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Sample records for fak-src mediated pathways

  1. Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways.

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    Xiong, Niya; Li, Shun; Tang, Kai; Bai, Hongxia; Peng, Yueting; Yang, Hong; Wu, Chunhui; Liu, Yiyao

    2017-01-01

    Tumor cells translocating to distant sites are subjected to hemodynamic shear forces during their passage in the blood vessels. Low shear stress (LSS) plays a critical role in the regulation of various aspects of tumor cells functions, including motility and adhesion. Beyond its structural role, caveolin-1 (Cav-1), the important component of caveolae, represents a modulator of several cancer-associated functions as tumor progression and metastasis. However, the role of Cav-1 in regulating tumor cells response to shear stress remains poorly explored. Here, we characterized the role of LSS and Cav-1 in mediating cell motility and adhesion on human breast carcinoma MDA-MB-231 cells. We first showed that LSS exposure promoted cell polarity and focal adhesion (FA) dynamics, thus indicating elevated cell migration. Silencing of Cav-1 leaded to a significantly lower formation of stress fibers. However, LSS exposure was able to rescue it via the alteration of actin-associated proteins expression, including ROCK, p-MLC, cofilin and filamin A. Time-lapse migration assay indicated that Cav-1 expression fostered MDA-MB-231 cells motility and LSS triggered cells to rapidly generate new lamellipodia. Furthermore, Cav-1 and LSS significantly influenced cell adhesion. Taken together, our findings provide insights into mechanisms underlying LSS triggered events mediated by downstream Cav-1, including FAK/Src and ROCK/p-MLC pathways, involved in the reorganization of the cytoskeleton, cell motility, FA dynamics and breast cancer cell adhesion.

  2. Ganoderiol A-enriched extract suppresses migration and adhesion of MDA-MB-231 cells by inhibiting FAK-SRC-paxillin cascade pathway.

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    Guo-Sheng Wu

    Full Text Available Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. Ganoderma lucidum, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, G. lucidum extract mainly contains ganoderiol A (GA, dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that G.lucidum triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.

  3. Abnormal cell properties and down-regulated FAK-Src complex signaling in B lymphoblasts of autistic subjects.

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    Wei, Hongen; Malik, Mazhar; Sheikh, Ashfaq M; Merz, George; Ted Brown, W; Li, Xiaohong

    2011-07-01

    Recent studies suggest that one of the major pathways to the pathogenesis of autism is reduced cell migration. Focal adhesion kinase (FAK) has an important role in neural migration, dendritic morphological characteristics, axonal branching, and synapse formation. The FAK-Src complex, activated by upstream reelin and integrin β1, can initiate a cascade of phosphorylation events to trigger multiple intracellular pathways, including mitogen-activated protein kinase-extracellular signal-regulated kinase and phosphatidylinositol 3-kinase-Akt signaling. In this study, by using B lymphoblasts as a model, we tested whether integrin β1 and FAK-Src signaling are abnormally regulated in autism and whether abnormal FAK-Src signaling leads to defects in B-lymphoblast adhesion, migration, proliferation, and IgG production. To our knowledge, for the first time, we show that protein expression levels of both integrin β1 and FAK are significantly decreased in autistic lymphoblasts and that Src protein expression and the phosphorylation of an active site (Y416) are also significantly decreased. We also found that lymphoblasts from autistic subjects exhibit significantly decreased migration, increased adhesion properties, and an impaired capacity for IgG production. The overexpression of FAK in autistic lymphoblasts countered the adhesion and migration defects. In addition, we demonstrate that FAK mediates its effect through the activation of Src, phosphatidylinositol 3-kinase-Akt, and mitogen-activated protein kinase signaling cascades and that paxillin is also likely involved in the regulation of adhesion and migration in autistic lymphoblasts. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  4. MICA Expression Is Regulated by Cell Adhesion and Contact in a FAK/Src-Dependent Manner

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    Moncayo, Gerald; Lin, Da; McCarthy, Michael T.; Watson, Aleksandra A.; O’Callaghan, Christopher A.

    2017-01-01

    MICA is a major ligand for the NKG2D immune receptor, which plays a key role in activating natural killer (NK) cells and cytotoxic T cells. We analyzed NKG2D ligand expression on a range of cell types and could demonstrate that MICA expression levels were closely linked to cellular growth mode. While the expression of other NKG2D ligands was largely independent of cell growth mode, MICA expression was mainly found on cells cultured as adherent cells. In addition, MICA surface expression was reduced through increase in cell–cell contact or loss of cell–matrix adherence. Furthermore, we found that the reduction in MICA expression was modulated by focal adhesion kinase (FAK)/Src signaling and associated with increased susceptibility to NK cell-mediated killing. While the mechanisms of tumor immune evasion are not fully understood, the reduction of MICA expression following loss of attachment poises a potential way by which metastasizing tumor cells avoid immune detection. The role of FAK/Src in this process indicates a potential therapeutic approach to modulate MICA expression and immune recognition of tumor cells during metastasis. PMID:28154561

  5. Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by alpha1-integrins during colon cancer cell invasion.

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    Van Slambrouck, Severine; Grijelmo, Clara; De Wever, Olivier; Bruyneel, Erik; Emami, Shahin; Gespach, Christian; Steelant, Wim F A

    2007-12-01

    Increased src tyrosine kinase expression and activity has been associated with colon cancer cell invasion and survival. Several signaling pathways are involved in the oncogenic activation of src during the adenoma to carcinoma progression and cellular invasion. In the present study, the synthetic ether lipid analog ET-18-OMe was shown to promote invasion of HCT-8/S11 colon cancer cells into collagen type I through the concomitant activation of src by phosphorylation at Tyr416 (5-30 min) in alpha1-integrin immunoprecipitates containing the integrin binding proteins talin and paxillin, as well as the phoshorylated and activated forms of focal adhesion kinase (FAK) at Tyr397 (a FAK kinase activation signal), Tyr576 and Tyr861. This was associated with the lateral redistribution of alpha1-integrins in focal aggregates and persistent activation of the p130Cas/JNK pathways at 5-30 min, with the subsequent induction and activation of the matrix metalloproteinases MMP-2 and MMP-9 (2-12 h). These activated molecular scaffolds and signaling cascades were not observed in immunoprecipitates of alpha2- and beta1-integrins, and tetraspanin CD9, an invasion and metastasis suppressor linked to integrins and FAK signaling. Our data demonstrate that the lateral redistribution and clustering of alpha1-integrins results in the recruitment of the FAK/src motility-promoting signaling complex involved in cancer cell invasion. Disruption of this proinvasive pathway was accomplished by the dominant negative mutant of src (K295R, kinase dead), src pharmacological inhibitor (PP1) and alpha1-integrin function blocking antibodies. These findings support the notion that the alpha1-integrin- and src-dependent signalosome is a relevant therapeutic target against tumor progression in colon cancer patients.

  6. LOXL2 catalytically inactive mutants mediate epithelial-to-mesenchymal transition

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    Eva P. Cuevas

    2014-01-01

    Lysyl-oxidase-like 2 (LOXL2 is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity.

  7. Ganglioside GM2 mediates migration of tumor cells by interacting with integrin and modulating the downstream signaling pathway.

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    Kundu, Manjari; Mahata, Barun; Banerjee, Avisek; Chakraborty, Sohini; Debnath, Shibjyoti; Ray, Sougata Sinha; Ghosh, Zhumur; Biswas, Kaushik

    2016-07-01

    The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor.

  8. DMPD: Signalling pathways mediating type I interferon gene expression. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17904888 Signalling pathways mediating type I interferon gene expression. Edwards M...csml) Show Signalling pathways mediating type I interferon gene expression. PubmedID 17904888 Title Signalli...ng pathways mediating type I interferon gene expression. Authors Edwards MR, Slat

  9. Endocytic pathways mediating oligomeric Aβ42 neurotoxicity

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    Laxton Kevin

    2010-05-01

    Full Text Available Abstract Background One pathological hallmark of Alzheimer's disease (AD is amyloid plaques, composed primarily of amyloid-β peptide (Aβ. Over-production or diminished clearance of the 42 amino acid form of Aβ (Aβ42 in the brain leads to accumulation of soluble Aβ and plaque formation. Soluble oligomeric Aβ (oAβ has recently emerged to be as a likely proximal cause of AD. Results Here we demonstrate that endocytosis is critical in mediating oAβ42-induced neurotoxicity and intraneuronal accumulation of Aβ. Inhibition of clathrin function either with a pharmacological inhibitor, knock-down of clathrin heavy chain expression, or expression of the dominant-negative mutant of clathrin-assembly protein AP180 did not block oAβ42-induced neurotoxicity or intraneuronal accumulation of Aβ. However, inhibition of dynamin and RhoA by expression of dominant negative mutants reduced neurotoxicity and intraneuronal Aβ accumulation. Pharmacologic inhibition of the dynamin-mediated endocytic pathway by genistein also reduced neurotoxicity. Conclusions These data suggest that dynamin-mediated and RhoA-regulated endocytosis are integral steps for oligomeric Aβ42-induced neurotoxicity and intraneuronal Aβ accumulation.

  10. KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

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    Binod Kumar

    2016-11-01

    Full Text Available Kaposi’s sarcoma associated herpesvirus (KSHV is etiologically associated with human endothelial cell hyperplastic Kaposi’s sarcoma and B-cell primary effusion lymphoma. KSHV infection of adherent endothelial and fibroblast cells are used as in vitro models for infection and KSHV enters these cells by host membrane bleb and actin mediated macropinocytosis or clathrin endocytosis pathways, respectively. Infection in endothelial and fibroblast cells is initiated by the interactions between multiple viral envelope glycoproteins and cell surface associated heparan sulfate (HS, integrins (α3β1, αVβ3 and αVβ5, and EphA2 receptor tyrosine kinase (EphA2R. This review summarizes the accumulated studies demonstrating that KSHV manipulates the host signal pathways to enter and traffic in the cytoplasm of the target cells, to deliver the viral genome into the nucleus, and initiate viral gene expression. KSHV interactions with the cell surface receptors is the key platform for the manipulations of host signal pathways which results in the simultaneous induction of FAK, Src, PI3-K, Rho-GTPase, ROS, Dia-2, PKC ζ, c-Cbl, CIB1, Crk, p130Cas and GEF-C3G signal and adaptor molecules that play critical roles in the modulation of membrane and actin dynamics, and in the various steps of the early stages of infection such as entry and trafficking towards the nucleus. The Endosomal Sorting Complexes Required for Transport (ESCRT proteins are also recruited to assist in viral entry and trafficking. In addition, KSHV interactions with the cell surface receptors also induces the host transcription factors NF-κB, ERK1/2, and Nrf2 early during infection to initiate and modulate viral and host gene expression. Nuclear delivery of the viral dsDNA genome is immediately followed by the host innate responses such as the DNA damage response (DDR, inflammasome and interferon responses. Overall, these studies form the initial framework for further studies of

  11. KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

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    Kumar, Binod; Chandran, Bala

    2016-01-01

    Kaposi’s sarcoma associated herpesvirus (KSHV) is etiologically associated with human endothelial cell hyperplastic Kaposi’s sarcoma and B-cell primary effusion lymphoma. KSHV infection of adherent endothelial and fibroblast cells are used as in vitro models for infection and KSHV enters these cells by host membrane bleb and actin mediated macropinocytosis or clathrin endocytosis pathways, respectively. Infection in endothelial and fibroblast cells is initiated by the interactions between multiple viral envelope glycoproteins and cell surface associated heparan sulfate (HS), integrins (α3β1, αVβ3 and αVβ5), and EphA2 receptor tyrosine kinase (EphA2R). This review summarizes the accumulated studies demonstrating that KSHV manipulates the host signal pathways to enter and traffic in the cytoplasm of the target cells, to deliver the viral genome into the nucleus, and initiate viral gene expression. KSHV interactions with the cell surface receptors is the key platform for the manipulations of host signal pathways which results in the simultaneous induction of FAK, Src, PI3-K, Rho-GTPase, ROS, Dia-2, PKC ζ, c-Cbl, CIB1, Crk, p130Cas and GEF-C3G signal and adaptor molecules that play critical roles in the modulation of membrane and actin dynamics, and in the various steps of the early stages of infection such as entry and trafficking towards the nucleus. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins are also recruited to assist in viral entry and trafficking. In addition, KSHV interactions with the cell surface receptors also induces the host transcription factors NF-κB, ERK1/2, and Nrf2 early during infection to initiate and modulate viral and host gene expression. Nuclear delivery of the viral dsDNA genome is immediately followed by the host innate responses such as the DNA damage response (DDR), inflammasome and interferon responses. Overall, these studies form the initial framework for further studies of simultaneous targeting of

  12. Molecular pathways of pannexin1-mediated neurotoxicity

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    Valery I. Shestopalov

    2014-02-01

    Full Text Available Pannexin1 (Panx1 forms nonselective membrane channels, structurally similar to gap junction hemichannels, that is permeable to ions, nucleotides and other small molecules below 900 Da. Panx1 activity is implicated in paracrine signaling and inflammasome regulation. Recent studies in different animal models showed that Panx1 overactivation correlates with a selective demise of several types of neurons, including retinal ganglion cells, brain pyramidal and enteric neurons. The list of Panx1 activators includes extracellular ATP, glutamate, high K+, Zn2+, fibroblast growth factors (FGFs, pro-inflammatory cytokines and elevation of intracellular Ca2+. Most of these molecules are released following mechanical, ischemic or inflammatory injury of the CNS, and rapidly activate this channel. As a result, prolonged opening of Panx1 channel induced by these danger signals trigger a cascade of neurotoxic events capable of killing cells. The most vulnerable cell type are neurons that express high levels of Panx1. Experimental evidence suggests that Panx1 channels mediate at least two distinct neurotoxic processes: increased permeability of the plasma membrane and activation of the inflammasome in neurons and glia. Importantly, either pharmacological or genetic inactivation of Panx1 suppresses both these processes, providing a marked protection in several disease and injury models. These findings indicate that external danger signals generated after diverse types of injuries converge to activate Panx1. In this review we discuss molecular mechanisms associated with Panx1 toxicity and the crosstalk between different pathways.

  13. A mitochondrial pathway for biosynthesis of lipid mediators

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    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-06-01

    The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury.

  14. Platycodin D inhibits tumor growth by antiangiogenic activity via blocking VEGFR2-mediated signaling pathway

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    Luan, Xin; Gao, Yun-Ge; Guan, Ying-Yun; Xu, Jian-Rong; Lu, Qin [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Mei [Department of Pharmacy, Shanghai Institute of Health Sciences and Health School Attached to SJTU-SM, 279 Zhouzhu Road, Shanghai 201318 (China); Liu, Ya-Rong; Liu, Hai-Jun [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Fang, Chao, E-mail: fangchao100@hotmail.com [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Hong-Zhuan, E-mail: hongzhuan_chen@hotmail.com [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China)

    2014-11-15

    Platycodin D (PD) is an active component mainly isolated from the root of Platycodon grandiflorum. Recent studies proved that PD exhibited inhibitory effect on proliferation, migration, invasion and xenograft growth of diverse cancer cell lines. However, whether PD is suppressive for angiogenesis, an important hallmark in cancer development, remains unknown. Here, we found that PD could dose-dependently inhibit human umbilical vein endothelial cell (HUVEC) proliferation, motility, migration and tube formation. PD also significantly inhibited angiogenesis in the chick embryo chorioallantoic membrane (CAM). Moreover, the antiangiogenic activity of PD contributed to its in vivo anticancer potency shown in the decreased microvessel density and delayed growth of HCT-15 xenograft in mice with no overt toxicity. Western blot analysis indicated that PD inhibited the phosphorylation of VEGFR2 and its downstream protein kinase including PLCγ1, JAK2, FAK, Src, and Akt in endothelial cells. Molecular docking simulation showed that PD formed hydrogen bonds and hydrophobic interactions within the ATP binding pocket of VEGFR2 kinase domain. The present study firstly revealed the high antiangiogenic activity and the underlying molecular basis of PD, suggesting that PD may be a potential antiangiogenic agent for angiogenesis-related diseases. - Highlights: • Platycodin D inhibits HUVEC proliferation, motility, migration and tube formation. • Platycodin D inhibits the angiogenesis in chick embryo chorioallantoic membrane. • Platycodin D suppresses the angiogenesis and growth of HCT-15 xenograft in mice. • Platycodin D inhibits the phosphorylation of VEGFR2 and downstream kinases in HUVEC.

  15. Protein-mediated energy-dissipating pathways in mitochondria.

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    Starkov, Anatoly A

    2006-10-27

    Mitochondrial production of reactive oxygen species (ROS) is a well-established fact of fundamental importance to aging and etiology of many pathologies with serious public health implications. The ROS production is an innate property of mitochondrial biochemistry inseparable from the oxidative metabolism. Recent discoveries indicate that in addition to several ROS-detoxifying enzyme systems, which remove ROS, mitochondria may also be able to limit their ROS production by the mechanism comprising several protein-mediated energy-dissipating ("uncoupling") pathways. Although the physiological significance and in vivo modus operandi of these pathways remain to be elucidated, several proteins potentially capable of energy dissipation are known. This mini-review addresses the identity of mitochondrial protein-mediated energy-dissipating pathways and the experimental evidence to their role in controlling ROS production.

  16. Validation of signalling pathways: Case study of the p16-mediated pathway.

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    Akçay, Nimet İlke; Bashirov, Rza; Tüzmen, Şükrü

    2015-04-01

    p16 is recognized as a tumor suppressor gene due to the prevalence of its genetic inactivation in all types of human cancers. Additionally, p16 gene plays a critical role in controlling aging, regulating cellular senescence, detection and maintenance of DNA damage. The molecular mechanism behind these events involves p16-mediated signaling pathway (or p16- Rb pathway), the focus of our study. Understanding functional dependence between dynamic behavior of biological components involved in the p16-mediated pathway and aforesaid molecular-level events might suggest possible implications in the diagnosis, prognosis and treatment of human cancer. In the present work, we employ reverse-engineering approach to construct the most detailed computational model of p16-mediated pathway in higher eukaryotes. We implement experimental data from the literature to validate the model, and under various assumptions predict the dynamic behavior of p16 and other biological components by interpreting the simulation results. The quantitative model of p16-mediated pathway is created in a systematic manner in terms of Petri net technologies.

  17. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis.

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    Gaits, F; Fourcade, O; Le Balle, F; Gueguen, G; Gaigé, B; Gassama-Diagne, A; Fauvel, J; Salles, J P; Mauco, G; Simon, M F; Chap, H

    1997-06-23

    From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.

  18. Intergenerational continuity in parenting behavior: mediating pathways and child effects.

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    Neppl, Tricia K; Conger, Rand D; Scaramella, Laura V; Ontai, Lenna L

    2009-09-01

    This prospective, longitudinal investigation examined mechanisms proposed to explain continuities in parenting behavior across 2 generations (G1, G2). Data came from 187 G2 adults, their mothers (G1), and their children (G3). Prospective information regarding G2 was collected both during adolescence and early adulthood. G1 data were collected during G2's adolescence, and G3 data were generated during the preschool years. Assessments included both observational and self-report measures. The results indicated a direct relationship between G1 and G2 harsh parenting, and between G1 and G2 positive parenting. As predicted, specific mediators accounted for intergenerational continuity in particular types of parenting behavior. G2 externalizing behavior mediated the relationship between G1 and G2 harsh parenting, whereas G2 academic attainment mediated the relationship between G1 and G2 positive parenting. In addition, the hypothesized mediating pathways remained statistically significant after taking into account possible G2 effects on G1 parenting and G3 effects on G2 parenting.

  19. Multiple parietal-frontal pathways mediate grasping in macaque monkeys

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    Gharbawie, Omar A.; Stepniewska, Iwona; Qi, Huixin; Kaas, Jon H.

