolecular nanoparticles exerted a temporally-sustained inhibition of phosphorylation of Akt, mTOR, S6K and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of supramolecular nanoparticles abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer treatment

  12. Carbamazepine enhances the activity of glutamate transporter type 3 via phosphatidylinositol 3-kinase.

    PubMed

    Lee, Gwanwoo; Huang, Yueming; Washington, Jacqueline M; Briggs, Nicole W; Zuo, Zhiyi

    2005-01-01

    Glutamate transporters (also called excitatory amino acid transporters, EAAT) participate in maintaining extracellular homeostasis of glutamate, a major excitatory neurotransmitter, and regulating glutamate neurotransmission. EAAT3, the major neuronal EAAT, may also regulate gamma-aminobutyric acid-mediated inhibitory neurotransmission. Dysfunction of EAAT3 has been shown to induce seizure in rats. We hypothesize that carbamazepine, a commonly used antiepileptic agent, enhances EAAT3 activity. We tested this hypothesis using oocytes artificially expressing EAAT3 and C6 rat glioma cells expressing endogenous EAAT3. In oocytes, carbamazepine dose-dependently enhanced EAAT3 activity. The EC50 of this carbamazepine effect was 12.2muM. The concentrations of carbamazepine to significantly enhance EAAT3 activity were within the therapeutic serum levels (17-51muM) of carbamazepine for the antiepileptic effect. Carbamazepine decreased the Km but did not change the maximal response of EAAT3 to glutamate. Carbamazepine-increased EAAT3 activity was inhibited by wortmannin or LY-294002, phosphatidylinositol 3-kinase (PI3K) inhibitors, but was not affected by staurosporine, chelerythrine or calphostin C, protein kinase C inhibitors. In C6 cells, carbamazepine also enhanced the endogenous EAAT3 activity. However, carbamazepine did not affect the activity of EAAT4 expressed in Cos7 cells. These results suggest that carbamazepine at clinically relevant concentrations specifically enhances the affinity of EAAT3 for glutamate to increase EAAT3 activity via a PI3K-dependent pathway. EAAT3 may be a therapeutic target for carbamazepine in the central nervous system.

  13. Supramolecular nanoparticles that target phosphoinositide-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy.

    PubMed

    Kulkarni, Ashish A; Roy, Bhaskar; Rao, Poornima S; Wyant, Gregory A; Mahmoud, Ayaat; Ramachandran, Madhumitha; Sengupta, Poulomi; Goldman, Aaron; Kotamraju, Venkata Ramana; Basu, Sudipta; Mashelkar, Raghunath A; Ruoslahti, Erkki; Dinulescu, Daniela M; Sengupta, Shiladitya

    2013-12-01

    The centrality of phosphoinositide-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intratumoral concentration, and an insulin resistance "class effect." This study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polythylene glycol)]. The supramolecular nanoparticles (SNP) that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-Ras(LSL/+)/Pten(fl/fl) ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the SNPs highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the SNPs exerted a temporally sustained inhibition of phosphorylation of Akt, mTOR, S6K, and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of SNPs abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer

  14. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    SciTech Connect

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li{sub 2}CO{sub 3} significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li{sub 2}CO{sub 3} did not affect PI3K-mediated PI(3,4,5)P{sub 3} production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li{sub 2}CO{sub 3} on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li{sub 2}CO{sub 3} significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li{sub 2}CO{sub 3} significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity.

  15. Dual roles of hemidesmosomal proteins in the pancreatic epithelium: the phosphoinositide 3-kinase decides.

    PubMed

    Laval, S; Laklai, H; Fanjul, M; Pucelle, M; Laurell, H; Billon-Galés, A; Le Guellec, S; Delisle, M-B; Sonnenberg, A; Susini, C; Pyronnet, S; Bousquet, C

    2014-04-10

    Given the failure of chemo- and biotherapies to fight advanced pancreatic cancer, one major challenge is to identify critical events that initiate invasion. One priming step in epithelia carcinogenesis is the disruption of epithelial cell anchorage to the basement membrane which can be provided by hemidesmosomes (HDs). However, the existence of HDs in pancreatic ductal epithelium and their role in carcinogenesis remain unexplored. HDs have been explored in normal and cancer pancreatic cells, and patient samples. Unique cancer cell models where HD assembly can be pharmacologically manipulated by somatostatin/sst2 signaling have been then used to investigate the role and molecular mechanisms of dynamic HD during pancreatic carcinogenesis. We surprisingly report the presence of mature type-1 HDs comprising the integrin α6β4 and bullous pemphigoid antigen BP180 in the human pancreatic ductal epithelium. Importantly, HDs are shown to disassemble during pancreatic carcinogenesis. HD breakdown requires phosphoinositide 3-kinase (PI3K)-dependent induction of the matrix-metalloprotease MMP-9, which cleaves BP180. Consequently, integrin α6β4 delocalizes to the cell-leading edges where it paradoxically promotes cell migration and invasion through S100A4 activation. As S100A4 in turn stimulates MMP-9 expression, a vicious cycle maintains BP180 cleavage. Inactivation of this PI3K-MMP-9-S100A4 signaling loop conversely blocks BP180 cleavage, induces HD reassembly and inhibits cell invasion. We conclude that mature type-1 HDs are critical anchoring structures for the pancreatic ductal epithelium whose disruption, upon PI3K activation during carcinogenesis, provokes pancreatic cancer cell migration and invasion.

  16. Role of phosphoinositide 3-kinase in the pathogenesis of acute pancreatitis

    PubMed Central

    Lupia, Enrico; Pigozzi, Luca; Goffi, Alberto; Hirsch, Emilio; Montrucchio, Giuseppe

    2014-01-01

    A large body of experimental and clinical data supports the notion that inflammation in acute pancreatitis has a crucial role in the pathogenesis of local and systemic damage and is a major determinant of clinical severity. Thus, research has recently focused on molecules that can regulate the inflammatory processes, such as phosphoinositide 3-kinases (PI3Ks), a family of lipid and protein kinases involved in intracellular signal transduction. Studies using genetic ablation or pharmacologic inhibitors of different PI3K isoforms, in particular the class I PI3Kδ and PI3Kγ, have contributed to a greater understanding of the roles of these kinases in the modulation of inflammatory and immune responses. Recent data suggest that PI3Ks are also involved in the pathogenesis of acute pancreatitis. Activation of the PI3K signaling pathway, and in particular of the class IB PI3Kγ isoform, has a significant role in those events which are necessary for the initiation of acute pancreatic injury, namely calcium signaling alteration, trypsinogen activation, and nuclear factor-κB transcription. Moreover, PI3Kγ is instrumental in modulating acinar cell apoptosis, and regulating local neutrophil infiltration and systemic inflammatory responses during the course of experimental acute pancreatitis. The availability of PI3K inhibitors selective for specific isoforms may provide new valuable therapeutic strategies to improve the clinical course of this disease. This article presents a brief summary of PI3K structure and function, and highlights recent advances that implicate PI3Ks in the pathogenesis of acute pancreatitis. PMID:25386068

  17. Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors.

    PubMed

    Singh, Abhay Kumar; Pantouris, Georgios; Borosch, Sebastian; Rojanasthien, Siripong; Cho, Thomas Yoonsang

    2017-01-01

    Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.

  18. Selective Inhibition of Phosphoinositide 3-Kinase p110α Preserves Lymphocyte Function*

    PubMed Central

    So, Lomon; Yea, Sung Su; Oak, Jean S.; Lu, Mengrou; Manmadhan, Arun; Ke, Qiao Han; Janes, Matthew R.; Kessler, Linda V.; Kucharski, Jeff M.; Li, Lian-Sheng; Martin, Michael B.; Ren, Pingda; Jessen, Katti A.; Liu, Yi; Rommel, Christian; Fruman, David A.

    2013-01-01

    Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors. PMID:23275335

  19. Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

    PubMed

    Aparicio, I M; Bragado, M J; Gil, M C; Garcia-Herreros, M; Gonzalez-Fernandez, L; Tapia, J A; Garcia-Marin, L J

    2007-08-01

    Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

  20. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    PubMed Central

    Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.

    2014-01-01

    Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity. PMID:27919038

  1. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    PubMed Central

    Leprince, Anne-Sophie; Magalhaes, Nelly; De Vos, Delphine; Bordenave, Marianne; Crilat, Emilie; Clément, Gilles; Meyer, Christian; Munnik, Teun; Savouré, Arnould

    2015-01-01

    Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose. PMID:25628629

  2. Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

    PubMed Central

    Higaki, Yasuki; Mikami, Toshio; Fujii, Nobuharu; Hirshman, Michael F.; Koyama, Katsuhiro; Seino, Tetsuya; Tanaka, Keitaro; Goodyear, Laurie J.

    2010-01-01

    We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 μmol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-L-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 μmol/l had no effect on ATP concentrations and did not increase the activities of either the α1 or α2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism. PMID:18303121

  3. Selective inhibition of phosphoinositide 3-kinase p110α preserves lymphocyte function.

    PubMed

    So, Lomon; Yea, Sung Su; Oak, Jean S; Lu, Mengrou; Manmadhan, Arun; Ke, Qiao Han; Janes, Matthew R; Kessler, Linda V; Kucharski, Jeff M; Li, Lian-Sheng; Martin, Michael B; Ren, Pingda; Jessen, Katti A; Liu, Yi; Rommel, Christian; Fruman, David A

    2013-02-22

    Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

  4. The Association of PI3 Kinase Signaling and Chemoresistance in Advanced Ovarian Cancer

    PubMed Central

    Carden, Craig P.; Stewart, Adam; Thavasu, Parames; Kipps, Emma; Pope, Lorna; Crespo, Mateus; Miranda, Susana; Attard, Gerhardt; Garrett, Michelle D.; Clarke, Paul A.; Workman, Paul; de Bono, Johann S.; Gore, Martin; Kaye, Stan B; Banerji, Udai

    2015-01-01

    Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN. PMID:22556379

  5. Enterococcus faecalis infection activates phosphatidylinositol 3-kinase signaling to block apoptotic cell death in macrophages.

    PubMed

    Zou, Jun; Shankar, Nathan

    2014-12-01

    Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.

  6. Adaptive vibration suppression system: an iterative control law for a piezoelectric actuator shunted by a negative capacitor.

    PubMed

    Kodejska, Milos; Mokry, Pavel; Linhart, Vaclav; Vaclavik, Jan; Sluka, Tomas

    2012-12-01

    An adaptive system for the suppression of vibration transmission using a single piezoelectric actuator shunted by a negative capacitance circuit is presented. It is known that by using a negative-capacitance shunt, the spring constant of a piezoelectric actuator can be controlled to extreme values of zero or infinity. Because the value of spring constant controls a force transmitted through an elastic element, it is possible to achieve a reduction of transmissibility of vibrations through the use of a piezoelectric actuator by reducing its effective spring constant. Narrow frequency range and broad frequency range vibration isolation systems are analyzed, modeled, and experimentally investigated. The problem of high sensitivity of the vibration control system to varying operational conditions is resolved by applying an adaptive control to the circuit parameters of the negative capacitor. A control law that is based on the estimation of the value of the effective spring constant of a shunted piezoelectric actuator is presented. An adaptive system which achieves a self-adjustment of the negative capacitor parameters is presented. It is shown that such an arrangement allows the design of a simple electronic system which offers a great vibration isolation efficiency under variable vibration conditions.

  7. Progress in the Preclinical Discovery and Clinical Development of Class I and Dual Class I/IV Phosphoinositide 3-Kinase (PI3K) Inhibitors

    PubMed Central

    Shuttleworth, S.J; Silva, F.A; Cecil, A.R.L; Tomassi, C.D; Hill, T.J; Raynaud, F.I; Clarke, P.A; Workman, P

    2011-01-01

    The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer. PMID:21649578

  8. Estradiol regulates the insulin-like growth factor-I (IGF-I) signalling pathway: A crucial role of phosphatidylinositol 3-kinase (PI 3-kinase) in estrogens requirement for growth of MCF-7 human breast carcinoma cells

    SciTech Connect

    Bernard, Laurence; Legay, Christine; Adriaenssens, Eric; Mougel, Alexandra; Ricort, Jean-Marc . E-mail: ricort@lbpa.ens-cachan.fr

    2006-12-01

    Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. With a view to investigating the molecular mechanisms implicated, we studied the effect of estradiol on the expression of proteins implicated in the insulin-like growth factor signalling pathway. Estradiol dose- and time-dependently increased the expression of insulin receptor substrate-1 and the p85/p110 subunits of phosphatidylinositol 3-kinase but did not change those of ERK2 and Akt/PKB. ICI 182,780 did not inhibit estradiol-induced IRS-1 and p85 expression. Moreover, two distinct estradiol-BSA conjugate compounds were as effective as estradiol in inducing IRS-1 and p85/p110 expression indicating the possible implication of an estradiol membrane receptor. Comparative analysis of steroids-depleted and steroids-treated cells showed that IGF-I only stimulates cell growth in the latter condition. Nevertheless, expression of a constitutively active form of PI 3-kinase in steroid-depleted cells triggers proliferation. These results demonstrate that estradiol positively regulates essential proteins of the IGF signalling pathway and put in evidence that phosphatidylinositol 3-kinase plays a central role in the synergistic pro-proliferative action of estradiol and IGF-I.

  9. Longitudinal Relations of Children's Effortful Control, Impulsivity, and Negative Emotionality to Their Externalizing, Internalizing, and Co-Occurring Behavior Problems

    ERIC Educational Resources Information Center

    Eisenberg, Nancy; Valiente, Carlos; Spinrad, Tracy L.; Cumberland, Amanda; Liew, Jeffrey; Reiser, Mark; Zhou, Qing; Losoya, Sandra H.

    2009-01-01

    The purpose of the study was to examine the relations of effortful control (EC), impulsivity, and negative emotionality to at least borderline clinical levels of symptoms and change in maladjustment over four years. Children's (N = 214; 77% European American; M age = 73 months) externalizing and internalizing symptoms were rated by parents and…

  10. Tentativeness and fervor in cell biology require negative and positive feedforward control.

    PubMed

    Sinclair, N R; Challis, J R

    1993-01-01

    End-product feedback regulates early steps in metabolic pathways, affecting activation and the rate of end-product synthesis. Early formation of end-product also modifies later steps in the synthesis of end-product. We designate this form of regulation feedforward. Negative feedback/feedforward by end-products may result in homeostasis, but also in physiologic tentativeness. Positive feedback/feedforward by end-product gives rise to fervid events. Tentativeness and fervor, due to negative and positive feedforward rather than feedback, explain otherwise puzzling aspects of immunology and endocrinology.

  11. Plasma glycine and serine levels in schizophrenia compared to normal controls and major depression: relation to negative symptoms.

    PubMed

    Sumiyoshi, Tomiki; Anil, A Elif; Jin, Dai; Jayathilake, Karu; Lee, Myung; Meltzer, Herbert Y

    2004-03-01

    Previous studies have suggested decreased N-methyl-D-aspartate (NMDA)-type glutamate receptor function may contribute to increased negative symptoms in patients with schizophrenia. Consistent with this hypothesis, glycine, a co-agonist at NMDA receptors, has been reported to improve negative symptoms associated with the illness. This study was performed to determine if plasma levels of glycine or its ratio to serine, a precursor of glycine, are decreased in patients with schizophrenia compared to normal control subjects or patients with major depression. We also tested the hypothesis that these amino acids were correlated with negative symptoms in subjects with schizophrenia. Plasma levels of glycine, serine, and their ratio, were compared in 144 patients with schizophrenia, 44 patients with major depression, and 49 normal control subjects. All subjects were medication-free. Psychopathology was evaluated using the Brief Psychiatric Rating Scale (BPRS). Plasma glycine levels and glycine/serine ratios were decreased in patients with schizophrenia relative to control subjects and patients with major depression. By contrast, serine levels were increased in patients with schizophrenia compared to normal subjects but not compared to major depression. Patients with major depression also had increased plasma serine levels and decreased glycine/serine ratios compared to normal controls, but glycine levels were not different from those of normal controls. In subjects with schizophrenia, glycine levels predicted the Withdrawal-Retardation score (BPRS), whereas no such correlation was found in subjects with major depression. These results provide additional evidence that decreased availability of glycine may be related to the pathophysiology of negative symptoms. The decreases in plasma glycine levels support the evidence for an abnormality in the glutamatergic system in schizophrenia, and provide additional support for efforts to improve negative symptoms by augmentation of

  12. Phosphoinositide 3-kinase signaling is critical for ErbB3-driven breast cancer cell motility and metastasis

    PubMed Central

    Smirnova, Tatiana; Zhou, Zhen Ni; Flinn, Rory J.; Wyckoff, Jeffrey; Boimel, Pamela J.; Pozzuto, Maria; Coniglio, Salvatore J.; Backer, Jonathan M.; Bresnick, Anne R.; Condeelis, John S.; Hynes, Nancy E.; Segall, Jeffrey E.

    2011-01-01

    Many malignancies show increased expression of the EGF receptor family member ErbB3 (HER3). ErbB3 binds beta-1 (HRGβ1), and forms a heterodimer with other ErbB family members, such as ErbB2 (HER2) or EGFR (HER1), enhancing phosphorylation of specific C terminal tyrosine residues and activation of downstream signaling pathways. ErbB3 contains six YXXM motifs that bind the p85 subunit of PI3-kinase. Previous studies demonstrated that overexpression of ErbB3 in mammary tumor cells can significantly enhance chemotaxis to HRGβ1 and overall metastatic potential. We tested the hypothesis that ErbB3-mediated PI3-kinase signaling is critical for heregulin-induced motility, and therefore crucial for ErbB3-mediated invasion, intravasation and metastasis. The tyrosines in the six YXXM motifs on the ErbB3 C-terminus were replaced with phenylalanine. In contrast to overexpression of the wild-type ErbB3, overexpression of the mutant ErbB3 did not enhance chemotaxis towards HRGβ1 in vitro or in vivo. We also observed reduced tumor cell motility in the primary tumor by multiphoton microscopy, as well as a dramatically reduced ability of these cells to cross the endothelium and intravasate into the circulation. Moreover, while mutation of the ErbB3 C-terminus had no effect on tumor growth, it had a dramatic effect on spontaneous metastatic potential. Treatment with the PI3-kinase inhibitor PIK-75 similarly inhibited motility and invasion in vitro and in vivo. Our results indicate that stimulation of the early metastatic steps of motility and invasion by ErbB3 requires activation of the PI3-kinase pathway by the ErbB3 receptor. PMID:21725367

  13. Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation–Induced Cell Death

    PubMed Central

    Sharlow, Elizabeth R.; Epperly, Michael W.; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M.; Wipf, Peter; Greenberger, Joel S.

    2013-01-01

    Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after

  14. PI3 kinase is involved in cocaine behavioral sensitization and its reversal with brain area specificity

    SciTech Connect

    Zhang Xiuwu . E-mail: xwzhang@duke.edu; Mi Jing; Wetsel, William C.; Davidson, Colin; Xiong Xieying; Chen Qiang; Ellinwood, Everett H.; Lee, Tong H.

    2006-02-24

    Phosphatidylinositol 3-kinase (PI3K) is an important signaling molecule involved in cell differentiation, proliferation, survival, and phagocytosis, and may participate in various brain functions. To determine whether it is also involved in cocaine sensitization, we measured the p85{alpha}/p110 PI3K activity in the nuclear accumbens (NAc) shell, NAc core, and prefrontal cortex (PFC) following establishment of cocaine sensitization and its subsequent reversal. Naive rats were rank-ordered and split into either daily cocaine or saline pretreatment group based on their locomotor responses to an acute cocaine injection (7.5 mg/kg, i.p.). These two groups were then injected with cocaine (40 mg/kg, s.c.) or saline for 4 consecutive days followed by 9-day withdrawal. Cocaine sensitization was subsequently reversed by 5 daily injections of the D{sub 1}/D{sub 2} agonist pergolide (0.1 mg/kg, s.c.) in combination with the 5-HT{sub 3} antagonist ondansetron (0.2 mg/kg, s.c., 3.5 h after pergolide injection). After another 9-day withdrawal, behavioral cocaine sensitization and its reversal were confirmed with an acute cocaine challenge (7.5 mg/kg, i.p.), and animals were sacrificed the next day for measurement of p85{alpha}/p110 PI3K activity. Cocaine-sensitized animals exhibited increased PI3K activity in the NAc shell, and this increase was reversed by combined pergolide/ondansetron treatment, which also reversed behavioral sensitization. In the NAc core and PFC, cocaine sensitization decreased and increased the PI3K activity, respectively. These changes, in contrast to that in the NAc shell, were not normalized following the reversal of cocaine-sensitization. Interestingly, daily injections of pergolide alone in saline-pretreated animals induced PI3K changes that were similar to the cocaine sensitization-associated changes in the NAc core and PFC but not the NAc shell; furthermore, these changes in saline-pretreated animals were prevented by ondansetron given 3.5 h after

  15. Phosphatidylinositol-3 kinase-dependent translational regulation of Id1 involves the PPM1G phosphatase

    PubMed Central

    Xu, Kaiming; Wang, Lanfang; Feng, Wei; Feng, Yue; Shu, Hui-Kuo G.

    2016-01-01

    Id1 is a helix-loop-helix transcriptional modulator that increases the aggressiveness of malignant glial neoplasms. Since most glioblastomas (GBMs) show increased phosphatidylinositol-3 kinase (PI-3K) signaling, we sought to determine whether this pathway regulates Id1 expression. Higher basal Id1 expression correlates with dysregulated PI-3K signaling in multiple established GBM cell lines. Further characterization of PI-3K-dependent Id1 regulation reveals that chemical or genetic inhibition of PI-3K signaling reduces Id1 protein but not mRNA expression. Overall, PI-3K signaling appears to enhance Id1 translation with no significant effect on its stability. PI-3K signaling is known to regulate protein translation through mTORC1-dependent phosphorylation of 4E-BP1, which reduces its association with and inhibition of the translation initiation factor eIF4E. Interestingly, while inhibition of PI-3K and AKT lowers 4E-BP1 phosphorylation and expression of Id1 in all cases, inhibition of TORC1 with rapamycin does not consistently have a similar effect suggesting an alternative mechanism for PI-3K-dependent regulation of Id1 translation. We now identify a potential role for the serine-threonine phosphatase PPM1G in translational regulation of Id1 protein expression. PPM1G knockdown by siRNA increase both 4E-BP1 phosphorylation and Id1 expression and PPM1G and 4E-BP1 co-associates in GBM cells. Furthermore, PPM1G is a phosphoprotein and this phosphorylation appears to be regulated by PI-3K activity. Finally, PI-3K inhibition increases PPM1G activity when assessed by an in vitro phosphatase assay. Our findings provide the first evidence that the PI-3K/AKT signaling pathway modulates PPM1G activity resulting in a shift in the balance between hyper- and hypo-phosphorylated 4E-BP1 and translational regulation of Id1 expression. PMID:27065332

  16. Negative Affectivity, Effortful Control, and Attention to Threat-Relevant Stimuli

    ERIC Educational Resources Information Center

    Lonigan, Christopher J.; Vasey, Michael W.

    2009-01-01

    There is increasing recognition of temperamental influences on risk for psychopathology. Whereas the link between the broad temperament construct of negative affectivity (NA) and problems associated with anxiety and depression is now well-established, the mechanisms through which this link operate are not well understood. One possibility involves…

  17. Teenagers: fertility control behavior and attitudes before and after abortion, childbearing or negative pregnancy test.

    PubMed

    Evans, J R; Selstad, G; Welcher, W H

    1976-01-01

    Following abortion or delivery, teenagers' knowledge and use of effective contraception improved markedly; but those who had negative pregnancy tests continued to take risks--and to get pregnant--subsequently. Most sexually active teenagers were poorly educated on the facts of reproduction, and began to have intercourse before seeking contraception.

  18. On the Control of Single-Prime Negative Priming: The Effects of Practice and Time Course

    ERIC Educational Resources Information Center

    Chao, Hsuan-Fu

    2009-01-01

    Single-prime negative priming refers to the phenomenon wherein repetition of a prime as the probe target results in delayed response. Sometimes this effect has been found to be contingent on participants' unawareness of the primes, and sometimes it has not. Further, sometimes this effect has been found to be eliminated when the prime could predict…

  19. Efficacy of Creative Clay Work for Reducing Negative Mood: A Randomized Controlled Trial

    ERIC Educational Resources Information Center

    Kimport, Elizabeth R.; Robbins, Steven J.

    2012-01-01

    Clay work has long been used in art therapy to achieve therapeutic goals. However, little empirical evidence exists to document the efficacy of such work. The present study randomly assigned 102 adult participants to one of four conditions following induction of a negative mood: (a) handling clay with instructions to create a pinch pot, (b)…

  20. The invasive American weed parthenium hysterophorus can negatively impact malaria control in Africa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The direct negative effects of invasive plant species on agriculture and biodiversity are well known, but their indirect effects on human health, and particularly their interactions with disease-transmitting vectors, remains poorly explored. This study sought to investigate the impact of the invasiv...

  1. Control of emergence of multi-resistant gram-negative bacilli by exclusive use of amikacin.

    PubMed

    Ruiz-Palacios, G M; Ponce de Leon, S; Sifuentes, J; Ponce de Leon, S; Calva, J J; Huazano, F; Ontiveros, C; Ojeda, F; Bobadilla, M

    1986-06-30

    Results of a three-year prospective study of amikacin as the only aminoglycoside used at the Instituto Nacional de la Nutrición "Salvador Zubirán" are presented. During the initial three-month baseline period, resistance to amikacin, gentamicin, and tobramycin among 870 gram-negative bacterial isolates was 3.2 percent, 17.4 percent, and 11.2 percent, respectively. In this period, the overall consumption of aminoglycosides was 69 percent for gentamicin, 30.5 percent for amikacin, and 0.5 percent for tobramycin. In the following period of exclusive amikacin use, sensitivity patterns of 9,344 gram-negative strains isolated over three years were recorded. During this period, amikacin constituted 99.3 percent of all aminoglycosides used. The percentage of gentamicin-resistant gram-negative strains declined to 7.4 percent (p less than 0.0001), whereas the percentage of amikacin-resistant strains did not change significantly. Quarterly trend analysis of aminoglycoside-resistant strains also demonstrated a significant decrease in gentamicin resistance (p less than 0.005) and an overall steady state of amikacin resistance. It is concluded that the exclusive use of amikacin was not accompanied by a significant increase in amikacin resistance during a three-year period, and may even lead to a decrease in resistance to gentamicin and tobramycin among most gram-negative organisms.

  2. Characteristics of a high-power RF source of negative hydrogen ions for neutral beam injection into controlled fusion devices

    NASA Astrophysics Data System (ADS)

    Abdrashitov, G. F.; Belchenko, Yu. I.; Gusev, I. A.; Ivanov, A. A.; Kondakov, A. A.; Sanin, A. L.; Sotnikov, O. Z.; Shikhovtsev, I. V.

    2017-01-01

    An injector of hydrogen atoms with an energy of 0.5-1 MeV and equivalent current of up to 1.5 A for purposes of controlled fusion research is currently under design at the Budker Institute of Nuclear Physics, Siberian Branch, Russian Academy of Sciences. Within this project, a multiple-aperture RF surface-plasma source of negative hydrogen ions is designed. The source design and results of experiments on the generation of a negative ion beam with a current of >1 A in the long-pulse mode are presented.

  3. Controllable negative and positive group delay in transmission through a single quantum well at finite magnetic fields

    NASA Astrophysics Data System (ADS)

    Ban, Yue; Chen, Xi; Li, Chun-Fang

    2007-04-01

    We investigate the controllable negative and positive group delay in transmission through a single quantum well at the finite longitudinal magnetic fields. It is shown that the magneto-coupling effect between the longitudinal motion component and the transverse Landau orbits plays an important role in the group delay. The group delay depends not only on the width of potential well and the incident energy, but also on the magnetic-field strengthen and the Landau quantum number. The results show that the group delay can be changed from positive to negative by the modulation of the magnetic field. These interesting phenomena may lead to the tunable quantum mechanical delay line.