    2011-01-01

    The nodes of a parietal-frontal pathway that mediates grasping in primates are in anterior intraparietal area (AIP) and ventral premotor cortex (PMv). Nevertheless, multiple somatosensory and motor representations of the hand, respectively in parietal and frontal cortex, suggest that additional pathways remain unrealized. We explored this possibility in macaque monkeys by injecting retrograde tracers into grasp zones identified in M1, PMv, and area 2 with long train electrical stimulation. The M1 grasp zone was densely connected with other frontal cortex motor regions. The remainder of the connections originated from somatosensory areas 3a and S2/PV, and from the medial bank and fundus of the intraparietal sulcus (IPS). The PMv grasp zone was also densely connected with frontal cortex motor regions, albeit to a lesser extent than the M1 grasp zone. The remainder of the connections originated from areas S2/PV and aspects of the inferior parietal lobe such as PF, PFG, AIP, and the tip of the IPS. The area 2 grasp zone was densely connected with the hand representations of somatosensory areas 3b, 1, and S2/PV. The remainder of the connections was with areas 3a and 5 and the medial bank and fundus of the IPS. Connections with frontal cortex were relatively weak and concentrated in caudal M1. Thus, the three grasp zones may be nodes of parallel parietal-frontal pathways. Differential points of origin and termination of each pathway suggest varying functional specializations. Direct and indirect connections between those parietal-frontal pathways likely coordinate their respective functions into an accurate grasp. PMID:21832196

  20. The Molecular Pathway of Argon-Mediated Neuroprotection

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    Felix Ulbrich

    2016-10-01

    Full Text Available The noble gas argon has attracted increasing attention in recent years, especially because of its neuroprotective properties. In a variety of models, ranging from oxygen-glucose deprivation in cell culture to complex models of mid-cerebral artery occlusion, subarachnoid hemorrhage or retinal ischemia-reperfusion injury in animals, argon administration after individual injury demonstrated favorable effects, particularly increased cell survival and even improved neuronal function. As an inert molecule, argon did not show signs of adverse effects in the in vitro and in vivo model used, while being comparably cheap and easy to apply. However, the molecular mechanism by which argon is able to exert its protective and beneficial characteristics remains unclear. Although there are many pieces missing to complete the signaling pathway throughout the cell, it is the aim of this review to summarize the known parts of the molecular pathways and to combine them to provide a clear insight into the cellular pathway, starting with the receptors that may be involved in mediating argons effects and ending with the translational response.

  1. Lipid-mediated muscle insulin resistance: different fat, different pathways?

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    Ritter, Olesja; Jelenik, Tomas; Roden, Michael

    2015-08-01

    Increased dietary fat intake and lipolysis result in excessive lipid availability, which relates to impaired insulin sensitivity. Over the last years, several mechanisms possibly underlying lipid-mediated insulin resistance evolved. Lipid intermediates such as diacylglycerols (DAG) associate with changes in insulin sensitivity in many models. DAG activate novel protein kinase C (PKC) isoforms followed by inhibitory serine phosphorylation of insulin receptor substrate 1 (IRS1). Activation of Toll-like receptor 4 (TLR4) raises another lipid class, ceramides (CER), which induce pro-inflammatory pathways and lead to inhibition of Akt phosphorylation. Inhibition of glucosylceramide and ganglioside synthesis results in improved insulin sensitivity and increased activatory tyrosine phosphorylation of IRS1 in the muscle. Incomplete fat oxidation can increase acylcarnitines (ACC), which in turn stimulate pro-inflammatory pathways. This review analyzed the effects of lipid metabolites on insulin action in skeletal muscle of humans and rodents. Despite the evidence for the association of both DAG and CER with insulin resistance, its causal relevance may differ depending on the subcellular localization and the tested cohorts, e.g., athletes. Nevertheless, recent data indicate that individual lipid species and their degree of fatty acid saturation, particularly membrane and cytosolic C18:2 DAG, specifically activate PKCθ and induce both acute lipid-induced and chronic insulin resistance in humans.

  2. The N-acylethanolamine-mediated regulatory pathway in plants.

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    Kilaru, Aruna; Blancaflor, Elison B; Venables, Barney J; Tripathy, Swati; Mysore, Kirankumar S; Chapman, Kent D

    2007-08-01

    While cannabinoids are secondary metabolites synthesized by just a few plant species, N-acylethanolamines (NAEs) are distributed widely in the plant kingdom, and are recovered in measurable, bioactive quantities in many plant-derived products. NAEs in higher plants are ethanolamides of fatty acids with acyl-chain lenghts of C12-C(18) and zero to three C=C bonds. Generally, the most-abundant NAEs found in plants and vertebrates are similar, including NAE 16 : 0, 18 : 1, 18 : 2, and 18 : 3. Like in animal systems, NAEs are formed in plants from N-acylphosphatidylethanolamines (NAPEs), and they are hydrolyzed by an amidase to yield ethanolamine and free fatty acids (FFA). Recently, a homologue of the mammalian fatty acid amide hydrolase (FAAH-1) was identified in Arabidopsis thaliana and several other plant species. Overexpression of Arabidopsis FAAH (AtFAAH) resulted in plants that grew faster, but were more sensitive to biotic and abiotic insults, suggesting that the metabolism of NAEs in plants resides at the balance between growth and responses to environmental stresses. Similar to animal systems, exogenously applied NAEs have potent and varied effects on plant cells. Recent pharmacological approaches combined with molecular-genetic experiments revealed that NAEs may act in certain plant tissues via specific membrane-associated proteins or by interacting with phospholipase D-alpha, although other, direct targets for NAE action in plants are likely to be discovered. Polyunsaturated NAEs can be oxidized via the lipoxygenase pathway in plants, producing an array of oxylipin products that have received little attention so far. Overall, the conservation of NAE occurrence and metabolic machinery in plants, coupled with the profound physiological effects of elevating NAE content or perturbing endogenous NAE metabolism, suggest that an NAE-mediated regulatory pathway, sharing similarities with the mammalian endocannabinoid pathway, indeed exists.

  3. The effect of chronic seaweed subsidies on herbivory: plant-mediated fertilization pathway overshadows lizard-mediated predator pathways.

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    Piovia-Scott, Jonah; Spiller, David A; Takimoto, Gaku; Yang, Louie H; Wright, Amber N; Schoener, Thomas W

    2013-08-01

    Flows of energy and materials link ecosystems worldwide and have important consequences for the structure of ecological communities. While these resource subsidies typically enter recipient food webs through multiple channels, most previous studies focussed on a single pathway of resource input. We used path analysis to evaluate multiple pathways connecting chronic marine resource inputs (in the form of seaweed deposits) and herbivory in a shoreline terrestrial ecosystem. We found statistical support for a fertilization effect (seaweed increased foliar nitrogen content, leading to greater herbivory) and a lizard numerical response effect (seaweed increased lizard densities, leading to reduced herbivory), but not for a lizard diet-shift effect (seaweed increased the proportion of marine-derived prey in lizard diets, but lizard diet was not strongly associated with herbivory). Greater seaweed abundance was associated with greater herbivory, and the fertilization effect was larger than the combined lizard effects. Thus, the bottom-up, plant-mediated effect of fertilization on herbivory overshadowed the top-down effects of lizard predators. These results, from unmanipulated shoreline plots with persistent differences in chronic seaweed deposition, differ from those of a previous experimental study of the short-term effects of a pulse of seaweed deposition: while the increase in herbivory in response to chronic seaweed deposition was due to the fertilization effect, the short-term increase in herbivory in response to a pulse of seaweed deposition was due to the lizard diet-shift effect. This contrast highlights the importance of the temporal pattern of resource inputs in determining the mechanism of community response to resource subsidies.

  4. Triglyceride-mediated pathways and coronary disease: collaborative analysis of 101 studies

    DEFF Research Database (Denmark)

    Sarwar, Nadeem; Sandhu, Manjinder S; Ricketts, Sally L

    2010-01-01

    Whether triglyceride-mediated pathways are causally relevant to coronary heart disease is uncertain. We studied a genetic variant that regulates triglyceride concentration to help judge likelihood of causality....

  5. PHLPP phosphatase:a key mediator integrating multiple signaling pathways

    Institute of Scientific and Technical Information of China (English)

    Hui ZHONG

    2010-01-01

    @@ Cellular responses to bacterial or viral infections and to stress require rapid and accurate transmission of signals from cell-surface receptors to the nucleus (Karin and Hunter, 1995).These signaling pathways, relying on extensive protein phosphorylation events, lead to the activation of specific transcription factors that induce the expression of appropriate target genes.Among the activated transcription factors, nuclear factor KB (NF-KB)is essential for inflammation, immunity, cell proliferation and apoptosis.NF-KB requires a signaling pathway for activation.Such NF-KB-activating pathways can be triggered by a variety of extracellular stimuli, which lead to the phosphorylation and subsequent proteasomemediated degradation of inhibitory molecules, the inhibitor of NF-KB (hcB) proteins (Karin and Ben-Neriah, 2000).Activated NF-KB migrates into the nucleus to regulate the expression of multiple target genes.

  6. New insights into Reelin-mediated signaling pathways

    Directory of Open Access Journals (Sweden)

    Gum Hwa eLee

    2016-05-01

    Full Text Available Reelin, a multifunctional extracellular protein that is important for mammalian brain development and function, is secreted by different cell types in the prenatal or postnatal brain. The spatiotemporal regulation of Reelin expression and distribution during development relates to its multifaceted function in the brain. Prenatally Reelin controls neuronal radial migration and proper positioning in cortical layers, whereas postnatally Reelin promotes neuronal maturation, synaptic formation and plasticity. The molecular mechanisms underlying the distinct biological functions of Reelin during and after brain development involve unique and overlapping signaling pathways that are activated following Reelin binding to its cell surface receptors. Distinct Reelin ligand isoforms, such as the full-length protein or fragments generated by proteolytic cleavage differentially affect the activity of downstream signaling pathways. In this review, we discuss recent advances in our understanding of the signaling transduction pathways activated by Reelin that regulate different aspects of brain development and function. A core signaling machinery, including ApoER2/VLDLR receptors, Src/Fyn kinases, and the adaptor protein Dab1, participates in all known aspects of Reelin biology. However, distinct downstream mechanisms, such as the Crk/Rap1 pathway and cell adhesion molecules, play crucial roles in the control of neuronal migration, whereas the PI3K/Akt/mTOR pathway appears to be more important for dendrite and spine development. Finally, the NMDAR and an unidentified receptor contribute to the activation of the MEK/Erk1/2 pathway leading to the upregulation of genes involved in synaptic plasticity and learning. This knowledge may provide new insight into neurodevelopmental or neurodegenerative disorders that are associated with Reelin dysfunction.

  7. Multiple host-cell recombination pathways act in Agrobacterium-mediated transformation of plant cells.

    Science.gov (United States)

    Mestiri, Imen; Norre, Frédéric; Gallego, Maria E; White, Charles I

    2014-02-01

    Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.

  8. Aromatase pathway mediates sex change in each direction

    OpenAIRE

    2005-01-01

    The enzyme aromatase controls the androgen/oestrogen ratio by catalysing the irreversible conversion of testosterone into oestradiol (E2). Therefore, the regulation of E2 synthesis by aromatase is thought to be critical in sexual development and differentiation. Here, we demonstrate for the first time that experimental manipulation of E2 levels via the aromatase pathway induces adult sex change in each direction in a hermaphroditic fish that naturally exhibits bidirectional sex change. Our re...

  9. BAD-mediated apoptotic pathway is associated with human cancer development.

    Science.gov (United States)

    Stickles, Xiaomang B; Marchion, Douglas C; Bicaku, Elona; Al Sawah, Entidhar; Abbasi, Forough; Xiong, Yin; Bou Zgheib, Nadim; Boac, Bernadette M; Orr, Brian C; Judson, Patricia L; Berry, Amy; Hakam, Ardeshir; Wenham, Robert M; Apte, Sachin M; Berglund, Anders E; Lancaster, Johnathan M

    2015-04-01

    The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, pcancers, as well as with ovarian endometriosis (n=20, pcancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, pcancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.

  10. DMPD: Signal transduction pathways mediated by the interaction of CpG DNA withToll-like receptor 9. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14751759 Signal transduction pathways mediated by the interaction of CpG DNA withTo...;16(1):17-22. (.png) (.svg) (.html) (.csml) Show Signal transduction pathways mediated by the interaction of... CpG DNA withToll-like receptor 9. PubmedID 14751759 Title Signal transduction pa

  11. Complement-Mediated Glomerular Diseases: A Tale of 3 Pathways

    Directory of Open Access Journals (Sweden)

    Andrew S. Bomback

    2016-09-01

    Full Text Available A renewed interest in the role of complement in the pathogenesis of glomerular diseases has improved our understanding of their basic, underlying physiology. All 3 complement pathways—classical, lectin, and alternative—have been implicated in glomerular lesions both rare (e.g., dense deposit disease and common (e.g., IgA nephropathy. Here we review the basic function of these pathways and highlight, with a disease-specific focus, how activation can lead to glomerular injury. We end by exploring the promise of complement-targeted therapies as disease-specific interventions for glomerular diseases.

  12. EETs mediate cardioprotection of salvianolic acids through MAPK signaling pathway

    Directory of Open Access Journals (Sweden)

    Shoubao Wang

    2013-02-01

    Full Text Available Salvianolic acids, including salvianolic acid A (SAA and salvianolic acid B (SAB, are the main water-soluble bioactive compounds isolated from the Chinese medicinal herb Danshen and have been shown to exert in vitro and in vivo cardiovascular protection. Recent studies suggest that epoxyeicosatrienoic acids (EETs, the primary cytochrome P450 2J (CYP2J epoxygenase metabolites of arachidonic acid, are involved in the progression of ischemic injury in diverse organs. Here, we investigated the relation between the protective effects of salvianolic acids and EETs/sEH as well as MAPK signaling pathway. In the present study, the rat acute myocardial infarction (AMI model was established by the left anterior descending coronary artery occlusion. Our results showed that salvianolic acids significantly reduced ST-segment elevation and serum levels of CK-MB, LDH, and ALT in AMI rats, and significantly attenuated the caspase 3 expression and reduced the ratio of Bax/Bcl-2. ELISA measurement showed that salvianolic acids significantly increased the 14,15-EET levels in blood and heart, and attenuated hydrolase activity of sEH in heart of AMI rat. Western blotting analysis suggested that salvianolic acids significantly attenuated the phosphorylation of JNK and p38, and increased phosphorylation of ERK in heart. In conclusion, these results indicate that EETs/sEH and MAPK signaling pathways are important processes in cardioprotection of salvianolic acids.

  13. RD26 mediates crosstalk between drought and brassinosteroid signalling pathways

    Science.gov (United States)

    Ye, Huaxun; Liu, Sanzhen; Tang, Buyun; Chen, Jiani; Xie, Zhouli; Nolan, Trevor M.; Jiang, Hao; Guo, Hongqing; Lin, Hung-Ying; Li, Lei; Wang, Yanqun; Tong, Hongning; Zhang, Mingcai; Chu, Chengcai; Li, Zhaohu; Aluru, Maneesha; Aluru, Srinivas; Schnable, Patrick S.; Yin, Yanhai

    2017-01-01

    Brassinosteroids (BRs) regulate plant growth and stress responses via the BES1/BZR1 family of transcription factors, which regulate the expression of thousands of downstream genes. BRs are involved in the response to drought, however the mechanistic understanding of interactions between BR signalling and drought response remains to be established. Here we show that transcription factor RD26 mediates crosstalk between drought and BR signalling. When overexpressed, BES1 target gene RD26 can inhibit BR-regulated growth. Global gene expression studies suggest that RD26 can act antagonistically to BR to regulate the expression of a subset of BES1-regulated genes, thereby inhibiting BR function. We show that RD26 can interact with BES1 protein and antagonize BES1 transcriptional activity on BR-regulated genes and that BR signalling can also repress expression of RD26 and its homologues and inhibit drought responses. Our results thus reveal a mechanism coordinating plant growth and drought tolerance. PMID:28233777

  14. Neurotransmitter receptor-mediated signaling pathways as modulators of carcinogenesis.

    Science.gov (United States)

    Schuller, Hildegard M

    2007-01-01

    The autonomic nervous system with its two antagonistic branches, the sympathicus and the parasympathicus, regulates the activities of all body functions that are not under voluntary control. While the autonomic regulation of organ functions has been extensively studied, little attention has been given to the potential role of neurohumoral transmission at the cellular level in the development of cancer. Studies conducted by our laboratory first showed that binding of the parasympathetic neurotransmitter, acetylcholine, as well as nicotine or its nitrosated cancer-causing derivative, NNK, to nicotinic acetylcholine receptors comprised of alpha7 subunits activated a mitogenic signal transduction pathway in normal and neoplastic pulmonary neuroendocrine cells. On the other hand, beta-adrenergic receptors (Beta-ARs), which transmit signals initiated by binding of the catecholamine neurotransmitters of the sympathicus, were identified by our laboratory as important regulators of cell proliferation in cell lines derived from human adenocarcinomas of the lungs, pancreas, and breast. The tobacco-specific carcinogen NNK bound with high affinity to Beta1- and Beta2-ARs, thus activating cAMP, protein kinase A, and the transcription factor CREB. Collectively, neurotransmitter receptors of the nicotinic and Beta-adrenergic families appear to regulate cellular functions essential for the development and survival of the most common human cancers.

  15. Identification of a novel Gnao-mediated alternate olfactory signaling pathway in murine OSNs

    Directory of Open Access Journals (Sweden)

    Paul eScholz

    2016-03-01

    Full Text Available It is generally agreed that in olfactory sensory neurons (OSNs, the binding of odorant molecules to their specific olfactory receptor (OR triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG and at least one other known weak Olfr73 agonist (Raspberry Ketone trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl- efflux; however, the activation of adenylyl cyclase III (ACIII, the recruitment of Ca2+ from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.

  16. Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs.

    Science.gov (United States)

    Scholz, Paul; Mohrhardt, Julia; Jansen, Fabian; Kalbe, Benjamin; Haering, Claudia; Klasen, Katharina; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl(-) efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca(2+) from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.

  17. EVIDENCE FOR EGFR PATHWAY MEDIATION OF CLEFT PALATE INDUCTION BY TCDD

    Science.gov (United States)

    EVIDENCE FOR EGFR PATHWAY MEDIATION OF CLEFT PALATE INDUCTION BY TCDD. B D Abbott, A R Buckalew, and K E Leffler. RTD, NHEERL, ORD,US EPA, RTP, NC, USA.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in C57BL/6J mice, producing cleft palate (CP) after exposure...

  18. Ficolin-3-mediated lectin complement pathway activation in patients with subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Zanier, Elisa R; Zangari, Rosalia; Munthe-Fog, Lea

    2014-01-01

    OBJECTIVES: To assess the involvement of ficolin-3, the main initiator of the lectin complement pathway (LCP), in subarachnoid hemorrhage (SAH) pathology and outcome. METHODS: In this preliminary exploratory study, plasma concentration of ficolin-3 and of ficolin-3-mediated functional LCP activit...

  19. Triglyceride-mediated pathways and coronary disease: collaborative analysis of 101 studies

    NARCIS (Netherlands)

    N. Sarwar; M.S. Sandhu; S.L. Ricketts; A.S. Butterworth; E. Di Angelantonio; S.M. Boekholdt; W. Ouwehand; H. Watkins; N.J. Samani; D. Saleheen; D. Lawlor; M.P. Reilly; A.D. Hingorani; P.J. Talmud; J. Danesh

    2010-01-01

    BACKGROUND: Whether triglyceride-mediated pathways are causally relevant to coronary heart disease is uncertain. We studied a genetic variant that regulates triglyceride concentration to help judge likelihood of causality. METHODS: We assessed the -1131T>C (rs662799) promoter polymorphism of the apo

  20. Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

    DEFF Research Database (Denmark)

    Whiteford, James; Behrends, Volker; Kirby, Hishani;

    2007-01-01

    to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence...

  1. Triglyceride-mediated pathways and coronary disease: collaborative analysis of 101 studies

    NARCIS (Netherlands)

    Kromhout, D.

    2010-01-01

    Background -Whether triglyceride-mediated pathways are causally relevant to coronary heart disease is uncertain. We studied a genetic variant that regulates triglyceride concentration to help judge likelihood of causality. Methods - We assessed the -1131T>C (rs662799) promoter polymorphism of the

  2. The XPB and XPD DNA helicases are components of the p53-mediated apoptosis pathway.

    NARCIS (Netherlands)

    X.W. Wang (Xin Wei); W. Vermeulen (Wim); J.D. Coursen; M.K. Gibson (Michael); S.E. Lupold; K. Forrester; G. Xu; L. Elmore; H. Yeh; J.H.J. Hoeijmakers (Jan); C.C. Harris

    1996-01-01

    textabstractThe molecular pathway of p53-dependent apoptosis (programmed cell death) is poorly understood. Because p53 binds to the basal transcription-repair complex TFIIH and modulates its DNA helicase activities, we hypothesized that TFIIH DNA helicases XPB and XPD are members of the p53-mediated

  3. Anxiety and disruptive behavior mediate pathways from attention-deficit/hyperactivity disorder to depression.

    Science.gov (United States)

    Roy, Arunima; Oldehinkel, Albertine J; Verhulst, Frank C; Ormel, Johan; Hartman, Catharina A

    2014-02-01

    The progression to depression in children with attention-deficit/hyperactivity disorder (ADHD) is not clearly understood. To clarify this relationship, we tested the following hypotheses in a population-based study: (1) children with ADHD have a higher risk of developing depression than children without ADHD; (2) the pathway from ADHD to depression is mediated (partly) through anxiety and disruptive behavior disorders; and (3) mediation through anxiety is more prevalent in girls, and mediation through disruptive behavior disorders is more prevalent in boys. From October 2008 to September 2010, the Composite International Diagnostic Interview was used to assess ADHD, major depressive episodes, anxiety disorders, and disruptive behavior disorders in 1,584 participants from the TRacking Adolescents' Individual Lives Survey (TRAILS) cohort. Cox regression was used to model the effects of ADHD, anxiety, and disruptive behaviors on depression. Risk of and pathways to depression were studied in both children with ADHD and children with subthreshold ADHD. Comorbid depression was present in 36% of children with a diagnosis of ADHD, 24% of children with subthreshold ADHD, and 14% of children with no ADHD. Anxiety and disruptive behaviors mediated 32% of depression in ADHD. Pathways through anxiety and disruptive behavior disorders were independent of gender. Disruptive behavior disorder was a stronger mediator than anxiety for both genders (all P disruptive behavior disorders are present in a child with ADHD. © Copyright 2014 Physicians Postgraduate Press, Inc.