  4. Ovarian expressed microsomal epoxide hydrolase: Role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling

    SciTech Connect

    Bhattacharya, Poulomi; Sen, Nivedita; Hoyer, Patricia B.; Keating, Aileen F.

    2012-01-01

    4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P < 0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P < 0.05) on day 4 of VCD (30 μM) exposure, followed by increased (P < 0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH. -- Highlights: ► Ovarian mEH functions to metabolize VCD to a less toxic compound. ► mEH expression is increased in a temporal pattern in response to VCD exposure. ► PI3K signaling is involved in regulation of ovarian mEH expression.

  5. Power-controlled transition from standard to negative refraction in reorientational soft matter.

    PubMed

    Piccardi, Armando; Alberucci, Alessandro; Kravets, Nina; Buchnev, Oleksandr; Assanto, Gaetano

    2014-11-25

    Refraction at a dielectric interface can take an anomalous character in anisotropic crystals, when light is negatively refracted with incident and refracted beams emerging on the same side of the interface normal. In soft matter subject to reorientation, such as nematic liquid crystals, the nonlinear interaction with light allows tuning of the optical properties. We demonstrate that in such material a beam of light can experience either positive or negative refraction depending on input power, as it can alter the spatial distribution of the optic axis and, in turn, the direction of the energy flow when traveling across an interface. Moreover, the nonlinear optical response yields beam self-focusing and spatial localization into a self-confined solitary wave through the formation of a graded-index waveguide, linking the refractive transition to power-driven readdressing of copolarized guided-wave signals, with a number of output ports not limited by diffraction.

  6. Transformation of Rat-1 fibroblasts with the v-src oncogene induces inositol 1,4,5-trisphosphate 3-kinase expression.

    PubMed Central

    Woodring, P J; Garrison, J C

    1996-01-01

    Transformation of Rat-1 fibroblasts with the v-src oncogene leads to a 6- to 8-fold enhancement of the activity of the Ins(1,4,5)P3 3-kinase in cytosolic extracts [Johnson, Wasilenko, Mattingly, Weber and Garrison (1989) Science 246, 121-124]. This study confirms these results using another v-src-transformed Rat-1 cell line (B31 cells) and investigates the molecular mechanism by which pp60v-src activates Ins(1,4,5)P3 3-kinase. The mRNA and protein levels for two rat isoforms of Ins(1,4,5)P3 3-kinase were determined in the v-src-transformed cell line. Both the mRNA and protein levels for isoform A were elevated in v-src-transformed Rat-1 cells while those for isoform B were not significantly affected. Moreover, stable expression of either form of Ins(1,4,5)P3 3-kinase in the B31 v-src-transformed Rat-1 cell line did not result in tyrosine phosphorylation of Ins(1,4,5)P3 3-kinase A or B. These results suggest that at least one mechanism by which the v-src oncogene increases the activity of the Ins(1,4,5)P3 3-kinase in the Rat-1 transformed fibroblast is by increasing the level of expression of Ins(1,4,5)P3 3-kinase A. PMID:8870651

  7. Overcoming Disembodiment: The Effect of Movement Therapy on Negative Symptoms in Schizophrenia—A Multicenter Randomized Controlled Trial

    PubMed Central

    Martin, Lily A. L.; Koch, Sabine C.; Hirjak, Dusan; Fuchs, Thomas

    2016-01-01

    Objective: Negative symptoms of patients with Schizophrenia are resistant to medical treatment or conventional group therapy. Understanding schizophrenia as a form of disembodiment of the self, a number of scientists have argued that the approach of embodiment and associated embodied therapies, such as Dance and Movement Therapy (DMT) or Body Psychotherapy (BPT), may be more suitable to explain the psychopathology underlying the mental illness and to address its symptoms. Hence the present randomized controlled trial (DRKS00009828, http://apps.who.int/trialsearch/) aimed to examine the effectiveness of manualized movement therapy (BPT/DMT) on the negative symptoms of patients with schizophrenia. Method:A total of 68 out-patients with a diagnosis of a schizophrenia spectrum disorder were randomly allocated to either the treatment (n = 44, 20 sessions of BPT/DMT) or the control condition [n = 24, treatment as usual (TAU)]. Changes in negative symptom scores on the Scale for the Assessment of Negative Symptoms (SANS) were analyzed using Analysis of Covariance (ANCOVA) with Simpson-Angus Scale (SAS) scores as covariates in order to control for side effects of antipsychotic medication. Results:After 20 sessions of treatment (BPT/DMT or TAU), patients receiving movement therapy had significantly lower negative symptom scores (SANS total score, blunted affect, attention). Effect sizes were moderate and mean symptom reduction in the treatment group was 20.65%. Conclusion:The study demonstrates that embodied therapies, such as BPT/DMT, are highly effective in the treatment of patients with schizophrenia. Results strongly suggest that BPT/DMT should be embedded in the daily clinical routine. PMID:27064347

  8. Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor.

    PubMed

    Hellyer, N J; Kim, M S; Koland, J G

    2001-11-09

    The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.

  9. Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not protein kinase B activation or glucose uptake, in rat muscle.

    PubMed Central

    Whitehead, J P; Soos, M A; Aslesen, R; O'rahilly, S; Jensen, J

    2000-01-01

    The initial stages of insulin-stimulated glucose uptake are thought to involve tyrosine phosphorylation of insulin receptor substrates (IRSs), which recruit and activate phosphoinositide 3-kinase (PI 3-kinase), leading to the activation of protein kinase B (PKB) and other downstream effectors. In contrast, contraction stimulates glucose uptake via a PI 3-kinase-independent mechanism. The combined effects of insulin and contraction on glucose uptake are additive. However, it has been reported that contraction causes a decrease in insulin-stimulated IRS-1-associated PI 3-kinase activity. To investigate this paradox, we have examined the effects of contraction on insulin-stimulated glucose uptake and proximal insulin-signalling events in isolated rat epitrochlearis muscle. Stimulation by insulin or contraction produced a 3-fold increase in glucose uptake, with the effects of simultaneous treatment by insulin and contraction being additive. Wortmannin completely blocked the additive effect of insulin in contracting skeletal muscle, indicating that this is a PI 3-kinase-dependent effect. Insulin-stimulated recruitment of PI 3-kinase to IRS-1 was unaffected by contraction; however, insulin produced no discernible increase in PI 3-kinase activity in IRS-1 or IRS-2 immunocomplexes in contracting skeletal muscle. Consistent with this, contraction inhibited insulin-stimulated p70(S6K) activation. In contrast, insulin-stimulated activation of PKB was unaffected by contraction. Thus, in contracting skeletal muscle, insulin stimulates glucose uptake and activates PKB, but not p70(S6K), by a PI 3-kinase-dependent mechanism that is independent of changes in IRS-1- and IRS-2-associated PI 3-kinase activity. PMID:10903138

  10. Negative regulation of geminin by CDK-dependent ubiquitination controls replication licensing.

    PubMed

    Li, Anatoliy; Blow, J Julian

    2004-04-01

    The replication licensing system ensures the precise duplication of chromosomal DNA in each cell cycle. In metazoans, a small protein called geminin plays a central role in negatively regulating licensing late in the cell cycle. Recent work using Xenopus egg extracts shows how geminin activity is downregulated on exit from metaphase in a process that requires mitotic cyclin-dependent kinases (CDKs). Geminin is polyubiquitinated by the Anaphase Promoting Complex, but instead of being proteolysed-the normal fate of polyubiquitinated proteins-much of the geminin is deubiquitinated, leaving it inactive. These results suggest a simple model for how precise chromosome duplication is ensured in the Xenopus model system.

  11. Gram-negative bacteria account for main differences between faecal microbiota from patients with ulcerative colitis and healthy controls.

    PubMed

    Vigsnæs, L K; Brynskov, J; Steenholdt, C; Wilcks, A; Licht, T R

    2012-12-01

    Detailed knowledge about the composition of the intestinal microbiota may be critical to unravel the pathogenesis of ulcerative colitis (UC), a human chronic inflammatory bowel disease, since the intestinal microbes are expected to influence some of the key mechanisms involved in the inflammatory process of the gut mucosa. The aim of this study was to investigate the faecal microbiota in patients either with UC in remission (n=6) or with active disease (n=6), and in healthy controls (n=6). The composition of Gram-negative bacteria and Gram-positive bacteria was examined. Antigenic structures of Gram-negative bacteria such as lipopolysaccharides have been related to the inflammatory responses and pathogenesis of inflammatory bowel disease. Dice cluster analysis and principal component analysis of faecal microbiota profiles obtained by denaturing gradient gel electrophoresis and quantitative PCR, respectively, revealed that the composition of faecal bacteria from UC patients with active disease differed from the healthy controls and that this difference should be ascribed to Gram-negative bacteria. The analysis did not show any clear grouping of UC patients in remission. Even with the relatively low number of subjects in each group, we were able to detect a statistically significant underrepresentation of Lactobacillus spp. and Akkermansia muciniphila in UC patients with clinically active disease compared to the healthy controls. In line with previous communications, we have shown that the microbiota in UC patients with active disease differ from that in healthy controls. Our findings indicate that alterations in the composition of the Gram-negative bacterial population, as well as reduced numbers of lactobacilli and A. muciniphila may play a role in UC.

  12. HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis

    PubMed Central

    Nawkar, Ganesh M.; Kang, Chang Ho; Maibam, Punyakishore; Park, Joung Hun; Jung, Young Jun; Chae, Ho Byoung; Chi, Yong Hun; Jung, In Jung; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2017-01-01

    Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box–like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression. PMID:28167764

  13. HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis.

    PubMed

    Nawkar, Ganesh M; Kang, Chang Ho; Maibam, Punyakishore; Park, Joung Hun; Jung, Young Jun; Chae, Ho Byoung; Chi, Yong Hun; Jung, In Jung; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2017-02-21

    Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box-like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression.

  14. Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3-kinase-dependent and -independent pathways.

    PubMed

    Reynolds, Lucinda F; Smyth, Lesley A; Norton, Trisha; Freshney, Norman; Downward, Julian; Kioussis, Dimitris; Tybulewicz, Victor L J

    2002-05-06

    Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4(+)CD8(+) double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-gamma1 (PLCgamma1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCgamma1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCgamma1 and the adaptor molecule Src homology 2 domain-containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.

  15. Vav1 Transduces T Cell Receptor Signals to the Activation of Phospholipase C-γ1 via Phosphoinositide 3-Kinase-dependent and -independent Pathways

    PubMed Central

    Reynolds, Lucinda F.; Smyth, Lesley A.; Norton, Trisha; Freshney, Norman; Downward, Julian; Kioussis, Dimitris; Tybulewicz, Victor L.J.

    2002-01-01

    Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4+CD8+ double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-γ1 (PLCγ1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCγ1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCγ1 and the adaptor molecule Src homology 2 domain–containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K. PMID:11994416

  16. ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway

    PubMed Central

    1996-01-01

    AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Since some RTKs act through Ras and phosphatidylinositol 3-kinase (PI3K), we investigated the role of these pathways in ARIA signaling. Expression of activated Ras or Raf mimicked ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocked the effect of ARIA. ARIA rapidly activated erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulated association of PI3K with erbB3, expression of an activated PI3K led to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolished the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways. PMID:8707830

  17. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  18. EFA6 controls Arf1 and Arf6 activation through a negative feedback loop.

    PubMed

    Padovani, Dominique; Folly-Klan, Marcia; Labarde, Audrey; Boulakirba, Sonia; Campanacci, Valérie; Franco, Michel; Zeghouf, Mahel; Cherfils, Jacqueline

    2014-08-26

    Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.

  19. Conceptual Design, Implementation and Commissioning of Data Acquisition and Control System for Negative Ion Source at IPR

    SciTech Connect

    Soni, Jignesh; Gahlaut, A.; Bansal, G.; Parmar, K. G.; Pandya, K.; Chakraborty, A.; Yadav, Ratnakar; Singh, M. J.; Bandyopadhyay, M.

    2011-09-26

    Negative ion Experimental facility has been setup at IPR. The facility consists of a RF based negative ion source (ROBIN)--procured under a license agreement with IPP Garching, as a replica of BATMAN, presently operating in IPP, 100 kW 1 MHz RF generators and a set of low and high voltage power supplies, vacuum system and diagnostics. 35 keV 10A H- beam is expected from this setup. Automated successful operation of the system requires an advanced, rugged, time proven and flexible control system. Further the data generated in the experimental phase needs to be acquired, monitored and analyzed to verify and judge the system performance. In the present test bed, this is done using a combination of PLC based control system and a PXI based data acquisition system. The control system consists of three different Siemens PLC systems viz. (1) S-7 400 PLC as a Master Control, (2) S-7 300 PLC for Vacuum system control and (3) C-7 PLC for RF generator control. Master control PLC directly controls all the subsystems except the Vacuum system and RF generator. The Vacuum system and RF generator have their own dedicated PLCs (S-7 300 and C-7 respectively). Further, these two PLC systems work as a slave for the Master control PLC system. Communication between PLC S-7 400, S-7 300 and central control room computer is done through Industrial Ethernet (IE). Control program and GUI are developed in Siemens Step-7 PLC programming software and Wincc SCADA software, respectively. There are approximately 150 analog and 200 digital control and monitoring signals required to perform complete closed loop control of the system. Since the source floats at high potential ({approx}35 kV); a combination of galvanic and fiber optic isolation has been implemented. PXI based Data Acquisition system (DAS) is a combination of PXI RT (Real time) system, front end signal conditioning electronics, host system and DAQ program. All the acquisition signals coming from various sub-systems are connected and

  20. Conceptual Design, Implementation and Commissioning of Data Acquisition and Control System for Negative Ion Source at IPR

    NASA Astrophysics Data System (ADS)

    Soni, Jignesh; Yadav, Ratnakar; Gahlaut, A.; Bansal, G.; Singh, M. J.; Bandyopadhyay, M.; Parmar, K. G.; Pandya, K.; Chakraborty, A.

    2011-09-01

    Negative ion Experimental facility has been setup at IPR. The facility consists of a RF based negative ion source (ROBIN)—procured under a license agreement with IPP Garching, as a replica of BATMAN, presently operating in IPP, 100 kW 1 MHz RF generators and a set of low and high voltage power supplies, vacuum system and diagnostics. 35 keV 10A H- beam is expected from this setup. Automated successful operation of the system requires an advanced, rugged, time proven and flexible control system. Further the data generated in the experimental phase needs to be acquired, monitored and analyzed to verify and judge the system performance. In the present test bed, this is done using a combination of PLC based control system and a PXI based data acquisition system. The control system consists of three different Siemens PLC systems viz. (1) S-7 400 PLC as a Master Control, (2) S-7 300 PLC for Vacuum system control and (3) C-7 PLC for RF generator control. Master control PLC directly controls all the subsystems except the Vacuum system and RF generator. The Vacuum system and RF generator have their own dedicated PLCs (S-7 300 and C-7 respectively). Further, these two PLC systems work as a slave for the Master control PLC system. Communication between PLC S-7 400, S-7 300 and central control room computer is done through Industrial Ethernet (IE). Control program and GUI are developed in Siemens Step-7 PLC programming software and Wincc SCADA software, respectively. There are approximately 150 analog and 200 digital control and monitoring signals required to perform complete closed loop control of the system. Since the source floats at high potential (˜35 kV); a combination of galvanic and fiber optic isolation has been implemented. PXI based Data Acquisition system (DAS) is a combination of PXI RT (Real time) system, front end signal conditioning electronics, host system and DAQ program. All the acquisition signals coming from various sub-systems are connected and

  1. A Application of Feedback Control to Stabilise a Specific Case of the Negative Buoyancy Flow Instability

    NASA Astrophysics Data System (ADS)

    Ffowcs Williams, J. E.; Morgans, A. S.

    2001-10-01

    This paper is one that David Maull would have enjoyed savaging. Firstly, because he always enjoyed lively discussions about hare-brained schemes for using feedback control to produce unnatural states of motion, and secondly, because he was never happier than when probing details of imaginative undergraduate projects. This paper results from such a project in the group where David spent most of his professional life, a project so far-fetched that it would have attracted his most critical attention. It would also have attracted his characteristically encouraging support. The idea that instabilities of inviscid vortex sheets, which prevent them from being naturally steady, might be controlled through suitably excited actuators is being tentatively advanced, and some peculiar features of the controlled state also attract attention. The project that produced this paper addressed one peculiarity that turned out to be an error [see Ffowcs Williams (1982)], the solution to the actively controlled sheet turning out to be different and much simpler; this solution is published in Ffowcs Williams (2001). The project went on to consider whether another planar interface that is naturally unstable might be made stable by the action of an adjustable nearby surface. The situation envisaged is an unstable density stratified arrangement where heavy fluid lies initially at rest above a lighter fluid, the interface between the two fluids being planar. Above the interface is an impervious structural surface, which can be moved by a small amount by actuators designed to induce in the fluid a motion that will prevent the growth of any gravitational mode of instability. We show that it is indeed possible to define a controller with the required performance, but its implementation would be a very demanding task. A finite body of fluid could, on the other hand, be held suspended against gravity by relatively straightforward control arrangements. A particular example is worked out in detail

  2. Bilingual Language Control and General Purpose Cognitive Control among Individuals with Bilingual Aphasia: Evidence Based on Negative Priming and Flanker Tasks

    PubMed Central

    Dash, Tanya; Kar, Bhoomika R.

    2014-01-01

    Background. Bilingualism results in an added advantage with respect to cognitive control. The interaction between bilingual language control and general purpose cognitive control systems can also be understood by studying executive control among individuals with bilingual aphasia. Objectives. The current study examined the subcomponents of cognitive control in bilingual aphasia. A case study approach was used to investigate whether cognitive control and language control are two separate systems and how factors related to bilingualism interact with control processes. Methods. Four individuals with bilingual aphasia performed a language background questionnaire, picture description task, and two experimental tasks (nonlinguistic negative priming task and linguistic and nonlinguistic versions of flanker task). Results. A descriptive approach was used to analyse the data using reaction time and accuracy measures. The cumulative distribution function plots were used to visualize the variations in performance across conditions. The results highlight the distinction between general purpose cognitive control and bilingual language control mechanisms. Conclusion. All participants showed predominant use of the reactive control mechanism to compensate for the limited resources system. Independent yet interactive systems for bilingual language control and general purpose cognitive control were postulated based on the experimental data derived from individuals with bilingual aphasia. PMID:24982591

  3. Surgency and negative affectivity, but not effortful control, are uniquely associated with obesogenic eating behaviors among low-income preschoolers.

    PubMed

    Leung, Christy Y Y; Lumeng, Julie C; Kaciroti, Niko A; Chen, Yu Pu; Rosenblum, Katherine; Miller, Alison L

    2014-07-01

    Despite increased attention to the role of temperament in children's obesogenic eating behaviors, there is a paucity of research examining whether different dimensions of temperament may be differentially associated with specific eating behaviors among preschool-age children. The purpose of the current study was to examine whether three temperament dimensions (surgency, negative affectivity, and effortful control) were uniquely associated with six obesogenic eating behaviors (caregiver-reported food responsiveness, enjoyment of food, emotional overeating, satiety responsiveness, and tantrums over food; and observed eating in the absence of hunger) among low-income preschool-age children, covarying home environment quality. Results showed that temperament dimensions were differentially associated with different eating behaviors. Specifically, preschoolers with higher surgency were more likely to overeat in response to external cues, have frequent desire to eat, derive pleasure from food, and eat in the absence of hunger. In contrast, preschoolers with higher negative affectivity were more likely to have tantrums over being denied food and less likely to eat in the absence of hunger. Effortful control was not uniquely associated with obesogenic eating behavior. Findings remained significant even when home chaos was accounted for, suggesting that child surgency and negative affectivity are important to consider, independent of home environment. Results are discussed with regard to theoretical implications for the study of childhood obesity and for applied prevention implications.

  4. The associations among maternal negative control, children's social information processing patterns, and teachers' perceptions of children's behavior in preschool.

    PubMed

    Ziv, Yair; Kupermintz, Haggai; Aviezer, Ora

    2016-02-01

    The links between social information processing (SIP) and social behavior in preschool are well documented. However, the antecedents of SIP in that age group are less clear. A number of influential theoretical models suggest that a major contributor to SIP is the quality of the child's relationships with the parent. Therefore, we examined the links among quality of the mother-child relationships (measured via direct observations of dyadic play interactions), the child's SIP patterns (measured via direct interview with the child), and the child's perceived behavior in preschool (measured via teacher reports) in a sample of 218 preschool and kindergarten children and their mothers. Applying structural equation modeling, we found support for our theoretical model with a specific emphasis on the negative nature of this association. Specifically, we found a strong indirect path from maternal negative control to the teacher's negative perception of the child's behavior in preschool and kindergarten via less competent SIP patterns. This empirical path remained intact after controlling for various variables such as the family income, the mother's education level, and the child's expressive language abilities, thereby providing further support for the robustness of this association.

  5. The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High-Throughput Cell-Based Screens

    DTIC Science & Technology

    2005-02-01

    cells. Psycho- Calmodulin antagonists inhibit insulin -stimulated GLUT4 ( glucose trans- pharmacology (Berl.) 150, 383-390. porter 4) translocation by...AD Award Number: W81XWH-04-1-0169 TITLE: The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High ...Phosphoinositide-3-Kinase Pathway Through High -Throughput Cell-Based Screens 6. AUTHOR(S) William R. Sellers, M.D. 7. PERFORMING ORGANIZA TION NAME(S) AND

  6. Effectively control negative thermal expansion of single-phase ferroelectrics of PbTiO3-(Bi,La)FeO3 over a giant range.

    PubMed

    Chen, Jun; Wang, Fangfang; Huang, Qingzhen; Hu, Lei; Song, Xiping; Deng, Jinxia; Yu, Ranbo; Xing, Xianran

    2013-01-01

    Control of negative thermal expansion is a fundamentally interesting topic in the negative thermal expansion materials in order for the future applications. However, it is a challenge to control the negative thermal expansion in individual pure materials over a large scale. Here, we report an effective way to control the coefficient of thermal expansion from a giant negative to a near zero thermal expansion by means of adjusting the spontaneous volume ferroelectrostriction (SVFS) in the system of PbTiO3-(Bi,La)FeO3 ferroelectrics. The adjustable range of thermal expansion contains most negative thermal expansion materials. The abnormal property of negative or zero thermal expansion previously observed in ferroelectrics is well understood according to the present new concept of spontaneous volume ferroelectrostriction. The present studies could be useful to control of thermal expansion of ferroelectrics, and could be extended to multiferroic materials whose properties of both ferroelectricity and magnetism are coupled with thermal expansion.

  7. Effectively control negative thermal expansion of single-phase ferroelectrics of PbTiO3-(Bi,La)FeO3 over a giant range

    PubMed Central

    Chen, Jun; Wang, Fangfang; Huang, Qingzhen; Hu, Lei; Song, Xiping; Deng, Jinxia; Yu, Ranbo; Xing, Xianran

    2013-01-01

    Control of negative thermal expansion is a fundamentally interesting topic in the negative thermal expansion materials in order for the future applications. However, it is a challenge to control the negative thermal expansion in individual pure materials over a large scale. Here, we report an effective way to control the coefficient of thermal expansion from a giant negative to a near zero thermal expansion by means of adjusting the spontaneous volume ferroelectrostriction (SVFS) in the system of PbTiO3-(Bi,La)FeO3 ferroelectrics. The adjustable range of thermal expansion contains most negative thermal expansion materials. The abnormal property of negative or zero thermal expansion previously observed in ferroelectrics is well understood according to the present new concept of spontaneous volume ferroelectrostriction. The present studies could be useful to control of thermal expansion of ferroelectrics, and could be extended to multiferroic materials whose properties of both ferroelectricity and magnetism are coupled with thermal expansion. PMID:23949238

  8. The impact of perceptions of health control and coping modes on negative affect among individuals with spinal cord injuries.

    PubMed

    Livneh, Hanoch; Martz, Erin

    2011-09-01

    A wide range of demographic, medical, and personality and coping variables have been implicated as predictors of psychosocial outcomes following the onset of spinal cord injuries (SCI). The primary purpose of this study was to examine the role that perceptions of health control (internality, chance-determined, and other persons-determined) and coping strategies play in predicting respondents' negative affect, namely, reactions of depression and anxiety [i.e., posttraumatic stress disorder (PTSD)], as outcomes of psychosocial adaptation to disability. A second purpose was to investigate the potential role that time since injury (TSI) plays in moderating the influence of coping on psychosocial outcomes related to SCI. Ninety five survivors of SCI participated in the study by completing a battery of self-report measures. Two sets of multiple regression analyses were employed to address the study's goals. Findings indicated that after controlling the influence of gender, age, time since injury, and number of prior life traumas: (a) the use of disengagement coping successfully predicted both respondents' levels of depression and PTSD; (b) none of the perceptions of control of one's health significantly influenced psychosocial reactions to SCI, as indicated by depression and PTSD, although perceptions of chance control showed a moderate positive trend; and (c) time since injury did not moderate the relationships between coping and negative affect related to the onset of SCI. The implications of these findings to rehabilitation professionals are discussed.

  9. Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

    PubMed Central

    He, Feng; Jacobson, Allan

    2015-01-01

    Decapping commits an mRNA to complete degradation and promotes general 5′ to 3′ decay, nonsense-mediated decay (NMD), and transcript-specific degradation. In Saccharomyces cerevisiae, a single decapping enzyme composed of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2) targets thousands of distinct substrate mRNAs. However, the mechanisms controlling this enzyme's in vivo activity and substrate specificity remain elusive. Here, using a genetic approach, we show that the large C-terminal domain of Dcp2 includes a set of conserved negative and positive regulatory elements. A single negative element inhibits enzymatic activity and controls the downstream functions of several positive elements. The positive elements recruit the specific decapping activators Edc3, Pat1, and Upf1 to form distinct decapping complexes and control the enzyme's substrate specificity and final activation. Our results reveal unforeseen regulatory mechanisms that control decapping enzyme activity and function in vivo, and define roles for several decapping activators in the regulation of mRNA decapping. PMID:26184073

  10. Effect of Art Production on Negative Mood: A Randomized, Controlled Trial

    ERIC Educational Resources Information Center

    Bell, Chloe E.; Robbins, Steven J.

    2007-01-01

    Art therapists have long held that art production causes reductions in stress and elevations in mood (Rubin, 1999). The authors examined this claim in a randomized, controlled trial. Fifty adults between the ages of 18 and 30 were randomly assigned to either create an art work or to view and sort a series of art prints. Three measures of overall…

  11. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor

    PubMed Central

    Holgado-Madruga, Marina; Moscatello, David K.; Emlet, David R.; Dieterich, Rebekka; Wong, Albert J.

    1997-01-01

    Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF. PMID:9356464

  12. Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin.

    PubMed

    Greenwood, J A; Theibert, A B; Prestwich, G D; Murphy-Ullrich, J E

    2000-08-07

    Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.

  13. Controllable transition from positive space charge to negative space charge in an inverted cylindrical magnetron

    NASA Astrophysics Data System (ADS)

    Rane, R.; Bandyopadhyay, M.; Ranjan, M.; Mukherjee, S.

    2016-01-01

    The combined effect of magnetic field (B), gas pressure (P), and the corresponding discharge voltage on the discharge properties of argon in inverted cylindrical magnetron has been investigated. In the experiment, anode is biased with continuous 10 ms sinusoidal half wave. It is observed that at a comparatively higher magnetic field (i.e., >200 gauss) and lower operating pressure (i.e., <1 × 10-3 mbar), the discharge extinguishes and demands a high voltage to reignite. Discharge current increases with increase in magnetic field and starts reducing at sufficiently higher magnetic field for a particular discharge voltage due to restricted electron diffusion towards the anode. It is observed that B/P ratio plays an important role in sustaining the discharge and is constant for a discharge voltage. The discharge is transformed to negative space charge regime from positive space charge regime at certain B/P ratio and this ratio varies linearly with the discharge voltage. The space charge reversal is indicated by the radial profile of the floating potential and plasma potential in between two electrodes for different magnetic fields. At a particular higher magnetic field (beyond 100 gauss), the floating potential increases gradually with the radial distance from cathode, whereas it remains almost constant at lower magnetic field.