  4. Acetylcholine produces contraction mediated by cyclooxigenase pathway in arterial vessels in the marine fish (Isacia conceptionis

    Directory of Open Access Journals (Sweden)

    FA. Moraga

    Full Text Available Preliminary studies showed that dorsal artery contraction mediated by acetylcholine (ACh is blocked with indomethacin in intertidal fish (G. laevifrons. Our objective was to characterize the cholinergic pathway in several artery vessels of the I. conceptionis. Afferent and efferent branchial, dorsal and mesenteric arteries were dissected of 6 juvenile specimens, isometric tension studies were done using doses response curves (DRC for Ach (10–13 to 10–3 M, and cholinergic pathways were obtained by blocking with atropine or indomethacin. CRC to ACh showed a pattern of high sensitivity only in efferente branchial artery and low sensibility in all vessels. Furthermore, these contractions were blocked in the presence of atropine and indomethacin in all vessels. Our results corroborate previous results observed in intertidal species that contraction induced by acetylcholine is mediated by receptors that activate a cyclooxygenase contraction pathway.

  5. The CEK1-mediated mitogen-activated protein kinase pathway in the fungal pathogen Candida albicans

    Directory of Open Access Journals (Sweden)

    Elvira Román

    2013-06-01

    Full Text Available Mitogen-activated protein kinases (MAPK mediated signal transduction pathways are essential for the adaptation of living organisms to environmental changes. In pathogenic fungi, these MAPK cascades govern the response to many types of situations, and are essential for the successful establishment of the fungus within the host. Therefore, they influence virulence and can be considered as promising therapeutic targets. In the opportunistic pathogen Candida albicans, the Cek1-mediated pathway was identified long time ago as an important virulence determinant in certain animal models. We will review here the recent work that reveals the role that this route plays in three important processes for the cell: osmotic adaptation, fungal morphogenesis and cell wall remodeling. We will also show the complementary (and sometimes opposite roles that under specific circumstances the high osmolarity glycerol and CEK1 pathways play in C. albicans biology, especially in the context of the interaction with the mammalian host.

  6. Therapeutic Potential of IL-17-Mediated Signaling Pathway in Autoimmune Liver Diseases

    Directory of Open Access Journals (Sweden)

    Haiyan Zhang

    2015-01-01

    Full Text Available Emerging evidence reveals that various cytokines and tissue microenvironments contribute to liver inflammation and autoimmunity, and IL-17 family is one of highlights acknowledged. Although the implication of IL-17 family in most common autoimmune diseases (such as psoriasis, inflammatory bowel disease, and rheumatoid arthritis has been extensively characterized, the role of this critical family in pathophysiology of autoimmune liver diseases (AILD still needs to be clarified. In the review, we look into the intriguing biology of IL-17 family and further dissect on the intricate role of IL-17-mediated pathway in AILD. Considering encouraging data from preclinical and clinical trials, IL-17 targeted therapy has shown promises in several certain autoimmune conditions. However, blocking IL-17-mediated pathway is just beginning, and more fully investigation and reflection are required. Taking together, targeting IL-17-mediated responses may open up new areas of potential clinical treatment for AILD.

  7. The Small Interfering RNA Pathway Is Not Essential for Wolbachia-Mediated Antiviral Protection in Drosophila melanogaster

    OpenAIRE

    Lauren M Hedges; Yamada, Ryuichi; O'Neill, Scott L; Karyn N. Johnson

    2012-01-01

    Wolbachia pipientis delays RNA virus-induced mortality in Drosophila spp. We investigated whether Wolbachia-mediated protection was dependent on the small interfering RNA (siRNA) pathway, a key antiviral defense. Compared to Wolbachia-free flies, virus-induced mortality was delayed in Wolbachia-infected flies with loss-of-function of siRNA pathway components, indicating that Wolbachia-mediated protection functions in the absence of the canonical siRNA pathway.

  8. Anti-adipogenic activity of berberine is not mediated by the WNT/β-catenin pathway.

    Science.gov (United States)

    Bae, Sungmin; Yoon, Yoosik

    2013-06-01

    Adipogenesis is a differentiation process from preadipocytes to adipocytes, accompanied by the inductions of adipogenic transcription factors and lipid metabolizing enzymes. Among cellular pathways regulating adipogenesis, the WNT/β-catenin pathway is well-known as a suppressor of adipogenesis. Berberine (BBR) is an isoquinoline alkaloid component of the medicinal plants including Coptis chinensis and Coptis japonica with diverse biological activities. This study was conducted to elucidate whether the anti-adipogenic effect of BBR is mediated by the WNT/β-catenin pathway. The results of the present study confirmed that BBR efficiently inhibited adipogenesis of 3T3-L1 cells. However, the anti-adipogenic effects of BBR were not accompanied by the modulations of the WNT/β-catenin pathway members including WNT10B, LRP6, DVL2, GSK3β and β-catenin. When β-catenin was knocked down by its siRNA transfection, the anti-adipogenic effects of BBR including the expression of adipogenic transcription factors and lipid metabolizing enzymes as well as the intracellular fat accumulation were not affected at all. The results of this study showed that the anti-adipogenic effect of BBR is not mediated by the WNT/β-catenin pathway.

  9. Newcomers to the WW Domain-Mediated Network of the Hippo Tumor Suppressor Pathway.

    Science.gov (United States)

    Sudol, Marius

    2010-11-01

    The Hippo tumor suppressor pathway regulates the size of organs by controlling 2 opposing processes: proliferation and apoptosis. The pathway was originally defined in Drosophila, but it is well conserved in mammals. One of the unique features of Hippo signaling is the unusually wide occurrence of WW domains and its cognate PPxY ligand motifs within components of this pathway. Recently, it was proposed that the prevalence of WW domain-mediated complexes in the Hippo signaling pathway should facilitate its molecular analysis and help in the identification of new components of the Hippo-centered network. Indeed, several new members of the Hippo pathway, which form functional complexes with WW domains of YAP and TAZ effectors, were recently described. We focus here on 2 families of such proteins, angiomotins and SMADs, plus 1 regulatory factor, WBP-2, which together shed new light on the rapidly expanding Hippo network. Since the Hippo pathway acts as a tumor suppressor pathway, the complexes described here, which assemble on WW domains of YAP and TAZ, represent potential targets of cancer therapy.

  10. How does MBCT for depression work? studying cognitive and affective mediation pathways.

    Directory of Open Access Journals (Sweden)

    Tim Batink

    Full Text Available Mindfulness based cognitive therapy (MBCT is a non-pharmacological intervention to reduce current symptoms and to prevent recurrence of major depressive disorder. At present, it is not well understood which underlying mechanisms during MBCT are associated with its efficacy. The current study (n = 130 was designed to examine the roles of mindfulness skills, rumination, worry and affect, and the interplay between those factors, in the mechanisms of change in MBCT for residual depressive symptoms. An exploratory but systematic approach was chosen using Sobel-Goodman mediation analyses to identify mediators on the pathway from MBCT to reduction in depressive symptoms. We replicated earlier findings that therapeutic effects of MBCT are mediated by changes in mindfulness skills and worry. Second, results showed that changes in momentary positive and negative affect significantly mediated the efficacy of MBCT, and also mediated the effect of worry on depressive symptoms. Third, within the group of patients with a prior history of ≤ 2 episodes of MDD, predominantly changes in cognitive and to a lesser extent affective processes mediated the effect of MBCT. However, within the group of patients with a prior history of ≥ 3 episodes of MDD, only changes in affect were significant mediators for the effect of MBCT.[corrected] Nederlands Trial Register NTR1084.

  11. Mediators, Receptors, and Signalling Pathways in the Anti-Inflammatory and Antihyperalgesic Effects of Acupuncture

    Directory of Open Access Journals (Sweden)

    John L. McDonald

    2015-01-01

    Full Text Available Acupuncture has been used for millennia to treat allergic diseases including both intermittent rhinitis and persistent rhinitis. Besides the research on the efficacy and safety of acupuncture treatment for allergic rhinitis, research has also investigated how acupuncture might modulate immune function to exert anti-inflammatory effects. A proposed model has previously hypothesized that acupuncture might downregulate proinflammatory neuropeptides, proinflammatory cytokines, and neurotrophins, modulating transient receptor potential vallinoid (TRPV1, a G-protein coupled receptor which plays a central role in allergic rhinitis. Recent research has been largely supportive of this model. New advances in research include the discovery of a novel cholinergic anti-inflammatory pathway activated by acupuncture. A chemokine-mediated proliferation of opioid-containing macrophages in inflamed tissues, in response to acupuncture, has also been demonstrated for the first time. Further research on the complex cross talk between receptors during inflammation is also helping to elucidate the mediators and signalling pathways activated by acupuncture.

  12. Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways.

    Science.gov (United States)

    Szymczak-Workman, Andrea L; Delgoffe, Greg M; Green, Douglas R; Vignali, Dario A A

    2011-11-01

    Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells. We show that Tregs are able to suppress the proliferation of conventional T cells that are resistant to apoptosis (Bim(-/-), Bim(-/-)Puma(-/-), Bcl-2 transgenic) or receptor-interacting serine-threonine kinase-dependent necrosis (also referred to as regulated necrosis or necroptosis) (Ripk3(-/-)) in several in vitro and in vivo assays. These data suggest that programmed cell death pathways, such as apoptosis and receptor-interacting serine-threonine kinase-dependent necrosis, are not required for Treg-mediated suppression.

  13. The Hedgehog signalling pathway mediates drug response of MCF-7 mammosphere cells in breast cancer patients.

    Science.gov (United States)

    He, Miao; Fu, Yingzi; Yan, Yuanyuan; Xiao, Qinghuan; Wu, Huizhe; Yao, Weifan; Zhao, Haishan; Zhao, Lin; Jiang, Qian; Yu, Zhaojin; Jin, Feng; Mi, Xiaoyi; Wang, Enhua; Cui, Zeshi; Fu, Liwu; Chen, Jianju; Wei, Minjie

    2015-11-01

    BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e., PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+ / CD24-, and the expression of SMO and Gli1 in CD44+ / CD24- tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy.

  14. The JNK pathway is a key mediator of Anopheles gambiae antiplasmodial immunity.

    Directory of Open Access Journals (Sweden)

    Lindsey S Garver

    Full Text Available The innate immune system of Anopheles gambiae mosquitoes limits Plasmodium infection through multiple molecular mechanisms. For example, midgut invasion by the parasite triggers an epithelial nitration response that promotes activation of the complement-like system. We found that suppression of the JNK pathway, by silencing either Hep, JNK, Jun or Fos expression, greatly enhanced Plasmodium infection; while overactivating this cascade, by silencing the suppressor Puckered, had the opposite effect. The JNK pathway limits infection via two coordinated responses. It induces the expression of two enzymes (HPx2 and NOX5 that potentiate midgut epithelial nitration in response to Plasmodium infection and regulates expression of two key hemocyte-derived immune effectors (TEP1 and FBN9. Furthermore, the An. gambiae L3-5 strain that has been genetically selected to be refractory (R to Plasmodium infection exhibits constitutive overexpression of genes from the JNK pathway, as well as midgut and hemocyte effector genes. Silencing experiments confirmed that this cascade mediates, to a large extent, the drastic parasite elimination phenotype characteristic of this mosquito strain. In sum, these studies revealed the JNK pathway as a key regulator of the ability of An. gambiae mosquitoes to limit Plasmodium infection and identified several effector genes mediating these responses.

  15. Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

    Science.gov (United States)

    Stępiński, Dariusz

    2016-08-01

    Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

  16. Dihydroartemisinin induces apoptosis preferentially via a Bim-mediated intrinsic pathway in hepatocarcinoma cells.

    Science.gov (United States)

    Qin, Guiqi; Zhao, ChuBiao; Zhang, Lili; Liu, Hongyu; Quan, Yingyao; Chai, Liuying; Wu, Shengnan; Wang, Xiaoping; Chen, Tongsheng

    2015-08-01

    This report is designed to dissect the detail molecular mechanism by which dihydroartemisinin (DHA), a derivative of artemisinin, induces apoptosis in human hepatocellular carcinoma (HCC) cells. DHA induced a loss of the mitochondrial transmemberane potential (ΔΨm), release of cytochrome c, activation of caspases, and externalization of phosphatidylserine indicative of apoptosis induction. Compared with the modest inhibitory effects of silencing Bax, silencing Bak largely prevented DHA-induced ΔΨm collapse and apoptosis though DHA induced a commensurable activation of Bax and Bak, demonstrating a key role of the Bak-mediated intrinsic apoptosis pathway. DHA did not induce Bid cleavage and translocation from cytoplasm to mitochondria and had little effects on the expressions of Puma and Noxa, but did increase Bim and Bak expressions and decrease Mcl-1 expression. Furthermore, the cytotoxicity of DHA was remarkably reduced by silencing Bim, and modestly but significantly reduced by silencing Puma or Noxa. Silencing Bim or Noxa preferentially reduced DHA-induced Bak activation, while silencing Puma preferentially reduced DHA-induced Bax activation, demonstrating that Bim and to a lesser extent Noxa act as upstream mediators to trigger the Bak-mediated intrinsic apoptosis pathway. In addition, silencing Mcl-1 enhanced DHA-induced Bak activation and apoptosis. Taken together, our data demonstrate a crucial role of Bim in preferentially regulating the Bak/Mcl-1 rheostat to mediate DHA-induced apoptosis in HCC cells.

  17. Exercise mediated protection of diabetic heart through modulation of microRNA mediated molecular pathways.

    Science.gov (United States)

    Lew, Jason Kar Sheng; Pearson, James T; Schwenke, Daryl O; Katare, Rajesh

    2017-01-13

    Hyperglycaemia, hypertension, dyslipidemia and insulin resistance collectively impact on the myocardium of people with diabetes, triggering molecular, structural and myocardial abnormalities. These have been suggested to aggravate oxidative stress, systemic inflammation, myocardial lipotoxicity and impaired myocardial substrate utilization. As a consequence, this leads to the development of a spectrum of cardiovascular diseases, which may include but not limited to coronary endothelial dysfunction, and left ventricular remodelling and dysfunction. Diabetic heart disease (DHD) is the term used to describe the presence of heart disease specifically in diabetic patients. Despite significant advances in medical research and long clinical history of anti-diabetic medications, the risk of heart failure in people with diabetes never declines. Interestingly, sustainable and long-term exercise regimen has emerged as an effective synergistic therapy to combat the cardiovascular complications in people with diabetes, although the precise molecular mechanism(s) underlying this protection remain unclear. This review provides an overview of the underlying mechanisms of hyperglycaemia- and insulin resistance-mediated DHD with a detailed discussion on the role of different intensities of exercise in mitigating these molecular alterations in diabetic heart. In particular, we provide the possible role of exercise on microRNAs, the key molecular regulators of several pathophysiological processes.

  18. A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea

    Science.gov (United States)

    Kono, Takunari; Mehrotra, Sandhya; Endo, Chikako; Kizu, Natsuko; Matusda, Mami; Kimura, Hiroyuki; Mizohata, Eiichi; Inoue, Tsuyoshi; Hasunuma, Tomohisa; Yokota, Akiho; Matsumura, Hiroyoshi; Ashida, Hiroki

    2017-01-01

    Two enzymes are considered to be unique to the photosynthetic Calvin–Benson cycle: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for CO2 fixation, and phosphoribulokinase (PRK). Some archaea possess bona fide RuBisCOs, despite not being photosynthetic organisms, but are thought to lack PRK. Here we demonstrate the existence in methanogenic archaea of a carbon metabolic pathway involving RuBisCO and PRK, which we term ‘reductive hexulose-phosphate' (RHP) pathway. These archaea possess both RuBisCO and a catalytically active PRK whose crystal structure resembles that of photosynthetic bacterial PRK. Capillary electrophoresis-mass spectrometric analysis of metabolites reveals that the RHP pathway, which differs from the Calvin–Benson cycle only in a few steps, is active in vivo. Our work highlights evolutionary and functional links between RuBisCO-mediated carbon metabolic pathways in methanogenic archaea and photosynthetic organisms. Whether the RHP pathway allows for autotrophy (that is, growth exclusively with CO2 as carbon source) remains unknown. PMID:28082747

  19. Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae*

    Science.gov (United States)

    Lazard, Myriam; Dauplais, Marc; Blanquet, Sylvain; Plateau, Pierre

    2015-01-01

    Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity. PMID:25745108

  20. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction

    Science.gov (United States)

    BRAVERMAN, ALAN S.; TALLARIDA, RONALD J.; RUGGIERI, MICHAEL R.

    2012-01-01

    M3 muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2 receptors participate in contraction because M3-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3 receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2 receptor and one by the M3 receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2 and M3 receptors can induce contraction. In the denervated bladder, the M2 and the M3 receptors interact in a facilitatory manner to mediate contraction. PMID:12185001

  1. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction.

    Science.gov (United States)

    Braverman, Alan S; Tallarida, Ronald J; Ruggieri, Michael R

    2002-09-01

    M(3) muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M(2) receptors participate in contraction because M(3)-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M(2)-selective antagonist methoctramine in the denervated bladder is consistent with M(3) receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M(2) receptor and one by the M(3) receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M(2) and M(3) receptors can induce contraction. In the denervated bladder, the M(2) and the M(3) receptors interact in a facilitatory manner to mediate contraction.

  2. The TEAD/TEF Family Protein Scalloped Mediates Transcriptional Output of the Hippo Growth-Regulatory Pathway

    National Research Council Canada - National Science Library

    Wu, Shian; Liu, Yi; Zheng, Yonggang; Dong, Jixin; Pan, Duojia

    2008-01-01

    .... Here we identify the TEAD/TEF family protein Scalloped (Sd) as a DNA-binding transcription factor that partners with Yki to mediate the transcriptional output of the Hpo growth-regulatory pathway. The diap1 (th...

  3. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Praveen Guleria

    Full Text Available BACKGROUND: Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. METHODOLOGY/PRINCIPAL FINDINGS: RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. CONCLUSIONS: SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  4. Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression

    Directory of Open Access Journals (Sweden)

    Mansukhani Mahesh

    2006-05-01

    Full Text Available Abstract Background Cervical Cancer (CC exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo. These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers. Results To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression. Conclusion Taken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i play a role in CC development, (ii further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii form epigenetic signatures to identify precancerous lesions at risk to progression.

  5. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    Science.gov (United States)

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  6. Natural Products Induce a G Protein-Mediated Calcium Pathway Activating p53 in Cancer Cells

    Science.gov (United States)

    van Ginkel, Paul R.; Yan, Michael B.; Bhattacharya, Saswati; Polans, Arthur S.; Kenealey, Jason D.

    2015-01-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. PMID:26341291

  7. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells.

    Science.gov (United States)

    van Ginkel, Paul R; Yan, Michael B; Bhattacharya, Saswati; Polans, Arthur S; Kenealey, Jason D

    2015-11-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death.

  8. Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways

    Science.gov (United States)

    Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

    2016-04-01

    Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and affecting of MAPKs and AKT signaling and DNA damage-mediated p53 phosphorylation to advance HePG-2 cells apoptosis. Therefore, our results show that the mechanism of ROS-mediated signaling pathways may provide useful information in AgNP-induced HePG-2 cell apoptosis.

  9. Functional diversification of the twin-arginine translocation pathway mediates the emergence of novel ecological adaptations.

    Science.gov (United States)

    Jiang, Xiaowei; Fares, Mario A

    2011-11-01

    Microorganisms occupy a myriad of ecological niches that show an astonishing diversity. The molecular mechanisms underlying microbes' ecological diversity remain a fundamental conundrum in evolutionary biology. Evidence points to that the secretion of a particular set of proteins mediates microbes' interaction with the environment. Several systems are involved in this secretion, including the Sec secretion system and the Tat pathway. Shifts in the functions of proteins from the secretion systems may condition the set of secreted proteins and can, therefore, mediate adaptations to new ecological niches. In this manuscript, we have investigated processes of functional divergence (FD)-a term used here to refer to the emergence of novel functions by the modification of ancestral ones-of Tat pathway proteins using a large set of microbes with different lifestyles. The application of a novel approach to identify FD allowed us to distinguish molecular changes in the three Tat proteins among different groups of archaea and bacteria. We found these changes as well as the composition of secreted proteins to be correlated with differences in microbe's lifestyles. We identified major signatures of FD in halophilic and thermophilic archaea as well as in pathogenic bacteria. The location of amino acids affected by FD in functionally important domains of Tat proteins made it possible to find the link between the molecular changes in Tat, the set of secreted proteins and the environmental features of the microbes. We present evidence that links specific molecular changes in secretion mediating proteins of microbes to their ecological adaptations.