  14. The Invasive American Weed Parthenium hysterophorus Can Negatively Impact Malaria Control in Africa

    PubMed Central

    Nyasembe, Vincent O.; Cheseto, Xavier; Kaplan, Fatma; Foster, Woodbridge A.; Teal, Peter E. A.; Tumlinson, James H.; Borgemeister, Christian; Torto, Baldwyn

    2015-01-01

    The direct negative effects of invasive plant species on agriculture and biodiversity are well known, but their indirect effects on human health, and particularly their interactions with disease-transmitting vectors, remains poorly explored. This study sought to investigate the impact of the invasive Neotropical weed Parthenium hysterophorus and its toxins on the survival and energy reserves of the malaria vector Anopheles gambiae. In this study, we compared the fitness of An. gambiae fed on three differentially attractive mosquito host plants and their major toxins; the highly aggressive invasive Neotropical weed Parthenium hysterophorus (Asteraceae) in East Africa and two other adapted weeds, Ricinus communis (Euphorbiaceae) and Bidens pilosa (Asteraceae). Our results showed that female An. gambiae fitness varied with host plants as females survived better and accumulated substantial energy reserves when fed on P. hysterophorus and R. communis compared to B. pilosa. Females tolerated parthenin and 1-phenylhepta-1, 3, 5-triyne, the toxins produced by P. hysterophorus and B. pilosa, respectively, but not ricinine produced by R. communis. Given that invasive plants like P. hysterophorus can suppress or even replace less competitive species that might be less suitable host-plants for arthropod disease vectors, the spread of invasive plants could lead to higher disease transmission. Parthenium hysterophorus represents a possible indirect effect of invasive plants on human health, which underpins the need to include an additional health dimension in risk-analysis modelling for invasive plants. PMID:26367123

  15. The Invasive American Weed Parthenium hysterophorus Can Negatively Impact Malaria Control in Africa.

    PubMed

    Nyasembe, Vincent O; Cheseto, Xavier; Kaplan, Fatma; Foster, Woodbridge A; Teal, Peter E A; Tumlinson, James H; Borgemeister, Christian; Torto, Baldwyn

    2015-01-01

    The direct negative effects of invasive plant species on agriculture and biodiversity are well known, but their indirect effects on human health, and particularly their interactions with disease-transmitting vectors, remains poorly explored. This study sought to investigate the impact of the invasive Neotropical weed Parthenium hysterophorus and its toxins on the survival and energy reserves of the malaria vector Anopheles gambiae. In this study, we compared the fitness of An. gambiae fed on three differentially attractive mosquito host plants and their major toxins; the highly aggressive invasive Neotropical weed Parthenium hysterophorus (Asteraceae) in East Africa and two other adapted weeds, Ricinus communis (Euphorbiaceae) and Bidens pilosa (Asteraceae). Our results showed that female An. gambiae fitness varied with host plants as females survived better and accumulated substantial energy reserves when fed on P. hysterophorus and R. communis compared to B. pilosa. Females tolerated parthenin and 1-phenylhepta-1, 3, 5-triyne, the toxins produced by P. hysterophorus and B. pilosa, respectively, but not ricinine produced by R. communis. Given that invasive plants like P. hysterophorus can suppress or even replace less competitive species that might be less suitable host-plants for arthropod disease vectors, the spread of invasive plants could lead to higher disease transmission. Parthenium hysterophorus represents a possible indirect effect of invasive plants on human health, which underpins the need to include an additional health dimension in risk-analysis modelling for invasive plants.

  16. Controllable transition from positive space charge to negative space charge in an inverted cylindrical magnetron

    SciTech Connect

    Rane, R. Ranjan, M.; Mukherjee, S.; Bandyopadhyay, M.

    2016-01-15

    The combined effect of magnetic field (B), gas pressure (P), and the corresponding discharge voltage on the discharge properties of argon in inverted cylindrical magnetron has been investigated. In the experiment, anode is biased with continuous 10 ms sinusoidal half wave. It is observed that at a comparatively higher magnetic field (i.e., >200 gauss) and lower operating pressure (i.e., <1 × 10{sup −3} mbar), the discharge extinguishes and demands a high voltage to reignite. Discharge current increases with increase in magnetic field and starts reducing at sufficiently higher magnetic field for a particular discharge voltage due to restricted electron diffusion towards the anode. It is observed that B/P ratio plays an important role in sustaining the discharge and is constant for a discharge voltage. The discharge is transformed to negative space charge regime from positive space charge regime at certain B/P ratio and this ratio varies linearly with the discharge voltage. The space charge reversal is indicated by the radial profile of the floating potential and plasma potential in between two electrodes for different magnetic fields. At a particular higher magnetic field (beyond 100 gauss), the floating potential increases gradually with the radial distance from cathode, whereas it remains almost constant at lower magnetic field.

  17. Hormonal regulation of the gravity s negative control of morphogenesis in cucumber seedlings

    NASA Astrophysics Data System (ADS)

    Takahashi, H.; Kamada, M.; Saito, Y.; Fujii, N.

    Just after germination, seedlings of most cucurbitaceous plants develop a peg to pull the cotyledons and plumule out from the seed coat. The peg usually develops on the concave side of the gravitropically bending transition zone between the hypocotyl and the root. Because cucumber seedlings grown in microgravity developed a peg on each side of the transition zone, it was suggested that peg formation was negatively regulated by gravity on Earth. It has also been suggested that auxin is an essential factor responsible for peg formation. To verify this hypothesis and to understand the molecular mechanism of the gravity-regulated peg formation, we measured the distribution of endogenous auxin in the transition zone, examined the expression patterns of an auxininducible genes (CS-IAAs), auxin response factor and auxin carrier genes (CS-ARFs, CS-AUX1, CS-PIN1). Because ethylene modifies peg development, we examined the expression of ACC synthase genes (CS-ACSs) and its relation to the auxin-mediated development of peg. Furthermore, we examined some other factors that might interact with auxin for peg formation. Based on the results of these studies, we propose a model for the mechanism of peg formation in cucumber seedlings.

  18. Contexts Control Negative Contrast and Restrict the Expression of Flavor Preference Conditioning

    PubMed Central

    2016-01-01

    Consumption of a high concentration of sucrose can have either a detrimental, negative contrast effect or a facilitatory, preference conditioning effect on subsequent consumption of a low concentration of sucrose, depending on the cues that are present during consumption. The role of context and flavor cues in determining these effects were studied using analysis of the microstructure of licking in mice. Exposure to a high concentration followed by exposure to a low concentration resulted in a transient reduction in mean lick cluster size, which was context dependent (Experiment 1). However, there was no change in the total number of licks or overall consumption. When a flavor that had previously been paired with a high concentration was paired with a low concentration, there was an increase in the total number of licks, and overall consumption, but no change in the mean lick cluster size (Experiment 2). Pairing a high concentration with a flavor in a particular context before pairing the context and flavor compound with a low concentration resulted in abolishing the expression of the flavor preference conditioning effect on the total number of licks and consumption (Experiment 3). These results demonstrate that although context and flavor cues have dissociable effects on licking behavior, their interaction has an antagonistic effect on the behavioral expression of memory. PMID:26752234

  19. Acadesine Inhibits Tissue Factor Induction and Thrombus Formation by Activating the Phosphoinositide 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Zhang, Weiyu; Wang, Jianguo; Wang, Huan; Tang, Rong; Belcher, John D.; Viollet, Benoit; Geng, Jian-Guo; Zhang, Chunxiang; Wu, Chaodong; Slungaard, Arne; Zhu, Chuhong; Huo, Yuqing

    2013-01-01

    Objective Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. Methods and Results Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A2A receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A2A receptor or α1–AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-κB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. Conclusion Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis. PMID:20185792

  20. Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study.

    PubMed

    Ul-Haq, Zaheer; Gul, Sana; Usmani, Saman; Wadood, Abdul; Khan, Waqasuddin

    2015-11-01

    The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10 ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues.

  1. Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway

    PubMed Central

    Jeong, Ae-Jin; Chung, Chung-Nam; Kim, Hye-Jin; Bae, Kil Soo; Choi, Song; Jun, Woo Jin; Shim, Sang In; Kang, Tae-Hong; Leem, Sun-Hee

    2012-01-01

    Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway. PMID:23118562

  2. Phosphoinositide 3-kinase p110δ promotes lumen formation through enhancement of apico-basal polarity and basal membrane organization

    PubMed Central

    Sar, Sokhavuth; Komaiha, Ola Hamze; Moyano, Romina; Rayal, Amel; Samuel, Didier; Shewan, Annette; Vanhaesebroeck, Bart; Mostov, Keith; Gassama-Diagne, Ama

    2016-01-01

    Signaling triggered by adhesion to the extracellular matrix plays a key role in the spatial orientation of epithelial polarity and formation of lumens in glandular tissues. Phosphoinositide 3-kinase signaling in particular is known to influence the polarization process during epithelial cell morphogenesis. Here, using Madin-Darby canine kidney epithelial cells grown in 3D culture, we show that the p110δ isoform of phosphoinositide 3-kinase colocalizes with focal adhesion proteins at the basal surface of polarized cells. Pharmacological, siRNA- or kinase-dead mediated inhibition of p110δ impair the early stages of lumen formation, resulting in inverted polarized cysts, with no laminin or type IV collagen assembly at cell/extracellular matrix contacts. p110δ also regulates the organization of focal adhesions and membrane localization of dystroglycan. Thus, we uncover a previously unrecognized role for p110δ in epithelial cells in the orientation of the apico-basal axis and lumen formation. PMID:25583025

  3. Hepatocyte growth factor (HGF) enhances cardiac commitment of differentiating embryonic stem cells by activating PI3 kinase

    SciTech Connect

    Roggia, Cristiana; Ukena, Christian; Boehm, Michael; Kilter, Heiko . E-mail: kilter@med-in.uni-saarland.de

    2007-03-10

    Hepatocyte growth factor (HGF) is a pleiotropic cytokine promoting proliferation, migration and survival in several cell types. HGF and its cognate receptor c-Met are expressed in cardiac cells during early cardiogenesis, but data concerning its role in cardiac differentiation of embryonic stem cells (ESCs) and the underlying molecular mechanisms involved are limited. In the present study we show that HGF significantly increases the number of beating embryoid bodies of differentiating ESCs without affecting beating frequency. Furthermore, HGF up-regulates the expression of the cardiac-specific transcription factors Nkx 2.5 and GATA-4 and of markers of differentiated cardiomyocytes, i.e. {alpha}-MHC, {beta}-MHC, ANF, MLC2v and Troponin T. The HGF-induced increase in Nkx 2.5 expression was inhibited by co-treatment with the PI3 kinase inhibitors Wortmannin and LY294002, but not by its inactive homolog LY303511, suggesting an involvement of the PI3 kinase/Akt pathway in this effect. We conclude that HGF is an important growth factor involved in cardiac differentiation and/or proliferation of ESCs and may therefore be critical for the in vitro generation of pre- or fully differentiated cardiomyocytes as required for clinical use of embryonic stem cells in cardiac diseases.

  4. Overlapping arrangement of the recF and dnaN operons of Escherichia coli; positive and negative control sequences.

    PubMed

    Armengod, M E; Lambíes, E

    1986-01-01

    The recF gene of Escherichia coli controls one of the recombination pathways and UV sensitivity, but its precise function and expression pattern are still largely unknown. We have characterized the promoter region of the recF gene by mapping for E. coli RNA polymerase binding sites, in vitro transcription experiments, cloning, and S1 mapping of in vivo mRNAs. It contains three overlapping promoters, two initiating transcription towards recF and one in the opposite direction. The recF promoter region is located about 600 bp upstream from the start codon of the recF structural gene and resides entirely within the translated region of the preceding gene, dnaN, which encodes for the beta subunit of DNA polymerase III. This unusual arrangement might provide discoordinate regulation of the recF and dnaN genes, thus controlling the level of DNA polymerase III holoenzyme. Expression of recF is also negatively controlled by sequences located upstream as well as inside the recF coding frame. Such negative regulation may serve to prevent toxic effects due to accumulation of an excessive number of copies of the recF gene product.

  5. Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

    PubMed

    Aziz, R; Beymer, M; Negrón, A L; Newshan, A; Yu, G; Rosati, B; McKinnon, D; Fukuda, M; Lin, R Z; Mayer, C; Boehm, U; Acosta-Martínez, M

    2014-07-01

    Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP

  6. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3β-dependent Cell–Cell Mechanical Adhesion

    PubMed Central

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1−/− mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and β-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated β-catenin through activation of GSK3β. Expression of a nondegradable form of β-catenin in PC-1 MDCK cells restores strong cell–cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

  7. Response Persistence: The Effects of Stimulus Control on Negatively Reinforced Problem Behavior in a Concurrent Operant

    PubMed Central

    McComas, Jennifer J

    2009-01-01

    In the context of instructional demands, compliance and problem behavior can be considered concurrent operants. Of applied interest is increasing one response (i.e., compliance) while decreasing the other (i.e., problem behavior). Strategic arrangement of reinforcement can alter response allocation accordingly. Such schedules can also influence response persistence and generalization. A case study is used to illustrate the effects of stimulus–reinforcer relations in a concurrent-operants arrangement involving an adult with developmental disabilities and problem behavior. Results are discussed in the context of basic operant research findings in the areas of stimulus control and behavioral persistence. PMID:22478529

  8. When to be skeptical of negative studies: pitfalls in evaluating occupational risks using population-based case-control studies.

    PubMed

    Hu, S W; Hertz-Picciotto, I; Siemiatycki, J

    1999-01-01

    This study investigated arsenic and lung cancer incidence in a community setting in the Montreal area. Job histories and sociodemographic factors were collected by interview from 857 lung cancer cases, 533 general population controls, and 1,360 controls with other cancers. Chemist-hygienists assessed each subject's life-time occupational exposure to 294 substances. Logistic regressions yielded arsenic/lung cancer odds ratios of 1.1 (95% confidence interval = 0.60, 1.7) based on cancer controls, and 0.82 (95% confidence interval = 0.41, 1.6) based on population controls. Risk did not rise with increasing level or probability of exposure. Worksite studies consistently show lung carcinogenicity from arsenic. Since confounding from other chemicals was well controlled, the most likely explanation is substantially lower exposures than in previous studies. The lack of association in this study demonstrates the need for caution in interpreting negative findings from population-based case-control studies, particularly when exposures are low or rare, as well as the difficulty in generating hypotheses from such studies.

  9. Conversation about Serostatus decreases risk of acquiring HIV: results from a case control study comparing MSM with recent HIV infection and HIV negative controls

    PubMed Central

    2014-01-01

    Background Data on knowledge, attitudes, behaviour and practices (KABP) of persons with recent HIV infection compared to controls with negative HIV test result provide information on current risk patterns and can help to re-focus HIV prevention strategies. Methods From March 2008 through May 2010, persons newly diagnosed with HIV (cases) and HIV-negative controls were recruited by physicians in Germany. To distinguish recent (< 5 months) from longstanding (> 5 months) infection, dried blood spots from people newly diagnosed with HIV were tested with the BED IgG-capture ELISA. Cases and controls completed a KABP-questionnaire. We compared cases with recent infection and controls among men having sex with men (MSM) regarding reported risk behaviour in the previous 6 months. To detect differences, unadjusted Odds Ratios (OR) were calculated and multivariate analysis was performed. Results Cases and controls did not differ in terms of knowledge on transmission risks, HIV testing frequency, partnership status, or regarding the frequency of any unprotected sex with partners known to be HIV-positive or assumed to be HIV-negative. Cases more often reported a shorter duration of partnership (< 6 months) with a primary partner than controls (OR = 3.9; p = 0.003) and indicated lower rates of condom use outside of primary relationships, with acquaintances (OR = 2.5; p = 0.01), and with persons met online (OR = 4.5; p = 0.04). Unprotected sex with persons of unknown HIV-serostatus was more often indicated by cases than controls (OR = 3.0; p = 0.003). Having a conversation about HIV serostatus before having sex was associated with a lower risk of infection (OR = 0.2; p = 0.01). In multivariate analysis “being always safe” (always using a condom when having sex in different situations outside of a relationship) and talking about serostatus before sex (OR = 0.23; p = 0.004; OR = 0.14; p = 0.014) were negatively

  10. High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

    PubMed Central

    Polachek, William S.; Moshrif, Hanan F.; Franti, Michael; Coen, Donald M.; Sreenu, Vattipally B.

    2016-01-01

    ABSTRACT High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. IMPORTANCE There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production

  11. Longitudinal relations among parents' reactions to children's negative emotions, effortful control, and math achievement in early elementary school.

    PubMed

    Swanson, Jodi; Valiente, Carlos; Lemery-Chalfant, Kathryn; Bradley, Robert H; Eggum-Wilkens, Natalie D

    2014-01-01

    Panel mediation models and fixed-effects models were used to explore longitudinal relations among parents' reactions to children's displays of negative emotions, children's effortful control (EC), and children's math achievement (N = 291; M age in fall of kindergarten = 5.66 years, SD = .39 year) across kindergarten through second grade. Parents reported their reactions and children's EC. Math achievement was assessed with a standardized achievement test. First-grade EC mediated the relation between parents' reactions at kindergarten and second-grade math achievement, beyond stability in constructs across study years. Panel mediation model results suggested that socialization of EC may be one method of promoting math achievement in early school; however, when all omitted time-invariant covariates of EC and math achievement were controlled, first-grade EC no longer predicted second-grade math achievement.

  12. Efficient synthesis of mosquitocidal toxins in Asticcacaulis excentricus demonstrates potential of gram-negative bacteria in mosquito control.

    PubMed

    Liu, J W; Yap, W H; Thanabalu, T; Porter, A G

    1996-03-01

    The control of mosquitoes with chemical insecticides pollutes the environment and leads to resistance in mosquito populations. Bacterial control of mosquito larvae with Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce protein toxins, has proved useful, safe, and nonpolluting. These bacteria do, however, suffer from disadvantages, including rapid setting, UV sensitivity, and lack of persistance of spores, proteolysis of toxins, narrow host range, and high production costs. Here we show that the Gram-negative bacterium Asticcacaulis excentricus is a promising host for delivering toxins to mosquito larvae. Plasmid-transformed A. excentricus cells expressing the binary toxin of B. sphaericus exhibited toxicity to Culex and Anopheles mosquito larvae similar to that of the high-toxicity strains of B. sphaericus which produce several toxins. A. excentricus has potential advantages as a larvicide compared with the bacilli, especially persistance in the larval feeding zone, resistance to UV light, lack of toxin-degrading proteases, and low production costs.

  13. Giant enhancement of the controllable in-plane anisotropy of biased isotropic noncentrosymmetric materials with epsilon-negative multilayers

    NASA Astrophysics Data System (ADS)

    Valagiannopoulos, C. A.; Tsitsas, N. L.; Lakhtakia, A.

    2017-02-01

    Giant in-plane anisotropy can be exhibited by a finitely thick periodic multilayer comprising bilayers of an isotropic noncentrosymmetric material and a non-dissipative isotropic medium of negative permittivity, when a dc electric field is applied in the thickness direction. Compared to a homogeneous layer of the noncentrosymmetric material with the same thickness as the periodic multilayer, the latter exhibits an effective in-plane anisotropy that can be three orders larger in magnitude. This enhancement gets more substantial at higher frequencies and is electrically controllable. The incorporation of dissipation reduces the enhancement of the effective in-plane anisotropy, which nevertheless remains significant. We expect the finitely thick periodic multilayer to be useful as a polarization transformer or a modulator in the terahertz regime fully controllable via external dc bias.

  14. Siglec-E Negatively Regulates the Activation of TLR4 by Controlling Its Endocytosis.

    PubMed

    Wu, Yin; Ren, Dongren; Chen, Guo-Yun

    2016-10-15

    TLR4 signaling is critical for providing effective immune protection, but it must be tightly controlled to avoid inflammation-induced pathology. Previously, we reported extensive and direct interactions between TLR and Siglec families of pattern recognition receptors. In this study, we examined the biological significance of this interaction during infection. We show that Siglec-E is required for Escherichia coli-induced endocytosis of TLR4. Siglec-E-deficient dendritic cells infected with E. coli fail to internalize TLR4. This leads to sustained TLR4 on the cell surface and activation of NF-κB and MAPK p38, resulting in high levels of TNF-α and IL-6 compared with wild-type dendritic cells. In contrast to the signaling events occurring at the plasma membrane, as a result of the inability to internalize TLR4, Siglec-E-deficient dendritic cells were also defective for TRIF-mediated IFN-β production in response to E. coli infection. Furthermore, we found that accumulation of ubiquitinated TLR4 and binding of E3 ubiquitin ligase Triad3A to TLR4 was increased significantly in bone marrow-derived dendritic cells from wild-type mice, but not from Siglec-E-deficient mice, after E. coli infection. This represents a newly discovered mechanism that regulates the signaling of TLR4 during E. coli infection.

  15. OCTOPUS Negatively Regulates BIN2 to Control Phloem Differentiation in Arabidopsis thaliana.

    PubMed

    Anne, Pauline; Azzopardi, Marianne; Gissot, Lionel; Beaubiat, Sébastien; Hématy, Kian; Palauqui, Jean-Christophe

    2015-10-05

    The phloem is a vascular strand that conducts photoassimilates and systemic signals throughout the plant to coordinate growth. To date, few molecular genetic determinants have been identified to control both specification and differentiation of this tissue [1-3]. Among them, OCTOPUS (OPS) protein was previously identified as a polarly localized plasma membrane-associated protein of unknown biochemical function whose broad provascular expression becomes restricted to the phloem upon differentiation [2]. OPS loss-of-function mutants showed an altered vascular network in cotyledons and an intermittent phloem differentiation in the root [2, 4]. Here, we demonstrate a role for OPS as a positive regulator of the brassinosteroid (BR) signaling pathway. Indeed, transgenic lines overexpressing OPS (OPS-OE) display the hallmarks of constitutively overactivated BR mutants. Physiological and genetic analyses place OPS as a positive regulator of the BR signaling pathway upstream of the key transcription factors BES1 and BZR1. Directed protein interactions with known BR signaling proteins identified BIN2, a GSK3 protein involved in multiple signaling pathways, as a partner of OPS. This interaction recruits BIN2 to the plasma membrane, thus preventing its inhibitory activity in the nucleus. Finally, both bikinin (a potent inhibitor of GSK3 [5]) treatment and downstream dominant mutants bes1-D [6] and bzr1-D [7] can rescue phloem defects of ops in the root. Together, our data show that OPS antagonizes BIN2 to promote phloem differentiation.

  16. YmoA negatively controls the expression of insecticidal genes in Yersinia enterocolitica.

    PubMed

    Starke, Mandy; Fuchs, Thilo M

    2014-04-01

    Yersinia enterocolitica is toxic towards invertebrates due to the presence of the toxin complex (tc) genes that are activated by the thermolabile regulator TcaR2. In the search for further regulatory factors involved in insecticidal gene expression, the modulator of yersinial virulence, YmoA, was identified to silence all tc genes of the Y. enterocolitica strain W22703 (biovar 2, serovar O:9). Using promoter fusions with the luciferase reporter, we found that the deletion of ymoA results in elevated transcription of tcaR1, tcaR2, tcaA, tcaB, tcaC, tccC1 and tccC2 at both 15 °C and 37 °C. Complementation by episomal ymoA significantly reduced tc gene expression, thus validating the inhibitory activity of YmoA on the production of insecticidal proteins. YmoA contributes to the binding properties of H-NS to the tc promoters by forming a complex with this nucleoid-associated protein, and this complex not only binds to the upstream regions of all tc genes, but also to intragenic sites of tcaA and tcaB that play an important role in controlling the expression of both genes. At low temperature, the intracellular amount of thermostable YmoA is not reduced, but the repressor is less functional. These data point to H-NS/YmoA as an antagonist of the inducer TcaR2.

  17. Improvements in Negative Symptoms and Functional Outcome After a New Generation Cognitive Remediation Program: A Randomized Controlled Trial

    PubMed Central

    Ojeda, Natalia

    2014-01-01

    Cognitive remediation improves cognition in patients with schizophrenia, but its effect on other relevant factors such as negative symptoms and functional outcome has not been extensively studied. In this hospital-based study, 84 inpatients with chronic schizophrenia were recruited from Alava Hospital (Spain). All of the subjects underwent a baseline and a 3-month assessment that examined neurocognition, clinical symptoms, insight, and functional outcome according to the Global Assessment of Functioning (GAF) scale and Disability Assessment Schedule from World Health Organization (DAS-WHO). In addition to receiving standard treatment, patients were randomly assigned either to receive neuropsychological rehabilitation (REHACOP) or to a control group. REHACOP is an integrative program that taps all basic cognitive functions. The program included experts’ latest suggestions about positive feedback and activities of daily living in the patients’ environment. The REHACOP group showed significantly greater improvements at 3 months in the areas of neurocognition, negative symptoms, disorganization, and emotional distress compared with the control group (Cohen’s effect size for these changes ranged from d = 0.47 for emotional distress to d = 0.58 for disorganization symptoms). The REHACOP group also improved significantly in both the GAF (d = 0.61) and DAS-WHO total scores (d = 0.57). Specifically, the patients showed significant improvement in vocational outcomes (d = 0.47), family contact (d = 0.50), and social competence (d = 0.56). In conclusion, neuropsychological rehabilitation may be useful for the reduction of negative symptoms and functional disability in schizophrenia. These findings support the integration of neuropsychological rehabilitation into standard treatment programs for patients with schizophrenia. PMID:23686130

  18. Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae.

    PubMed

    Waite, Christopher; Schumacher, Jörg; Jovanovic, Milija; Bennett, Mark; Buck, Martin

    2017-01-24

    The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses.

  19. Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator.

    PubMed

    Font, Joan; López-Cano, Marc; Notartomaso, Serena; Scarselli, Pamela; Di Pietro, Paola; Bresolí-Obach, Roger; Battaglia, Giuseppe; Malhaire, Fanny; Rovira, Xavier; Catena, Juanlo; Giraldo, Jesús; Pin, Jean-Philippe; Fernández-Dueñas, Víctor; Goudet, Cyril; Nonell, Santi; Nicoletti, Ferdinando; Llebaria, Amadeu; Ciruela, Francisco

    2017-04-11

    Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely targeting, in time and space, endogenous receptors, which may allow a better management of difficult-to-treat disorders.

  20. Turning a Negative into a Positive: Ascending GABAergic Control of Cortical Activation and Arousal

    PubMed Central

    Brown, Ritchie E.; McKenna, James T.

    2015-01-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. Recent technological advances have illuminated the role of GABAergic neurons in control of cortical arousal and sleep. Sleep-promoting GABAergic neurons in the preoptic hypothalamus are well-known. Less well-appreciated are GABAergic projection neurons in the brainstem, midbrain, hypothalamus, and basal forebrain, which paradoxically promote arousal and fast electroencephalographic (EEG) rhythms. Thus, GABA is not purely a sleep-promoting neurotransmitter. GABAergic projection neurons in the brainstem nucleus incertus and ventral tegmental nucleus of Gudden promote theta (4–8 Hz) rhythms. Ventral tegmental area GABAergic neurons, neighboring midbrain dopamine neurons, project to the frontal cortex and nucleus accumbens. They discharge faster during cortical arousal and regulate reward. Thalamic reticular nucleus GABAergic neurons initiate sleep spindles in non-REM sleep. In addition, however, during wakefulness, they tonically regulate the activity of thalamocortical neurons. Other GABAergic inputs to the thalamus arising in the globus pallidus pars interna, substantia nigra pars reticulata, zona incerta, and basal forebrain regulate motor activity, arousal, attention, and sensory transmission. Several subpopulations of cortically projecting GABAergic neurons in the basal forebrain project to the thalamus and neocortex and preferentially promote cortical gamma-band (30–80 Hz) activity and wakefulness. Unlike sleep-active GABAergic neurons, these ascending GABAergic neurons are fast-firing neurons which disinhibit and synchronize the activity of their forebrain targets, promoting the fast EEG rhythms typical of conscious states. They are prominent targets of GABAergic hypnotic agents. Understanding the properties of ascending GABAergic neurons may lead to novel treatments for diseases involving disorders of cortical activation and wakefulness. PMID:26124745

  1. Phosphatidylinositol 3-kinase and NF-kappa B/Rel are at the divergence of CD40-mediated proliferation and survival pathways.

    PubMed

    Andjelic, S; Hsia, C; Suzuki, H; Kadowaki, T; Koyasu, S; Liou, H C

    2000-10-01

    CD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-kappaB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-kappaB/Rel activation. This is achieved via activation of AKT and the degradation of IkappaBalpha. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-kappaB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-kappaB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-kappaB/Rel and PI-3K are crucial for CD40-induced proliferation.

  2. Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

    PubMed Central

    Baer, Romain; Cintas, Célia; Dufresne, Marlène; Cassant-Sourdy, Stéphanie; Schönhuber, Nina; Planque, Laetitia; Lulka, Hubert; Couderc, Bettina; Bousquet, Corinne; Garmy-Susini, Barbara; Vanhaesebroeck, Bart; Pyronnet, Stéphane; Saur, Dieter; Guillermet-Guibert, Julie

    2014-01-01

    Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110β, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment. PMID:25452273

  3. Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo.

    PubMed Central

    Vanhaesebroeck, B; Higashi, K; Raven, C; Welham, M; Anderson, S; Brennan, P; Ward, S G; Waterfield, M D

    1999-01-01

    Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules. PMID:10064595

  4. Diacylglycerol kinase theta is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries.

    PubMed Central

    Walker, A J; Draeger, A; Houssa, B; van Blitterswijk , W J; Ohanian, V; Ohanian, J

    2001-01-01

    Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKtheta, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKtheta was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKtheta translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKtheta activity in immunoprecipitates (126% at 60 s). Membrane translocation and DGKtheta activation were regulated differently: NA-induced DGKtheta activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKtheta suggests that this pathway may have a physiological role in vascular smooth-muscle responses. PMID:11115406

  5. WNT16B is a new marker of cellular senescence that regulates p53 activity and the phosphoinositide 3-kinase/AKT pathway.

    PubMed

    Binet, Romuald; Ythier, Damien; Robles, Ana I; Collado, Manuel; Larrieu, Delphine; Fonti, Claire; Brambilla, Elisabeth; Brambilla, Christian; Serrano, Manuel; Harris, Curtis C; Pedeux, Rémy

    2009-12-15

    Senescence is a tumor suppression mechanism that is induced by several stimuli, including oncogenic signaling and telomere shortening, and controlled by the p53/p21(WAF1) signaling pathway. Recently, a critical role for secreted factors has emerged, suggesting that extracellular signals are necessary for the onset and maintenance of senescence. Conversely, factors secreted by senescent cells may promote tumor growth. By using expression profiling techniques, we searched for secreted factors that were overexpressed in fibroblasts undergoing replicative senescence. We identified WNT16B, a member of the WNT family of secreted proteins. We found that WNT16B is overexpressed in cells undergoing stress-induced premature senescence and oncogene-induced senescence in both MRC5 cell line and the in vivo murine model of K-Ras(V12)-induced senescence. By small interfering RNA experiments, we observed that both p53 and WNT16B are necessary for the onset of replicative senescence. WNT16B expression is required for the full transcriptional activation of p21(WAF1). Moreover, WNT16B regulates activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overall, we identified WNT16B as a new marker of senescence that regulates p53 activity and the PI3K/AKT pathway and is necessary for the onset of replicative senescence.

  6. Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway.

    PubMed

    Chen, Jiun-Han; Hsiao, George; Lee, An-Rong; Wu, Chin-Chen; Yen, Mao-Hsiung

    2004-04-01

    Andrographolide (Andro), an active component isolated from the Chinese official herbal Andrographis paniculata, which has been reported to prevent oxygen radical production and thus prevent inflammatory diseases. In this study, we investigated the molecular mechanisms and signaling pathways by which Andro protects human umbilical vein endothelial cells (HUVECs) from growth factor (GF) deprivation-induced apoptosis. Results demonstrated that HUVECs undergo apoptosis after 18 hr of GF deprivation but that this cell death was suppressed by the addition of Andro in a concentration-dependent manner (1-100 microM). Andro suppresses the mitochondrial pathway of apoptosis by inhibiting release of cytochrome c into the cytoplasm and dissipation of mitochondrial potential (Deltapsi(m)), as a consequence, prevented caspase-3 and -9 activation. Treatment of endothelial cells with Andro-induced activation of the protein kinase Akt, an anti-apoptotic signal, and phosphorylation of BAD, a down-stream target of Akt. Suppression of Akt activity by wortmannin, by LY-294002 and by using a dominant negative Akt mutant abolished the anti-apoptotic effect of Andro. In contrast, the ERK1/2 activities were not affected by Andro. The ERK1/2 inhibitor, PD98059 failed to antagonize the protective effect of Andro. In conclusion, Andro exerts its anti-apoptotic potential via activation of the Akt-BAD pathway in HUVECs and thus may represent a candidate of therapeutic agent for atherosclerosis.

  7. Inhibition of phosphoinositol 3 kinase contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity

    PubMed Central

    Ilinskaya, Anna N; Man, Sonny; Patri, Anil K; Clogston, Jeffrey D; Crist, Rachael M; Cachau, Raul E; McNeil, Scott E; Dobrovolskaia, Marina A

    2014-01-01

    Aim Disseminated intravascular coagulation is an increasing concern for certain types of engineered nanomaterials. Recent studies have shed some light on the nanoparticle physicochemical properties contributing to this toxicity; however, the mechanisms are poorly understood. Leukocyte procoagulant activity (PCA) is a key factor contributing to the initiation of this toxicity. We have previously reported on the exaggeration of endotoxin-induced PCA by cationic dendrimers. Herein, we report an effort to discern the mechanism. Materials & methods Poly(amidoamine) dendrimers with various sizes and surface functionalities were studied in vitro by the recalcification test, flow cytometry and other relevant assays. Results & conclusion Cationic dendrimers exaggerated endotoxin-induced PCA, but their anionic or neutral counterparts did not; the cationic charge prompts this phenomenon, but different cationic surface chemistries do not influence it. Cationic dendrimers and endotoxin differentially affect the PCA complex. The inhibition of phosphoinositol 3 kinase by dendrimers contributes to the exaggeration of the endotoxin-induced PCA. PMID:24279459

  8. Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase.

    PubMed

    Miller, Michelle S; Pinson, Jo-Anne; Zheng, Zhaohua; Jennings, Ian G; Thompson, Philip E

    2013-02-01

    Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.

  9. Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

    SciTech Connect

    Smith, Adrian L.; D’Angelo, Noel D.; Bo, Yunxin Y.; Booker, Shon K.; Cee, Victor J.; Herberich, Brad; Hong, Fang-Tsao; Jackson, Claire L.M.; Lanman, Brian A.; Liu, Longbin; Nishimura, Nobuko; Pettus, Liping H.; Reed, Anthony B.; Tadesse, Seifu; Tamayo, Nuria A.; Wurz, Ryan P.; Yang, Kevin; Andrews, Kristin L.; Whittington, Douglas A.; McCarter, John D.; Miguel, Tisha San; Zalameda, Leeanne; Jiang, Jian; Subramanian, Raju; Mullady, Erin L.; Caenepeel, Sean; Freeman, Daniel J.; Wang, Ling; Zhang, Nancy; Wu, Tian; Hughes, Paul E.; Norman, Mark H.

    2012-09-17

    A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

  10. Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

    PubMed Central

    Kontos, Christopher D.; Stauffer, Thomas P.; Yang, Wen-Pin; York, John D.; Huang, Liwen; Blanar, Michael A.; Meyer, Tobias; Peters, Kevin G.

    1998-01-01

    Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival. PMID:9632797

  11. Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt.

    PubMed

    Kontos, C D; Stauffer, T P; Yang, W P; York, J D; Huang, L; Blanar, M A; Meyer, T; Peters, K G

    1998-07-01

    Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3, 4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

  12. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    PubMed Central

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes. PMID:8611143

  13. In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

    PubMed Central

    Cooper, J A; Kashishian, A

    1993-01-01

    We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

  14. Metacognitive deficits predict future levels of negative symptoms in schizophrenia controlling for neurocognition, affect recognition, and self-expectation of goal attainment.

    PubMed

    Lysaker, Paul H; Kukla, Marina; Dubreucq, Julien; Gumley, Andrew; McLeod, Hamish; Vohs, Jenifer L; Buck, Kelly D; Minor, Kyle S; Luther, Lauren; Leonhardt, Bethany L; Belanger, Elizabeth A; Popolo, Raffaele; Dimaggio, Giancarlo

    2015-10-01

    The recalcitrance of negative symptoms in the face of pharmacologic treatment has spurred interest in understanding the psychological factors that contribute to their formation and persistence. Accordingly, this study investigated whether deficits in metacognition, or the ability to form integrated ideas about oneself, others, and the world, prospectively predicted levels of negative symptoms independent of deficits in neurocognition, affect recognition and defeatist beliefs. Participants were 53 adults with a schizophrenia spectrum disorder. Prior to entry into a rehabilitation program, all participants completed concurrent assessments of metacognition with the Metacognitive Assessment Scale-Abbreviated, negative symptoms with the Positive and Negative Syndrome Scale, neurocognition with the MATRICS battery, affect recognition with the Bell Lysaker Emotion Recognition Task, and one form of defeatist beliefs with the Recovery Assessment Scale. Negative symptoms were then reassessed one week, 9weeks, and 17weeks after entry into the program. A mixed effects regression model revealed that after controlling for baseline negative symptoms, a general index of neurocognition, defeatist beliefs and capacity for affect recognition, lower levels of metacognition predicted higher levels of negative symptoms across all subsequent time points. Poorer metacognition was able to predict later levels of elevated negative symptoms even after controlling for initial levels of negative symptoms. Results may suggest that metacognitive deficits are a risk factor for elevated levels of negative symptoms in the future. Clinical implications are also discussed.

  15. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    PubMed Central

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-01-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing. PMID:27541856

  16. Nitric oxide decreases subventricular zone stem cell proliferation by inhibition of epidermal growth factor receptor and phosphoinositide-3-kinase/Akt pathway.

    PubMed

    Torroglosa, Ana; Murillo-Carretero, Maribel; Romero-Grimaldi, Carmen; Matarredona, Esperanza R; Campos-Caro, Antonio; Estrada, Carmen

    2007-01-01

    Nitric oxide (NO) inhibits proliferation of subventricular zone (SVZ) neural precursor cells in adult mice in vivo under physiological conditions. The mechanisms underlying this NO effect have now been investigated using SVZ-derived neural stem cells, which generate neurospheres in vitro when stimulated by epidermal growth factor (EGF). In these cultures, NO donors decreased the number of newly formed neurospheres as well as their size, which indicates that NO was acting on the neurosphere-forming neural stem cells and the daughter neural progenitors. The effect of NO was cytostatic, not proapoptotic, and did not involve cGMP synthesis. Neurosphere cells expressed the neuronal and endothelial isoforms of NO synthase (NOS) and produced NO in culture. Inhibition of NOS activity by N(omega)-nitro-L-arginine methylester (L-NAME) promoted neurosphere formation and growth, thus revealing an autocrine/paracrine action of NO on the neural precursor cells. Both exogenous and endogenous NO impaired the EGF-induced activation of the EGF receptor (EGFR) tyrosine kinase and prevented the EGF-induced Akt phosphorylation in neurosphere cells. Inhibition of the phosphoinositide-3-kinase (PI3-K)/Akt pathway by LY294002 significantly reduced the number of newly formed neurospheres, which indicates that this is an essential pathway for neural stem cell self-renewal. Chronic administration of l-NAME to adult mice enhanced phospho-Akt staining in the SVZ and reduced nuclear p27(Kip1) in the SVZ and olfactory bulb. The inhibition of EGFR and PI3-K pathway by NO explains, at least in part, its antimitotic effect on neurosphere cells and may be a mechanism involved in the physiological role of NO as a negative regulator of SVZ neurogenesis in adult mice.

  17. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

    PubMed

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-11-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

  18. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    not for its insulin secretory response with respect to major initiators and modulators of insulin release. The data indicate that MAP kinase is active and under the control of MAP kinase. PKC is upstream of a genisteinsensitive tyrosine kinase and probably downstream of a PI3-kinase in INS-1 cells. PMID:12369712

  19. Inhibitory Role of α6β4-Associated Erbb-2 and Phosphoinositide 3-Kinase in Keratinocyte Haptotactic Migration Dependent on α3β1 Integrin

    PubMed Central

    Hintermann, Edith; Bilban, Martin; Sharabi, Andrew; Quaranta, Vito

    2001-01-01

    Keratinocytes and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins α3β1 and α6β4. While α3β1 mediates adhesion, spreading, and migration (Kreidberg, J.A. 2000. Curr. Opin. Cell Biol. 12:548–553), α6β4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 112:411–418). We investigated a possible regulatory interplay between α3β1 and α6β4 in cell motility using HaCaT keratinocytes as a model. We found that α6β4 antibodies inhibit α3β1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by α3β1 activation), and not when chemotactic (i.e., triggered by epidermal growth factor receptor). Inhibition of migration by α6β4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti–α6β4 antibodies, activation of PI3-K is mediated by α6β4-associated erbB-2, as indicated by erbB-2 autophosphorylation and erbB-2/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-2 abolishes inhibition of haptotaxis by anti–α6β4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of α3β1 and α6β4 integrins, (b) α6β4-associated erbB-2 and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by α6β4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM. PMID:11331299

  20. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

    PubMed Central

    Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

    2006-01-01

    Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

  1. Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae

    PubMed Central

    Waite, Christopher; Schumacher, Jörg; Jovanovic, Milija; Bennett, Mark

    2017-01-01

    ABSTRACT   The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae. T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL. In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses. PMID:28119474

  2. Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.

    PubMed Central

    McIlroy, J; Chen, D; Wjasow, C; Michaeli, T; Backer, J M

    1997-01-01

    We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway. PMID:8972205

  3. Effect of selective expression of dominant-negative PPARγ in pro-opiomelanocortin neurons on the control of energy balance.

    PubMed

    Stump, Madeliene; Guo, Deng-Fu; Lu, Ko-Ting; Mukohda, Masashi; Liu, Xuebo; Rahmouni, Kamal; Sigmund, Curt D

    2016-07-01

    Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions.

  4. Effects of neck flexion on contingent negative variation and anticipatory postural control during arm movement while standing.

    PubMed

    Fujiwara, Katsuo; Tomita, Hidehito; Maeda, Kaoru; Kunita, Kenji

    2009-02-01

    We investigated the effects of neck flexion on contingent negative variation (CNV) and anticipatory postural control using an arm flexion task in standing. CNV was adopted to evaluate the state of activation of brain areas related to anticipatory postural control. Subjects were required to flex the arms in response to a sound stimulus preceded by a warning sound stimulus. Two different intervals (2.0 and 3.5s) between these two stimuli were used in neck position in quiet standing (neck resting) and neck position at 80% angle of maximal neck flexion. The mean amplitude of CNV 100-ms before the response stimulus, recorded from a Cz electrode, was calculated. Onset timing of activation of the postural muscles (lumbar paraspinal, biceps femoris and gastrocnemius) with respect to the anterior deltoid was analyzed. Reaction time at the anterior deltoid was significantly shorter in the 2.0s period than in the 3.5s period, and in the neck flexion than in the neck resting in both periods. In the 2.0s, but not in the 3.5s period, neck flexion resulted in an increased CNV amplitude and an increased duration of preceding activation of the postural muscles, and the correlation between these increases was significant.

  5. Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

    PubMed

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

    2009-06-01

    Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM.

  6. Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

    PubMed Central

    Cafferkey, R; Young, P R; McLaughlin, M M; Bergsma, D J; Koltin, Y; Sathe, G M; Faucette, L; Eng, W K; Johnson, R K; Livi, G P

    1993-01-01

    Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the

  7. Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

    PubMed Central

    Crljen, Vladiana; Volinia, Stefano; Banfic, Hrvoje

    2002-01-01

    Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and

  8. A pilot randomised controlled trial of negative pressure wound therapy to treat grade III/IV pressure ulcers [ISRCTN69032034

    PubMed Central

    2012-01-01

    Background Negative pressure wound therapy (NPWT) is widely promoted as a treatment for full thickness wounds; however, there is a lack of high-quality research evidence regarding its clinical and cost effectiveness. A trial of NPWT for the treatment of grade III/IV pressure ulcers would be worthwhile but premature without assessing whether such a trial is feasible. The aim of this pilot randomised controlled trial was to assess the feasibility of conducting a future full trial of NPWT for the treatment of grade III and IV pressure ulcers and to pilot all aspects of the trial. Methods This was a two-centre (acute and community), pilot randomised controlled trial. Eligible participants were randomised to receive either NPWT or standard care (SC) (spun hydrocolloid, alginate or foam dressings). Outcome measures were time to healing of the reference pressure ulcer, recruitment rates, frequency of treatment visits, resources used and duration of follow-up. Results Three hundred and twelve patients were screened for eligibility into this trial over a 12-month recruitment period and 12/312 participants (3.8%) were randomised: 6 to NPWT and 6 to SC. Only one reference pressure ulcer healed (NPWT group) during follow-up (time to healing 79 days). The mean number of treatment visits per week was 3.1 (NPWT) and 5.7 (SC); 6/6 NPWT and 1/6 SC participants withdrew from their allocated trial treatment. The mean duration of follow-up was 3.8 (NPWT) and 5.0 (SC) months. Conclusions This pilot trial yielded vital information for the planning of a future full study including projected recruitment rate, required duration of follow-up and extent of research nurse support required. Data were also used to inform the cost-effectiveness and value of information analyses, which were conducted alongside the pilot trial. Trial registration Current Controlled Trials ISRCTN69032034. PMID:22839453

  9. Impact of attention biases to threat and effortful control on individual variations in negative affect and social withdrawal in very young children.

    PubMed

    Cole, Claire E; Zapp, Daniel J; Fettig, Nicole B; Pérez-Edgar, Koraly

    2016-01-01

    Early temperamental sensitivity may form the basis for the later development of socioemotional maladjustment. In particular, temperamental negative affect places children at risk for the development of anxiety. However, not all children who show negative affect go on to develop anxiety or extreme social withdrawal. Recent research indicates that reactive control, in the form of attention to threat, may serve as a bridge between early temperament and the development of later social difficulties. In addition, variation in effortful control may also modulate this trajectory. Children (mean age=5.57 years) were assessed for attention bias to threatening and pleasant faces using a dot-probe paradigm. Attention bias to threatening (but not happy) faces moderated the direct positive relation between negative affect and social withdrawal. Children with threat biases showed a significant link between negative affect and social withdrawal, whereas children who avoided threat did not. In contrast, effortful control did not moderate the relation between negative affect and social withdrawal. Rather, there was a direct negative relation between effortful control and social withdrawal. The findings from this short report indicate that the relations among temperament, attention bias, and social withdrawal appears early in life and point to early emerging specificity in reactive and regulatory functioning.

  10. Platelet-derived growth factor triggers translocation of the insulin-regulatable glucose transporter (type 4) predominantly through phosphatidylinositol 3-kinase binding sites on the receptor.

    PubMed Central

    Kamohara, S; Hayashi, H; Todaka, M; Kanai, F; Ishii, K; Imanaka, T; Escobedo, J A; Williams, L T; Ebina, Y

    1995-01-01

    Insulin is the only known hormone which rapidly stimulates glucose uptake in target tissues, mainly by translocation to the cell surface of the intracellular insulin-regulatable glucose transporter (glucose transporter type 4, GLUT4). We have developed a cell line for direct, sensitive detection of GLUT4 on the cell surface. We have suggested that insulin-activated phosphatidylinositol (PI) 3-kinase may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report that platelet-derived growth factor (PDGF), which stimulates PI 3-kinase activity, triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the PDGF receptor and in 3T3-L1 mouse adipocytes. Using mutant PDGF receptors that cannot bind to Ras-GTPase-activating protein, phospholipase C-gamma, and PI 3-kinase, respectively, we obtained evidence that PI 3-kinase binding sites play a key role in the signaling pathway of PDGF-stimulated GLUT4 translocation in the CHO cell system. Images Fig. 1 Fig. 4 PMID:7862637

  11. In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARgamma in L6 myotubes.

    PubMed

    Anandharajan, R; Jaiganesh, S; Shankernarayanan, N P; Viswakarma, R A; Balakrishnan, A

    2006-06-01

    The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.

  12. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML.

    PubMed

    Park, S; Chapuis, N; Bardet, V; Tamburini, J; Gallay, N; Willems, L; Knight, Z A; Shokat, K M; Azar, N; Viguié, F; Ifrah, N; Dreyfus, F; Mayeux, P; Lacombe, C; Bouscary, D

    2008-09-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways are frequently activated in acute myelogenous leukemia (AML). mTORC1 inhibition with RAD001 induces PI3K/Akt activation and both pathways are activated independently, providing a rationale for dual inhibition of both pathways. PI-103 is a new potent PI3K/Akt and mTOR inhibitor. In human leukemic cell lines and in primary blast cells from AML patients, PI-103 inhibited constitutive and growth factor-induced PI3K/Akt and mTORC1 activation. PI-103 was essentially cytostatic for cell lines and induced cell cycle arrest in the G1 phase. In blast cells, PI-103 inhibited leukemic proliferation, the clonogenicity of leukemic progenitors and induced mitochondrial apoptosis, especially in the compartment containing leukemic stem cells. In contrast, apoptosis was not induced with RAD001 and IC87114 association, which specifically inhibits mTORC1 and p110delta activity, respectively. PI-103 had additive proapoptotic effects with etoposide in blast cells and in immature leukemic cells. Interestingly, PI-103 did not induce apoptosis in normal CD34(+) cells and had moderate effects on their clonogenic and proliferative properties. Here, we demonstrate that multitargeted therapy against PI3K/Akt and mTOR with PI-103 may be of therapeutic value in AML.

  13. MST3 Kinase Phosphorylates TAO1/2 to Enable Myosin Va Function in Promoting Spine Synapse Development

    PubMed Central

    Ultanir, Sila K.; Yadav, Smita; Hertz, Nicholas T.; Oses-Prieto, Juan A.; Claxton, Suzanne; Burlingame, Alma L.; Shokat, Kevan M.; Jan, Lily Y.; Jan, Yuh-Nung

    2014-01-01

    Summary Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS. PMID:25456499

  14. MST3 kinase phosphorylates TAO1/2 to enable Myosin Va function in promoting spine synapse development.

    PubMed

    Ultanir, Sila K; Yadav, Smita; Hertz, Nicholas T; Oses-Prieto, Juan A; Claxton, Suzanne; Burlingame, Alma L; Shokat, Kevan M; Jan, Lily Y; Jan, Yuh-Nung

    2014-12-03

    Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS.

  15. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex.

    PubMed

    Zhong, Yun; Wang, Qing Jun; Li, Xianting; Yan, Ying; Backer, Jonathan M; Chait, Brian T; Heintz, Nathaniel; Yue, Zhenyu

    2009-04-01

    Beclin 1, a mammalian autophagy protein that has been implicated in development, tumour suppression, neurodegeneration and cell death, exists in a complex with Vps34, the class III phosphatidylinositol-3-kinase (PI(3)K) that mediates multiple vesicle-trafficking processes including endocytosis and autophagy. However, the precise role of the Beclin 1-Vps34 complex in autophagy regulation remains to be elucidated. Combining mouse genetics and biochemistry, we have identified a large in vivo Beclin 1 complex containing the known proteins Vps34, p150/Vps15 and UVRAG, as well as two newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein). Characterization of the new proteins revealed that Atg14L enhances Vps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces Vps34 activity and downregulates autophagy. We show that Beclin 1 and Atg14L synergistically promote the formation of double-membraned organelles that are associated with Atg5 and Atg12, whereas forced expression of Rubicon results in aberrant late endosomal/lysosomal structures and impaired autophagosome maturation. We hypothesize that by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of the class III PI(3)K in regulating autophagy at multiple steps.

  16. Force engages vinculin and promotes tumor progression by enhancing PI3-kinase activation of phosphatidylinositol (3,4,5)-triphosphate

    PubMed Central

    Rubashkin, MG; Cassereau, L; Bainer, R; DuFort, CC; Yui, Y; Ou, G; Paszek, MJ; Davidson, MW; Chen, YY; Weaver, VM

    2014-01-01

    Extracellular matrix stiffness induces focal adhesion assembly to drive malignant transformation and tumor metastasis. Nevertheless, how force alters focal adhesions to promote tumor progression remains unclear. Here, we explored the role of the focal adhesion protein vinculin, a force-activated mechano-transducer, in mammary epithelial tissue transformation and invasion. We found that extracellular matrix stiffness stabilizes the assembly of a vinculin-talin-actin scaffolding complex that facilitates PI3-kinase mediated phosphatidylinositol (3,4,5)-triphosphate phosphorylation. Using defined two and three dimensional matrices, a mouse model of mammary tumorigenesis with vinculin mutants and a novel super resolution imaging approach, we established that ECM stiffness, per se, promotes the malignant progression of a mammary epithelium by activating and stabilizing vinculin and enhancing Akt signaling at focal adhesions. Our studies also revealed that vinculin strongly co-localizes with activated Akt at the invasive border of human breast tumors, where the ECM is stiffest and we detected elevated mechano-signaling. Thus, extracellular matrix stiffness could induce tumor progression by promoting the assembly of signaling scaffolds; a conclusion underscored by the significant association we observed between highly expressed focal adhesion plaque proteins and malignant transformation across multiple types of solid cancer. PMID:25183785

  17. Novel Anti-Microbial Peptide SR-0379 Accelerates Wound Healing via the PI3 Kinase/Akt/mTOR Pathway

    PubMed Central

    Tomioka, Hideki; Nakagami, Hironori; Tenma, Akiko; Saito, Yoshimi; Kaga, Toshihiro; Kanamori, Toshihide; Tamura, Nao; Tomono, Kazunori; Kaneda, Yasufumi; Morishita, Ryuichi

    2014-01-01

    We developed a novel cationic antimicrobial peptide, AG30/5C, which demonstrates angiogenic properties similar to those of LL-37 or PR39. However, improvement of its stability and cost efficacy are required for clinical application. Therefore, we examined the metabolites of AG30/5C, which provided the further optimized compound, SR-0379. SR-0379 enhanced the proliferation of human dermal fibroblast cells (NHDFs) via the PI3 kinase-Akt-mTOR pathway through integrin-mediated interactions. Furthermore SR-0379 promoted the tube formation of human umbilical vein endothelial cells (HUVECs) in co-culture with NHDFs. This compound also displays antimicrobial activities against a number of bacteria, including drug-resistant microbes and fungi. We evaluated the effect of SR-0379 in two different would-healing models in rats, the full-thickness defects under a diabetic condition and an acutely infected wound with full-thickness defects and inoculation with Staphylococcus aureus. Treatment with SR-0379 significantly accelerated wound healing when compared to fibroblast growth factor 2 (FGF2). The beneficial effects of SR-0379 on wound healing can be explained by enhanced angiogenesis, granulation tissue formation, proliferation of endothelial cells and fibroblasts and antimicrobial activity. These results indicate that SR-0379 may have the potential for drug development in wound repair, even under especially critical colonization conditions. PMID:24675668

  18. Endoglin regulates PI3-kinase/Akt trafficking and signaling to alter endothelial capillary stability during angiogenesis

    PubMed Central

    Lee, Nam Y.; Golzio, Christelle; Gatza, Catherine E.; Sharma, Arun; Katsanis, Nicholas; Blobe, Gerard C.