  10. Calmodulin-mediated suppression of 2-ketoisovalerate reductase in Beauveria bassiana beauvericin biosynthetic pathway.

    Science.gov (United States)

    Kim, Jiyoung; Yoon, Deok-Hyo; Oh, Junsang; Hyun, Min-Woo; Han, Jae-Gu; Sung, Gi-Ho

    2016-11-01

    Ketoisovalerate reductase (KIVR, E.C. 1.2.7.7) mediates the specific reduction of 2-ketoisovalerate (2-Kiv) to d-hydroxyisovalerate (d-Hiv), a precursor for beauvericin biosynthesis. Beauvericin, a famous mycotoxin produced by many fungi, is a cyclooligomer depsipeptide, which has insecticidal, antimicrobial, antiviral, and cytotoxic activities. In this report, we demonstrated that Beauveria bassiana 2-ketoisovalerate reductase (BbKIVR) acts as a typical KIVR enzyme in the entomopathogenic fungus B. bassiana. In addition, we found that BbKIVR interacts with calmodulin (CaM) in vitro and in vivo. The functional role of CaM-binding to BbKIVR was to negatively regulate the BbKIVR activity in B. bassiana. Environmental stimuli such as light and salt stress suppressed BbKIVR activity in B. bassiana. Interestingly, this negative effect of BbKIVR activity by light and salt stress was recovered by CaM inhibitors, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbKIVR plays an important role in the beauvericin biosynthetic pathway mediated by environmental stimuli such as light and salt stress via the CaM signaling pathway.

  11. Positive and negative affect as predictors of urge to smoke: temporal factors and mediational pathways.

    Science.gov (United States)

    Leventhal, Adam M; Greenberg, Jodie B; Trujillo, Michael A; Ameringer, Katherine J; Lisha, Nadra E; Pang, Raina D; Monterosso, John

    2013-03-01

    Elucidating interrelations between prior affective experience, current affective state, and acute urge to smoke could inform affective models of addiction motivation and smoking cessation treatment development. This study tested the hypothesis that prior levels of positive (PA) and negative (NA) affect predict current smoking urge via a mediational pathway involving current state affect. We also explored if tobacco deprivation moderated affect-urge relations and compared the effects of PA and NA on smoking urge to one another. At a baseline session, smokers reported affect experienced over the preceding few weeks. At a subsequent experimental session, participants were randomly assigned to 12-hr tobacco deprived (n = 51) or nondeprived (n = 69) conditions and reported state affect and current urge. Results revealed a mediational pathway whereby prior NA reported at baseline predicted state NA at the experimental session, which in turn predicted current urge. This mediational pathway was found primarily for an urge subtype indicative of urgent need to smoke and desire to smoke for NA relief, was stronger in the deprived (vs. nondeprived) condition, and remained significant after controlling for PA. Prior PA and current state PA were inversely associated with current urge; however, these associations were eliminated after controlling for NA. These results cohere with negative reinforcement models of addiction and with prior research and suggest that: (a) NA plays a stronger role in smoking motivation than PA; (b) state affect is an important mechanism linking prior affective experience to current urge; and (c) affect management interventions may attenuate smoking urge in individuals with a history of affective disturbance.

  12. TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To determine whether transforming growth factor betal ( TGF-β1 )/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines. Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study. TGF-β31-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay. For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements. After transfection, cells were treated with TGF-β1, then assayed for luciferase activity. Results The apoptosis rate of HepG2 cell lines (48.51% ± 8.21% ) was significantly higher than control (12. 72% ±2. 18%, P <0. 05 ). But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines. The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4. 38) was significantly higher than control (1.00, P <0. 05). But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control. Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines. Smad4 is a central mediator of TGF-β1 signaling transdution pathway. TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

  13. Macrophages Mediate a Switch between Canonical and Non-Canonical Wnt Pathways in Canine Mammary Tumors

    Science.gov (United States)

    Król, Magdalena; Mucha, Joanna; Majchrzak, Kinga; Homa, Agata; Bulkowska, Małgorzata; Majewska, Alicja; Gajewska, Małgorzata; Pietrzak, Marta; Perszko, Mikołaj; Romanowska, Karolina; Pawłowski, Karol; Manuali, Elisabetta; Hellmen, Eva; Motyl, Tomasz

    2014-01-01

    Objective According to the current hypothesis, tumor-associated macrophages (TAMs) are “corrupted” by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell–TAM interactions are complicated and controversial we aimed to better define this phenomenon. Methods and Results Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial–mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs. Conclusions We demonstrated that TAMs mediate a “switch” between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis. Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being “corrupted” by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis. These data challenge the conventional understanding of TAM–cancer cell interactions. PMID:24404146

  14. Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity.

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    Jun Wu

    2004-07-01

    Full Text Available The Frizzled (Fz; called here Fz1 and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/beta-catenin pathway or Fz/planar cell polarity (PCP signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/beta-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.

  15. Synchronization of cells with activator-inhibitor pathways through adaptive environment-mediated coupling.

    Science.gov (United States)

    Ghomsi, P Guemkam; Kakmeni, F M Moukam; Tchawoua, C; Kofane, T C

    2015-01-01

    In this paper, we report the synchronized dynamics of cells with activator-inhibitor pathways via an adaptive environment-mediated coupling scheme with feedbacks and control mechanisms. The adaptive character of the extracellular medium is modeled via its damping parameter as a physiological response aiming at annihilating the cellular differentiation existing between the chaotic biochemical pathways of the cells, in order to preserve homeostasis. We perform an investigation on the existence and stability of the synchronization manifold of the coupled system under the proposed coupling pattern. Both mathematical and computational tools suggest the accessibility of conducive prerequisites (conditions) for the emergence of a robust synchronous regime. The relevance of a phase-synchronized dynamics is appraised and several numerical indicators advocate for the prevalence of this fascinating phenomenon among the interacting cells in the phase space.

  16. LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis

    Science.gov (United States)

    Odena, Gemma; Chen, Jiegen; Lozano, Juan Jose; Altamirano, Jose; Rodrigo-Torres, Daniel; Affo, Silvia; Morales-Ibanez, Oriol; Matsushita, Hiroshi; Zou, Jian; Dumitru, Raluca; Caballeria, Juan; Gines, Pere; Arroyo, Vicente; You, Min; Rautou, Pierre-Emmanuel; Valla, Dominique; Crews, Fulton; Seki, Ekihiro; Sancho-Bru, Pau; Bataller, Ramon

    2016-01-01

    Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development. PMID:27752144

  17. Synchronization of cells with activator-inhibitor pathways through adaptive environment-mediated coupling

    Science.gov (United States)

    Ghomsi, P. Guemkam; Moukam Kakmeni, F. M.; Tchawoua, C.; Kofane, T. C.

    2015-11-01

    In this paper, we report the synchronized dynamics of cells with activator-inhibitor pathways via an adaptive environment-mediated coupling scheme with feedbacks and control mechanisms. The adaptive character of the extracellular medium is modeled via its damping parameter as a physiological response aiming at annihilating the cellular differentiation existing between the chaotic biochemical pathways of the cells, in order to preserve homeostasis. We perform an investigation on the existence and stability of the synchronization manifold of the coupled system under the proposed coupling pattern. Both mathematical and computational tools suggest the accessibility of conducive prerequisites (conditions) for the emergence of a robust synchronous regime. The relevance of a phase-synchronized dynamics is appraised and several numerical indicators advocate for the prevalence of this fascinating phenomenon among the interacting cells in the phase space.

  18. Exosomes mediated pentose phosphate pathway in ovarian cancer metastasis: a proteomics analysis.

    Science.gov (United States)

    Yi, Huan; Zheng, Xiangqin; Song, Jianrong; Shen, Rongkai; Su, Yanzhao; Lin, Danmei

    2015-01-01

    Epithelial ovarian cancer is the most lethal gynecological malignancies for readily metastasis. Exosomes have played an influential role in carcinogenicity and cancer progression. Our aim is to discover exosome-related mechanisms in ovarian cancer progress and explore potential diagnostic biomarkers and therapeutic targets of ovarian cancer. We initially presented the proteomic profiles of exosomes derived from two late-stage ovarian cell lines, OVCA429 and HO8910PM. A total of 2940 exosomal proteins were recorded by MS. FunRich appropriately processed these exosomal proteins, manifesting some superiority in contrast to Blast2go. Moreover, we demonstrated the pentose phosphate pathway was a dominant mechanism in exosome mediated intracellular communication. Glucose-6-phosphate dehydrogenase, transketolase and transaldolase 1, three key enzymes regulated pentose phosphate pathway, were all marked in the same exosomal parts of proteins between two ovarian cell lines. Moreover, these key proteins might become diagnostic, prognostic biomarkers and therapeutic targets of ovarian cancer.

  19. Reactive oxygen species-mediated unfolded protein response pathways in preimplantation embryos

    Science.gov (United States)

    Ali, Ihsan; Shah, Syed Zahid Ali; Jin, Yi; Li, Zhong-Shu; Ullah, Obaid

    2017-01-01

    Excessive production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress-mediated responses are critical to embryonic development in the challenging in vitro environment. ROS production increases during early embryonic development with the increase in protein requirements for cell survival and growth. The ER is a multifunctional cellular organelle responsible for protein folding, modification, and cellular homeostasis. ER stress is activated by a variety of factors including ROS. Such stress leads to activation of the adaptive unfolded protein response (UPR), which restores homeostasis. However, chronic stress can exceed the toleration level of the ER, resulting in cellular apoptosis. In this review, we briefly describe the generation and impact of ROS in preimplantation embryo development, the ROS-mediated activation mechanism of the UPR via the ER, and the subsequent activation of signaling pathways following ER stress in preimplantation embryos. PMID:28057903

  20. Taraxerol Induces Cell Apoptosis through A Mitochondria-Mediated Pathway in HeLa Cells.

    Science.gov (United States)

    Yaoi, Xiangyang; Lu, Binyu; Lü, Chaotian; Bai, Qin; Yan, Dazhong; Xu, Hui

    2017-10-01

    Taraxerol acetate has potent anti-cancer effects via the induction of apoptosis, autophagy, cell cycle arrest, and inhibition of cell migration. However, whether taraxerol induced apoptosis and its underlying mechanisms of action is not clear. In the present study, we assess the effects of taraxerol on the mitochondrial apoptotic pathway and determine the release of cytochrome c to the cytosol and activation of caspases. In this experimental study, we mainly investigated the effect of taraxerol on HeLa cells. We tested cell viability by the MTT assay and morphologic changes, analyzed apoptosis by DAPI staining and flow cytometry. We also determined reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) using a Microplate Reader. In addition, the apoptotic proteins were tested by Western blot. Taraxerol enhanced ROS levels and attenuated the MMP (Δψm) in HeLa cells. Taraxerol induced apoptosis mainly via the mitochondrial pathway including the release of cytochrome c to the cytosol and activation of caspases 9 and 3, and anti-poly (ADPribose) polymerase (PARP). Taraxerol could induce the down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax. It suppressed the PI3K/ Akt signaling pathway. These results demonstrated that taraxerol induced cell apoptosis through a mitochondria-mediated pathway in HeLa cells. Thus, taraxerol might be a potential anticervical cancer candidate.

  1. Fusion tyrosine kinase mediated signalling pathways in the transformation of haematopoietic cells.

    Science.gov (United States)

    Turner, S D; Alexander, D R

    2006-04-01

    The fusion tyrosine kinases (FTKs) are generated by chromosomal translocations creating bipartite proteins in which the kinase is hyperactivated by an adjoining oligomerization domain. Autophosphorylation of the FTK generates a 'signalosome', an ensemble of signalling proteins that transduce signals to downstream pathways. At the earliest stages of oncogenesis, FTKs can mimic mitogenic cytokine signalling pathways involving the GAB-2 adaptor protein and signal transducers and activators of transcription (STAT) factors, generating replicative stress and thereby promoting a mutator phenotype. In parallel, FTKs couple to survival pathways that upregulate prosurvival proteins such as Bcl-xL, so preventing DNA-damage-induced apoptosis. Following transformation, FTKs induce resistance to genotoxic attack by upregulating DNA repair mechanisms such as STAT5-dependent RAD51 transcription. The phenomenon of 'oncogene addiction' reflects the continued requirement of an active FTK 'signalosome' to mediate survival and mitogenic signals involving the PI 3-kinase and mitogen-activated protein stress-activated protein kinase pathways, and the nuclear factor-kappa B, activator protein 1 and STAT transcription factors. The available data so far suggest that FTKs, with some possible exceptions, induce and maintain the transformed state using similar panoplies of signals, a finding with important therapeutic implications. The FTK signalling field has matured to an exciting phase in which rapid advances are facilitating rational drug design.

  2. The nonsense-mediated RNA decay pathway is disrupted in inflammatory myofibroblastic tumors.

    Science.gov (United States)

    Lu, JingWei; Plank, Terra-Dawn; Su, Fang; Shi, XiuJuan; Liu, Chen; Ji, Yuan; Li, ShuaiJun; Huynh, Andrew; Shi, Chao; Zhu, Bo; Yang, Guang; Wu, YanMing; Wilkinson, Miles F; Lu, YanJun

    2016-08-01

    Inflammatory myofibroblastic tumors (IMTs) are characterized by myofibroblast proliferation and an inflammatory cell infiltrate. Little is known about the molecular pathways that precipitate IMT formation. Here, we report the identification of somatic mutations in UPF1, a gene that encodes an essential component of the nonsense-mediated RNA decay (NMD) pathway, in 13 of 15 pulmonary IMT samples. The majority of mutations occurred in a specific region of UPF1 and triggered UPF1 alternative splicing. Several mRNA targets of the NMD pathway were upregulated in IMT samples, indicating that the UPF1 mutations led to reduced NMD magnitude. These upregulated NMD targets included NIK mRNA, which encodes a potent activator of NF-κB. In human lung cells, UPF1 depletion increased expression of chemokine-encoding genes in a NIK-dependent manner. Elevated chemokines and IgE class switching events were observed in IMT samples, consistent with NIK upregulation in these tumors. Together, these results support a model in which UPF1 mutations downregulate NMD, leading to NIK-dependent NF-κB induction, which contributes to the immune infiltration that is characteristic of IMTs. The molecular link between the NMD pathway and IMTs has implications for the diagnosis and treatment of these tumors.

  3. A Radical-Mediated Pathway for the Formation of [M + H]+ in Dielectric Barrier Discharge Ionization

    Science.gov (United States)

    Wolf, Jan-Christoph; Gyr, Luzia; Mirabelli, Mario F.; Schaer, Martin; Siegenthaler, Peter; Zenobi, Renato

    2016-09-01

    Active capillary plasma ionization is a highly efficient ambient ionization method. Its general principle of ion formation is closely related to atmospheric pressure chemical ionization (APCI). The method is based on dielectric barrier discharge ionization (DBDI), and can be constructed in the form of a direct flow-through interface to a mass spectrometer. Protonated species ([M + H]+) are predominantly formed, although in some cases radical cations are also observed. We investigated the underlying ionization mechanisms and reaction pathways for the formation of protonated analyte ([M + H]+). We found that ionization occurs in the presence and in the absence of water vapor. Therefore, the mechanism cannot exclusively rely on hydronium clusters, as generally accepted for APCI. Based on isotope labeling experiments, protons were shown to originate from various solvents (other than water) and, to a minor extent, from gaseous impurities and/or self-protonation. By using CO2 instead of air or N2 as plasma gas, additional species like [M + OH]+ and [M - H]+ were observed. These gas-phase reaction products of CO2 with the analyte (tertiary amines) indicate the presence of a radical-mediated ionization pathway, which proceeds by direct reaction of the ionized plasma gas with the analyte. The proposed reaction pathway is supported with density functional theory (DFT) calculations. These findings add a new ionization pathway leading to the protonated species to those currently known for APCI.

  4. Mediation pathways and effects of green structures on respiratory mortality via reducing air pollution

    Science.gov (United States)

    Shen, Yu-Sheng; Lung, Shih-Chun Candice

    2017-01-01

    Previous studies have shown both health and environmental benefits of green spaces, especially in moderating temperature and reducing air pollution. However, the characteristics of green structures have been overlooked in previous investigations. In addition, the mediation effects of green structures on respiratory mortality have not been assessed. This study explores the potential mediation pathways and effects of green structure characteristics on respiratory mortality through temperature, primary and secondary air pollutants separately using partial least squares model with data from Taiwan. The measurable characteristics of green structure include the largest patch percentage, landscape proportion, aggregation, patch distance, and fragmentation. The results showed that mortality of pneumonia and chronic lower respiratory diseases could be reduced by minimizing fragmentation and increasing the largest patch percentage of green structure, and the mediation effects are mostly through reducing air pollutants rather than temperature. Moreover, a high proportion of but fragmented green spaces would increase secondary air pollutants and enhance health risks; demonstrating the deficiency of traditional greening policy with primary focus on coverage ratio. This is the first research focusing on mediation effects of green structure characteristics on respiratory mortality, revealing that appropriate green structure planning can be a useful complementary strategy in environmental health management. PMID:28230108

  5. Mediation pathways and effects of green structures on respiratory mortality via reducing air pollution

    Science.gov (United States)

    Shen, Yu-Sheng; Lung, Shih-Chun Candice

    2017-02-01

    Previous studies have shown both health and environmental benefits of green spaces, especially in moderating temperature and reducing air pollution. However, the characteristics of green structures have been overlooked in previous investigations. In addition, the mediation effects of green structures on respiratory mortality have not been assessed. This study explores the potential mediation pathways and effects of green structure characteristics on respiratory mortality through temperature, primary and secondary air pollutants separately using partial least squares model with data from Taiwan. The measurable characteristics of green structure include the largest patch percentage, landscape proportion, aggregation, patch distance, and fragmentation. The results showed that mortality of pneumonia and chronic lower respiratory diseases could be reduced by minimizing fragmentation and increasing the largest patch percentage of green structure, and the mediation effects are mostly through reducing air pollutants rather than temperature. Moreover, a high proportion of but fragmented green spaces would increase secondary air pollutants and enhance health risks; demonstrating the deficiency of traditional greening policy with primary focus on coverage ratio. This is the first research focusing on mediation effects of green structure characteristics on respiratory mortality, revealing that appropriate green structure planning can be a useful complementary strategy in environmental health management.

  6. MAPK signaling pathways regulate mitochondrial-mediated apoptosis induced by isoorientin in human hepatoblastoma cancer cells.

    Science.gov (United States)

    Yuan, Li; Wang, Jing; Xiao, Haifang; Wu, Wanqiang; Wang, Yutang; Liu, Xuebo

    2013-03-01

    Isoorientin (ISO) (CAS RN: 4261-42-1) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum. ISO is able to induce apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cells, however, the effects of ISO on MAPK signaling pathways remain unknown. The present study investigated the effects of ISO on this pathway, and the roles of MAPK kinases on mitochondrial-mediated apoptosis in HepG2 cells. The results showed that ISO induced cell death in a dose- and time-dependent manner, and induction apoptosis is main cause for ISO-induced cytotoxicity in HepG2 cells. ISO significantly inhibited the levels of ERK1/2 kinase and increased the expression of JNK and p38 kinases. Furthermore, U0126 (an ERK1/2 inhibitor) significantly enhanced the ISO-induced the Bax/Bcl-2 ratio, the release of cytochrome c to the cytosol fraction, and the levels of cleaved caspase-3. While SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) markedly prevented the expression of these proteins induced by ISO. Furthermore, the ROS inhibitor (NAC) notably promoted the inhibited effect of ISO on the ERK1/2 kinase. NAC also suppressed the p-JNK and p-p38, but failed to reverse the effects of ISO. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells through inactivating ERK1/2 kinase and activating JNK and p38 kinases, and ROS stimulated by ISO is able to activate the MAPK singaling pathway as the upstream signaling molecules. Initiating event of the mitochondrial-mediated apoptosis induced by ISO is MAPK signals.

  7. A novel aminothiazole KY-05009 with potential to inhibit Traf2- and Nck-interacting kinase (TNIK attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells.

    Directory of Open Access Journals (Sweden)

    Jiyeon Kim

    Full Text Available Transforming growth factor (TGF-β triggers the epithelial-to-mesenchymal transition (EMT of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido-2-(phenylaminothiazole-4-carboxamide (KY-05009 with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (Ki, 100 nM. In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

  8. Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development.