    2012-01-01

    Endoglin (CD105) is an endothelial-specific transforming growth factor β (TGF-β) coreceptor essential for angiogenesis and vascular homeostasis. Although endoglin dysfunction contributes to numerous vascular conditions, the mechanism of endoglin action remains poorly understood. Here we report a novel mechanism in which endoglin and Gα-interacting protein C-terminus–interacting protein (GIPC)–mediated trafficking of phosphatidylinositol 3-kinase (PI3K) regulates endothelial signaling and function. We demonstrate that endoglin interacts with the PI3K subunits p110α and p85 via GIPC to recruit and activate PI3K and Akt at the cell membrane. Opposing ligand-induced effects are observed in which TGF-β1 attenuates, whereas bone morphogenetic protein-9 enhances, endoglin/GIPC-mediated membrane scaffolding of PI3K and Akt to alter endothelial capillary tube stability in vitro. Moreover, we employ the first transgenic zebrafish model for endoglin to demonstrate that GIPC is a critical component of endoglin function during developmental angiogenesis in vivo. These studies define a novel non-Smad function for endoglin and GIPC in regulating endothelial cell function during angiogenesis. PMID:22593212

  19. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    PubMed Central

    Li, Jie; Zhang, Chao; Jiang, Hongchuan; Cheng, Jiao

    2015-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro) was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10−7 mol/L), by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF) gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future. PMID:25709476

  20. Andrographolide inhibits osteopontin expression and breast tumor growth through down regulation of PI3 kinase/Akt signaling pathway.

    PubMed

    Kumar, S; Patil, H S; Sharma, P; Kumar, D; Dasari, S; Puranik, V G; Thulasiram, H V; Kundu, G C

    2012-09-01

    Breast cancer is one of the most common cancers among women in India and around the world. Despite recent advancement in the treatment of breast cancer, the results of chemotherapy to date remain unsatisfactory, prompting a need to identify natural agents that could target cancer efficiently with least side effects. Andrographolide (Andro) is one such molecule which has been shown to possess inhibitory effect on cancer cell growth. In this study, Andro, a natural diterpenoid lactone isolated from Andrographis paniculata has been shown to inhibit breast cancer cell proliferation, migration and arrest cell cycle at G2/M phase and induces apoptosis through caspase independent pathway. Our experimental evidences suggest that Andro attenuates endothelial cell motility and tumor-endothelial cell interaction. Moreover, Andro suppresses breast tumor growth in orthotopic NOD/SCID mice model. The anti-tumor activity of Andro in both in vitro and in vivo model was correlated with down regulation of PI3 kinase/Akt activation and inhibition of pro-angiogenic molecules such as OPN and VEGF expressions. Collectively, these results demonstrate that Andro may act as an effective anti-tumor and anti-angiogenic agent for the treatment of breast cancer.

  1. Puquitinib mesylate, an inhibitor of phosphatidylinositol 3-kinase p110δ, for treating relapsed or refractory non-Hodgkin's lymphoma

    PubMed Central

    Zhan, Jing; Xia, Yi; Sun, Peng; Bi, Xi-Wen; Liu, Pan-Pan; Li, Zhi-Ming; Li, Su; Zou, Ben-Yan; Jiang, Wen-Qi

    2015-01-01

    Objectives To determine the safety of Puquitinib Mesylate (XC-302), an oral inhibitor of phosphatidylinositol 3-kinase, in treating relapsed or refractory non-Hodgkin's lymphoma (NHL). Methods Between October 2013 and July 2015, 21 patients from Sun Yat-sen University Cancer Center were treated twice daily on each day of a 28-day cycle (median number of cycles, 2; maximum, 20) with XC-302 at a post prandial dose of 25 mg, 37.5 mg, or 50 mg. Adverse events (AEs), AUClast and Cmax, response rates, and overall survival were assessed. Results Patients had received a median (range) of 1 (1 to 3) previous cancer treatments. At the latest follow-up, two patients were still benefitting from the study. The most common drug-related AEs were elevations in alanine transaminase (ALT, 14 of 21 patients) and aspartate transaminase (AST, 7 of 21 patients). Four patients, both in the-50-mg group, had dose-limiting toxicities, and therapy was discontinued in a fifth because of persistent abnormal liver function. The overall response rate was 2 of19. Serum concentrations of XC-302 increased in a dose-dependent pattern. Median progression-free survival in all patients was 1.9 (95% CI, 1.7 to 2.0) months. Conclusion XC-302 has an acceptable safety profile and offers potential therapeutic value to patients with relapsed or refractory non-Hodgkin lymphoma. PMID:26510909

  2. Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

    PubMed Central

    ZHANG, Zong-Kang; LI, Jie; LIU, Jin; GUO, Baosheng; LEUNG, Albert; ZHANG, Ge; ZHANG, Bao-Ting

    2016-01-01

    Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment could significantly attenuate the decreases in the phosphorylation level of PI3K at p110 catalytic subunit and its downstream markers related to protein synthesis, and inhibit the increases in protein degradation markers at mRNA and protein levels in rat soleus muscle following 28-day hindlimb unloading. In addition, the decreases in soleus muscle mass, muscle fiber cross-sectional area, twitch force, specific force, contraction time and half relaxation time could be significantly attenuated by the high dose ICT treatment. The low dose ICT treatment could moderately attenuate the above changes induced by unloading. Wortmannin, a specific inhibitor of PI3K at p110 catalytic subunit, could abolish the above effects of ICT in vitro and in vivo, indicating that PI3K/Akt signaling could be required by ICT to counteract skeletal muscle atrophy following mechanical unloading. PMID:26831566

  3. The role of inositol 1,4,5-trisphosphate 3-kinase A in regulating emotional behavior and amygdala function

    PubMed Central

    Chung, Sooyoung; Kim, Il Hwan; Lee, Dongmin; Park, Kyungjoon; Kim, Joo Yeon; Lee, Yeon Kyung; Kim, Eun Joo; Lee, Hyun Woo; Choi, June-seek; Son, Gi Hoon; Sun, Woong; Shin, Ki Soon; Kim, Hyun

    2016-01-01

    Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) is a molecule enriched in the brain and neurons that regulates intracellular calcium levels via signaling through the inositol trisphosphate receptor. In the present study, we found that IP3K-A expression is highly enriched in the central nucleus of the amygdala (CeA), which plays a pivotal role in the processing and expression of emotional phenotypes in mammals. Genetic abrogation of IP3K-A altered amygdala gene expression, particularly in genes involved in key intracellular signaling pathways and genes mediating fear- and anxiety-related behaviors. In agreement with the changes in amygdala gene expression profiles, IP3K-A knockout (KO) mice displayed more robust responses to aversive stimuli and spent less time in the open arms of the elevated plus maze, indicating high levels of innate fear and anxiety. In addition to behavioral phenotypes, decreased excitatory and inhibitory postsynaptic current and reduced c-Fos immunoreactivity in the CeA of IP3K-A KO mice suggest that IP3K-A has a profound influence on the basal activities of fear- and anxiety-mediating amygdala circuitry. In conclusion, our findings collectively demonstrate that IP3K-A plays an important role in regulating affective states by modulating metabotropic receptor signaling pathways and neural activity in the amygdala. PMID:27053114

  4. Phosphoinositide-3-Kinase Is the Primary Mediator of Phosphoinositide-Dependent Inhibition in Mammalian Olfactory Receptor Neurons

    PubMed Central

    Ukhanov, Kirill; Corey, Elizabeth; Ache, Barry W.

    2016-01-01

    Odorants inhibit as well as excite primary olfactory receptor neurons (ORNs) in many animal species. Growing evidence suggests that inhibition of mammalian ORNs is mediated by phosphoinositide (PI) signaling through activation of phosphoinositide 3-kinase (PI3K), and that canonical adenylyl cyclase III signaling and PI3K signaling interact to provide the basis for ligand-induced selective signaling. As PI3K is known to act in concert with phospholipase C (PLC) in some cellular systems, the question arises as to whether they work together to mediate inhibitory transduction in mammalian ORNs. The present study is designed to test this hypothesis. While we establish that multiple PLC isoforms are expressed in the transduction zone of rat ORNs, that odorants can activate PLC in ORNs in situ, and that pharmacological blockade of PLC enhances the excitatory response to an odorant mixture in some ORNs in conjunction with PI3K blockade, we find that by itself PLC does not account for an inhibitory response. We conclude that PLC does not make a measurable independent contribution to odor-evoked inhibition, and that PI3K is the primary mediator of PI-dependent inhibition in mammalian ORNs. PMID:27147969

  5. ERK and phosphoinositide 3-kinase temporally coordinate different modes of actin-based motility during embryonic wound healing.

    PubMed

    Li, Jingjing; Zhang, Siwei; Soto, Ximena; Woolner, Sarah; Amaya, Enrique

    2013-11-01

    Embryonic wound healing provides a perfect example of efficient recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic wound healing requires two functionally distinct actin structures: a contractile actomyosin cable and actin protrusions at the leading edge. Here, we report that the discrete formation and function of these two structures is achieved by the temporal segregation of two intracellular upstream signals and distinct downstream targets. The sequential activation of ERK and phosphoinositide 3-kinase (PI3K) signalling divides Xenopus embryonic wound healing into two phases. In the first phase, activated ERK suppresses PI3K activity, and is responsible for the activation of Rho and myosin-2, which drives actomyosin cable formation and constriction. The second phase is dominated by restored PI3K signalling, which enhances Rac and Cdc42 activity, leading to the formation of actin protrusions that drive migration and zippering. These findings reveal a new mechanism for coordinating different modes of actin-based motility in a complex tissue setting, namely embryonic wound healing.

  6. Emergence of the PI3-kinase pathway as a central modulator of normal and aberrant B cell differentiation.

    PubMed

    Baracho, G V; Miletic, A V; Omori, S A; Cato, M H; Rickert, R C

    2011-04-01

    Phosphoinositide 3-kinase (PI3K) defines a family of lipid kinases that direct a wide range of cellular processes and cell fate decisions. Since its discovery, and that of its enzymatic antagonist PTEN, much of the focus on PI3K has been on its oncogenic potential. In recent years, studies on PI3K signaling in B lymphocytes have established the importance of this pathway in effecting B cell differentiation and associated molecular events such as V(D)J recombination and class switch recombination. Intriguing new findings also indicate that there is specificity in the PI3K pathway in B cells, including preferential expression or usage of particular PI3K isoforms and counter-regulation by the PTEN and SHIP phosphatases. The role of PI3K adaptor proteins (CD19, BCAP, and TC21) has also undergone revision to reflect both shared and unique properties. The emergence of Foxo1 as a critical PI3K regulatory target for B cell differentiation has united membrane proximal regulatory events orchestrated by PI3K/PTEN/SHIP with key transcriptional targets. Insights into the regulation and impact of PI3K signaling have been brought to bear in new treatments for B cell malignancies, and will also be an important topic of consideration for B cell-dependent autoimmune diseases.

  7. Cobalt chloride stimulates phosphoinositide 3-kinase/Akt signaling through the epidermal growth factor receptor in oral squamous cell carcinoma.

    PubMed

    Ryu, Mi Heon; Park, Jeong Hee; Park, Ji Eun; Chung, Jin; Lee, Chang Hun; Park, Hae Ryoun

    2010-04-01

    Tumor cells are often found under hypoxic conditions due to the rapid outgrowth of their vascular supply, and, in order to survive hypoxia, these cells induce numerous signaling factors. Akt is an important kinase in cell survival, and its activity is regulated by the upstream phosphoinositide 3-kinase (PI3K) and receptor tyrosine kinases (RTKs). In this study, we examined Akt activation and RTKs/PI3K/Akt signaling using the hypoxia-mimetic cobalt chloride in oral squamous carcinoma cells. Cobalt chloride increases Akt phosphorylation in both a dose- and time-dependent manner. Blocking the activation of the PI3K/Akt pathway using LY294002 abolished Akt activation in response to cobalt chloride, suggesting that Akt phosphorylation by cobalt chloride is dependent on PI3K. In addition, activation of the PI3K/Akt pathway seems to rely on the epidermal growth factor receptor (EGFR), since the inhibition of EGFR attenuated cobalt chloride-induced Akt activation. The results in this study also demonstrate that cobalt chloride increases EGFR protein levels and induces oral squamous cell carcinoma cells to enter S phase.

  8. Novel roles for class II Phosphoinositide 3-Kinase C2β in signalling pathways involved in prostate cancer cell invasion

    PubMed Central

    Mavrommati, Ioanna; Cisse, Ouma; Falasca, Marco; Maffucci, Tania

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) regulate several cellular functions such as proliferation, growth, survival and migration. The eight PI3K isoforms are grouped into three classes and the three enzymes belonging to the class II subfamily (PI3K-C2α, β and γ) are the least investigated amongst all PI3Ks. Interest on these isoforms has been recently fuelled by the identification of specific physiological roles for class II PI3Ks and by accumulating evidence indicating their involvement in human diseases. While it is now established that these isoforms can regulate distinct cellular functions compared to other PI3Ks, there is still a limited understanding of the signalling pathways that can be specifically regulated by class II PI3Ks. Here we show that PI3K-C2β regulates mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2) activation in prostate cancer (PCa) cells. We further demonstrate that MEK/ERK and PI3K-C2β are required for PCa cell invasion but not proliferation. In addition we show that PI3K-C2β but not MEK/ERK regulates PCa cell migration as well as expression of the transcription factor Slug. These data identify novel signalling pathways specifically regulated by PI3K-C2β and they further identify this enzyme as a key regulator of PCa cell migration and invasion. PMID:26983806

  9. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration.

    PubMed

    Dise, Rebecca S; Frey, Mark R; Whitehead, Robert H; Polk, D Brent

    2008-01-01

    Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.

  10. Phosphoinositide-3-kinase and mitogen activated protein kinase signaling pathways mediate acute NGF sensitization of TRPV1.

    PubMed

    Zhu, Weiguo; Oxford, Gerry S

    2007-04-01

    Nerve growth factor (NGF) induces an acute sensitization of nociceptive DRG neurons, in part, through sensitization of the capsaicin receptor TRPV1 via the high affinity trkA receptor. The mechanisms linking trkA and TRPV1 remain controversial with several candidate signaling pathways proposed. Utilizing adult rat and mouse DRG neurons and CHO cells co-expressing trkA and TRPV1, we have investigated the signaling events underlying acute TRPV1 sensitization by NGF combining biochemical, electrophysiological, pharmacological, mutational and genetic knockout approaches. Pharmacological interference with p42/p44 mitogen activated protein kinase (MAPK) or phosphoinositide-3-kinase (PI3K), but not PLC abrogated sensitization of capsaicin responses. Co-expression of TRPV1 with wild-type or Y785F (PLC signal deficient) mutant human trkA reconstituted NGF sensitization. In contrast, TRPV1 co-expressed with MAPK signaling deficient Y490A or PI3K signaling deficient Y751F trkA mutants exhibited weaker sensitization. Biochemical analysis of p42/p44 and Akt phosphorylation confirmed the specificity of pharmacological agents and trkA mutants. Finally, NGF sensitization of capsaicin responses was greatly reduced in neurons from p85alpha (regulatory subunit of PI3K) null mice. These data strongly suggest that PI3K and MAPK pathways, but not the PLC pathway underlie the acute sensitization of TRPV1 by NGF.

  11. The novel PI3 kinase inhibitor, BAY 80-6946, impairs melanoma growth in vivo and in vitro.

    PubMed

    Schneider, Philine; Schön, Margarete; Pletz, Nadin; Seitz, Cornelia S; Liu, Ningshu; Ziegelbauer, Karl; Zachmann, Karolin; Emmert, Steffen; Schön, Michael P

    2014-08-01

    Due to its almost universal resistance to chemotherapy, metastasized melanoma remains a major challenge in clinical oncology. Given that phosphatidyl inositol-3 kinase (PI3K) activation in melanoma cells is associated with poor prognosis, disease progression and resistance to chemotherapy, the PI3K-Akt signalling pathway is a promising therapeutic target for melanoma treatment. We analysed six human melanoma cell lines for their constitutive activation of Akt and then tested two representative lines, A375 and LOX, for their susceptibility to PI3K-inhibition by the highly specific small molecule inhibitor, BAY 80-6946. In addition, the effect of BAY 80-6946 on A375 and LOX melanoma cells was assessed in vivo in a xenotransplantation mouse model. We provide experimental evidence that specifically inhibiting the PI3K pathway and phosphorylation of Akt by this novel compound results in antitumoral activities including inhibition of proliferation, induction of apoptosis and cell cycle arrest in vitro and in vivo. However, the susceptibility did not show a clear-cut pattern and differed between the melanoma cell lines tested, resulting in in vivo growth inhibition of A375 but not LOX melanoma cells. Thus, in some cases BAY 80-6946 or related compounds may be a valuable addition to the therapeutic armamentarium.

  12. PI3 Kinase Disease

    MedlinePlus

    ... investigate other treatment options, such as PI3K-specific drugs. Featured Research Treating PASLI Disease Watch an NIAID video about ... Translational Research Other Resources Clinical Agents Repository ... NIAID Contributes to New TB Drug With Broad Antibiotic Potential NIAID-Developed Technology Helps ...

  13. Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

    PubMed Central

    Baxter, Paul; Kassubek, Rebecca; Albrecht, Philipp; Van Liefferinge, Joeri; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Karpel-Massler, Georg; Meakin, Paul J.; Hayes, John D.; Aronica, Eleonora; Smolders, Ilse; Ludolph, Albert C.; Methner, Axel; Conrad, Marcus; Massie, Ann; Hardingham, Giles E.

    2014-01-01

    Abstract Aims: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc− imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc− and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. Results: PI3Ks induce system xc− through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. Innovation: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc−. Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. Conclusion: PI3K-regulated system xc− activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate. Antioxid. Redox Signal. 20: 2907–2922. PMID:24219064

  14. The Role of Phosphatidylinositol-3-Kinase and AMP-Activated Kinase in the Rapid Estrogenic Attenuation of Cannabinoid-Induced Changes in Energy Homeostasis

    PubMed Central

    Jeffery, Garrett S.; Peng, Kelly C.; Wagner, Edward J.

    2011-01-01

    We sought to determine the involvement of phosphatidyl inositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK) in the estrogenic antagonism of the cannabinoid regulation of energy homeostasis. Food intake and body weight were evaluated in ovariectomized female guinea pigs treated s.c. with estradiol benzoate (EB) or its sesame oil vehicle, or the CB1 receptor antagonist AM251 or its cremephor/ethanol/0.9% saline vehicle. AMPK catalytic subunit, PI3K p85α regulatory subunit and proopiomelanocortin (POMC) gene expression was assessed via quantitative RT-PCR in microdissected hypothalamic tissue. Whole-cell patch clamp recordings were performed in hypothalamic slices. Both EB and AM251 decreased food intake and weight gain, and increased AMPKα1, AMPKα2 and PI3K p85α gene expression in the mediobasal hypothalamus. 17β-Estradiol rapidly and markedly attenuated the decreases in glutamatergic miniature excitatory postsynaptic current (mEPSC) frequency caused by the cannabinoid receptor agonist WIN 55,212-2 in POMC neurons. This rapid estrogenic diminution of cannabinoid-induced decreases in mEPSC frequency was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and the PI3K inhibitor PI 828, the latter of which also prevented the AM251-induced increase in mEPSC frequency. In addition, the AMPK activator metformin reversed the EB-induced decreases in food intake and weight gain and restored the ability of WIN 55,212-2 to reduce mEPSC frequency. These data reveal that estrogens physiologically antagonize cannabinoid-induced changes in appetite and POMC neuronal activity by activating PI3K and inhibiting AMPK. As such, they provide insight into the neuroanatomical substrates and signal transduction mechanisms upon which these counter-regulatory factors converge in the control of energy homeostasis.

  15. Dexmedetomidine increases the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes: the involvement of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Do, Sang-Hwan; Park, Seong-Joo; Shin, Hyun-Jung; Paik, Hye-Sun; Zuo, Zhiyi; Yoon, Hea-Jo; Ryu, Jung-Hee

    2014-09-05

    Dexmedetomidine, an α2 adrenergic agonist, has neuroprotective and anticonvulsant properties in addition to its sedative and anxiolytic effects. We hypothesized that dexmedetomidine would increase the activity of excitatory amino acid transporter type 3 (EAAT3) and that this effect would involve protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two protein kinases known to regulate EAAT3 activity. EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after application of 30 μM l-glutamate in the presence of 0.1-30 nM dexmedetomidine. Dexmedetomidine-treated oocytes were also exposed to a PKC activator (phorbol-12-myristate-13-acetate [PMA]), PKC inhibitors (chelerythrine, staurosporine, and calphostin C), and PI3K inhibitors (wortmannin and LY294002) before current measurement. Dexmedetomidine application resulted in a concentration-dependent increase in the EAAT3 activity in response to l-glutamate. The kinetic study showed that dexmedetomidine significantly increased the Vmax without changing Km. Treatment of oocytes with PMA significantly increased transporter currents compared with controls, but treatment with dexmedetomidine plus PMA did not further increase the response compared with PMA or dexmedetomidine alone. In addition, pre-treatment of oocytes with PKC inhibitors and PI3K inhibitors significantly abolished the dexmedetomidine-enhanced EAAT3 activity. These results suggest that dexmedetomidine increases the activity of EAAT3 expressed in Xenopus oocytes. PKC and PI3K seem to mediate this effect. These findings may explain the neuroprotective and anticonvulsant effects of dexmedetomidine.

  16. Ellagic acid prevents rat colon carcinogenesis induced by 1, 2 dimethyl hydrazine through inhibition of AKT-phosphoinositide-3 kinase pathway.

    PubMed

    Umesalma, Syed; Sudhandiran, Ganapasam

    2011-06-25

    Colon cancer is the third most malignant neoplasm in the world and chemoprevention through dietary intervention is an emerging option to reduce its mortality. Ellagic acid (EA) a major component of berries possesses attractive biological deeds. This study is aimed to investigate the effect of ellagic acid in fostering apoptosis in 1,2-dimethyl hydrazine (DMH) mediated experimental colon carcinogenesis model. Wistar male rats were segregated into four groups: group I-control rats, group II-rats received ellagic acid (60 mg/kg body weight p.o. every day), rats in group III-induced with DMH (20 mg/kg body weight, s.c.) for 15 weeks, DMH-induced group IV rats were initiated with ellagic acid treatment. The present study is designed to explore the significance of phosphoinositide-3-kinase (PI3K)/Akt molecular pathway as well as ellagic acid's chemopreventive effect in colon cancer. DMH-induced rats exhibited elevated expressions of PI3K and Akt as confirmed by immunofluorescence, immunoblot and confocal microscopic analysis. Mechanistically, ellagic acid was found to prevent PI3K/Akt activation that in turn, results in modulation of its downstream Bcl-2 family proteins. Bax expression and caspase-3 activation was noted after ellagic acid supplementation leading to elevation of cytochrome c (cyt c) levels and finally cell death. These observations were supported by the DNA fragmentation results, which showed the occurrence of apoptosis. This study reveals the involvement of PI3K-Akt signaling through which ellagic acid induces apoptosis and subsequently suppresses colon cancer during DMH-induced rat colon carcinogenesis. In conclusion, our findings demonstrate that ellagic acid begets apoptosis in DMH-induced colon carcinoma.

  17. Phosphoinositide 3-kinase alpha-dependent regulation of branching morphogenesis in murine embryonic lung: evidence for a role in determining morphogenic properties of FGF7.

    PubMed

    Carter, Edward; Miron-Buchacra, Gabriela; Goldoni, Silvia; Danahay, Henry; Westwick, John; Watson, Malcolm L; Tosh, David; Ward, Stephen G

    2014-01-01

    Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.

  18. Modulation in Activation and Expression of PTEN, Akt1, and PDK1: Further Evidence Demonstrating Altered Phosphoinositide 3-kinase Signaling in Postmortem Brain of Suicide Subjects

    PubMed Central

    Dwivedi, Yogesh; Rizavi, Hooriyah S.; Zhang, Hui; Roberts, Rosalinda C.; Conley, Robert R.; Pandey, Ghanshyam N.

    2010-01-01

    Background Phosphoinositide 3-kinase (PI 3-K) signaling plays a crucial role in neuronal growth and plasticity. Recently, we demonstrated that suicide brain is associated with decreased activation and expression of selective catalytic and regulatory subunits of PI 3-K. The present investigation examined the regulation and functional significance of compromised PI 3-K in suicide brain at the level of upstream phosphatase and tensin homolog on chromosome ten (PTEN) and downstream substrates 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt. Method mRNA expression of Akt1, Akt3, PTEN, and PDK1 by competitive RT-PCR; protein expression of Akt1, Akt3, PTEN, PDK1, phosphorylated-Akt1 (Ser473), phosphorylated-Akt1(Thr308), phosphorylated-PDK1, and phosphorylated-PTEN by Western blot; and catalytic activities of Akt1, Akt3, and PDK1 by enzymatic assays were determined in prefrontal cortex (PFC) and hippocampus obtained from suicide subjects and nonpsychiatric controls. Results No significant changes in the expression of Akt1 or Akt3 were observed; however, catalytic activity of Akt1, but not of Akt3, was decreased in PFC and hippocampus of suicide subjects, which was associated with decreased phosphorylation of Akt1 at Ser473 and Thr308. The catalytic activity of PDK1 and the level of phosphorylated-PDK1 were also decreased in both brain areas without any change in expression levels of PDK1. On the other hand, mRNA and protein expression of PTEN was increased, whereas the level of phosphorylated-PTEN was decreased. Conclusion Our study demonstrates abnormalities in PI 3-K signaling at several levels in brain of suicide subjects and suggests the possible involvement of aberrant PI 3-K/Akt signaling in the pathogenic mechanisms of suicide. PMID:20163786

  19. Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex.

    PubMed

    Xia, Hui-Jun; Brearley, Charles; Elge, Stephan; Kaplan, Boaz; Fromm, Hillel; Mueller-Roeber, Bernd

    2003-02-01

    Inositol 1,4,5-trisphosphate 3-kinase, and more generally inositol polyphosphate kinases (Ipk), play important roles in signal transduction in animal cells; however, their functions in plant cells remain to be elucidated. Here, we report the molecular cloning of a cDNA (AtIpk2beta) from a higher plant, Arabidopsis. Arabidopsis AtIpk2beta is a 33-kD protein that exhibits weak homology ( approximately 25% identical amino acids) with Ipk proteins from animals and yeast and lacks a calmodulin binding site, as revealed by sequence analysis and calmodulin binding assays. However, recombinant AtIpk2beta phosphorylates inositol 1,4,5-trisphosphate to inositol 1,4,5,6-tetrakisphosphate and also converts it to inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P(5)]. AtIpk2beta also phosphorylates inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4,5,6)P(5). Thus, the enzyme is a D3/D6 dual-specificity inositol phosphate kinase. AtIpk2beta complements a yeast ARG82/IPK2 mutant lacking a functional ArgR-Mcm1 transcription complex. This complex is involved in regulating Arg metabolism-related gene expression and requires inositol polyphosphate kinase activity to function. AtIpk2beta was found to be located predominantly in the nucleus of plant cells, as demonstrated by immunolocalization and fusion to green fluorescent protein. RNA gel blot analysis and promoter-beta-glucuronidase reporter gene studies demonstrated AtIpk2beta gene expression in various organs tested. These data suggest a role for AtIpk2beta as a transcriptional control mediator in plants.

  20. Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling.