    Science.gov (United States)

    Al-Shanti, Nasser; Stewart, Claire E

    2009-11-01

    The loss of muscle mass with age and disuse has a significant impact on the physiological and social well-being of the aged; this is an increasingly important problem as the population becomes skewed towards older age. Exercise has psychological benefits but it also impacts on muscle protein synthesis and degradation, increasing muscle tissue volume in both young and older individuals. Skeletal muscle hypertrophy involves an increase in muscle mass and cross-sectional area and associated increased myofibrillar protein content. Attempts to understand the molecular mechanisms that underlie muscle growth, development and maintenance, have focused on characterising the molecular pathways that initiate, maintain and regenerate skeletal muscle. Such understanding may aid in improving targeted interventional therapies for age-related muscle loss and muscle wasting associated with diseases. Two major routes through which skeletal muscle development and growth are regulated are insulin-like growth factor I (IGF-I) and Ca(2+)/calmodulin-dependent transcriptional pathways. Many reviews have focused on understanding the signalling pathways of IGF-I and its receptor, which govern skeletal muscle hypertrophy. However, alternative molecular signalling pathways such as the Ca(2+)/calmodulin-dependent transcriptional pathways should also be considered as potential mediators of muscle growth. These latter pathways have received relatively little attention and the purpose herein is to highlight the progress being made in the understanding of these pathways and associated molecules: calmodulin, calmodulin kinases (CaMKs), calcineurin and nuclear factor of activated T-cell (NFAT), which are involved in skeletal muscle regulation. We describe: (1) how conformational changes in the Ca(2+) sensor calmodulin result in the exposure of binding pockets for the target proteins (CaMKs and calcineurin). (2) How Calmodulin consequently activates either the Ca(2+)/calmodulin-dependent kinases

  9. Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation.

    Science.gov (United States)

    Wang, Jianbo; Hamblet, Natasha S; Mark, Sharayne; Dickinson, Mary E; Brinkman, Brendan C; Segil, Neil; Fraser, Scott E; Chen, Ping; Wallingford, John B; Wynshaw-Boris, Anthony

    2006-05-01

    The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension.

  10. HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

    Science.gov (United States)

    Deng, Lin; Chen, Ming; Tanaka, Motofumi; Ku, Yonson; Itoh, Tomoo; Shoji, Ikuo; Hotta, Hak

    2015-09-01

    We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

  11. Buyang Huanwu Decoction Ameliorates Poststroke Depression via Promoting Neurotrophic Pathway Mediated Neuroprotection and Neurogenesis

    Science.gov (United States)

    Luo, Lin; Deng, Shuhua; Yi, Jian; Zhou, Sainan; She, Yan

    2017-01-01

    Objective. The aim of the present research is to investigate the therapeutic effect of Buyang Huanwu Decoction (BHD) in poststroke depression (PSD) animal model and illustrate its underlying mechanism via promoting neurotrophic pathway mediated neuroprotection and neurogenesis. Methods. To induce PSD rat model, isolation housed rats that received middle cerebral artery occlusion (MCAO) surgery successively suffered from chronic mild stress (CMS) treatment for consecutive twenty-one days. Meanwhile, rats were correspondingly given vehicle, BHD, and fluoxetine. Then, neurologic function was scored and depressive-like behaviors were assessed by sucrose preference test, locomotor activity, novelty-suppressed feeding test, and forced swim test. Thereafter, the neuroprotection and neurogenesis related molecular markers and signaling were detected. Results. We firstly observed a significant neurological function recovery and antidepressants effect of BHD after MCAO together with CMS treatment. Our study also found that treatment with BHD and fluoxetine can significantly rescue neurons from apoptosis and promote neurogenesis in the CA3 and DG regions in the hippocampus. Notably, BHD and fluoxetine treatment can activate BDNF/ERK/CREB signaling. Conclusion. The results suggest that BHD is a promising candidate for treating PSD. Its curative effects can be attributed to neurotrophic pathway mediated neuroprotection and neurogenesis.

  12. Tryptanthrin inhibits angiogenesis by targeting the VEGFR2-mediated ERK1/2 signalling pathway.

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    Xuemei Liao

    Full Text Available Angiogenesis is a key step for tumour growth and metastasis, and anti-angiogenesis has been proposed as an important strategy for cancer therapy. Tryptanthrin is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants and has been shown to possess anti-tumour activities on various cancer cell types. This study aims to investigate the in vitro and in vivo anti-angiogenic activities of tryptanthrin and to unravel its underlying molecular action mechanisms. Our results show that tryptanthrin inhibited the in vitro proliferation, migration, and tube formation of the human microvascular endothelial cells (HMEC-1 in a concentration-dependent manner and significantly suppressed angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that tryptanthrin reduced the expression of several pro-angiogenic factors (Ang-1, PDGFB and MMP2. Tryptanthrin was also found to suppress the VEGFR2-mediated ERK1/2 signalling pathway in HMEC-1 cells and molecular docking simulation indicated that tryptanthrin could bound to the ATP-binding site of VEGFR2. Collectively, the present study demonstrated that tryptanthrin exhibited both in vitro and in vivo anti-angiogenic activities by targeting the VEGFR2-mediated ERK1/2 signalling pathway and might have therapeutic potential for the treatment of angiogenesis-related diseases.

  13. Tryptanthrin inhibits angiogenesis by targeting the VEGFR2-mediated ERK1/2 signalling pathway.

    Science.gov (United States)

    Liao, Xuemei; Zhou, Xuelin; Mak, Nai-ki; Leung, Kwok-nam

    2013-01-01

    Angiogenesis is a key step for tumour growth and metastasis, and anti-angiogenesis has been proposed as an important strategy for cancer therapy. Tryptanthrin is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants and has been shown to possess anti-tumour activities on various cancer cell types. This study aims to investigate the in vitro and in vivo anti-angiogenic activities of tryptanthrin and to unravel its underlying molecular action mechanisms. Our results show that tryptanthrin inhibited the in vitro proliferation, migration, and tube formation of the human microvascular endothelial cells (HMEC-1) in a concentration-dependent manner and significantly suppressed angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that tryptanthrin reduced the expression of several pro-angiogenic factors (Ang-1, PDGFB and MMP2). Tryptanthrin was also found to suppress the VEGFR2-mediated ERK1/2 signalling pathway in HMEC-1 cells and molecular docking simulation indicated that tryptanthrin could bound to the ATP-binding site of VEGFR2. Collectively, the present study demonstrated that tryptanthrin exhibited both in vitro and in vivo anti-angiogenic activities by targeting the VEGFR2-mediated ERK1/2 signalling pathway and might have therapeutic potential for the treatment of angiogenesis-related diseases.

  14. Matrine inhibits proliferation and induces apoptosis via BID-mediated mitochondrial pathway in esophageal cancer cells.

    Science.gov (United States)

    Wang, Qiao; Du, Haoxin; Geng, Guojun; Zhou, Huan; Xu, Minying; Cao, Hanwei; Zhang, Bing; Song, Gang; Hu, Tianhui

    2014-05-01

    Matrine, as a member of Sophora family, is an alkaloid found in plants, and produces plethora pharmacological effects, including anti-cancer effects. However, the mechanism involved remains largely unknown. This study is conducted to investigate the anti-cancer mechanisms of matrine in human esophageal cancer in vitro and in vivo. In human esophageal cancer cell Eca-109, matrine significantly decreased the cell viability in a dose-dependent manner, and induced apoptosis as well as cell cycle arrest in G0/G1 phase by up-regulation of P53 and P21. The expression of several apoptosis-related proteins in cells and tumor tissues were evaluated by Western blot analysis. We found that matrine induced cell apoptosis by down-regulation of the ratio of BCL-2/BID and increasing activation of caspase-9. Further studies indicated that matrine induced apoptosis of Eca-109 was through the mitochondria-mediated internal pathway, but not by death receptor-mediated extrinsic apoptotic pathway, which was confirmed by the fact that Bid translocated from the nucleus to mitochondria during the process of the apoptosis induced by matrine. In vivo study found that matrine effectively inhibited the tumor formation of Eca-109 cells in nude mice. Our study suggests that matrine could serve as a potential novel agent from natural products to treat esophageal cancer.

  15. Cherry Valley ducks mitochondrial antiviral-signaling protein (MAVS mediated signaling pathway and antiviral activity research

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    Ning Li

    2016-09-01

    Full Text Available Mitochondrial antiviral-signaling protein (MAVS, an adaptor protein of retinoic acid-inducible gene I (RIG-I like receptors (RLRs-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline rich domain and a transmembrane domain at C-terminal. Quantitative real time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly up-regulated after infection with duck Tembusu virus. Overexpression of duMAVS could drive the activation of interferon-β, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8 in duck embryo fibroblast cells. What’s more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-β activation. The antiviral assay showed that duMAVS could suppress the various viral replications (duck Tembusu virus, novel reovirus, and duck plague virus at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks.

  16. MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation--divergence in pathways leading to aggrecanase and MMP-mediated articular cartilage degradation

    DEFF Research Database (Denmark)

    Sondergaard, B-C; Schultz, N; Madsen, S H;

    2010-01-01

    Matrix metalloproteinases (MMPs) and aggrecanases are essential players in cartilage degradation. However, the signaling pathways that results in MMP and/or aggrecanase synthesis and activation are not well understood. We investigated the molecular events leading to MMP- and aggrecanase-mediated ...

  17. Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina.

    Science.gov (United States)

    Borghuis, Bart G; Looger, Loren L; Tomita, Susumu; Demb, Jonathan B

    2014-04-30

    A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.

  18. MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

    Science.gov (United States)

    Kostyrko, Kaja; Neuenschwander, Samuel; Junier, Thomas; Regamey, Alexandre; Iseli, Christian; Schmid-Siegert, Emanuel; Bosshard, Sandra; Majocchi, Stefano; Le Fourn, Valérie; Girod, Pierre-Alain; Xenarios, Ioannis; Mermod, Nicolas

    2017-02-01

    Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

  19. Chronic hypobaric hypoxia mediated skeletal muscle atrophy: role of ubiquitin-proteasome pathway and calpains.

    Science.gov (United States)

    Chaudhary, Pooja; Suryakumar, Geetha; Prasad, Rajendra; Singh, Som Nath; Ali, Shakir; Ilavazhagan, Govindsamy

    2012-05-01

    The most frequently reported symptom of exposure to high altitude is loss of body mass and decreased performance which has been attributed to altered protein metabolism affecting skeletal muscles mass. The present study explores the mechanism of chronic hypobaric hypoxia mediated skeletal muscle wasting by evaluating changes in protein turnover and various proteolytic pathways. Male Sprague-Dawley rats weighing about 200 g were exposed to hypobaric hypoxia (7,620 m) for different durations of exposure. Physical performance of rats was measured by treadmill running experiments. Protein synthesis, protein degradation rates were determined by (14)C-Leucine incorporation and tyrosine release, respectively. Chymotrypsin-like enzyme activity of the ubiquitin-proteasome pathway and calpains were studied fluorimetrically as well as using western blots. Declined physical performance by more than 20%, in terms of time taken in exhaustion on treadmill, following chronic hypobaric hypoxia was observed. Compared to 1.5-fold increase in protein synthesis, the increase in protein degradation was much higher (five-folds), which consequently resulted in skeletal muscle mass loss. Myofibrillar protein level declined from 46.79 ± 1.49 mg/g tissue at sea level to 37.36 ± 1.153 (P calpains (three-fold) has been found to be important factors for the enhanced protein degradation rate. The study provided strong evidences suggesting that elevated protein turnover rate lead to skeletal muscle atrophy under chronic hypobaric hypoxia via ubiquitin-proteasome pathway and calpains.

  20. Rosuvastatin inhibits the smooth muscle cell proliferation by targeting TNFα mediated Rho kinase pathway

    Institute of Scientific and Technical Information of China (English)

    Xiao Sun; Hao Tong; Man Zhang; Xiao-Hang Wang

    2012-01-01

    Objective To investigate whether Tumor Necrosis Factor-alpha (TNFα) is capable of activating Rho kinase pathway which leads to smooth muscle cell proliferation and the intervention function of Rosuvastatin, and clarify the mechanism and intervention manner of anti-atherosclerosis by Rosuvastatin. Methods Wistar neonate rat smooth muscle cells were cultured, and the activity of cell proliferation was determined by methyl thiazolyl tetrazolium (MTT). The expression of Rho kinase genes after the stimulation of TNFα was evaluated by RT-PCR. Western blot method was used to measure the protein expression of proliferating cell nuclear antigen (PCNA) after TNFα stimulation and Rosuvastatin intervention in smooth muscle cell. Results The TNFα stimulation significantly enhanced the expression of Rho kinase and increased the expression of PCNA protein in smooth muscle cells (P < 0.05). These effects were positively correlated with prolonged treatment whereas additional Rosuvastatin administration inhibited the above-mentioned effects (P < 0.05). Conclusions The activation of TNFα mediated Rho kinase signaling pathway can significantly promote smooth muscle cell proliferation, and Rosuvastatin can not only inhibit this pathway but also the induced proliferation.

  1. Hijacking the Hexosamine Biosynthetic Pathway to Promote EMT-Mediated Neoplastic Phenotypes.

    Science.gov (United States)

    Taparra, Kekoa; Tran, Phuoc T; Zachara, Natasha E

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a highly conserved program necessary for orchestrating distant cell migration during embryonic development. Multiple studies in cancer have demonstrated a critical role for EMT during the initial stages of tumorigenesis and later during tumor invasion. Transcription factors (TFs) such as SNAIL, TWIST, and ZEB are master EMT regulators that are aberrantly overexpressed in many malignancies. Recent evidence correlates EMT-related transcriptomic alterations with metabolic reprograming in cancer. Metabolic alterations may allow cancer to adapt to environmental stressors, supporting the irregular macromolecular demand of rapid proliferation. One potential metabolic pathway of increasing importance is the hexosamine biosynthesis pathway (HBP). The HBP utilizes glycolytic intermediates to generate the metabolite UDP-GlcNAc. This and other charged nucleotide sugars serve as the basis for biosynthesis of glycoproteins and other glycoconjugates. Recent reports in the field of glycobiology have cultivated great curiosity within the cancer research community. However, specific mechanistic relationships between the HBP and fundamental pathways of cancer, such as EMT, have yet to be elucidated. Altered protein glycosylation downstream of the HBP is well positioned to mediate many cellular changes associated with EMT including cell-cell adhesion, responsiveness to growth factors, immune system evasion, and signal transduction programs. Here, we outline some of the basics of the HBP and putative roles the HBP may have in driving EMT-related cancer processes. With novel appreciation of the HBP's connection to EMT, we hope to illuminate the potential for new therapeutic targets of cancer.

  2. Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway.

    Science.gov (United States)

    Lee, J-H; Jeong, J-K; Park, S-Y

    2014-10-10

    Prion diseases are neurodegenerative and infectious disorders that involve accumulation of misfolded scrapie prion protein, and which are characterized by spongiform degeneration. Autophagy, a major homeostatic process responsible for the degradation of cytoplasmic components, has garnered attention as the potential target for neurodegenerative diseases such as prion disease. We focused on protective effects of sulforaphane found in cruciferous vegetables on prion-mediated neurotoxicity and the mechanism of sulforaphane related to autophagy. In human neuroblastoma cells, sulforaphane protected prion protein (PrP) (106-126)-mediated neurotoxicity and increased autophagy flux marker microtubule-associated protein 1 light chain 3-II protein levels, following a decrease of p62 protein level. Pharmacological and genetical inhibition of autophagy by 3MA, wortmannin and knockdown of autophagy-related 5 (ATG5) led to block the effect of sulforaphane against PrP (106-126)-induced neurotoxicity. Furthermore we demonstrated that both sulforaphane-induced autophagy and protective effect of sulforaphane against PrP (106-126)-induced neurotoxicity are dependent on the AMP-activated protein kinase (AMPK) signaling. The present results indicated that sulforaphane of cruciferous vegetables enhanced autophagy flux led to the protection effects against prion-mediated neurotoxicity, which was regulated by AMPK signaling pathways in human neuron cells. Our data also suggest that sulforaphane has a potential value as a therapeutic tool in neurodegenerative disease including prion diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Gastric relaxation induced by hyperglycemia is mediated by vagal afferent pathways in the rat.

    Science.gov (United States)

    Zhou, Shi-Yi; Lu, Yuan-Xu; Owyang, Chung

    2008-05-01

    Hyperglycemia has a profound effect on gastric motility. However, little is known about the site and mechanism that sense alteration in blood glucose level. The identification of glucose-sensing neurons in the nodose ganglia led us to hypothesize that hyperglycemia acts through vagal afferent pathways to inhibit gastric motility. With the use of a glucose-clamp rat model, we showed that glucose decreased intragastric pressure in a dose-dependent manner. In contrast to intravenous infusion of glucose, intracisternal injection of glucose at 250 and 500 mg/dl had little effect on intragastric pressure. Pretreatment with hexamethonium, as well as truncal vagotomy, abolished the gastric motor responses to hyperglycemia (250 mg/dl), and perivagal and gastroduodenal applications of capsaicin significantly reduced the gastric responses to hyperglycemia. In contrast, hyperglycemia had no effect on the gastric contraction induced by electrical field stimulation or carbachol (10(-5) M). To rule out involvement of serotonergic pathways, we showed that neither granisetron (5-HT(3) antagonist, 0.5 g/kg) nor pharmacological depletion of 5-HT using p-chlorophenylalanine (5-HT synthesis inhibitor) affected gastric relaxation induced by hyperglycemia. Lastly, N(G)-nitro-L-arginine methyl ester (L-NAME) and a VIP antagonist each partially reduced gastric relaxation induced by hyperglycemia and, in combination, completely abolished gastric responses. In conclusion, hyperglycemia inhibits gastric motility through a capsaicin-sensitive vagal afferent pathway originating from the gastroduodenal mucosa. Hyperglycemia stimulates vagal afferents, which, in turn, activate vagal efferent cholinergic pathways synapsing with intragastric nitric oxide- and VIP-containing neurons to mediate gastric relaxation.

  4. Expression of Wnt pathway mediators in metaplasic tissue in animal model and clinical samples of tendinopathy.

    Science.gov (United States)

    Lui, Pauline Po Yee; Lee, Yuk Wa; Wong, Yin Mei; Zhang, Xiaoling; Dai, Kerong; Rolf, Christer Gustav

    2013-09-01

    Tissue metaplasia is observed in both ossified failed healing animal model and clinical samples of tendinopathy. The Wnt signalling pathway plays a vital role in pathological calcification. We hypothesized that the Wnt signalling pathway might contribute to tissue metaplasia and failed healing in tendinopathy. This study aimed to examine the spatial-temporal expression of Wnt pathway mediators in an ossified failed tendon healing animal model and clinical samples of tendinopathy. The effect of Wnt3a on the osteogenic differentiation of tendon-derived stem cells (TDSCs) was also examined. Ossified failed tendon healing was induced by the injection of collagenase into the patellar tendon of rats. At various times the tendons were harvested for immunohistochemical staining of Wnt3a, β-catenin, Lrp5 and Tcf1. Patellar tendon samples were obtained from 13 patients with patellar tendinopathy (11 unossified and 2 ossified) and 10 controls. Immunohistochemical staining of Wnt3a, β-catenin, Lrp5 and Tcf1 was similarly performed. Rat patellar TDSCs were treated with Wnt3a. The osteogenic differentiation of TDSCs was examined by ALP activity, alizarin red S staining and mRNA expression of osteogenic markers. There was increased expression of Wnt3a, β-catenin, Lrp5 and Tcf1 in the healing fibroblast-like cells, chondrocyte-like cells and ossified deposits in the animal model and in some clinical samples of tendinopathy. Wnt3a increased ALP activity, calcium nodule formation and expression of osteogenic markers in TDSCs. Activation of the Wnt signalling pathway and its effect on TDSCs might contribute to tissue metaplasia and failed healing in some cases of tendinopathy.

  5. A glutamatergic network mediates lithium response in bipolar disorder as defined by epigenome pathway analysis.

    Science.gov (United States)

    Higgins, Gerald A; Allyn-Feuer, Ari; Barbour, Edward; Athey, Brian D

    2015-01-01

    A regulatory network in the human brain mediating lithium response in bipolar patients was revealed by analysis of functional SNPs from genome-wide association studies (GWAS) and published gene association studies, followed by epigenome mapping. An initial set of 23,312 SNPs in linkage disequilibrium with lead SNPs, and sub-threshold GWAS SNPs rescued by pathway analysis, were studied in the same populations. These were assessed using our workflow and annotation by the epigenome roadmap consortium. Twenty-seven percent of 802 SNPs that were associated with lithium response (13 published studies gene association studies and two GWAS) were shared in common with 1281 SNPs from 18 GWAS examining psychiatric disorders and adverse events associated with lithium treatment. Nineteen SNPs were annotated as active regulatory elements such as enhancers and promoters in a tissue-specific manner. They were located within noncoding regions of ten genes: ANK3, ARNTL, CACNA1C, CACNG2, CDKN1A, CREB1, GRIA2, GSK3B, NR1D1 and SLC1A2. Following gene set enrichment and pathway analysis, these genes were found to be significantly associated (p = 10(-27); Fisher exact test) with an AMPA2 glutamate receptor network in human brain. Our workflow results showed concordance with annotation of regulatory elements from the epigenome roadmap. Analysis of cognate mRNA and enhancer RNA exhibited patterns consistent with an integrated pathway in human brain. This pharmacoepigenomic regulatory pathway is located in the same brain regions that exhibit tissue volume loss in bipolar disorder. Although in silico analysis requires biological validation, the approach provides value for identification of candidate variants that may be used in pharmacogenomic testing to identify bipolar patients likely to respond to lithium.