    PubMed

    Vijapurkar, Ulka; Kim, Myong-Soo; Koland, John G

    2003-04-01

    ErbB2/HER2 and ErbB3/HER3, two members of the ErbB/HER family, together constitute a heregulin coreceptor complex that elicits a potent mitogenic and transforming signal. Among known intracellular effectors of the ErbB2/ErbB3 heregulin coreceptor are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. Activation of the distinct MAPK and PI 3-kinase signaling pathways by the ErbB2/ErbB3 coreceptor in response to heregulin and their relative contributions to the mitogenic and transformation potentials of the activated coreceptor were investigated here. To this end, cDNAs encoding the wild-type ErbB3 protein (ErbB3-WT) and ErbB3 proteins with amino acid substitutions in either the Shc-binding site (ErbB3-Y1325F), the six putative PI 3-kinase-binding sites (ErbB3-6F), or both (ErbB3-7F) were generated and expressed in NIH-3T3 cells to form functional ErbB2/ErbB3 heregulin coreceptors. While the coreceptor incorporating ErbB3-WT activated both the MAPK and the PI 3-kinase signaling pathways, those incorporating ErbB3-Y1325F or ErbB3-6F activated either PI 3-kinase or MAPK, respectively. The ErbB2/ErbB3-7F coreceptor activated neither. Elimination of either signaling pathway lowered basal and eliminated heregulin-dependent expression of cyclin D1, which was in each case accompanied by an attenuated mitogenic response. Selective elimination of the PI 3-kinase pathway severely impaired the ability of heregulin to transform cells expressing the coreceptor, whereas attenuation of the MAPK pathway had a lesser effect. Thus, while both pathways contributed in a roughly additive manner to the mitogenic response elicited by the activated ErbB2/ErbB3 coreceptor, the PI 3-kinase pathway predominated in the induction of cellular transformation.

  1. Cortisol Response to an Induction of Negative Affect among Adolescents with and without Loss of Control Eating

    PubMed Central

    Kelly, Nichole R.; Pickworth, Courtney K.; Thompson, Katherine A.; Brady, Sheila M.; Demidowich, Andrew; Galescu, Ovidiu; Altschul, Anne M.; Shank, Lisa M.; Yanovski, Susan Z.; Tanofsky-Kraff, Marian; Yanovski, Jack A.

    2015-01-01

    Background Adults with binge-eating disorder may have an exaggerated or blunted cortisol response to stress. Yet, limited data exist among youth who report loss of control (LOC) eating, a developmental precursor to binge-eating disorder. Methods We studied cortisol reactivity among 178 healthy adolescents with and without LOC eating. Following a buffet lunch meal adolescents were randomly assigned to watch a neutral or sad film clip. After, they were offered snacks from a multi-item array to assess eating in the absence of hunger. Salivary cortisol was collected at −80, 0, 30, and 50 minutes relative to film administration, and state mood ratings were reported before and after the film. Results Adolescents with LOC had greater increases in negative affect during the experimental paradigm in both conditions (ps>.05). Depressive symptoms, but not LOC, related to a greater cortisol response in the sad film condition (ps>.05). Depressive symptoms and state LOC were related to different aspects of eating behavior, independent of film condition or cortisol response (ps>.05). Conclusions A film clip that induced depressed state affect increased salivary cortisol only in adolescents with more elevated depressive symptoms. Adolescents with and without LOC were differentiated by greater increases in state depressed affect during laboratory test meals, but had no difference in cortisol reactivity. Future studies are required to determine if adolescents with LOC manifest alterations in stress reactivity to alternative stress-inducing situations. PMID:26667312

  2. Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

    SciTech Connect

    Gaur, Naseem Akhtar; Manoharlal, Raman; Saini, Preeti; Prasad, Tulika; Mukhopadhyay, Gauranga; Hoefer, Milan; Morschhaeuser, Joachim; Prasad, Rajendra . E-mail: rp47@hotmail.com

    2005-06-24

    Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.

  3. Sensitivity improvement of an electrical sensor achieved by control of biomolecules based on the negative dielectrophoretic force.

    PubMed

    Kim, Hye Jin; Kim, Jinsik; Yoo, Yong Kyoung; Lee, Jeong Hoon; Park, Jung Ho; Hwang, Kyo Seon

    2016-11-15

    Effective control of nano-scale biomolecules can enhance the sensitivity and limit of detection of an interdigitated microelectrode (IME) sensor. Manipulation of the biomolecules by dielectrophoresis (DEP), especially the negative DEP (nDEP) force, so that they are trapped between electrodes (sensing regions) was predicted to increase the binding efficiency of the antibody and target molecules, leading to a more effective reaction. To prove this concept, amyloid beta 42 (Aβ42) and prostate specific antigen (PSA) protein were respectively trapped between the sensing region owing to the nDEP force under 5V and 0.05V, which was verified with COMSOL simulation. Using the simulation value, the resistance change (ΔR/Rb) of the IME sensor from the specific antibody-antigen reaction of the two biomolecules and the change in fluorescence intensity were compared in the reference (pDEP) and nDEP conditions. The ΔR/Rb value improved by about 2-fold and 1.66-fold with nDEP compared to the reference condition with various protein concentrations, and these increases were confirmed with fluorescence imaging. Overall, nDEP enhanced the detection sensitivity for Aβ42 and PSA by 128% and 258%, respectively, and the limit of detection improved by up to 2-orders of magnitude. These results prove that DEP can improve the biosensor's performance.

  4. An Nkx2-5/Bmp2/Smad1 negative feedback loop controls second heart field progenitor specification and proliferation

    PubMed Central

    Prall, Owen WJ; Menon, Mary K; Solloway, Mark J; Watanabe, Yusuke; Zaffran, Stéphane; Bajolle, Fanny; Biben, Christine; McBride, Jim J; Robertson, Bronwyn R; Chaulet, Hervé; Stennard, Fiona A; Wise, Natalie; Schaft, Daniel; Wolstein, Orit; Furtado, Milena B; Shiratori, Hidetaka; Chien, Kenneth R; Hamada, Hiroshi; Black, Brian L; Saga, Yumiko; Robertson, Elizabeth J; Buckingham, Margaret E; Harvey, Richard P

    2007-01-01

    Summary During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were up-regulated, leading initially to progenitor over-specification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD. PMID:17350578

  5. Stereo and photometric image sequence interpretation for detecting negative obstacles using active gaze control and performing an autonomous jink

    NASA Astrophysics Data System (ADS)

    Hofmann, Ulrich; Siedersberger, Karl-Heinz

    2003-09-01

    Driving cross-country, the detection and state estimation relative to negative obstacles like ditches and creeks is mandatory for safe operation. Very often, ditches can be detected both by different photometric properties (soil vs. vegetation) and by range (disparity) discontinuities. Therefore, algorithms should make use of both the photometric and geometric properties to reliably detect obstacles. This has been achieved in UBM's EMS-Vision System (Expectation-based, Multifocal, Saccadic) for autonomous vehicles. The perception system uses Sarnoff's image processing hardware for real-time stereo vision. This sensor provides both gray value and disparity information for each pixel at high resolution and framerates. In order to perform an autonomous jink, the boundaries of an obstacle have to be measured accurately for calculating a safe driving trajectory. Especially, ditches are often very extended, so due to the restricted field of vision of the cameras, active gaze control is necessary to explore the boundaries of an obstacle. For successful measurements of image features the system has to satisfy conditions defined by the perception expert. It has to deal with the time constraints of the active camera platform while performing saccades and to keep the geometric conditions defined by the locomotion expert for performing a jink. Therefore, the experts have to cooperate. This cooperation is controlled by a central decision unit (CD), which has knowledge about the mission and the capabilities available in the system and of their limitations. The central decision unit reacts dependent on the result of situation assessment by starting, parameterizing or stopping actions (instances of capabilities). The approach has been tested with the 5-ton van VaMoRs. Experimental results will be shown for driving in a typical off-road scenario.

  6. Negative Affect Prior to and Following Overeating-Only, Loss of Control Eating-Only, and Binge Eating Episodes in Obese Adults

    PubMed Central

    Berg, Kelly C.; Crosby, Ross D.; Cao, Li; Crow, Scott J.; Engel, Scott G.; Wonderlich, Stephen A.; Peterson, Carol B.

    2015-01-01

    Objective The objective was to examine the trajectory of five types of negative affect (global negative affect, fear, guilt, hostility, sadness) prior to and following three types of eating episodes (overeating in the absence of loss of control [OE-only], loss of control eating in the absence of overeating [LOC-only], and binge eating) among obese adults using ecological momentary assessment (EMA). Method Fifty obese adults (84% female) completed a two-week EMA protocol during which they were asked to record all eating episodes and rate each episode on continua of overeating and loss of control. Momentary measures of global negative affect, fear, guilt, hostility, and sadness were assessed using an abbreviated version of the Positive and Negative Affect Schedule (PANAS). Trajectories for each of the five types of negative affect were modeled prior to and following episodes of OE-only, LOC-only, and binge eating. Results Consistent with previous findings, global negative affect and Guilt increased prior to and decreased following binge eating episodes (all ps<.05). Guilt also decreased following OE-only episodes (p<.05). Discussion These results are consistent with the affect regulation model of binge eating and suggest that binge eating may function to regulate global negative affect, and more specifically, guilt among obese adults. These data suggest that the relationship between negative affect and binge eating may not be unique to individuals with clinical eating disorders and indicate that targeting negative affect may be an effective strategy for the treatment of binge eating in the context of obesity. PMID:25808854

  7. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

  8. Negative Emotions and Alcohol Use Initiation in High-Risk Boys: The Moderating Effect of Good Inhibitory Control

    ERIC Educational Resources Information Center

    Pardini, Dustin; Lochman, John; Wells, Karen

    2004-01-01

    Studies on the relation between negative affect and later alcohol use have provided mixed results. Because definitions of negative affect often include diverse emotions, researchers have begun to dismantle this higher-order construct in an attempt to explain these inconsistent findings. More recent evidence also indicates that good inhibitory…

  9. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    PubMed

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

  10. Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

    PubMed Central

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

  11. A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica.

    PubMed

    Koushik, Amrita B; Welter, Brenda H; Rock, Michelle L; Temesvari, Lesly A

    2014-03-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

  12. Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or phosphatidylinositol 3 kinase.

    PubMed

    DeMali, K A; Whiteford, C C; Ulug, E T; Kazlauskas, A

    1997-04-04

    Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) beta receptor mutants (beta-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the beta-PDGFR but no PDGF-alpha receptor (alpha-PDGFR). Hence, this cell system can be used to study signaling of mutant alphaPDGFRs or alpha/beta chimeras. We constructed chimeric receptors containing the alphaPDGFR extracellular domain and the betaPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type beta-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-gamma or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-gamma are key initiators of such signal relay cascades.

  13. Promotion of melanoma cell invasion and tumor metastasis by microcystin-LR via phosphatidylinositol 3-kinase/AKT pathway.

    PubMed

    Xu, Pengfei; Zhang, Xu-Xiang; Miao, Chen; Fu, Ziyi; Li, Zhengrong; Zhang, Gen; Zheng, Maqing; Liu, Yuefang; Yang, Liuyan; Wang, Ting

    2013-08-06

    Recently, we have indicated that microcystin-LR, a cyanobacterial toxin produced in eutrophic lakes or reservoirs, can increase invasive ability of melanoma MDA-MB-435 cells; however, the stimulatory effect needs identification by in vivo experiment and the related molecular mechanism is poorly understood. In this study, in vitro and in vivo experiments were conducted to investigate the effect of microcystin-LR on invasion and metastasis of human melanoma cells, and the underlying molecular mechanism was also explored. MDA-MB-435 xenograft model assay showed that oral administration of nude mice with microcystin-LR at 0.001-0.1 mg/kg/d posed no significant effect on tumor weight. Histological examination demonstrated that microcystin-LR could promote lung metastasis, which is confirmed by Matrigel chamber assay suggesting that microcystin-LR treatment at 25 nM can increase the invasiveness of MDA-MB-435 cells. In vitro and in vivo experiments consistently showed that microcystin-LR exposure increased mRNA and protein levels of matrix metalloproteinases (MMP-2/-9) by activating phosphatidylinositol 3-kinase (PI3-K)/AKT. Additionally, microcystin-LR treatment at low doses (≤25 nM) decreased lipid phosphatase PTEN expression, and the microcystin-induced invasiveness enhancement and MMP-2/-9 overexpression were reversed by the PI3-K/AKT chemical inhibitor LY294002 and AKT siRNA, indicating that microcystin-LR promotes invasion and metastasis of MDA-MB-435 cells via the PI3-K/AKT pathway.

  14. Investigation into the Role of PI3K and JAK3 Kinase Inhibitors in Murine Models of Asthma.

    PubMed

    Wagh, Akshaya D; Sharma, Manoranjan; Mahapatra, Jogeshwar; Chatterjee, Abhijeet; Jain, Mukul; Addepalli, Veeranjaneyulu

    2017-01-01

    Asthma is a clinical disorder commonly characterized by chronic eosinophilic inflammation, remodeling and hyper responsiveness of the airways. However, the kinases like Phosphoinositide 3 kinase (PI3K) and Janus kinase 3 (JAK3) are involved in mast cell proliferation, activation, recruitment, migration, and prolonged survival of inflammatory cells. The present study was designed to evaluate the in-vivo comparative effects of two kinase inhibitors on airway inflammation and airway remodeling in acute and chronic models of asthma. Mice were sensitized twice intra-peritoneally and then challenged by inhalation with ovalbumin (OVA). They developed an extensive inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening similar to pathologies observed in human asthma. The effects of PI3K inhibitor (30 mg/kg, p.o), JAK3 inhibitor (30 mg/kg, p.o) and Dexamethasone (0.3 mg/kg) on airway inflammation and remodeling in OVA sensitized/challenged BALB/c mice were evaluated. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage (BAL) and histological examinations were carried out. It was observed that kinase inhibitors significantly reduced airway inflammation as evidenced by the decrease in pro inflammatory cytokines in BALF and lung homogenate and inflammatory cell count in sensitized mice after allergen challenge. Lung histological analysis showed increased infiltration of inflammatory cells, hyperplasia of goblet cells and the collagen deposition, which were significantly reduced with kinase inhibitor. In conclusion, our data suggest that PI3K and JAK3 inhibitors showed promising alternative therapeutic activity in asthma, which might significantly counteract the airway inflammation in patients with allergic asthma.

  15. Identification of upregulated phosphoinositide 3-kinase γ as a target to suppress breast cancer cell migration and invasion

    PubMed Central

    Xie, Yan; Abel, Peter W.; Kirui, Joseph K.; Deng, Caishu; Sharma, Poonam; Wolff, Dennis W.; Toews, Myron L.; Tu, Yaping

    2013-01-01

    Metastasis is the major cause of breast cancer mortality. We recently reported that aberrant G-protein coupled receptor (GPCR) signaling promotes breast cancer metastasis by enhancing cancer cell migration and invasion. Phosphatidylinositol 3-kinase γ (PI3Kγ) is specifically activated by GPCRs. The goal of the present study was to determine the role of PI3Kγ in breast cancer cell migration and invasion. Immunohistochemical staining showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma when compared to adjacent benign breast tissue or ductal carcinoma in situ. PI3Kγ was also detected in metastatic breast cancer cells, but not in normal breast epithelial cell line or in non-metastatic breast cancer cells. In contrast, PI3K isoforms α, β and δ were ubiquitously expressed in these cell lines. Overexpression of recombinant PI3Kγ enhanced the metastatic ability of non-metastatic breast cancer cells. Conversely, migration and invasion of metastatic breast cancer cells were inhibited by a PI3Kγ inhibitor or by siRNA knockdown of PI3Kγ but not by inhibitors or siRNAs of PI3Kα or PI3Kβ. Lamellipodia formation is a key step in cancer metastasis, and PI3Kγ blockade disrupted lamellipodia formation induced by the activation of GPCRs such as CXC chemokine receptor 4 and protease-activated receptor 1, but not by the epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that upregulated PI3Kγ conveys the metastatic signal initiated by GPCRs in breast cancer cells, and suggest that PI3Kγ may be a novel therapeutic target for development of chemotherapeutic agents to prevent breast cancer metastasis. PMID:23500535

  16. Phosphatidylinositol-3-kinase pathway aberrations in gastric and colorectal cancer: meta-analysis, co-occurrence and ethnic variation.

    PubMed

    Chong, Mei-Ling; Loh, Marie; Thakkar, Bhavin; Pang, Brendan; Iacopetta, Barry; Soong, Richie

    2014-03-01

    Inhibition of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a cancer treatment strategy that has entered into clinical trials. We performed a meta-analysis on the frequency of prominent genetic (PIK3CA mutation, PIK3CA amplification and PTEN deletion) and protein expression (high PI3K, PTEN loss and high pAkt) aberrations in the PI3K pathway in gastric cancer (GC) and colorectal cancer (CRC). We also performed laboratory analysis to investigate the co-occurrence of these aberrations. The meta-analysis indicated that East Asian and Caucasian GC patients differ significantly for the frequencies of PIK3CA Exon 9 and 20 mutations (7% vs. 15%, respectively), PTEN deletion (21% vs. 4%) and PTEN loss (47% vs. 78%), while CRC patients differed for PTEN loss (57% vs. 26%). High study heterogeneity (I(2) > 80) was observed for all aberrations except PIK3CA mutations. Laboratory analysis of tumors from East Asian patients revealed significant differences between GC (n = 79) and CRC (n = 116) for the frequencies of PIK3CA amplification (46% vs. 4%) and PTEN loss (54% vs. 78%). The incidence of GC cases with 0, 1, 2 and 3 concurrent aberrations was 14%, 52%, 27% and 8%, respectively, while for CRC it was 10%, 60%, 25% and 4%, respectively. Our study consolidates knowledge on the frequency, co-occurrence and clinical relevance of PI3K pathway aberrations in GC and CRC. Up to 86% of GC and 90% of CRC have at least one aberration in the PI3K pathway, and there are significant differences in the frequencies of these aberrations according to cancer type and ethnicity.

  17. Euphorbia fischeriana Steud inhibits malignant melanoma via modulation of the phosphoinositide-3-kinase/Akt signaling pathway

    PubMed Central

    DONG, MENG-HUA; ZHANG, QIAN; WANG, YUAN-YUAN; ZHOU, BAI-SUI; SUN, YU-FEI; FU, QIANG

    2016-01-01

    Euphorbia fischeriana Steud, a traditional Chinese medicine, has been shown to inhibit the growth of various cancers by the induction of apoptosis and cell cycle arrest. The purpose of the present study was to investigate the association between the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and the inhibitory effect of Euphorbia fischeriana Steud on the growth and metastasis of melanoma B16 cells in vitro, and the underlying mechanisms. MTT assay results indicated that Euphorbia fischeriana Steud inhibited the growth of B16 cells in a time- and dose-dependent manner. Flow cytometric analysis revealed that Euphorbia fischeriana Steud markedly induced apoptosis of the B16 cells, with arrest at the G0/G1 phase of the cell cycle. In addition, in a Transwell assay Euphorbia fischeriana Steud significantly suppressed the migration of B16 cells. Western blot analysis revealed that the expression levels of phosphatase and tensin homolog (PTEN) were upregulated, and the phosphorylation of Akt was downregulated, which resulted in inhibition of the PI3K/Akt signaling pathway and the eventual suppression of its downstream targets, such as matrix metalloproteinase-2 mRNA, in B16 cells. The results demonstrated that Euphorbia fischeriana Steud inhibited the growth and migration of B16 cells, possibly via modulation of the PI3K/Akt signaling pathway and upregulation of PTEN expression levels, in addition to downregulation of p-Akt expression. The aforementioned findings suggest that Euphorbia fischeriana Steud may have broad therapeutic applications in the treatment of malignant melanoma. PMID:27073468

  18. Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity.

    PubMed

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination.

  19. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

    PubMed

    Campbell, Paul M; Groehler, Angela L; Lee, Kwang M; Ouellette, Michel M; Khazak, Vladimir; Der, Channing J

    2007-03-01

    Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

  20. Involvement of PI 3 kinase/Akt-dependent Bad phosphorylation in Toxoplasma gondii-mediated inhibition of host cell apoptosis.

    PubMed

    Quan, Juan-Hua; Cha, Guang-Ho; Zhou, Wei; Chu, Jia-Qi; Nishikawa, Yoshifumi; Lee, Young-Ha

    2013-04-01

    Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.

  1. Thrombopoietin enhances the alpha IIb beta 3-dependent adhesion of megakaryocytic cells to fibrinogen or fibronectin through PI 3 kinase.

    PubMed

    Zauli, G; Bassini, A; Vitale, M; Gibellini, D; Celeghini, C; Caramelli, E; Pierpaoli, S; Guidotti, L; Capitani, S

    1997-02-01

    The effect of thrombopoietin (TPO) on the functional activity of surface alpha IIb beta 3 (GPIIbIIIa) was investigated in both primary human megakaryocytic cells, derived from peripheral blood CD34+ cells, and HEL hematopoietic cell line. TPO (100 ng/mL) induced a sixfold to ninefold enhancement of adhesion of both primary megakaryocytic and HEL cells to plates coated with either fibrinogen or fibronectin and a parallel increase of immunoreactivity to the PAC1 monoclonal antibody (MoAb) and fluorescein isothiocyanate-fibrinogen, both of which recognize an activated state of alpha IIb beta 3. The enhanced adhesion to fibrinogen or fibronectin was mediated by the Arg-Gly-Asp (RGD) recognition sequence of alpha IIb beta 3, as it was abolished by pretreatment of cells with saturating concentrations of RGDS peptide. A MoAb specific for the alpha IIb beta subunit of alpha IIb beta 3 also inhibited cell attachment to fibrinogen or fibronectin, while MoAb to anti-alpha v beta 3 or anti-alpha 5 integrins were completely ineffective, clearly indicating that alpha IIb beta 3 participates in this association. A role for PI 3 kinase (PI 3-K) in the TPO-mediated increase in alpha IIb beta 3 function in megakaryocytic cells was suggested by the ability of the PI 3-K inhibitor wortmannin (100 nmol/L) and antisense oligonucleotides directed against the p85 regulatory subunit of PI 3-K to completely block the TPO-induced increase in alpha IIb beta 3 integrin activity upon TPO stimulation. The modulation of adhesiveness to extracellular matrix proteins containing the RGD motif mediated by TPO likely plays a physiologic role in megakaryocytopoiesis, as pretreatment of CD34+ cells with RGDS or anti-alpha IIb MoAb significantly reduced the number of megakaryocytic colonies obtained in a fibrinclot semisolid assay.

  2. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    PubMed

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA.

  3. Investigation into the Role of PI3K and JAK3 Kinase Inhibitors in Murine Models of Asthma

    PubMed Central

    Wagh, Akshaya D.; Sharma, Manoranjan; Mahapatra, Jogeshwar; Chatterjee, Abhijeet; Jain, Mukul; Addepalli, Veeranjaneyulu

    2017-01-01

    Asthma is a clinical disorder commonly characterized by chronic eosinophilic inflammation, remodeling and hyper responsiveness of the airways. However, the kinases like Phosphoinositide 3 kinase (PI3K) and Janus kinase 3 (JAK3) are involved in mast cell proliferation, activation, recruitment, migration, and prolonged survival of inflammatory cells. The present study was designed to evaluate the in-vivo comparative effects of two kinase inhibitors on airway inflammation and airway remodeling in acute and chronic models of asthma. Mice were sensitized twice intra-peritoneally and then challenged by inhalation with ovalbumin (OVA). They developed an extensive inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening similar to pathologies observed in human asthma. The effects of PI3K inhibitor (30 mg/kg, p.o), JAK3 inhibitor (30 mg/kg, p.o) and Dexamethasone (0.3 mg/kg) on airway inflammation and remodeling in OVA sensitized/challenged BALB/c mice were evaluated. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage (BAL) and histological examinations were carried out. It was observed that kinase inhibitors significantly reduced airway inflammation as evidenced by the decrease in pro inflammatory cytokines in BALF and lung homogenate and inflammatory cell count in sensitized mice after allergen challenge. Lung histological analysis showed increased infiltration of inflammatory cells, hyperplasia of goblet cells and the collagen deposition, which were significantly reduced with kinase inhibitor. In conclusion, our data suggest that PI3K and JAK3 inhibitors showed promising alternative therapeutic activity in asthma, which might significantly counteract the airway inflammation in patients with allergic asthma. PMID:28293189

  4. Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor

    SciTech Connect

    Van Aller, Glenn S.; Carson, Jeff D.; Tang, Wei; Peng, Hao; Zhao, Lin; Copeland, Robert A.; Tummino, Peter J.; Luo, Lusong

    2011-03-11

    Research highlights: {yields} Epigallocatechin-3-gallate (EGCG) is an ATP-competitive inhibitor of PI3K and mTOR with Ki values around 300 nM. {yields} EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231and A549 cells. {yields} Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site. {yields} These results suggest another important molecular mechanism for the anticancer activities of EGCG. -- Abstract: The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K{sub i} values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

  5. Phosphatidylinositol 3-kinase, MEK-1 and p38 mediate leptin/interferon-gamma synergistic NOS type II induction in chondrocytes.

    PubMed

    Otero, Miguel; Lago, Rocío; Gómez, Rodolfo; Lago, Francisca; Gomez-Reino, Juan Jesús; Gualillo, Oreste

    2007-10-27

    In a previous study, we established that leptin acts synergistically with interferon-gamma in inducing nitric oxide synthase type II in cultured chondrocytes via Janus kinase-2 activation. However, the exact molecular mechanism that accounts for this synergism is not completely understood. The aim of the present study was to further delineate the signalling pathway used by leptin/interferon-gamma in the nitric oxide synthase type II induction in chondrocytes. Consequently, the roles of PI-3 kinase, MEK1 and p38 kinase were investigated using specific pharmacological inhibitors (Wortmannin, LY 294002, PD 098,059 and SB 203580). For this purpose, the amount of stable nitrite, the end product of NO generation by activated chondrocytes, has been evaluated by Griess colorimetric reaction in culture medium of human primary chondrocytes and in the murine ATDC5 cell line stimulated with leptin (400 nM) and interferon-gamma (1 ng/ml), alone or in combination. Specific inhibitors for PI-3K, MEK1 and p38 were added 1 h before stimulation. Nitric oxide synthase type II mRNA was investigated by real-time RT-PCR and NOS type II protein expression has been evaluated by western blot analysis. Our results showed that, as expected, leptin synergizes with IFN-gamma in inducing NO accumulation in the supernatant of co-stimulated cells. Pre-treatment with Wortmannin, LY 294002, PD 098,059 and SB 203580 caused a significant decrease in nitrite production, NOS type II protein expression and NOS type II mRNA expression induced by leptin and interferon-gamma co-stimulation. These findings were confirmed in 15 and 21-day differentiated ATDC5 cells, and in normal human primary chondrocytes. This is the first report showing that NOS type II induction triggered by co-stimulation with leptin and interferon-gamma is mediated by a signaling pathway involving PI-3K, MEK1 and p38.

  6. Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization.

    PubMed

    Zhuang, Zhi-Ye; Xu, Haoxing; Clapham, David E; Ji, Ru-Rong

    2004-09-22

    Although the PI3K (phosphatidylinositol 3-kinase) pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in neural plasticity. It is unknown whether the PI3K pathway can mediate pain hypersensitivity. Intradermal injection of capsaicin and NGF produce heat hyperalgesia by activating their respective TRPV1 (transient receptor potential vanilloid receptor-1) and TrkA receptors on nociceptor sensory nerve terminals. We examined the activation of PI3K in primary sensory DRG neurons by these inflammatory agents and the contribution of PI3K activation to inflammatory pain. We further investigated the correlation between the PI3K and the ERK (extracellular signal-regulated protein kinase) pathway. Capsaicin and NGF induce phosphorylation of the PI3K downstream target AKT (protein kinase B), which is blocked by the PI3K inhibitors LY294002 and wortmannin, indicative of the activation of PI3K by both agents. ERK activation by capsaicin and NGF was also blocked by PI3K inhibitors. Similarly, intradermal capsaicin in rats activated PI3K and ERK in C-fiber DRG neurons and epidermal nerve fibers. Injection of PI3K or MEK (ERK kinase) inhibitors into the hindpaw attenuated capsaicin- and NGF-evoked heat hyperalgesia but did not change basal heat sensitivity. Furthermore, PI3K, but not ERK, inhibition blocked early induction of hyperalgesia. In acutely dissociated DRG neurons, the capsaicin-induced TRPV1 current was strikingly potentiated by NGF, and this potentiation was completely blocked by PI3K inhibitors and primarily suppressed by MEK inhibitors. Therefore, PI3K induces heat hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent manner. The PI3K pathway also appears to play a role that is distinct from ERK by regulating the early onset of inflammatory pain.

  7. PI3-kinase signaling contributes to orientation in shallow gradients and enhances speed in steep chemoattractant gradients.

    PubMed

    Bosgraaf, Leonard; Keizer-Gunnink, Ineke; Van Haastert, Peter J M

    2008-11-01

    Dictyostelium cells that chemotax towards cAMP produce phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] at the leading edge, which has been implicated in actin reorganization and pseudopod extension. However, in the absence of PtdIns(3,4,5)P(3) signaling, cells will chemotax via alternative pathways. Here we examined the potential contribution of PtdIns(3,4,5)P(3) to chemotaxis of wild-type cells. The results show that steep cAMP gradients (larger than 10% concentration difference across the cell) induce strong PtdIns(3,4,5)P(3) patches at the leading edge, which has little effect on the orientation but strongly enhances the speed of the cell. Using a new sensitive method for PtdIns(3,4,5)P(3) detection that corrects for the volume of cytosol in pixels at the boundary of the cell, we show that, in shallow cAMP gradient (less than 5% concentration difference across the cell), PtdIns(3,4,5)P(3) is still somewhat enriched at the leading edge. Cells lacking PI3-kinase (PI3K) activity exhibit poor chemotaxis in these shallow gradients. Owing to the reduced speed and diminished orientation of the cells in steep and shallow gradients, respectively, cells lacking PtdIns(3,4,5)P(3) signaling require two- to six-fold longer times to reach a point source of chemoattractant compared with wild-type cells. These results show that, although PI3K signaling is dispensable for chemotaxis, it gives the wild type an advantage over mutant cells.

  8. Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation.

    PubMed

    Diez, Héctor; Benitez, Ma José; Fernandez, Silvia; Torres-Aleman, Ignacio; Garrido, Juan José; Wandosell, Francisco

    2016-11-01

    PI3K proteins family have multiple and essential functions in most cellular events. This family is composed of class I, class II and class III PI3Ks, which upstream and downstream elements are not completely elucidated. Previous studies using the broad PI3K inhibitor, LY294002 allowed to propose that PI3 kinase>Akt pathway is a key element in the determination of axonal polarity in hippocampal neurons. Recently, new inhibitors with a higher selectivity for class I PI3K have been characterized. In the present study we have examined this widely accepted theory using a new class I PI3K inhibitor (GDC-0941), as well as Akt inhibitors, and PTEN phosphatase constructs to reduce PIP3 levels. Our present data show that both, class I PI3K inhibitor and Akt inhibitor did not alter axon specification in hippocampal neurons, but greatly reduced axon length. However, in the same experiments LY294002 effectively impeded axonal polarization, as previously reported. Our biochemical data show that both, class I PI3K and Akt inhibitors, effectively block downstream elements from Akt to S6K1 activity. Both inhibitors are stable in culture medium along the time period analysed, maintaining the inhibition better than LY294002. Besides, we found evidence that LY294002 directly inhibits mTORC1. However, further analysis using an mTORC1 inhibitor showed no change in neuron polarity. Same result was obtained using a general class III PI3K inhibitor. Interestingly, we found that either, wild-type PTEN, or a phosphatase-dead form of PTEN, disrupted axonal polarization, strongly suggesting that the role of PTEN in axonal polarity can be independent of PIP3.

  9. Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

    PubMed

    Göckeritz, Elisa; Kerwien, Susan; Baumann, Michael; Wigger, Marion; Vondey, Verena; Neumann, Lars; Landwehr, Thomas; Wendtner, Clemens M; Klein, Christian; Liu, Ningshu; Hallek, Michael; Frenzel, Lukas P; Krause, Günter

    2015-11-01

    Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

  10. Biodiversity vs. biocontrol: positive and negative effects of alternative prey on control of slugs by carabid beetles.

    PubMed

    Symondson, W O C; Cesarini, S; Dodd, P W; Harper, G L; Bruford, M W; Glen, D M; Wiltshire, C W; Harwood, J D

    2006-12-01

    Environment-friendly farming techniques seek to increase invertebrate biodiversity in part with the intention of encouraging greater numbers of predators that will help to control crop pests. However, in theory, this effect may be negated if the availability of a greater abundance and diversity of alternative prey diverts predators away from feeding on pests. The hypothesis that access to alternative prey can lead to reduced pest suppression under semi-field conditions was tested. Alternative prey type and diversity were manipulated in 70 mesocosms over 7+ weeks in the presence of the carabid Pterostichus melanarius (Illiger), a known predator of slugs, and reproducing populations of the slug Deroceras reticulatum (Müller). Significantly fewer slugs survived where no alternative prey were provided. Maximum slug numbers and biomass were found in treatments containing either carabids plus a high diversity of alternative prey (many species of earthworm and three of Diptera larvae) or a single additional prey (blowfly larvae, Calliphora vomitoria Linnaeus). In these treatments slug numbers and biomass were as high as in plots lacking predators. The effects of alternative prey were taxon-specific. Alternative prey strongly affected carabid fitness in terms of biomass and egg load. The fittest predators (those with access to high alternative prey diversity or C. vomitoria larvae) reduced slug numbers the least. The mean individual slug weights were greater in treatments with alternative prey than where no alternative prey were provided to the carabids. These results suggest that pests may survive and reproduce more rapidly in patches where predators have access to alternative prey.

  11. Thai Negation.

    ERIC Educational Resources Information Center

    Alam, Samsul

    A study analyzed the structure of negative sentences in the Thai language, based on data gathered from two native speakers. It is shown that the Thai negative marker generally occurs between the noun phrase (subject) and the verb phrase in simple active sentences and in passive sentences. Negation of noun phrases is also allowed in Thai, with a…

  12. The roles of internal locus of control and neighborhood affluence in predicting the continuity of negative self-feelings from adolescence to young adulthood.

    PubMed

    Pals, Heili; Kaplan, Howard B

    2013-10-01

    The present study examines the moderating effect of locus of control on the continuity in negative self-feelings from adolescence to young adulthood. We use longitudinal data of 1296 respondents from adolescence (11-13 years old) to young adulthood (20-24 years old). Using interaction effects in linear regression with robust standard errors we find that those with an internal locus of control experience stronger continuity in negative self-feelings from adolescence to young adulthood. Furthermore, based on both self-reported and objective measures of neighborhood status, internal locus of control increases the continuity in negative self-feelings in the transition to adulthood in disadvantaged neighborhoods but not in advantaged neighborhoods. Thus, locus of control acts as a maladaptive mechanism in less affluent neighborhoods. This result demonstrates the importance of considering both the individual social psychological and the contextual sociological perspectives when investigating the role of internal locus of control on the perpetuation of negative self-feelings.

  13. Concordance of self- and observer-rated motivation and pleasure in patients with negative symptoms and healthy controls.

    PubMed

    Engel, Maike; Lincoln, Tania M

    2017-01-01

    This study examined the validity of using a self-rating scale for assessing the motivation and pleasure domain of negative symptoms in the general population by examining the concordance of self- and observer-rated negative symptoms in a healthy sample and by comparing it with a patient sample. The motivation and pleasure domain of negative symptoms was assessed using the observer-rated Clinical Assessment Interview for Negative Symptoms (CAINS) and the self-rated Motivation and Pleasure Scale-Self-Report (MAP-SR). We found 52.9% of the healthy individuals and 46% of the patients to have relatively equal self- and observer-ratings. Despite the absence of extreme discrepancies, 31.4% of the healthy individuals and 14% of the patients rated their negative symptoms as more severe, whereas 15.7% of the healthy individuals and 40% of the patients rated them as less severe than the observers. By using self-ratings in combination with observer-ratings, possible discrepancies can be uncovered, which may be relevant for the successful treatment of negative symptoms.

  14. A new method with flexible and balanced control of false negatives and false positives for hit selection in RNA interference high-throughput screening assays.

    PubMed

    Zhang, Xiaohua Douglas

    2007-08-01

    The z-score method and its variants for testing mean difference are commonly used for hit selection in high-throughput screening (HTS) assays. Strictly standardized mean difference (SSMD) offers a way to measure and classify the short interfering RNA (siRNA) effects. In this article, based on SSMD, the authors propose a new testing method for hit selection in RNA interference (RNAi) HTS assays. This SSMD-based method allows the differentiation between siRNAs with large and small effects on the assay output and maintains flexible and balanced control of both the false-negative rate, in which the siRNAs with strong effects are not selected as hits, and the restricted false-positive rate, in which the siRNAs with weak or no effects are selected as hits. This method directly addresses the size of siRNA effects represented by the strength of difference between an siRNA and a negative reference, whereas the classic z-score method and t-test of testing no mean difference address whether the mean of an siRNA is exactly the same as the mean of a negative reference. This method can readily control the false-negative rate, whereas it is nontrivial for the classic z-score method and t-test to control the false-negative rate. Therefore, theoretically, the SSMD-based method offers better control of the sizes of siRNA effects and the associated false-positive and false-negative rates than the commonly used z-score method and t-test for hit selection in HTS assays. The SSMD-based method should generally be applicable to any assay in which the end point is a difference in signal compared to a reference sample, including those for RNAi, receptor, enzyme, and cellular function.

  15. Predictors of Change Following Cognitive-Behavioral Treatment of Children with Anxiety Problems: A Preliminary Investigation on Negative Automatic Thoughts and Anxiety Control

    ERIC Educational Resources Information Center

    Muris, Peter; Mayer, Birgit; den Adel, Madelon; Roos, Tamara; van Wamelen, Julie

    2009-01-01

    The purpose of the present study was to evaluate negative automatic thoughts and anxiety control as predictors of change produced by cognitive-behavioral treatment of youths with anxiety disorders. Forty-five high-anxious children aged between 9 and 12 years who were selected from the primary school population, received a standardized CBT…

  16. NEGATIVE SYMPTOMS AND NEGATIVE SCHIZOPHRENIA

    PubMed Central

    Chaturvedi, S.K.; Gopinath, P.S.; Mathai, P. John; Michael, Albert

    1984-01-01

    SUMMARY This study determines the frequency distribution of prominent negative symptoms in a group of chronic, hospitalised schizophrenics. Thirty chronic Schizophrenic (D.S.M. III) patients were rated on the scale for Assessment of Negative Symptoms (SANS) and the prominent negative symptoms were correlated with age, sex and certain illness variables. Majority (80%) of patients had some or the other negative symptom, except thought blocking which was found in none. The subjective awareness of the symptoms was poor. Most negative symptoms were present to a severe degree in about 40% of cases. However, no significant correlation was found between severe negative symptoms and age or sex. Similarly, duration of illness, duration of hospitalisation or current medications did not influence negative symptoms to any appreciable degree. The implications are discussed. PMID:21965985

  17. Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae.

    PubMed

    Seeds, Andrew M; Bastidas, Robert J; York, John D

    2005-07-29

    The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.

  18. Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

    PubMed Central

    Holt, K H; Olson, L; Moye-Rowley, W S; Pessin, J E

    1994-01-01

    Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110. Images PMID:8264609

  19. Combining TRAIL with PI3 Kinase or HSP90 inhibitors enhances apoptosis in colorectal cancer cells via suppression of survival signaling

    PubMed Central

    Saturno, Grazia; Valenti, Melanie; De Haven Brandon, Alexis; Thomas, George V.; Eccles, Suzanne; Clarke, Paul A.; Workman, Paul

    2013-01-01

    TRAIL has been shown to induce apoptosis in cancer cells, but in some cases they fail to respond to this ligand. We explored the ability of representative phosphatidylinositol-3-kinase (PI3 Kinase)/mTOR and HSP90 inhibitors to overcome TRAIL resistance by increasing apoptosis in colorectal cancer models. We determined the sensitivity of 27 human colorectal cancer and 2 non-transformed colon epithelial cell lines to TRAIL treatment. A subset of the cancer cell lines with a range of responses to TRAIL was selected from the panel for treatment with TRAIL combined with the PI3 Kinase/mTOR inhibitor PI-103 or the HSP90 inhibitor 17-AAG (tanespimycin). Two TRAIL-resistant cell lines were selected for in vivo combination studies with TRAIL and 17-AAG. We found that 13 colorectal cancer cell lines and the 2 non-transformed colon epithelial cell lines were resistant to TRAIL. We demonstrated that co-treatment of TRAIL and PI-103 or 17-AAG was synergistic or additive and significantly enhanced apoptosis in colorectal cancer cells. This was associated with decreased expression or activity of survival protein biomarkers such as ERBB2, AKT, IKKα and XIAP. In contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We show here for the first time that co-treatment in vivo with TRAIL and 17-AAG in two TRAIL-resistant human colorectal cancer xenograft models resulted in significantly greater tumor growth inhibition compared to single treatments. We propose that combining TRAIL with PI3 Kinase/mTOR or HSP90 inhibitors has therapeutic potential in the treatment of TRAIL-resistant colorectal cancers. PMID:23852390

  20. Class II phosphoinositide 3-kinase C2β regulates a novel signaling pathway involved in breast cancer progression

    PubMed Central

    Abbott, Jonathan J.; Piñeiro, Roberto; Buus, Richard; Iezzi, Manuela; Ricci, Francesca; Bergamaschi, Daniele; Ostano, Paola; Chiorino, Giovanna; Lattanzio, Rossano; Broggini, Massimo; Piantelli, Mauro; Maffucci, Tania; Falasca, Marco

    2016-01-01

    It is now well established that the enzymes phosphoinositide 3-kinases (PI3Ks) have a key role in the development and progression of many cancer types and indeed PI3Ks inhibitors are currently being tested in clinical trials. Although eight distinct PI3K isoforms exist, grouped into three classes, most of the evidence currently available are focused on one specific isoform with very little known about the potential role of the other members of this family in cancer. Here we demonstrate that the class II enzyme PI3K-C2β is overexpressed in several human breast cancer cell lines and in human breast cancer specimens. Our data indicate that PI3K-C2β regulates breast cancer cell growth in vitro and in vivo and that PI3K-C2β expression in breast tissues is correlated with the proliferative status of the tumor. Specifically we show that downregulation of PI3K-C2β in breast cancer cell lines reduces colony formation, induces cell cycle arrest and inhibits tumor growth, in particular in an estrogen-dependent in vivo xenograft. Investigation of the mechanism of the PI3K-C2β-dependent regulation of cell cycle progression and cell growth revealed that PI3K-C2β regulates cyclin B1 protein levels through modulation of microRNA miR-449a levels. Our data further demonstrate that downregulation of PI3K-C2β inhibits breast cancer cell invasion in vitro and breast cancer metastasis in vivo. Consistent with this, PI3K-C2β is highly expressed in lymph-nodes metastases compared to matching primary tumors. These data demonstrate that PI3K-C2β plays a pivotal role in breast cancer progression and in metastasis development. Our data indicate that PI3K-C2β may represent a key molecular switch that regulates a rate-limiting step in breast tumor progression and therefore it may be targeted to limit breast cancer spread. PMID:26934321

  1. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed Central

    Hellyer, N J; Cheng, K; Koland, J G

    1998-01-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3. PMID:9677338

  2. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed

    Hellyer, N J; Cheng, K; Koland, J G

    1998-08-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

  3. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

    PubMed Central

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2010-01-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa α-subunit (p110α) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H2O2 stimulates the expression of p110α in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton. PMID:16204406

  4. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD.

    PubMed

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2006-04-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa alpha-subunit (p110alpha) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H(2)O(2) stimulates the expression of p110alpha in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

  5. Low [Mg2+]e enhances arterial spontaneous tone via phosphatidylinositol 3-kinase in DOCA-salt hypertension.

    PubMed

    Northcott, Carrie A; Watts, Stephanie W

    2004-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been implicated in low extracellular Mg2+ concentration ( [Mg2+]e)-induced aortic contraction, and Mg2+ deficiency has been associated with hypertension. Moreover, arterial PI3K activity is increased in hypertensive deoxycorticosterone (DOCA)-salt rats. We hypothesized that low [Mg2+]e activates PI3K, eliciting enhanced vascular contraction, PI3K activity, and norepinephrine (NE)-induced contraction. Spontaneous tone was monitored in endothelium-denuded aortic strips from sham and DOCA-salt rats exposed to low Mg2+ (0.15 mmol/L), high Mg2+ (4.8 mmol/L), or normal (1.17 mmol/L) physiologic salt solution (PSS) in isolated tissue baths. LY294002 (20 micromol/L), a PI3K inhibitor, or vehicle was added (30 minutes), followed by NE (10(-9) to 3 x10(-5) mol/L). Low [Mg2+]e significantly enhanced tone in aortas from DOCA-salt and sham rats compared with normal PSS (DOCA-salt low [Mg2+]e, +51.5 +7.0 vs DOCA-salt normal PSS, +7.1 +1.4 % of initial phenylephrine [PE] contraction). LY294002 and incubation with high Mg2+ PSS decreased tone in aortas from DOCA-salt rats (low [Mg2+]e LY294002, --87.5 +8.8; normal PSS LY294002, -81.7 +13.7; and high [Mg2+]e, -31.2 +10.8 % of initial PE contraction). Low [Mg2+]e leftward-shifted NE-induced aortic contractions in sham and thus matched the shift observed with DOCA (-log EC50 mol/L: sham PSS, -7.7 +0.1; DOCA-salt PSS, -8.2 +0.1; sham low [Mg2+]e, -8.2 +0.1; and DOCA-salt low [Mg2+]e, -8.1 +0.1). Moreover, this shift was inhibited by LY294002. In conclusion, low [Mg2+]e might activate PI3K, leading to enhanced tone and agonist-induced contraction observed in aortas from DOCA-salt hypertensive rats.

  6. Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

    PubMed

    Sekar, N; Veldhuis, J D

    2001-07-01

    Insulin and insulin-like growth factor I (IGF-I) can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, viz. low density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein, and P450 cholesterol side-chain cleavage enzyme (CYP11A). The mechanisms underlying the foregoing bihormonal interactions are not known. Accordingly, in relation to the LDL receptor gene, the present study tests the hypothesis that insulin/IGF-I and LH can act via concerted transcriptional control of promoter expression. To this end, we transiently transfected primary monolayer cultures of porcine granulosa-luteal cells with a reporter vector containing the putative 5'-upstream full-length (pLDLR1076/luc) regulatory region (-1076 to +11 bp) of the homologous LDL receptor gene driving firefly luciferase in the presence or absence of insulin (or IGF-I) and/or LH (each 100 ng/ml). Combined exposure to LH and insulin (or IGF-I) stimulated LDL receptor transcriptional activity maximally at 4 h by 8- to 20-fold, as normalized by coexpression of Renilla luciferase. Further analysis of multiple 5'-nested deletional constructs of the LDL receptor gene promoter showed that deletion of -139 bp upstream of the transcriptional start site virtually abolished basal expression and promoter responsiveness to LH and insulin/IGF-I. In contrast, full basal activity and 60-80% of maximal monohormonal and bihormonal drive were retained by the -255 to +11 bp fragment. As LDL receptor gene expression in other tissues is negatively regulated by the abundance of intracellular free cholesterol, we assessed the impact of concomitant pretreatment of granulosa-luteal cells with an exogenous soluble sterol (25-hydroxycholesterol, 1 and 10 microM). Excess sterol markedly (50-70%) attenuated bihormonally and, in lesser measure, LH-stimulated and basal LDL receptor promoter expression, thus affirming a feedback-sensitive sterol

  7. Molecular modeling study of CP-690550 derivatives as JAK3 kinase inhibitors through combined 3D-QSAR, molecular docking, and dynamics simulation techniques.

    PubMed

    Wang, Jing Li; Cheng, Li Ping; Wang, Tian Chi; Deng, Wei; Wu, Fan Hong

    2017-03-01

    To develop more potent JAK3 kinase inhibitors, a series of CP-690550 derivatives were investigated using combined molecular modeling techniques, such as 3D-QSAR, molecular docking and molecular dynamics (MD). The leave-one-out correlation (q(2)) and non-cross-validated correlation coefficient (r(2)) of the best CoMFA model are 0.715 and 0.992, respectively. The q(2) and r(2) values of the best CoMSIA model are 0.739 and 0.995, respectively. The steric, electrostatic, and hydrophobic fields played important roles in determining the inhibitory activity of CP-690550 derivatives. Some new JAK3 kinase inhibitors were designed. Some of them have better inhibitory activity than the most potent Tofacitinib (CP-690550). Molecular docking was used to identify some key amino acid residues at the active site of JAK3 protein. 10ns MD simulations were successfully performed to confirm the detailed binding mode and validate the rationality of docking results. The calculation of the binding free energies by MMPBSA method gives a good correlation with the predicted biological activity. To our knowledge, this is the first report on MD simulations and free energy calculations for this series of compounds. The combination results of this study will be valuable for the development of potent and novel JAK3 kinase inhibitors.

  8. [Effects of phosphatidylinositol-3 kinase/protein kinase b/bone morphogenetic protein-15 pathway on the follicular development in the mammalian ovary].

    PubMed

    Wu, Yan-qing; Chen, Li-yun; Zhang, Zheng-hong; wang, Zheng-chao

    2013-04-01

    In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.

  9. Gastrin decreases Na+,K+-ATPase activity via a PI 3-kinase- and PKC-dependent pathway in human renal proximal tubule cells.

    PubMed

    Liu, Tianbing; Konkalmatt, Prasad R; Yang, Yu; Jose, Pedro A

    2016-04-01

    The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes.

  10. Adenosine A{sub 2A} receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

    SciTech Connect

    Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama

    2013-05-10

    Highlights: •A{sub 2A} receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A{sub 2A} receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A{sub 2A} receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A{sub 2A} receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A{sub 2A} receptor. A{sub 2A} receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A{sub 2A} receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A{sub 2A} receptor-overexpressing HLMVECs. Adenoviral-mediated A{sub 2A} receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A{sub 2A} receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A{sub 2A} receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A{sub 2A} receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A{sub 2A}-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A{sub 2A} receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.

  11. Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation.

    PubMed

    Krajewski, Stefanie; Kurz, Julia; Geisler, Tobias; Peter, Karlheinz; Wendel, Hans Peter; Straub, Andreas

    2012-01-01

    Extracorporeal circulation (ECC) and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K) p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2)Y(12) and P(2)Y(1) blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2)Y(12) antagonist 2-MeSAMP, the P(2)Y(1) antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls). Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2)Y blockers (p<0.05), while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2)Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P(2)Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05). Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2)Y and PI3K blockade (p<0.05). Combined blockade of P

  12. Negative Feedback Control of Pituitary Thyroid-stimulating Hormone Synthesis and Secretion by Thyroid Hormones during Metamorphosis in Xenopus laevis

    EPA Science Inventory

    A basic understanding of the endocrinology of the hypothalamic-pituitary-thyroid (HPT) axis of anuran larvae is necessary for predicting the consequences of HPT perturbation by thyroid-disrupting chemicals (TDCs) on the whole organism. This project examined negative feedback con...

  13. CONTROL OF LASER RADIATION PARAMETERS: Method for calculating a negative-dispersion resonator-type multilayer mirror

    NASA Astrophysics Data System (ADS)

    Kholokhonova, Polina A.; Erg, G. V.

    2005-11-01

    A method is proposed for the calculation of negative-dispersion mirrors with resonator cavities. The mirror optimisation algorithm combines the capabilities of the gradient method and the random search method. A multilayer mirror structure with a reflectivity R>99.9% and a group delay dispersion of -60±10 fs2 in the 930-1070 nm wavelength range was calculated. The sensitivity of the obtained structure to random variations of layer thicknesses was analysed.

  14. Negative-Pressure Wound Therapy: A Hemostatic Adjunct for Control of Coagulopathic Hemorrhage in Large Soft Tissue Wounds

    DTIC Science & Technology

    2012-01-01

    such as large soft tissue injuries, high-energy pene- trating trauma and open fractures have also been reported.6Y10 NPWT has been used for evacuating...injuries associated with high-energy open tibial shaft fractures . J Orthop Trauma. 2007;21:11 17. 10. Stannard JP, Robinson JT, Anderson ER, et al...Negative pressure wound therapy to treat hematomas and surgical incisions following high-energy trauma. J Trauma. 2006;60;1301 1306. 11. Kakagia D

  15. Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathway

    PubMed Central

    Wu, Mengrui; Chen, Wei; Lu, Yun; Zhu, Guochun; Hao, Liang; Li, Yi-Ping

    2017-01-01

    Many positive signalling pathways of osteoclastogenesis have been characterized, but negative signalling pathways are less well studied. Here we show by microarray and RNAi that guanine nucleotide-binding protein subunit α13 (Gα13) is a negative regulator of osteoclastogenesis. Osteoclast-lineage-specific Gna13 conditional knockout mice have a severe osteoporosis phenotype. Gna13-deficiency triggers a drastic increase in both osteoclast number and activity (hyper-activation), mechanistically through decreased RhoA activity and enhanced Akt/GSK3β/NFATc1 signalling. Consistently, Akt inhibition or RhoA activation rescues hyper-activation of Gna13-deficient osteoclasts, and RhoA inhibition mimics the osteoclast hyperactivation resulting from Gna13-deficiency. Notably, Gα13 gain-of-function inhibits Akt activation and osteoclastogenesis, and protects mice from pathological bone loss in disease models. Collectively, we reveal that Gα13 is a master endogenous negative switch for osteoclastogenesis through regulation of the RhoA/Akt/GSK3β/NFATc1 signalling pathway, and that manipulating Gα13 activity might be a therapeutic strategy for bone diseases. PMID:28102206

  16. Effect of basic physical parameters to control plasma meniscus and beam halo formation in negative ion sources

    SciTech Connect

    Miyamoto, K.; Okuda, S.; Nishioka, S.; Hatayama, A.

    2013-09-14

    Our previous study shows that the curvature of the plasma meniscus causes the beam halo in the negative ion sources: the negative ions extracted from the periphery of the meniscus are over-focused in the extractor due to the electrostatic lens effect, and consequently become the beam halo. In this article, the detail physics of the plasma meniscus and beam halo formation is investigated with two-dimensional particle-in-cell simulation. It is shown that the basic physical parameters such as the H{sup −} extraction voltage and the effective electron confinement time significantly affect the formation of the plasma meniscus and the resultant beam halo since the penetration of electric field for negative ion extraction depends on these physical parameters. Especially, the electron confinement time depends on the characteristic time of electron escape along the magnetic field as well as the characteristic time of electron diffusion across the magnetic field. The plasma meniscus penetrates deeply into the source plasma region when the effective electron confinement time is short. In this case, the curvature of the plasma meniscus becomes large, and consequently the fraction of the beam halo increases.

  17. Longitudinal Relations among Parents' Reactions to Children's Negative Emotions, Effortful Control, and Math Achievement in Early Elementary School

    ERIC Educational Resources Information Center

    Swanson, Jodi; Valiente, Carlos; Lemery-Chalfant, Kathryn; Bradley, Robert H.; Eggum-Wilkens, Natalie D.

    2014-01-01

    Panel mediation models and fixed-effects models were used to explore longitudinal relations among parents' reactions to children's displays of negative emotions, children's effortful control (EC), and children's math achievement (N = 291; M age in fall of kindergarten = 5.66 years, SD = 0.39 year) across kindergarten through…

  18. The effects of neuroticism, extraversion, and positive and negative life events on a one-year course of depressive symptoms in euthymic previously depressed patients versus healthy controls.