  6. The Notch pathway mediates the angiotensin II-induced synthesis of extracellular matrix components in podocytes.

    Science.gov (United States)

    Yao, Min; Wang, Xiaomei; Wang, Xiaomeng; Zhang, Tao; Chi, Yanqing; Gao, Feng

    2015-07-01

    The Notch pathway is known to contribute to the development of glomerular disease. Angiotensin II (Ang II), an important member of the renin-angiotensin system, stimulates the accumulation of extracellular matrix components in glomerular disease; however, the exact mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of the Notch pathway on the synthesis of extracellular matrix components in Ang II-stimulated podocytes. Mouse podocytes were stimulated with Ang II (10-6 mol/l). The activation of the Notch pathway was inhibited by a vector carrying short hairpin RNA (shRNA) targeting Notch1 (sh-Notch1) or by γ-secretase inhibitor (GSI). The protein levels of Notch1, Notch intracellular domain 1 (NICD1), hairy and enhancer of split-1 (Hes1), matrix metalloproteinase (MMP)-2, MMP-9, transforming growth factor-β1 (TGF-β1), type IV collagen and laminin were determined by western blot analysis. The Notch1, Hes1, MMP-2, MMP-9, TGF-β1, type IV collagen and laminin mRNA levels were detected by RT-PCR. The MMP-2 and MMP-9 activity was measured using a cell active fluorescence assay kit. The levels of TGF-β1, type IV collagen and laminin were determined in the culture medium of the podocytes by enzyme-linked immunosorbent assay (ELISA). Our results revealed that Ang II upregulated Notch1, NICD1, Hes1, TGF-β1, type IV collagen and laminin expression and downregulated MMP-2 and MMP-9 expression in the cultured podocytes. The inhibition of the Notch pathway by sh-Notch1 or GSI increased MMP-2 and MMP-9 expression, decreased the TGF-β1 level and suppressed type IV collagen and laminin expression. The inhibition of the Notch pathway by sh-Notch1 or GSI also increased MMP-2 and MMP-9 activity, and decreased TGF-β1 levels, type IV collagen levels and laminin secretion. These findings indicate that the Notch pathway potentially mediates the Ang II-induced synthesis of extracellular matrix components in podocytes through the

  7. Eight paths of ERK1/2 signalling pathway regulating hepatocyte proliferation in rat liver regeneration

    Indian Academy of Sciences (India)

    J. W. Li; G. P. Wang; J. Y. Fan; C. F. Chang; C. S. Xu

    2011-12-01

    Although it is known that hormones, growth factors and integrin promote hepatocyte proliferation in liver regeneration (LR) through ERK1/2 signalling pathway, reports about regulating processes of its intracellular paths in hepatocytes of LR are limited. This study aims at exploring which paths of ERK1/2 signalling pathway participate in the regulation of rat LR, especially in hepatocyte proliferation, and how they do so. In all, 14 paths and 165 genes are known to be involved in ERK1/2 signalling pathway. Of them, 161 genes are included in Rat Genome 230 2.0 Array. This array was used to detect expression changes of genes related to ERK1/2 signalling pathway in isolated hepatocytes of rat LR, showing that 60 genes were related to hepatocytes of LR. In addition, bioinformatics and systems biology methods were used to analyse the roles of 14 above paths in regenerating hepatocytes. We found that three paths, RTK → SHC → GRB2/SOS → RAS → RAF, Integrin → FAK → RAC → PAK → RAF and G → PI3K → RAC → PAK → RAF, promoted the G1 phase progression of hepatocytes by activating ERK1/2. A further four paths, Gq → PLC → PKC → SRC/PYK2 → GRB2/SOS → RAS → RAF, RTK → PLC → PKC → SRC/PYK2 → GRB2/SOS → RAS → RAF, Integrin → FAK/SRC → GRB2/SOS → RAS → RAF and Integrin → FAK → RAC → PAK → RAF, advanced the cell progression of S phase and G2/M checkpoint by activating ERK1/2, and so did PP1/2 → Mek1/2 by decreasing the negative influence on ERK1/2. At the late phase of LR, Gs → AC → EPAC → Rap1 → Raf blocked hepatocyte proliferation by decreasing the activity of ERK1/2 and so did PP1/2 → Mek1/2. In summary, 60 genes and 8 paths of ERK1/2 signalling pathway regulated hepatocyte proliferation in rat LR.

  8. Promising Targets for Cancer Immunotherapy: TLRs, RLRs, and STING-Mediated Innate Immune Pathways

    Science.gov (United States)

    Li, Kai; Qu, Shuai; Chen, Xi; Wu, Qiong; Shi, Ming

    2017-01-01

    Malignant cancers employ diverse and intricate immune evasion strategies, which lead to inadequately effective responses of many clinical cancer therapies. However, emerging data suggest that activation of the tolerant innate immune system in cancer patients is able, at least partially, to counteract tumor-induced immunosuppression, which indicates triggering of the innate immune response as a novel immunotherapeutic strategy may result in improved therapeutic outcomes for cancer patients. The promising innate immune targets include Toll-like Receptors (TLRs), RIG-I-like Receptors (RLRs), and Stimulator of Interferon Genes (STING). This review discusses the antitumor properties of TLRs, RLRs, and STING-mediated innate immune pathways, as well as the promising innate immune targets for potential application in cancer immunotherapy. PMID:28216575

  9. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius

    2013-01-01

    of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies......Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent...... advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently...

  10. Genistein induces apoptosis by stabilizing intracellular p53 protein through an APE1-mediated pathway.

    Science.gov (United States)

    Zhu, Jianwu; Zhang, Chong; Qing, Yi; Cheng, Yi; Jiang, Xiaolin; Li, Mengxia; Yang, Zhenzhou; Wang, Dong

    2015-09-01

    Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1-p53 protein-protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway.

  11. Inhibiton of neurons in the amygdala by dorsal raphe stimulation: mediation through a direct serotonergic pathway.

    Science.gov (United States)

    Wang, R Y; Aghajanian, G K

    1977-01-14

    This study presents data showing that the dorsal raphe nucleus (DRN) has a marked inhibitory influence upon neurons in the amygdala and that this inhibitory effect is mediated by a direct DRN-amygdala serotonergic pathway. The evidence may be briefly summarized as follows:(1) on the same amygdaloid cells, both iontophoresis of serotonin (5-HT) and electrical stimulation of the DRN markedly inhibited spontaneous single unit activities; (2) the latency of DRN-induced inhibition was relatively short and is compatible with the conduction velocities (which were determined by antidromic activation of the 5-HT pathway) of unmyelinated 5-HT fibers; (3) destruction of 5-HT projections by 5,7-dihydroxytryptamine (5,7-DHT) or pharmacological depletion of 5-HT by parachlorophenylalanine (PCPA) prevented the inhibitory responsed to DRN stimulation in the great majority of cells studied; (4) in PCPA-pretreated animals, injection of 5-hydroxytryptophan (5-HTP) reversed the PCPA effect, restoring the responses of amygdaloid cells to DRN stimulation. In the amygdala, the presumptive 5-HT antagonists which we tested did not block the inhibitory effects of 5-HT except that intravenously administered LSD blocked the inhibitory responses produced by submaximal DRN stimulation. The implications of these results for the possible functions of 5-HT in the amygdala is discussed.

  12. The role of miR-100-mediated Notch pathway in apoptosis of gastric tumor cells.

    Science.gov (United States)

    Yang, Geng; Gong, Yi; Wang, Qizhi; Wang, Yumeng; Zhang, Xiaobo

    2015-06-01

    MicroRNAs (miRNAs) are small non-coding regulatory molecules that influence many biological functions, including apoptosis, but their role in the regulation of apoptosis in gastric tumor cells has not been intensively investigated. Here, we showed that miR-100 was specifically upregulated in human epithelium-derived gastric cancer cells and that silencing miR-100 expression in human gastric epithelial cancer cells initiated a robust apoptotic response in vitro. Our in vivo assays indicated that the development of gastric cancer was inhibited by the miR-100 antagonism via initiating apoptosis of tumor. The results presented that antagonism of miR-100 increased the expression level of HS3ST2, the target gene of miR-100, and further resulted in the activation of the Notch-apoptosis pathway in tumor cells. The data also revealed that silencing of miR-100 expression sensitized gastric cancer cells to chemotherapy. Therefore our study presented a novel miR-100 mediated Notch pathway in apoptosis of tumor cells.

  13. Effects of tributyltin chloride in ascidian embryos: modulation of kinase-mediated signalling pathways

    Directory of Open Access Journals (Sweden)

    F Damiani

    2009-03-01

    Full Text Available We studied the effects of various TBT concentrations by assaying the activity of ERK 1/2 (p44/42 and phospho-ERK1/2 (phospho-p44/42, proteins with a key role in ascidian development, and tyrosine kinase-dependent pathway. The effects of this xenobiotic and the role of some signalling mechanisms on ascidian embryos were examined by using Western immunoblotting. The tyrosine phosphorylation pattern in the ascidians Ciona intestinalis and Phallusia mammillata development was examined and different levels of protein phosphorylation were found as a response to TBT at μM range. To determine whether another key signalling pathway was activated, the effects of TBT on the phosphorylation state of a component of tyrosine kinase-mediated signal transduction MAPK, ERK 1/2 (p44/42 were evaluated. Embryos of Ciona intestinalis exposed to 0.1, 0.25 and 0.5 μM TBT showed a slight decrement in the level of phosphorylated ERK, while a remarkable decrement in level of phopshorylated ERK were observed at higher TBT concentrations (0.5 μM to 10 μM. These data indicated that exposures to TBT induced changes in the total pattern of phosphotyrosine and in the phosphorylation levels of ERK 1/2 but there were no changes on the overall level of total ERK in ascidian embryos.

  14. Elimination pathways of cyclosarin (GF) mediated by β-cyclodextrin in vitro: pharmacokinetic and toxicokinetic aspects.

    Science.gov (United States)

    Kranawetvogl, Andreas; Schüler, Jan; Müller, Susanne; Thiermann, Horst; Worek, Franz; Reiter, Georg

    2013-10-24

    Cyclodextrins (CD) are promising small molecular scavengers showing favourable degradation of extremely toxic organophosphorus compounds (OP) such as tabun (GA), soman (GD) or cyclosarin (GF). For β-CD derivatives as potential OP antidotes with low intrinsic toxicity it is of great interest to completely understand the modes of interaction of both compounds in terms of OP detoxification. The mechanisms of CD action are not completely understood which prompted us to investigate the interactions of GF and β-cyclodextrin (β-CD) as model compounds. Using positive electrospray ionization mass spectrometry (ESI/MS), the formation of covalent conjugates of β-CD with O-cyclohexylmethylphosphonate (CHMP) residue was detected for the first time and was examined in vitro. With a newly developed LC-MS method the formation of O-cyclohexylmethylphosphonic acid (CHMPA) (i.e. GF hydrolysis) and covalent CHMP-β-CD conjugates was analyzed. Compared to water, tris(hydroxymethyl)aminomethane (TRIS) reduced the formation of covalent conjugates but amplified formation of CHMPA. Depending on experimental conditions the degradation of GF by β-CD may be preferably catalytic or stoichiometric. For illustrating different possible reaction pathways a scheme was established that could support the idea of β-CD acting as an artificial enzyme. These results provide an important insight into the β-CD mediated detoxification pathways of GF. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Mechanisms of RON-mediated epithelial-mesenchymal transition in MDCK cells through the MAPK pathway

    Directory of Open Access Journals (Sweden)

    Xu Xiangming

    2011-07-01

    Full Text Available The epithelial-mesenchymal transition (EMT is involved in neoplastic metastasis, and the RON protein may be involved. In the present study, we determined the role and the mechanisms of action of RON in EMT in Madin-Darby canine kidney (MDCK cells by Western blot and cell migration analysis. Activation of RON by macrophage stimulating protein (MSP results in cell migration and initiates changes in the morphology of RON-cDNA-transfected MDCK cells. The absence of E-cadherin, the presence of vimentin and an increase in Snail were observed in RE7 cells, which were derived from MDCK cells transfected with wt-RON, compared with MDCK cells. Stimulation of RE7 cells with MSP resulted in increased migration (about 69% of the wounded areas were covered as well as increased activation of extracellular signal-regulated kinase 1/2 (Erk1/2 and glycogen synthase kinase-3β (GSK-3β; the percent of the activation ratio was 143.6/599.8% and 512.4%, respectively, which could be inhibited with an individual chemical inhibitor PD98059 (50 μM specific to MAPK/ERK kinase (the percent inhibition was 98.9 and 81.2%, respectively. Thus, the results indicated that RON protein could mediate EMT in MDCK cells via the Erk1/2 pathway. Furthermore, GSK-3β regulates the function of Snail in controlling EMT by this pathway.

  16. Differential expression of extracellular matrix-mediated pathways in single-suture craniosynostosis.

    Directory of Open Access Journals (Sweden)

    Brendan D Stamper

    Full Text Available Craniosynostosis is a disease defined by premature fusion of one or more cranial sutures. The mechanistic pathology of single-suture craniosynostosis is complex and while a number of genetic biomarkers and environmental predispositions have been identified, in many cases the causes remain controversial and inconclusive. In this study, gene expression data from 199 patients with isolated sagittal (n = 100, unilateral coronal (n = 50, and metopic (n = 49 synostosis are compared against both a control population (n = 50, as well as each other. After controlling for variables contributing to potential bias, FGF7, SFRP4, and VCAM1 emerged as genes associated with single-suture craniosynostosis due to their significantly large changes in gene expression compared to the control population. Pathway analysis implicated focal adhesion and extracellular matrix (ECM-receptor interaction as differentially regulated gene networks when comparing all cases of single-suture synostosis and controls. Lastly, overall gene expression was found to be highly conserved between coronal and metopic cases, as evidenced by the fact that WNT2 and IGFBP2 were the only genes differentially regulated to a significantly large extent in a direct comparison. The identification of genes and gene networks associated with Fgf/Igf/Wnt signaling and ECM-mediated focal adhesion not only support the involvement of biomarkers previously reported to be related to craniosynostosis, but also introduce novel transcripts and pathways that may play critical roles in its pathogenesis.

  17. Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells.

    Science.gov (United States)

    Clark, Sara; Rainville, Jennifer; Zhao, Xing; Katzenellenbogen, Benita S; Pfaff, Donald; Vasudevan, Nandini

    2014-01-01

    While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17β-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Gαq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERα phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERα is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

  18. Visualization of oxytocin release that mediates paired pulse facilitation in hypothalamic pathways to brainstem autonomic neurons.

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    Ramón A Piñol

    Full Text Available Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2 expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV. We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs, neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection.

  19. Visualization of Oxytocin Release that Mediates Paired Pulse Facilitation in Hypothalamic Pathways to Brainstem Autonomic Neurons

    Science.gov (United States)

    Piñol, Ramón A.; Jameson, Heather; Popratiloff, Anastas; Lee, Norman H.; Mendelowitz, David

    2014-01-01

    Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN) contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2) expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV). We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs), neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection. PMID:25379676

  20. The TGF-β pathway mediates doxorubicin effects on cardiac endothelial cells.

    Science.gov (United States)

    Sun, Zuyue; Schriewer, Jill; Tang, Mingxin; Marlin, Jerry; Taylor, Frederick; Shohet, Ralph V; Konorev, Eugene A

    2016-01-01

    Elevated ALK4/5 ligands including TGF-β and activins have been linked to cardiovascular remodeling and heart failure. Doxorubicin (Dox) is commonly used as a model of cardiomyopathy, a condition that often precedes cardiovascular remodeling and heart failure. In 7-8-week-old C57Bl/6 male mice treated with Dox we found decreased capillary density, increased levels of ALK4/5 ligand and Smad2/3 transcripts, and increased expression of Smad2/3 transcriptional targets. Human cardiac microvascular endothelial cells (HCMVEC) treated with Dox also showed increased levels of ALK4/5 ligands, Smad2/3 transcriptional targets, a decrease in proliferation and suppression of vascular network formation in a HCMVEC and human cardiac fibroblasts co-culture assay. Our hypothesis is that the deleterious effects of Dox on endothelial cells are mediated in part by the activation of the TGF-β pathway. We used the inhibitor of ALK4/5 kinases SB431542 (SB) in concert with Dox to ascertain the role of TGF-β pathway activation in doxorubicin induced endothelial cell defects. SB prevented the suppression of HCMVEC proliferation in the presence of TGF-β2 and activin A, and alleviated the inhibition of HCMVEC proliferation by Dox. SB also prevented the suppression of vascular network formation in co-cultures of HCMVEC and human cardiac fibroblasts treated with Dox. Our results show that the inhibition of the TGF-β pathway alleviates the detrimental effects of Dox on endothelial cells in vitro.

  1. MDA-7/IL-24 inhibits Nrf2-mediated antioxidant response through activation of p38 pathway and inhibition of ERK pathway involved in cancer cell apoptosis.

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    Tian, H; Zhang, D; Gao, Z; Li, H; Zhang, B; Zhang, Q; Li, L; Cheng, Q; Pei, D; Zheng, J

    2014-10-01

    Reactive oxygen species (ROS) have a crucial role in melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24)-induced cancer cell apoptosis. However, cancer cell has a series of protective mechanisms to resist ROS damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates antioxidant response element (ARE)-mediated gene expression involved in cellular protection against oxidative stress. As the Nrf2 repressor, Kelch-like ECH-associated protein-1 (Keap1) sequesters Nrf2 in cytoplasm to block Nrf2 nuclear translocation. In the present study, administration of MDA-7/IL-24 by means of tumor-selective replicating adenovirus (ZD55-IL-24) was used to investigate whether ZD55-IL-24 could attenuate Nrf2-mediated oxidative stress response in cancer cell. We found that ZD55-IL-24 effectively strengthened the association between Nrf2 and Keap1 to restrict Nrf2 nuclear translocation, thereby inhibiting ARE-dependent transcriptional response. To evaluate the detailed mechanism underlying the suppression of ZD55-IL-24 on Nrf2-mediated oxidative stress response, we further tested three different mitogen-activated protein kinase (MAPK) signaling pathways in A549 and HeLa cells transfected by ZD55-IL-24. Our data showed that ZD55-IL-24 inhibited extracellular signal-regulated kinase (ERK) signal pathway but activated p38 and c-Jun-NH2-kinase (JNK) signal pathways to exert the tumor-specific apoptosis. Moreover, ERK pathway inhibitor U0126 prevented Nrf2 phosphorylation at Ser40 to retard Nrf2 nuclear translocation, thus decreasing antioxidant gene transcription. In contrast, p38 pathway inhibitor SB203580 obviously promoted the dissociation of Nrf2 from Keap1 to promote antioxidant gene transcription. However, JNK pathway had no effect on Nrf2 subcellular localization or the association of Nrf2 with Keap1. Conclusively, our results indicate that ZD55-IL-24 inhibits Nrf2-mediated oxidative stress response not only by activating p38 signal pathway to

  2. Chlorpromazine-induced hepatotoxicity during inflammation is mediated by TIRAP-dependent signaling pathway in mice

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    Gandhi, Adarsh, E-mail: adarsh.gandhi@nih.gov [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Guo, Tao, E-mail: tguo4@jhu.edu [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Shah, Pranav [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Moorthy, Bhagavatula [Baylor College of Medicine, Department of Pediatrics, 1102 Bates Avenue, Suite 530, Houston, TX 77030 (United States); Ghose, Romi, E-mail: rghose@uh.edu [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States)

    2013-02-01

    Inflammation is a major component of idiosyncratic adverse drug reactions (IADRs). To understand the molecular mechanism of inflammation-mediated IADRs, we determined the role of the Toll-like receptor (TLR) signaling pathway in idiosyncratic hepatotoxicity of the anti-psychotic drug, chlorpromazine (CPZ). Activation of TLRs recruits the first adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to the TIR domain of TLRs leading to the activation of the downstream kinase, c-Jun-N-terminal kinase (JNK). Prolonged activation of JNK leads to cell-death. We hypothesized that activation of TLR2 by lipoteichoic acid (LTA) or TLR4 by lipopolysaccharide (LPS) will augment the hepatotoxicity of CPZ by TIRAP-dependent mechanism involving prolonged activation of JNK. Adult male C57BL/6, TIRAP{sup +/+} and TIRAP{sup −/−} mice were pretreated with saline, LPS (2 mg/kg) or LTA (6 mg/kg) for 30 min or 16 h followed by CPZ (5 mg/kg) or saline (vehicle) up to 24 h. We found that treatment of mice with CPZ in presence of LPS or LTA leads to ∼ 3–4 fold increase in serum ALT levels, a marked reduction in hepatic glycogen content, significant induction of serum tumor necrosis factor (TNF) α and prolonged JNK activation, compared to LPS or LTA alone. Similar results were observed in TIRAP{sup +/+} mice, whereas the effects of LPS or LTA on CPZ-induced hepatotoxicity were attenuated in TIRAP{sup −/−} mice. For the first time, we show that inflammation-mediated hepatotoxicity of CPZ is dependent on TIRAP, and involves prolonged JNK activation in vivo. Thus, TIRAP-dependent pathways may be targeted to predict and prevent inflammation-mediated IADRs. -- Highlights: ► Inflammation augments the toxicity of an idiosyncratic hepatotoxin chlorpromazine. ► Activation of Toll-like receptors by LPS or LTA induces chlorpromazine toxicity. ► Sustained stress kinase (JNK) activation is associated with chlorpromazine toxicity. ► These studies

  3. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    Science.gov (United States)

    Shah, Dhiral Ashwin

    Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

  4. Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

    Directory of Open Access Journals (Sweden)

    Jessica Mühlhaus

    2014-11-01

    Full Text Available The thyroid hormone derivative 3-iodothyronamine (3-T1AM exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1 agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8. By RT-qPCR and locked-nucleic-acid (LNA in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs.