    PubMed

    Spinhoven, Philip; Elzinga, Bernet; Roelofs, Karin; Hovens, Jacqueline G F M; van Oppen, Patricia; Zitman, Frans G; Penninx, Brenda W J H

    2011-09-01

    We investigated a) the concurrent impact of positive and negative life events on the course of depressive symptoms in persons remitted from depression and healthy controls, b) whether the impact of life events on symptom course is moderated by the history of depression and the personality traits of neuroticism and extraversion, and c) whether life events mediate possible relationships of history of depression and personality traits with symptom course. Using data from the Netherlands Study of Depression and Anxiety, we examined 239 euthymic participants with a previous depressive disorder based on DSM-IV and 450 healthy controls who completed a) baseline assessments of personality dimensions (NEO Five-Factor Inventory) and depression severity (Inventory of Depressive Symptoms [IDS]) and b) 1-year follow-up assessments of depression severity and the occurrence of positive and negative life events during the follow-up period (List of Threatening Events Questionnaire). Remitted persons reported higher IDS scores at 1-year follow-up than did the controls. Extraversion and positive and negative life events independently predicted the course of depressive symptoms. The impact of life events on symptom course was not moderated by history of depression or personality traits. The effect of extraversion on symptom course was partly caused by differential engagement in positive life events.

  19. Piloted simulator study of allowable time delay in pitch flight control system of a transport airplane with negative static stability

    NASA Technical Reports Server (NTRS)

    Grantham, William D.; Smith, Paul M.; Person, Lee H., Jr.; Meyer, Robert T.; Tingas, Stephen A.

    1987-01-01

    A piloted simulation study was conducted to determine the permissible time delay in the flight control system of a 10-percent statically unstable transport airplane during cruise flight conditions. The math model used for the simulation was a derivative Lockheed L-1011 wide-body jet transport. Data were collected and analyzed from a total of 137 cruising flights in both calm- and turbulent-air conditions. Results of this piloted simulation study verify previous findings that show present military specifications for allowable control-system time delay may be too stringent when applied to transport-size airplanes. Also, the degree of handling-qualities degradation due to time delay is shown to be strongly dependent on the source of the time delay in an advanced flight control system. Maximum allowable time delay for each source of time delay in the control system, in addition to a less stringent overall maximum level of time delay, should be considered for large aircraft. Preliminary results also suggest that adverse effects of control-system time delay may be at least partially offset by variations in control gearing. It is recommended that the data base include different airplane baselines, control systems, and piloting tasks with many pilots participating, so that a reasonable set of limits for control-system time delay can be established to replace the military specification limits currently being used.

  20. Social Information Processing of Positive and Negative Hypothetical Events in Children with ADHD and Conduct Problems and Controls

    ERIC Educational Resources Information Center

    Andrade, Brendan F.; Waschbusch, Daniel A.; Doucet, Amelie; King, Sara; MacKinnon, Maura; McGrath, Patrick J.; Stewart, Sherry H.; Corkum, Penny

    2012-01-01

    Objective: This study examined social information processing (SIP) of events with varied outcomes in children with ADHD and conduct problems (CPs; defined as oppositional defiant disorder [ODD] or conduct disorder [CD]) and controls. Method: Participants were 64 children (46 boys, 18 girls) aged 6 to 12, including 39 with ADHD and 25 controls.…

  1. Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

    PubMed

    Horn, S; Endl, E; Fehse, B; Weck, M M; Mayr, G W; Jücker, M

    2004-11-01

    The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

  2. Identification of gram-negative bacteria from critical control points of raw and pasteurized cow milk consumed at Gondar town and its suburbs, Ethiopia

    PubMed Central

    2012-01-01

    Background Milk is highly prone to contamination and can serve as an efficient vehicle for human transmission of foodborne pathogens, especially gram-negative bacteria, as these are widely distributed in the environment. Methods This cross-sectional study of gram-negative staining bacterial contamination of milk meant for human consumption was carried out from October 2010 to May 2011 in Gondar town, Ethiopia. Milk samples were collected from critical control points, from production to consumption, that were hypothesized to be a source of potential contamination. Milk sampling points included smallholder’s milk producers, dairy co-operatives, a milk processing plant, and supermarkets. The hygienic procedures applied during milking, milk collection, transportation, pasteurization, and postpasteurization storage conditions at these specified critical control points were evaluated. Standard bacteriological cultivation and biochemical assays were used to isolate and identify bacterial pathogens in the milk samples. Results The results of the current study showed that conditions for contamination of raw milk at different critical points were due to less hygienic practices in pre-milking udder preparation, sub-optimal hygiene of milk handlers, and poor sanitation practices associated with milking and storage equipments. Among all critical control points considered, transportation containers at milk collection centers and at processing plants were found to be the most heavily contaminated with gram-negative staining bacterial species. Overall, 54 different bacterial species were indentified, and Escherichia coli (29.6%), Pseudomonas aeruginosa (18.5%), and Klebsiella pneumoniae (16.7%), were the most commonly identified gram-negative staining bacterial pathogens. Of particular interest was that no gram-negative staining bacteria were isolated from pasteurized milk samples with varying shelf life. Conclusion This study showed the presence of diverse pathogenic gram-negative

  3. Translational control by negative-strand RNA viruses: methods for the study of a crucial virus/host interaction.

    PubMed

    Hodges, Erin N; Connor, John H

    2013-02-01

    Protein synthesis is a vital step in the successful replication of negative-strand RNA viruses. Protein synthesis is also a critical step in the development of a successful antiviral response from the host. This makes understanding the interplay between host and viral translation an important aspect of defining the virus/host interaction. For the negative-strand RNA viruses there are disparate mechanism of how viruses interact with the host protein synthesis apparatus, ranging from the complete takeover of all protein synthesis to the subtle insertion of viral mRNAs into an otherwise unchanged protein synthesis pattern. In this article, we discuss different ways to investigate protein synthesis in virus-infected cells, ranging from the use of metabolic labeling for the study of general translation changes to using fluorescence-coupled labeling techniques that allow the pinpointing of any subcellular localization of protein synthesis during virus replication. We also discuss methods for analyzing the translation initiation factors that are frequently modified in virus-infected cells.

  4. Role of erbB-2 and erbB-3 in the Activation of Phosphatidylinositol 3-Kinase

    DTIC Science & Technology

    1998-06-01

    Culouscou, J-M., et al. Heregulin induces tyrosine phosphorylation of HER4/p 180erbB4 . Nature 366:473-475, 1993. 9. Goldman, R., Ben Levy , R., Peles, E...of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66:257- 270, 1991. 53. Lofts, F. J., Hurst, H

  5. Negative birefringent polyimide films

    NASA Technical Reports Server (NTRS)

    Harris, Frank W. (Inventor); Cheng, Stephen Z. D. (Inventor)

    1994-01-01

    A negative birefringent film, useful in liquid crystal displays, and a method for controlling the negative birefringence of a polyimide film is disclosed which allows the matching of an application to a targeted amount of birefringence by controlling the degree of in-plane orientation of the polyimide by the selection of functional groups within both the diamine and dianhydride segments of the polyimide which affect the polyimide backbone chain rigidity, linearity, and symmetry. The higher the rigidity, linearity and symmetry of the polyimide backbone, the larger the value of the negative birefringence of the polyimide film.

  6. Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    PubMed Central

    Hussain, Azhar R.; Ahmed, Saeeda O.; Ahmed, Maqbool; Khan, Omar S.; Al AbdulMohsen, Sally; Platanias, Leonidas C.; Al-Kuraya, Khawla S.; Uddin, Shahab

    2012-01-01

    Background A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Methodology/Principal Findings We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. Conclusion/Significance These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways. PMID:22768179

  7. The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma

    SciTech Connect

    Baumann, Philipp Mandl-Weber, Sonja; Oduncu, Fuat; Schmidmaier, Ralf

    2009-02-01

    NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.

  8. PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

    PubMed

    Deng, Youping; Xu, Hu; Riedel, Heimo

    2007-02-15

    The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

  9. Artesunate Protected Blood-Brain Barrier via Sphingosine 1 Phosphate Receptor 1/Phosphatidylinositol 3 Kinase Pathway After Subarachnoid Hemorrhage in Rats.

    PubMed

    Zuo, Shilun; Ge, Hongfei; Li, Qiang; Zhang, Xuan; Hu, Rong; Hu, Shengli; Liu, Xin; Zhang, John H; Chen, Yujie; Feng, Hua

    2017-03-01

    Blood-brain barrier preservation plays an important role in attenuating vasogenic brain edema after subarachnoid hemorrhage (SAH). This study was designed to investigate the protective effect and mechanism of artesunate, a traditional anti-malaria drug, on blood-brain barrier after SAH. Three hundred and seventy-seven (377) male Sprague-Dawley rats were subjected to endovascular perforation model for SAH. The rats received artesunate alone or in combination with Sphingosine-1-phosphate receptor-1 (S1P1) small interfering RNA (siRNA), antagonist VPC23019, or phosphatidylinositol 3-kinase inhibitor wortmannin after SAH. Modified Garcia score, SAH grades, brain water content, Evans blue leakage, transmission electron microscope, immunohistochemistry staining, Western blot, and cultured endothelial cells were used to investigate the optimum concentration and the therapeutic mechanism of artesunate. We found that artesunate (200 mg/kg) could do better in raising modified Garcia score, reducing brain water content and Evans blue leakage than other groups after SAH. Moreover, artesunate elevated S1P1 expression, enhanced phosphatidylinositol 3-kinase activation, lowered GSK-3β activation, stabilized β-catenin, and improved the expression of Claudin-3 and Claudin-5 after SAH in rats. These effects were eliminated by S1P1 siRNA, VPC23019, and wortmannin. This study revealed that artesunate could preserve blood-brain barrier integrity and improve neurological outcome after SAH, possibly through activating S1P1, enhancing phosphatidylinositol 3-kinase activation, stabilizing β-catenin via GSK-3β inhibition, and then effectively raising the expression of Claudin-3 and Claudin-5. Therefore, artesunate may be favorable for the blood-brain barrier (BBB) protection after SAH and become a potential candidate for the treatment of SAH patients.

  10. When seeing outweighs feeling: a role for prefrontal cortex in passive control of negative affect in blindsight

    PubMed Central

    Eippert, Falk; Wiens, Stefan; Birbaumer, Niels; Lotze, Martin; Wildgruber, Dirk

    2009-01-01

    Affective neuroscience has been strongly influenced by the view that a ‘feeling’ is the perception of somatic changes and has consequently often neglected the neural mechanisms that underlie the integration of somatic and other information in affective experience. Here, we investigate affective processing by means of functional magnetic resonance imaging in nine cortically blind patients. In these patients, unilateral postgeniculate lesions prevent primary cortical visual processing in part of the visual field which, as a result, becomes subjectively blind. Residual subcortical processing of visual information, however, is assumed to occur in the entire visual field. As we have reported earlier, these patients show significant startle reflex potentiation when a threat-related visual stimulus is shown in their blind visual field. Critically, this was associated with an increase of brain activity in somatosensory-related areas, and an increase in experienced negative affect. Here, we investigated the patients’ response when the visual stimulus was shown in the sighted visual field, that is, when it was visible and cortically processed. Despite the fact that startle reflex potentiation was similar in the blind and sighted visual field, patients reported significantly less negative affect during stimulation of the sighted visual field. In other words, when the visual stimulus was visible and received full cortical processing, the patients’ phenomenal experience of affect did not closely reflect somatic changes. This decoupling of phenomenal affective experience and somatic changes was associated with an increase of activity in the left ventrolateral prefrontal cortex and a decrease of affect-related somatosensory activity. Moreover, patients who showed stronger left ventrolateral prefrontal cortex activity tended to show a stronger decrease of affect-related somatosensory activity. Our findings show that similar affective somatic changes can be associated with

  11. Negative Certainty

    ERIC Educational Resources Information Center

    Ariso, José María

    2017-01-01

    The definitions of "negative knowledge" and the studies in this regard published to date have not considered the categorial distinction Wittgenstein established between knowledge and certainty. Hence, the important role that certainty, despite its omission, should have in these definitions and studies has not yet been shown. In this…

  12. Negative Numbers

    ERIC Educational Resources Information Center

    Galbraith, Mary J.

    1974-01-01

    Examination of models for representing integers demonstrates that formal operational thought is required for establishing the operations on integers. Advocated is the use of many models for introducing negative numbers but, apart from addition, it is recommended that operations on integers be delayed until the formal operations stage. (JP)

  13. Enhancement of negative hydrogen ion production at low pressure by controlling the electron kinetics property with transverse magnetic field

    NASA Astrophysics Data System (ADS)

    Kim, June Young; Cho, Won-Hwi; Dang, Jeong-Jeung; Kim, Seongcheol; Chung, Kyoung-Jae; Hwang, Y. S.

    2016-12-01

    In a volume production H- ion source, independent control of electron energy distribution between the driver region and the extraction region is crucial for the efficient production of H- ions due to its unique volume production mechanism. However, at the low pressure regime compatible to ITER operation, it is difficult to control electron energy distribution separately because the nonlocal property dominates the electron kinetics. In this work, we suggest a new method to control the locality of electron kinetics. In this method, an additional pair of permanent magnets is introduced in the vicinity of the skin layer, differently from the conventional method in which the magnetic filter field was strengthened in the extraction region. This magnetic field shortens the energy relaxation length and changes the electron kinetics from nonlocal to local even for low pressure discharges. In this paper, we show that the locality of electron kinetics can be effectively controlled by the additional magnetic field near the skin layer by measuring the electron temperature profile along the center of the discharge chamber as well as by comparing electron energy probability function shapes for different strengths of magnetic field. Using this new method, we demonstrate that control of locality of electron kinetics can greatly enhance the production of H- ions in the extraction region by measuring H- ion beam current extracted from the plasma source.

  14. Emotion Risk-Factor in Patients With Cardiac Diseases: The Role of Cognitive Emotion Regulation Strategies, Positive Affect and Negative Affect (A Case-Control Study)

    PubMed Central

    Bahremand, Mostafa; Alikhani, Mostafa; Zakiei, Ali; Janjani, Parisa; Aghaei, Abbas

    2016-01-01

    Application of psychological interventions is essential in classic treatments for patient with cardiac diseases. The present study compared cognitive emotion regulation strategies, positive affect, and negative affect for cardiac patients with healthy subjects. This study was a case-control study. Fifty subjects were selected using convenient sampling method from cardiac (coronary artery disease) patients presenting in Imam Ali medical center of Kermanshah, Iran in the spring 2013. Fifty subjects accompanied the patients to the medical center, selected as control group, did not have any history of cardiac diseases. For collecting data, the cognitive emotion regulation questionnaire and positive and negative affect scales were used. For data analysis, multivariate analysis of variance (MANOVA) was applied using the SPSS statistical software (ver. 19.0). In all cognitive emotion regulation strategies, there was a significant difference between the two groups. A significant difference was also detected regarding positive affect between the two groups, but no significant difference was found regarding negative affect. We found as a result that, having poor emotion regulation strategies is a risk factor for developing heart diseases. PMID:26234976

  15. Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway.

    PubMed

    Yang, Heping; Wu, Dapeng; Zhang, Xiaojie; Wang, Xiang; Peng, Yi; Hu, Zhiping

    2012-10-05

    Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

  16. Transcriptional regulation of genes encoding the selenium-free [NiFe]-hydrogenases in the archaeon Methanococcus voltae involves positive and negative control elements.

    PubMed Central

    Noll, I; Müller, S; Klein, A

    1999-01-01

    Methanococcus voltae harbors genetic information for two pairs of homologous [NiFe]-hydrogenases. Two of the enzymes contain selenocysteine, while the other two gene groups encode apparent isoenzymes that carry cysteinyl residues in the homologous positions. The genes coding for the selenium-free enzymes, frc and vhc, are expressed only under selenium limitation. They are transcribed out of a common intergenic region. A series of deletions made in the intergenic region localized a common negative regulatory element for the vhc and frc promoters as well as two activator elements that are specific for each of the two transcription units. Repeated sequences, partially overlapping the frc promoter, were also detected. Mutations in these repeated heptanucleotide sequences led to a weak induction of a reporter gene under the control of the frc promoters in the presence of selenium. This result suggests that the heptamer repeats contribute to the negative regulation of the frc transcription unit. PMID:10430564

  17. A Study of Longitudinal Control Problems at Low and Negative Damping and Stability with Emphasis on Effects of Motion Cues

    NASA Technical Reports Server (NTRS)

    Sadoff, Melvin; McFadden, Norman M.; Heinle, Donovan R.

    1961-01-01

    As part of a general investigation to determine the effects of simulator motions on pilot opinion and task performance over a wide range of vehicle longitudinal dynamics, a cooperative NASA-AMAL program was conducted on the centrifuge at Johnsville, Pennsylvania. The test parameters and measurements for this program duplicated those of earlier studies made at Ames Research Center with a variable-stability airplane and with a pitch-roll chair flight simulator. Particular emphasis was placed on the minimum basic damping and stability the pilots would accept and on the minimum dynamics they considered controllable in the event of stability-augmentation system failure. Results of the centrifuge-simulator program indicated that small positive damping was required by the pilots over most of the frequency range covered for configurations rated acceptable for emergency conditions only (e.g., failure of a pitch damper). It was shown that the pilot's tolerance for unstable dynamics was dependent primarily on the value of damping. For configurations rated acceptable for emergency operation only, the allowable instability and damping corresponded to a divergence time to double amplitude of about 1 second. Comparisons were made of centrifuge, pitch-chair and fixed-cockpit simulator tests with flight tests. Pilot ratings indicated that the effects of incomplete or spurious motion cues provided by these three modes of simulation were important only for high-frequency, lightly damped dynamics or unstable, moderately damped dynamics. The pitch- chair simulation, which provided accurate angular-acceleration cues to the pilot, compared most favorably with flight. For the centrifuge simulation, which furnished accurate normal accelerations but spurious pitching and longitudinal accelerations, there was a deterioration of pilots' opinion relative to flight results. Results of simulator studies with an analog pilot replacing the human pilot illustrated the adaptive capability of human

  18. A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling

    PubMed Central

    Vullhorst, Detlef; Mitchell, Robert M.; Keating, Carolyn; Roychowdhury, Swagata; Karavanova, Irina; Tao-Cheng, Jung-Hwa; Buonanno, Andres

    2015-01-01

    The neuregulin receptor ErbB4 is an important modulator of GABAergic interneurons and neural network synchronization. However, little is known about the endogenous ligands that engage ErbB4, the neural processes that activate them or their direct downstream targets. Here we demonstrate, in cultured neurons and in acute slices, that the NMDA receptor is both effector and target of neuregulin 2 (NRG2)/ErbB4 signalling in cortical interneurons. Interneurons co-express ErbB4 and NRG2, and pro-NRG2 accumulates on cell bodies atop subsurface cisternae. NMDA receptor activation rapidly triggers shedding of the signalling-competent NRG2 extracellular domain. In turn, NRG2 promotes ErbB4 association with GluN2B-containing NMDA receptors, followed by rapid internalization of surface receptors and potent downregulation of NMDA but not AMPA receptor currents. These effects occur selectively in ErbB4-positive interneurons and not in ErbB4-negative pyramidal neurons. Our findings reveal an intimate reciprocal relationship between ErbB4 and NMDA receptors with possible implications for the modulation of cortical microcircuits associated with cognitive deficits in psychiatric disorders. PMID:26027736

  19. Anomalous negative electrocaloric effect in a relaxor/normal ferroelectric polymer blend with controlled nano- and meso-dipolar couplings

    NASA Astrophysics Data System (ADS)

    Qian, Xiaoshi; Yang, Tiannan; Zhang, Tian; Chen, Long-Qing; Zhang, Q. M.

    2016-04-01

    In general, a dielectric material will eject (or absorb) heat when an electric field is applied and absorb (or eject) heat when the field is removed, under isothermal condition, which is known as the normal (or negative) electrocaloric (EC) effect. For some applications, it is highly desired that an EC material will absorb heat (cooling the surrounding) without subsequent heating under an electric pulse. Here, we show that such an EC material can be realized in a properly designed hybrid normal ferroelectric/relaxor ferroelectric polymer blend in which the normal ferroelectric component induces dipole ordering in the relaxor polymer in the poled state, which can be switched to a de-poled state by an external field. More importantly, the de-poled state can be maintained by the relaxor component when the de-poling field is removed. Consequently, the hybrid blend exhibits a large cooling (an isothermal entropy change ΔS = 11.5 J kg-1 K-1) without the subsequent heating upon the application of an electric pulse.

  20. Rheumatoid Arthritis Was Negatively Associated with Alzheimer’s Disease: A Population-Based Case-Control Study

    PubMed Central

    Kao, Li-Ting; Kang, Jiunn-Horng; Lin, Herng-Ching

    2016-01-01

    Some of the prior literature investigated the potential association between rheumatoid arthritis (RA) and Alzheimer's disease (AD) because these two diseases may share similar inflammatory mechanisms. Nevertheless, to date, findings of the previous literature are still controversial, and some methodological limitations were observed in those studies. The aim of this case-control study was to investigate the relationship between prior RA and AD using a large population-based dataset. This study used the Taiwan Longitudinal Health Insurance Database 2005. We included 2271 patients with AD who had received prescriptions for acetylcholinesterase inhibitors (AChEIs) as cases and 6813 patients without AD as controls in this study. In addition, we performed a conditional logistic regression to examine the odds ratio (OR) and 95% confidence interval (CI) for prior RA between cases and controls. The study found that 330 (3.63%) of the total sampled patients had an RA diagnosis before the index date. Additionally, prior RA was found in 60 (2.64%) cases and in 270 (3.96%) controls. The conditional logistic regression analysis showed that the crude OR of prior RA for cases was 0.66 (95% confidence interval (CI): 0.49~0.87) compared to controls. After adjusting for patients’ geographic location, urbanization level, and comorbidities, the adjusted OR of prior RA for patients with AD was 0.73 (95% CI: 0.55~0.98) compared to those without AD. We concluded that there was an inverse association between prior RA and AD even after adjusting for potential confounders. PMID:27997574

  1. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.

    PubMed

    Pene, Frédéric; Claessens, Yann-Erick; Muller, Odile; Viguié, Franck; Mayeux, Patrick; Dreyfus, François; Lacombe, Catherine; Bouscary, Didier

    2002-09-26

    Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that constitutive activation of signaling pathways occurs. One of the signaling pathways whose deregulation may play an oncogenic role in MM is the phosphatidylinositol 3-kinase (PI 3-K) pathway. In human growth factor-independent MM cell lines OPM2 and RPMI8226, we show that the PI 3-K inhibitors LY294002 and Wortmannin strongly inhibited cell proliferation, whereas inhibition of the mammalian Target Of Rapamycin (mTOR)/P70-S6-kinase (P70(S6K)) pathway with rapamycin or of the Mitogen-Activated Protein Kinase (MAPK) pathway with PD98059 had minimal effect on proliferation. In both cell lines, constitutive activation of the PI 3-K/Akt/FKHRL-1, mTOR/P70(S6K) and MAPK pathways was detected. LY294002 inhibited phosphorylation of Akt, FKHRL-1 and P70(S6K) but had no effect on ERK1/2 phosphorylation, indicating that the PI 3-K and MAPK pathways are independent. IGF-1 but not IL-6 increased phosphorylation of Akt, FKHRL-1 and P70(S6K). Purified plasmocytes from four patients with MM and two patients with primary PCL were studied. In three of them including the two patients with PCL, constitutive phosphorylation of Akt, FKHRL-1 and P70(S6K) was present, inhibited by LY294002 and enhanced by IGF-1. In these patients with constitutive Akt activation, normal PTEN expression was detected. PI 3-K inhibition induced caspase-dependent apoptosis as confirmed by inhibition with the large spectrum caspase inhibitor Z-VAD-FMK and cleavage of pro-caspase-3. Both cell lines spontaneously expressed Skp2 and cyclin D1 proteins at high levels but no p27(Kip1) protein. In the

  2. The σ1 Receptor Engages the Redox-Regulated HINT1 Protein to Bring Opioid Analgesia Under NMDA Receptor Negative Control

    PubMed Central

    Rodríguez-Muñoz, María; Sánchez-Blázquez, Pilar; Herrero-Labrador, Raquel; Martínez-Murillo, Ricardo; Merlos, Manuel; Vela, José Miguel

    2015-01-01

    Abstract Aims: The in vivo pharmacology of the sigma 1 receptor (σ1R) is certainly complex; however, σ1R antagonists are of therapeutic interest, because they enhance mu-opioid receptor (MOR)-mediated antinociception and reduce neuropathic pain. Thus, we investigated whether the σ1R is involved in the negative control that glutamate N-methyl-d-aspartate acid receptors (NMDARs) exert on opioid antinociception. Results: The MOR C terminus carries the histidine triad nucleotide-binding protein 1 (HINT1) coupled to the regulator of G-protein signaling RGSZ2-neural nitric oxide synthase assembly. Activated MORs stimulate the production of nitric oxide (NO), and the redox zinc switch RGSZ2 converts this signal into free zinc ions that are required to recruit the redox sensor PKCγ to HINT1 proteins. Then, PKCγ impairs HINT1-RGSZ2 association and enables σ1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. The inhibition of NOS or the absence of σ1Rs prevents HINT1-PKCγ interaction, and MOR-NMDAR cross-regulation fails. The σ1R antagonists transitorily remove the binding of σ1Rs to NR1 subunits, facilitate the entrance of negative regulators of NMDARs, likely Ca2+-CaM, and prevent NR1 interaction with HINT1, thereby impairing the negative feedback of glutamate on opioid analgesia. Innovation: A redox-regulated process situates MOR signaling under NMDAR control, and in this context, the σ1R binds to the cytosolic C terminal region of the NMDAR NR1 subunit. Conclusion: The σ1R antagonists enhance opioid analgesia in naïve mice by releasing MORs from the negative influence of NMDARs, and they also reset antinociception in morphine tolerant animals. Moreover, σ1R antagonists alleviate neuropathic pain, probably by driving the inhibition of up-regulated NMDARs. Antioxid. Redox Signal. 22, 799–818. PMID:25557043

  3. Variants in the vitamin D pathway, serum levels of vitamin D, and estrogen receptor negative breast cancer among African-American women: a case-control study

    PubMed Central

    2012-01-01

    Introduction American women of African ancestry (AA) are more likely than European Americans (EA) to have estrogen receptor (ER)-negative breast cancer. 25-hydroxyvitamin D (25OHD) is low in AAs, and was associated with ER-negative tumors in EAs. We hypothesized that racial differences in 25OHD levels, as well as in inherited genetic variations, may contribute, in part, to the differences in tumor characteristics. Methods In a case (n = 928)-control (n = 843) study of breast cancer in AA and EA women, we measured serum 25OHD levels in controls and tested associations between risk and tag single nucleotide polymorphisms (SNPs) in VDR, CYP24A1 and CYP27B1, particularly by ER status. Results More AAs had severe vitamin D deficiency (< 10 ng/ml) than EAs (34.3% vs 5.9%), with lowest levels among those with the highest African ancestry. Associations for SNPs differed by race. Among AAs, VDR SNP rs2239186, associated with higher serum levels of 25OHD, decreased risk after correction for multiple testing (OR = 0.53, 95% CI = 0.31-0.79, p by permutation = 0.03), but had no effect in EAs. The majority of associations were for ER-negative breast cancer, with seven differential associations between AA and EA women for CYP24A1 (p for interaction < 0.10). SNP rs27622941 was associated with a > twofold increased risk of ER-negative breast cancer among AAs (OR = 2.62, 95% CI = 1.38-4.98), but had no effect in EAs. rs2209314 decreased risk among EAs (OR = 0.38, 95% CI = 0.20-0.73), with no associations in AAs. The increased risk of ER-negative breast cancer in AAs compared to EAs was reduced and became non-significant (OR = 1.20, 95% CI = 0.80-1.79) after adjusting for these two CYP24A1 SNPs. Conclusions These data suggest that genetic variants in the vitamin D pathway may be related to the higher prevalence of ER-negative breast cancer in AA women. PMID:22480149

  4. MGMT-independent temozolomide resistance in pediatric glioblastoma cells associated with a PI3-kinase-mediated HOX/stem cell gene signature.

    PubMed

    Gaspar, Nathalie; Marshall, Lynley; Perryman, Lara; Bax, Dorine A; Little, Suzanne E; Viana-Pereira, Marta; Sharp, Swee Y; Vassal, Gilles; Pearson, Andrew D J; Reis, Rui M; Hargrave, Darren; Workman, Paul; Jones, Chris

    2010-11-15

    Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screen