  5. The Che4 pathway of Myxococcus xanthus regulates type IV pilus-mediated motility.

    Science.gov (United States)

    Vlamakis, Hera C; Kirby, John R; Zusman, David R

    2004-06-01

    Myxococcus xanthus co-ordinates cell movement during its complex life cycle using multiple chemotaxis-like signal transduction pathways. These pathways regulate both type IV pilus-mediated social (S) motility and adventurous (A) motility. During a search for new chemoreceptors, we identified the che4 operon, which encodes homologues to a MCP (methyl-accepting chemotaxis protein), two CheWs, a hybrid CheA-CheY, a response regulator and a CheR. Deletion of the che4 operon did not cause swarming or developmental defects in either the wild-type (A(+)S(+)) strain or in a strain sustaining only A motility (A(+)S(-)). However, in a strain displaying only S motility (A(-)S(+)), deletion of the che4 operon or the gene encoding the response regulator, cheY4, caused enhanced vegetative swarming and prevented aggregation and sporulation. In contrast, deletion of mcp4 caused reduced vegetative swarming and enhanced development compared with the parent strain. Single-cell analysis of the motility of the A(-)S(+) parent strain revealed a previously unknown inverse correlation between velocity and reversal frequency. Thus, cells that moved at higher velocities showed a reduced reversal frequency. This co-ordination of reversal frequency and velocity was lost in the mcp4 and cheY4 mutants. The structural components of the S motility apparatus were unaffected in the che4 mutants, suggesting that the Che4 system affects reversal frequency of cells by modulating the function of the type IV pilus.

  6. Constitutive Photomorphogensis Protein1 (COP1 mediated p53 pathway and its oncogenic role

    Directory of Open Access Journals (Sweden)

    Md. Golam Rabbani

    2014-05-01

    Full Text Available We have reviewed the COP1 mediated tumor suppressor protein p53 pathway and its oncogenic role. COP1 is a negative regulator of p53 and acts as a pivotal controller of p53-Akt death-live switch (Protein kinase B. In presence of p53, COP1 is overexpressed in breast, ovarian, gastric cancers, even without MDM2 (Mouse double minute-2 amplification. Following DNA damage, COP1 is phosphorylated instantly by ATM (Ataxia telangiectasia mutated and degraded by 14-3-3 and #963; following nuclear export and enhancing ubiquitination. In ATM lacking cell, other kinases, i.e. ATR (ataxia telangiectasia and Rad3-related protein, Jun kinases and DNA-PK (DNA-dependent protein kinase cause COP1 and CSN3 (COP9 signalosome complex subunit-3 phosphorylation and initiate COP1's down regulation. Although, it has been previously found that co-knockout of MDM2 and COP1 enhance p53's half life by eight fold, the reason is still unknown. Additionally, while interacting with p53, COP1 upregulate MDM2's E3 ubiquitin ligase, Akt, CSN6 (COP9 signalosome 6 activity and inhibit 14-3-3 and #963;'s negative regulation on MDM2 and COP1 itself. Conclusively, there persists an amplification loop among COP1, MDM2, Akt and 14-3-3 and #963; to regulate p53's stability and activity. However, the role of another tumor suppressor PTEN (phosphatase and tensin homologue is yet to be discovered. This study provides insight on the molecular genetic pathways related to cancer and might be helpful for therapeutic inventions. [Biomed Res Ther 2014; 1(5.000: 142-151

  7. Nanoparticle-Mediated Expression of a Wnt Pathway Inhibitor Ameliorates Ocular Neovascularization

    Science.gov (United States)

    Wang, Zhongxiao; Cheng, Rui; Lee, Kyungwon; Puneet, Tyagi; Ding, Lexi; Kompella, Uday B.; Chen, Jing; Xu, Xun; Ma, Jian-xing

    2015-01-01

    Objective The deficiency of very low-density lipoprotein receptor (VLDLR) resulted in Wnt signaling activation and neovascularization (NV) in the retina. The present study sought to determine if the VLDLR extracellular domain (VLN) is responsible for the inhibition of Wnt signaling in ocular tissues. Approach and Results A plasmid expressing the soluble VLN was encapsulated with poly (lactide-co-glycolide acid) (PLGA) to form VLN nanoparticles (VLN-NP). Nanoparticles containing a plasmid expressing the low-density lipoprotein receptor extracellular domain (LN-NP) were used as negative control. MTT, modified Boyden chamber and Matrigel (™) assays were used to evaluate the inhibitory effect of VLN-NP on Wnt3a-stimulated endothelial cell (EC) proliferation, migration and tube formation. Vldlr−/− mice, oxygen-induced retinopathy (OIR) and alkali burn-induced corneal NV models were used to evaluate the effect of VLN-NP on ocular NV. Wnt reporter mice (BAT-gal), Western blotting and luciferase assay were used to evaluate Wnt pathway activity. Our results showed that VLN-NP specifically inhibited Wnt3a-induced EC proliferation, migration and tube formation. Intravitreal injection of VLN-NP inhibited abnormal NV in Vldlr−/−, OIR and alkali burn-induced corneal NV models, compared with LN-NP. VLN-NP significantly inhibited the phosphorylation of LRP6, the accumulation of β-catenin and the expression of VEGF in vivo and in vitro. Conclusions Taken together, these results suggest that the soluble VLN is a negative regulator of the Wnt pathway and has anti-angiogenic activities. Nanoparticle-mediated expression of VLN may thus represent a novel therapeutic approach to treat pathologic ocular angiogenesis and potentially other vascular diseases impacted by Wnt signaling. PMID:25657312

  8. Piracetam inhibits ethanol (EtOH)-induced memory deficit by mediating multiple pathways.

    Science.gov (United States)

    Yang, Yifan; Feng, Jian; Xu, Fangyuan; Wang, Jianglin

    2017-09-11

    Excessive ethanol (EtOH) intake, especially to prenatal exposure, can significantly affect cognitive function and cause permanent learning and memory injures in children. As a result, how to protect children from EtOH neurotoxicity has gained increasing attention in recent years. Piracetam (Pir) is a nootropic drug derived from c-aminobutyric acid and can manage cognition impairments in multiple neurological disorders. Studies have shown that Pir can exert therapeutic effects on EtOH-induced memory impairments, but the underlying mechanism is still unknown. In this study, we found that Pir inhibited ethanol-induced memory deficit by mediating multiple pathways. Treatment with EtOH could cause cognitive deficit in juvenile rats, and triggered the alteration of synaptic plasticity. Administration with Pir significantly increased long-term potentiation and protected hippocampus neurons from EtOH neurotoxicity. Pir intervention ameliorated EtOH-induced cell apoptosis and inhibited the activation of Caspase-3 in vitro, suggesting that Pir protected neurons by anti-apoptotic effects. Pir could decrease the expression of LC3-II and Beclin-1 induced by EtOH, and increase the phosphorylation of mTOR and reduce the phosphorylation of Akt, which suggested that the protective effect of Pir was involved in regulation of autophagic process and mTOR/Akt pathways. In conclusion, we speculate that Pir reduces EtOH-induced neuronal damage by regulation of apoptotic action and autophagic action, and our research offers preclinical evidence for the application of Pir in ethanol toxicity. Copyright © 2017. Published by Elsevier B.V.

  9. Nitric Oxide Mediated Transcriptome Profiling Reveals Activation of Multiple Regulatory Pathways in Arabidopsis thaliana.

    Science.gov (United States)

    Hussain, Adil; Mun, Bong-Gyu; Imran, Qari M; Lee, Sang-Uk; Adamu, Teferi A; Shahid, Muhammad; Kim, Kyung-Min; Yun, Byung-Wook

    2016-01-01

    Imbalance between the accumulation and removal of nitric oxide and its derivatives is a challenge faced by all plants at the cellular level, and is especially important under stress conditions. Exposure of plants to various biotic and abiotic stresses causes rapid changes in cellular redox tone potentiated by the rise in reactive nitrogen species that serve as signaling molecules in mediating defensive responses. To understand mechanisms mediated by these signaling molecules, we performed a large-scale analysis of the Arabidopsis transcriptome induced by nitrosative stress. We generated an average of 84 and 91 million reads from three replicates each of control and 1 mM S-nitrosocysteine (CysNO)-infiltrated Arabidopsis leaf samples, respectively. After alignment, more than 95% of all reads successfully mapped to the reference and 32,535 genes and 55,682 transcripts were obtained. CysNO infiltration caused differential expression of 6436 genes (3448 up-regulated and 2988 down-regulated) and 6214 transcripts (3335 up-regulated and 2879 down-regulated) 6 h post-infiltration. These differentially expressed genes were found to be involved in key physiological processes, including plant defense against various biotic and abiotic stresses, hormone signaling, and other developmental processes. After quantile normalization of the FPKM values followed by student's T-test (P pathways were verified using quantitative real-time PCR. This study provides comprehensive information about plant responses to nitrosative stress at transcript level and would prove helpful in understanding and incorporating mechanisms associated with nitrosative stress responses in plants.

  10. A convergent and essential interneuron pathway for Mauthner-cell-mediated escapes.

    Science.gov (United States)

    Lacoste, Alix M B; Schoppik, David; Robson, Drew N; Haesemeyer, Martin; Portugues, Ruben; Li, Jennifer M; Randlett, Owen; Wee, Caroline L; Engert, Florian; Schier, Alexander F

    2015-06-01

    The Mauthner cell (M-cell) is a command-like neuron in teleost fish whose firing in response to aversive stimuli is correlated with short-latency escapes [1-3]. M-cells have been proposed as evolutionary ancestors of startle response neurons of the mammalian reticular formation [4], and studies of this circuit have uncovered important principles in neurobiology that generalize to more complex vertebrate models [3]. The main excitatory input was thought to originate from multisensory afferents synapsing directly onto the M-cell dendrites [3]. Here, we describe an additional, convergent pathway that is essential for the M-cell-mediated startle behavior in larval zebrafish. It is composed of excitatory interneurons called spiral fiber neurons, which project to the M-cell axon hillock. By in vivo calcium imaging, we found that spiral fiber neurons are active in response to aversive stimuli capable of eliciting escapes. Like M-cell ablations, bilateral ablations of spiral fiber neurons largely eliminate short-latency escapes. Unilateral spiral fiber neuron ablations shift the directionality of escapes and indicate that spiral fiber neurons excite the M-cell in a lateralized manner. Their optogenetic activation increases the probability of short-latency escapes, supporting the notion that spiral fiber neurons help activate M-cell-mediated startle behavior. These results reveal that spiral fiber neurons are essential for the function of the M-cell in response to sensory cues and suggest that convergent excitatory inputs that differ in their input location and timing ensure reliable activation of the M-cell, a feedforward excitatory motif that may extend to other neural circuits.

  11. Cytoplasmic inositol hexakisphosphate production is sufficient for mediating the Gle1-mRNA export pathway.

    Science.gov (United States)

    Miller, Aimee L; Suntharalingam, Mythili; Johnson, Sylvia L; Audhya, Anjon; Emr, Scott D; Wente, Susan R

    2004-12-03

    Production of inositol hexakisphosphate (IP6) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116Deltaipk1Delta and nup42Deltaipk1Delta double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP6 production is sufficient for mediating the Gle1-mRNA export pathway.

  12. 25-Hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB dependent pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ichikawa, Tomohiro [Third Department of Internal Medicine, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8509 (Japan); Sugiura, Hisatoshi, E-mail: sugiura@rm.med.tohoku.ac.jp [Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 (Japan); Koarai, Akira; Kikuchi, Takashi; Hiramatsu, Masataka; Kawabata, Hiroki; Akamatsu, Keiichiro; Hirano, Tsunahiko; Nakanishi, Masanori; Matsunaga, Kazuto; Minakata, Yoshiaki [Third Department of Internal Medicine, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8509 (Japan); Ichinose, Masakazu [Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 (Japan)

    2013-05-01

    Abnormal structural alterations termed remodeling, including fibrosis and alveolar wall destruction, are important features of the pathophysiology of chronic airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma. 25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase (CH25H) in macrophages and is reported to be involved in the formation of arteriosclerosis. We previously demonstrated that the expression of CH25H and production of 25HC were increased in the lungs of COPD. However, the role of 25-HC in lung tissue remodeling is unknown. In this study, we investigated the effect of 25-HC on fibroblast-mediated tissue remodeling using human fetal lung fibroblasts (HFL-1) in vitro. 25-HC significantly augmented α-smooth muscle actin (SMA) (P<0.001) and collagen I (P<0.001) expression in HFL-1. 25-HC also significantly enhanced the release and activation of matrix metallaoproteinase (MMP)-2 (P<0.001) and MMP-9 (P<0.001) without any significant effect on the production of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. 25-HC stimulated transforming growth factor (TGF)-β{sub 1} production (P<0.01) and a neutralizing anti-TGF-β antibody restored these 25-HC-augmented pro-fibrotic responses. 25-HC significantly promoted the translocation of nuclear factor (NF)-κB p65 into the nuclei (P<0.01), but not phospholylated-c-jun, a complex of activator protein-1. Pharmacological inhibition of NF-κB restored the 25-HC-augmented pro-fibrotic responses and TGF-β{sub 1} release. These results suggest that 25-HC could contribute to fibroblast-mediated lung tissue remodeling by promoting myofibroblast differentiation and the excessive release of extracellular matrix protein and MMPs via an NF-κB-TGF-β dependent pathway.

  13. Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

    Directory of Open Access Journals (Sweden)

    Michelle R. Campbell

    2013-01-01

    Full Text Available Nuclear factor- (erythroid-derived 2 like 2 (NFE2L2, NRF2 is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1, and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

  14. VAMP-8 segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways.

    Science.gov (United States)

    Tiwari, Neeraj; Wang, Cheng-Chun; Brochetta, Cristiana; Ke, Gou; Vita, Francesca; Qi, Zeng; Rivera, Juan; Soranzo, Maria Rosa; Zabucchi, Giuliano; Hong, Wanjin; Blank, Ulrich

    2008-04-01

    Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow-derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8-deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8-deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3-positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

  15. Divergence of mechanistic pathways mediating cardiovascular aging and developmental programming of cardiovascular disease.

    Science.gov (United States)

    Allison, Beth J; Kaandorp, Joepe J; Kane, Andrew D; Camm, Emily J; Lusby, Ciara; Cross, Christine M; Nevin-Dolan, Rhianon; Thakor, Avnesh S; Derks, Jan B; Tarry-Adkins, Jane L; Ozanne, Susan E; Giussani, Dino A

    2016-05-01

    Aging and developmental programming are both associated with oxidative stress and endothelial dysfunction, suggesting common mechanistic origins. However, their interrelationship has been little explored. In a rodent model of programmed cardiovascular dysfunction we determined endothelial function and vascular telomere length in young (4 mo) and aged (15 mo) adult offspring of normoxic or hypoxic pregnancy with or without maternal antioxidant treatment. We show loss of endothelial function [maximal arterial relaxation to acetylcholine (71 ± 3 vs. 55 ± 3%) and increased vascular short telomere abundance (4.2-1.3 kb) 43.0 ± 1.5 vs. 55.1 ± 3.8%) in aged vs. young offspring of normoxic pregnancy (P programming of cardiovascular disease, and aging being decelerated by antioxidants even prior to birth.-Allison, B. J., Kaandorp, J. J., Kane, A. D., Camm, E. J., Lusby, C., Cross, C. M., Nevin-Dolan, R., Thakor, A. S., Derks, J. B., Tarry-Adkins, J. L., Ozanne, S. E., Giussani, D. A. Divergence of mechanistic pathways mediating cardiovascular aging and developmental programming of cardiovascular disease.

  16. Curcumin induces apoptosis through the mitochondria-mediated apoptotic pathway in HT-29 cells

    Institute of Scientific and Technical Information of China (English)

    Jin-bo WANG; Li-li QI; Shui-di ZHENG; Tian-xing WU

    2009-01-01

    Objective:To investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2,Bax,Bad,Bcl-xL,caspase-3,poly ADP-ribose polymerase (PARP),and survivin of HT-29 cells.Methods:HT-29 cells were treated with curcumin (0~80 μmol/L) for 24 h.The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax,Bcl-2,Bci-xL,Bad,caspase-3,PARP,and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR).Results:Curcumin significantly induced the growth inhibition and apoptosis of HT-29 ceils.A decrease in expressions of Bcl-2,Bci-xL and survivin was observed after exposure to 10~80 μmol/L curcumin,while the levels of Bax and Bad increased in the curcumin-treated cells.Curcumin also induced the release of cytochrome c,the activation ofcaspase-3,and the cleavage of PARP in a dose-dependent manner.Conclusion:These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway.

  17. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  18. Fipronil promotes adipogenesis via AMPKα-mediated pathway in 3T3-L1 adipocytes.

    Science.gov (United States)

    Sun, Quancai; Qi, Weipeng; Yang, Jeremy J; Yoon, Kyong Sup; Clark, John M; Park, Yeonhwa

    2016-06-01

    Emerging evidence suggests that organochlorine, organophosphorus and neonicotinoid insecticide exposure may be linked to the development of obesity and type 2 diabetes. However, there is no knowledge of the potential influence of fipronil, which belongs to the phenylpyrazole chemical family, on obesity. Thus, the goal of this study was to determine the role of fipronil in adipogenesis using 3T3-L1 adipocytes. Fipronil treatment, at 10 μM, increased fat accumulation in 3T3-L1 adipocytes as well as promoted key regulators of adipocyte differentiation (CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor gamma-γ), and key regulators of lipogenesis (acetyl-CoA carboxylase and fatty acid synthase). The activation of AMPKα with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) abolished effects of fipronil on increased adipogenesis. These results suggest that fipronil alters adipogenesis and results in increased lipid accumulation through a AMPKα-mediated pathway. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Towards Direct Synthesis of Alane: A Predicted Defect-Mediated Pathway Confirmed Experimentally.

    Science.gov (United States)

    Wang, Lin-Lin; Herwadkar, Aditi; Reich, Jason M; Johnson, Duane D; House, Stephen D; Peña-Martin, Pamela; Rockett, Angus A; Robertson, Ian M; Gupta, Shalabh; Pecharsky, Vitalij K

    2016-09-01

    Alane (AlH3 ) is a unique energetic material that has not found a broad practical use for over 70 years because it is difficult to synthesize directly from its elements. Using density functional theory, we examine the defect-mediated formation of alane monomers on Al(111) in a two-step process: (1) dissociative adsorption of H2 and (2) alane formation, which are both endothermic on a clean surface. Only with Ti dopant to facilitate H2 dissociation and vacancies to provide Al adatoms, both processes become exothermic. In agreement, in situ scanning tunneling microscopy showed that during H2 exposure, alane monomers and clusters form primarily in the vicinity of Al vacancies and Ti atoms. Moreover, ball milling of the Al samples with Ti (providing necessary defects) showed a 10 % conversion of Al into AlH3 or closely related species at 344 bar H2 , indicating that the predicted pathway may lead to the direct synthesis of alane from elements at pressures much lower than the 10(4)  bar expected from bulk thermodynamics.

  20. PGE2 Modulates GABAA Receptors via an EP1 Receptor-Mediated Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Guang Yang

    2015-07-01

    Full Text Available Aims: PGE2 is one of the most abundant prostanoids in mammalian tissues, but its effect on neuronal receptors has not been well investigated. This study examines the effect of PGE2 on GABAA receptor currents in rat cerebellar granule neurons. Methods: GABAA currents were recorded using a patch-clamp technique. Cell surface and total protein of GABAA β1/2/3 subunits was carried out by Western blot analysis. Results: Upon incubation of neurons with PGE2 (1 µM for 60 minutes, GABAA currents were significantly potentiated. This PGE2-driven effect could be blocked by PKC or CaMKII inhibitors as well as EP1 receptor antagonist, and mimicked by PMA or EP1 receptor agonist. Furthermore, Western blot data showed that PGE2 did not increase the total expression level of GABAA receptors, but significantly increased surface levels of GABAA β1/2/3 subunits after 1 h of treatment. Consistently, both PKC and CaMKII inhibitors were able to reduce PGE2-induced increases in cell surface expression of GABAA receptors. Conclusion: Activation of either the PKC or CaMKII pathways by EP1 receptors mediates the PGE2-induced increase in GABAA currents. This suggests that upregulation of postsynaptic GABAA receptors by PGE2 may have profound effects on cerebellar functioning under physiological and pathological conditions.

  1. Gut microbiome remodeling induces depressive-like behaviors through a pathway mediated by the host's metabolism.

    Science.gov (United States)

    Zheng, P; Zeng, B; Zhou, C; Liu, M; Fang, Z; Xu, X; Zeng, L; Chen, J; Fan, S; Du, X; Zhang, X; Yang, D; Yang, Y; Meng, H; Li, W; Melgiri, N D; Licinio, J; Wei, H; Xie, P

    2016-06-01

    Major depressive disorder (MDD) is the result of complex gene-environment interactions. According to the World Health Organization, MDD is the leading cause of disability worldwide, and it is a major contributor to the overall global burden of disease. However, the definitive environmental mechanisms underlying the pathophysiology of MDD remain elusive. The gut microbiome is an increasingly recognized environmental factor that can shape the brain through the microbiota-gut-brain axis. We show here that the absence of gut microbiota in germ-free (GF) mice resulted in decreased immobility time in the forced swimming test relative to conventionally raised healthy control mice. Moreover, from clinical sampling, the gut microbiotic compositions of MDD patients and healthy controls were significantly different with MDD patients characterized by significant changes in the relative abundance of Firmicutes, Actinobacteria and Bacteroidetes. Fecal microbiota transplantation of GF mice with 'depression microbiota' derived from MDD patients resulted in depression-like behaviors compared with colonization with 'healthy microbiota' derived from healthy control individuals. Mice harboring 'depression microbiota' primarily exhibited disturbances of microbial genes and host metabolites involved in carbohydrate and amino acid metabolism. This study demonstrates that dysbiosis of the gut microbiome may have a causal role in the development of depressive-like behaviors, in a pathway that is mediated through the host's metabolism.

  2. MOF Phosphorylation by ATM Regulates 53BP1-Mediated Double-Strand Break Repair Pathway Choice

    Directory of Open Access Journals (Sweden)

    Arun Gupta

    2014-07-01

    Full Text Available Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ or homologous recombination (HR. Here, we report that double-strand breaks (DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase phosphorylation (p-T392-MOF and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  3. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  4. Pathways of influence in school-based mentoring: the mediating role of parent and teacher relationships.

    Science.gov (United States)

    Chan, Christian S; Rhodes, Jean E; Howard, Waylon J; Lowe, Sarah R; Schwartz, Sarah E O; Herrera, Carla

    2013-02-01

    This study explores the pathways through which school-based mentoring relationships are associated with improvements in elementary and high school students' socio-emotional, academic, and behavioral outcomes. Participants in the study (N=526) were part of a national evaluation of the Big Brothers Big Sisters school-based mentoring programs, all of whom had been randomly assigned to receive mentoring at their schools over the course of one academic year. Students were assessed at the beginning and end of the school year. The results of structural equation modeling showed that mentoring relationship quality, as measured by the Youth-Centered Relationship scale and the Youth's Emotional Engagement scale, was significantly associated with positive changes in youths' relationships with parents and teachers, as measured by subscales of the Inventory of Parent and Peer Attachment, the Teacher Relationship Quality scale, and the Hemingway Measure of Adolescent Connectedness. Higher quality relationships with parents and teachers, in turn, were significantly associated with better youth outcomes, including self-esteem, academic attitudes, prosocial behaviors, and misconduct. The effect sizes of the associations ranged from 0.12 to 0.52. Mediation analysis found that mentoring relationship quality was indirectly associated with some of the outcomes through its association with improved parent and teacher relationships. Implications of the findings for theory and research are discussed.

  5. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium.

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, M. P.; Gorman-Lewis, D.; Aryal, B. P.; Paunesku, T.; Vogt, S.; Rickert, P. G.; Seifert, S.; Lai, B.; Woloschak, G. E.; Soderholm, L. (Chemical Sciences and Engineering Division); ( XSD); (Univ. of Chicago); (Northwestern Univ.)

    2011-08-01

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway -- receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu{sub c}Fe{sub N}Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.

  6. Class 3 semaphorin mediates dendrite growth in adult newborn neurons through Cdk5/FAK pathway.

    Directory of Open Access Journals (Sweden)

    Teclise Ng

    Full Text Available Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397 and serine phosphorylation (Ser 732 of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.

  7. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium.

    Science.gov (United States)

    Jensen, Mark P; Gorman-Lewis, Drew; Aryal, Baikuntha; Paunesku, Tatjana; Vogt, Stefan; Rickert, Paul G; Seifert, Soenke; Lai, Barry; Woloschak, Gayle E; Soderholm, L

    2011-06-26

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.

  8. Inositol Hexakisphosphate Mediates Apoptosis in Human Breast Adenocarcinoma MCF-7 Cell Line via Intrinsic Pathway

    Science.gov (United States)

    Agarwal, Rakhee; Ali, Nawab

    2010-04-01

    Inositol polyphosphates (InsPs) are naturally occurring compounds ubiquitously present in plants and animals. Inositol hexakisphosphate (InsP6) is the most abundant among all InsPs and constitutes the major portion of dietary fiber in most cereals, legumes and nuts. Certain derivatives of InsPs also regulate cellular signaling mechanisms. InsPs have also been shown to reduce tumor formation and induce apoptosis in cancerous cells. Therefore, in this study, the effects of InsPs on apoptosis were studied in an attempt to investigate their potential anti-cancer therapeutic application and understand their mechanism of action. Acridine orange and ethidium bromide staining suggested that InsP6 dose dependently induced apoptosis in human breast adenocarcinoma MCF-7 cells. Among InsPs tested (InsP3, InsP4, InsP5, and InsP6), InsP6 was found to be the most effective in inducing apoptosis. Furthermore, effects of InsP6 were found most potent inducing apoptosis. Etoposide, the drug known to induce apoptosis in both in vivo and in vitro, was used as a positive control. Western blotting experiments using specific antibodies against known apoptotic markers suggested that InsP6 induced apoptotic changes were mediated via an intrinsic apoptotic pathway.

  9. O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George, E-mail: pwang@nankai.edu.cn; Zhang, Lianwen, E-mail: lianwen@nankai.edu.cn

    2016-03-18

    O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. - Highlights: • Reduced NEDD4-1 correlates with increased overall O-GlcNAc level. • OGT negatively regulates NEDD4-1 stability. • O-GlcNAc regulates NEDD4-1 through caspase-mediated pathway.

  10. Triglyceride-mediated pathways and coronary disease: collaborative analysis of 101 studies.

    Science.gov (United States)

    Sarwar, Nadeem; Sandhu, Manjinder S; Ricketts, Sally L; Butterworth, Adam S; Di Angelantonio, Emanuele; Boekholdt, S Matthijs; Ouwehand, Willem; Watkins, Hugh; Samani, Nilesh J; Saleheen, Danish; Lawlor, Debbie; Reilly, Muredach P; Hingorani, Aroon D; Talmud, Philippa J; Danesh, John

    2010-05-08

    Whether triglyceride-mediated pathways are causally relevant to coronary heart disease is uncertain. We studied a genetic variant that regulates triglyceride concentration to help judge likelihood of causality. We assessed the -1131T>C (rs662799) promoter polymorphism of the apolipoprotein A5 (APOA5) gene in relation to triglyceride concentration, several other risk factors, and risk of coronary heart disease. We compared disease risk for genetically-raised triglyceride concentration (20,842 patients with coronary heart disease, 35,206 controls) with that recorded for equivalent differences in circulating triglyceride concentration in prospective studies (302 430 participants with no history of cardiovascular disease; 12,785 incident cases of coronary heart disease during 2.79 million person-years at risk). We analysed -1131T>C in 1795 people without a history of cardiovascular disease who had information about lipoprotein concentration and diameter obtained by nuclear magnetic resonance spectroscopy. The minor allele frequency of -1131T>C was 8% (95% CI 7-9). -1131T>C was not significantly associated with several non-lipid risk factors or LDL cholesterol, and it was modestly associated with lower HDL cholesterol (mean difference per C allele 3.5% [95% CI 2.6-4.6]; 0.053 mmol/L [0.039-0.068]), lower apolipoprotein AI (1.3% [0.3-2.3]; 0.023 g/L [0.005-0.041]), and higher apolipoprotein B (3.2% [1.3-5.1]; 0.027 g/L [0.011-0.043]). By contrast, for every C allele inherited, mean triglyceride concentration was 16.0% (95% CI 12.9-18.7), or 0.25 mmol/L (0.20-0.29), higher (p=4.4x10(-24)). The odds ratio for coronary heart disease was 1.18 (95% CI 1.11-1.26; p=2.6x10(-7)) per C allele, which was concordant with the hazard ratio of 1.10 (95% CI 1.08-1.12) per 16% higher triglyceride concentration recorded in prospective studies. -1131T>C was significantly associated with higher VLDL particle concentration (mean difference per C allele 12.2 nmol/L [95% CI 7.7-16.7]; p=9.3x

  11. Cell invasion by Neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin.

    Directory of Open Access Journals (Sweden)

    Heiko Slanina

    Full Text Available Entry of Neisseria meningitidis (the meningococcus into human brain microvascular endothelial cells (HBMEC is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK, which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells.

  12. Obesity Suppresses Estrogen Receptor Beta Expression in Breast Cancer Cells via a HER2-Mediated Pathway.

    Science.gov (United States)

    Bowers, Laura W; Wiese, Megan; Brenner, Andrew J; Rossi, Emily L; Tekmal, Rajeshwar R; Hursting, Stephen D; deGraffenried, Linda A

    2015-01-01

    Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ) expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB): ≥30 kg/m2; normal weight (N): 18.5-24.9 kg/m2). Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231) and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu) following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s) mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease.

  13. Obesity Suppresses Estrogen Receptor Beta Expression in Breast Cancer Cells via a HER2-Mediated Pathway.

    Directory of Open Access Journals (Sweden)

    Laura W Bowers

    Full Text Available Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB: ≥30 kg/m2; normal weight (N: 18.5-24.9 kg/m2. Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231 and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease.

  14. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

    Science.gov (United States)

    Modi, Souvik; Halder, Saheli; Nizak, Clément; Krishnan, Yamuna

    2013-12-01

    DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing

  15. DMPD: IRAK1: a critical signaling mediator of innate immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17890055 IRAK1: a critical signaling mediator of innate immunity. Gottipati S, Rao ...IRAK1: a critical signaling mediator of innate immunity. PubmedID 17890055 Title IRAK1: a critical signaling media

  16. Berbamine selectively induces apoptosis of human acute promyelocytic leukemia cells via survivin-mediated pathway

    Institute of Scientific and Technical Information of China (English)

    ZHAO Xiao-ying; HE Zhi-wen; WU Dong; XU Rong-zhen

    2007-01-01

    Background Currently, resistance and relapse are still major problems in acute promyelocytic leukemia (APL) cases.Thus, new agents that override the resistance are crucial to the development of curative therapies for APL. In this study,we investigated the effects of berbamine on the proliferation of APL cell line NB4 and its possible mechanisms.Methods NB4 cells were treated with berbamine at different concentrations (0-64 μg/ml) for 72 hours. MTT assay was used to determine proliferation inhibition of NB4 cells. Cell apoptosis was evaluated by both flow cytometry (FCM) and morphological examination. PML/RAR-α and survivin mRNAs were measured by nested-RT-PCR and RT-PCR,respectively. Activated-caspase 3 was determined by FCM.Results Berbamine greatly inhibited the proliferation of NB4 cells in dose- and time-dependent manners, and its IC50 value was 3.86 μg/ml at 48 hours. Both morphological observations and FCM results showed that berbamine induced apoptosis of NB4 cells with concomitant increase of activated caspase-3 and decrease of survivin mRNA. After treatment with berbamine at 8 μg/ml for 48 hours, the percentage of apoptotic cells increased from 2.83% to 58.44% (P<0.01), and the percentage of cells with activated-caspase 3 elevated from 2.06% to 70.89% (P<0.01), whereas, level of survivin mRNA was reduced to 38.24% of control (P<0.01). However, no significant change was observed in PML/RAR-α mRNA.Conclusions Berbamine induces caspase-3-dependent apoptosis of leukemia NB4 cells via survivin-mediated pathway, suggesting that berbamine may be a novel potential agent against APL with a mechanism distinct from that of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO).

  17. Molecular pathways: adipose inflammation as a mediator of obesity-associated cancer.

    Science.gov (United States)

    Howe, Louise R; Subbaramaiah, Kotha; Hudis, Clifford A; Dannenberg, Andrew J

    2013-11-15

    The increasing rate of obesity worldwide is predicted to be associated with a surge in diseases. Notably, obesity has been linked to approximately 20% of cancer cases in the United States; obesity is associated with both increased risk and worse outcomes after diagnosis. Altered levels of circulating factors are strongly implicated, including insulin, insulin-like growth factor 1, leptin, adiponectin, and interleukin-6 (IL-6). In addition, increasing attention has focused on the consequences of local adipose inflammation. Inflammatory foci characterized by crown-like structures consisting of dead adipocytes encircled by macrophages occur in white adipose depots, including the breast tissue, of most overweight and obese women. Saturated fatty acids, released as a consequence of obesity-associated lipolysis, induce macrophage activation via Toll-like receptor 4, thereby stimulating NF-κB signaling. This, in turn, activates transcription of proinflammatory genes including COX-2, IL-6, IL-1β, and TNFα. Elevated levels of proinflammatory mediators cause both local and systemic effects. Of particular relevance with regard to breast cancer is increased transcription of the CYP19 gene encoding aromatase, the rate-limiting enzyme for estrogen synthesis. Notably, this obesity-inflammation-aromatase axis provides a plausible explanation for increased rates of postmenopausal, hormone receptor-positive breast cancer associated with obesity and hence may offer targets for interventions to attenuate risk or improve prognosis. Potential approaches include weight reduction, exercise, and suppression of obesity-driven signaling pathways using pharmaceutical or dietary agents. A key future goal is to identify biomarkers that accurately report adipose inflammation, both for identification of at-risk individuals and to assess the efficacy of interventions. Clin Cancer Res; 19(22); 6074-83. ©2013 AACR.

  18. Siomycin A targets brain tumor stem cells partially through a MELK-mediated pathway.

    Science.gov (United States)

    Nakano, Ichiro; Joshi, Kaushal; Visnyei, Koppany; Hu, Bin; Watanabe, Momoko; Lam, Diana; Wexler, Eric; Saigusa, Kuniyasu; Nakamura, Yuko; Laks, Dan R; Mischel, Paul S; Viapiano, Mariano; Kornblum, Harley I

    2011-06-01

    Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.

  19. The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP.

    Science.gov (United States)

    Kimura, Tomomi E; Duggirala, Aparna; Smith, Madeleine C; White, Stephen; Sala-Newby, Graciela B; Newby, Andrew C; Bond, Mark

    2016-01-01

    Inhibition of vascular smooth muscle cell (VSMC) proliferation by intracellular cAMP prevents excessive neointima formation and hence angioplasty restenosis and vein-graft failure. These protective effects are mediated via actin-cytoskeleton remodelling and subsequent regulation of gene expression by mechanisms that are incompletely understood. Here we investigated the role of components of the growth-regulatory Hippo pathway, specifically the transcription factor TEAD and its co-factors YAP and TAZ in VSMC. Elevation of cAMP using forskolin, dibutyryl-cAMP or the physiological agonists, Cicaprost or adenosine, significantly increased phosphorylation and nuclear export YAP and TAZ and inhibited TEAD-luciferase report gene activity. Similar effects were obtained by inhibiting RhoA activity with C3-transferase, its downstream kinase, ROCK, with Y27632, or actin-polymerisation with Latrunculin-B. Conversely, expression of constitutively-active RhoA reversed the inhibitory effects of forskolin on TEAD-luciferase. Forskolin significantly inhibited the mRNA expression of the pro-mitogenic genes, CCN1, CTGF, c-MYC and TGFB2 and this was reversed by expression of constitutively-active YAP or TAZ phospho-mutants. Inhibition of YAP and TAZ function with RNAi or Verteporfin significantly reduced VSMC proliferation. Furthermore, the anti-mitogenic effects of forskolin were reversed by overexpression of constitutively-active YAP or TAZ. Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling inhibits YAP/TAZ-TEAD dependent expression of pro-mitogenic genes in VSMC. This mechanism contributes novel insight into the anti-mitogenic effects of cAMP in VSMC and suggests a new target for intervention. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. RANTES/CCL5 mediated-biological effects depend on the syndecan-4/PKCα signaling pathway

    Directory of Open Access Journals (Sweden)

    Loïc Maillard

    2014-09-01

    Full Text Available The perpetuation of angiogenesis is involved in certain chronic inflammatory diseases. The accelerated neovascularisation may result from an inflammatory status with a response of both endothelial cells and monocytes to inflammatory mediators such as chemokines. We have previously described in vitro and in vivo the pro-angiogenic effects of the chemokine Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES/CCL5. The effects of RANTES/CCL5 may be related to its binding to G protein-coupled receptors and to proteoglycans such as syndecan-1 and -4. The aim of this study was to evaluate the functionality of syndecan-4 as a co-receptor of RANTES/CCL5 by the use of mutated syndecan-4 constructs. Our data demonstrate that site-directed mutations in syndecan-4 modify RANTES/CCL5 biological activities in endothelial cells. The SDC4S179A mutant, associated with an induced protein kinase C (PKCα activation, leads to higher RANTES/CCL5 pro-angiogenic effects, whereas the SDC4L188QQ and the SDC4A198del mutants, leading to lower phosphatidylinositol 4,5-bisphosphate (PIP2 binding or to lower PDZ protein binding respectively, are associated with reduced RANTES/CCL5 cellular effects. Moreover, our data highlight that the intracellular domain of SDC-4 is involved in RANTES/CCL5-induced activation of the PKCα signaling pathway and biological effect. As RANTES/CCL5 is involved in various physiopathological processes, the development of a new therapeutic strategy may be reliant on the mechanism by which RANTES/CCL5 exerts its biological activities, for example by targeting the binding of the chemokine to its proteoglycan receptor.

  1. Heterogenous vasodilator pathways underlie flow-mediated dilation in men and women.

    Science.gov (United States)

    Parker, Beth A; Tschakovsky, Michael E; Augeri, Amanda L; Polk, Donna M; Thompson, Paul D; Kiernan, Francis J

    2011-09-01

    This study investigated the sex differences in the contribution of nitric oxide (NO) and prostaglandins (PGs) to flow-mediated dilation (FMD). Radial artery (RA) FMD, assessed as the dilatory response to 5-min distal cuff occlusion, was repeated after three separate brachial artery infusions of saline (SAL), N(G)-monomethyl-L-arginine (L-NMMA), and ketorolac (KETO) + L-NMMA in healthy younger men (M; n = 8) and women (W; n = 8). In eight subjects (4 M, 4W) RA FMD was reassessed on a separate day with drug order reversed (SAL, KETO, and L-NMMA + KETO). RA FMD was calculated as the peak dilatory response observed relative to baseline (%FMD) and expressed relative to the corresponding area under the curve shear stress (%FMD/AUC SS). L-NMMA reduced %FMD similarly and modestly (P = 0.68 for sex * trial interaction) in M and W (all subjects: 10.0 ± 3.8 to 7.6 ± 4.7%; P = 0.03) with no further effect of KETO (P = 0.68). However, all sex * trial and trial effects on %FMD/AUC SS for l-NMMA and KETO + l-NMMA were insignificant (all P > 0.20). There was also substantial heterogeneity of the magnitude and direction of dilator responses to blockade. After l-NMMA infusion, subjects exhibited both reduced (n = 14; range: 11 to 78% decrease) and augmented (n = 2; range: 1 to 96% increase) %FMD. Following KETO + l-NMMA, seven subjects exhibited reduced dilation (range: 10 to 115% decrease) and nine subjects exhibited augmented dilation (range: 1 to 212% increase). Reversing drug order did not change the nature of the findings. These findings suggest that RA FMD is not fully or uniformly NO dependent in either men or women, and that there is heterogeneity in the pathways underlying the conduit dilatory response to ischemia.

  2. Feedback regulation on PTEN/AKT pathway by the ER stress kinase PERK mediated by interaction with the Vault complex

    DEFF Research Database (Denmark)

    Zhang, Wei; Neo, Suat Peng; Gunaratne, Jayantha

    2015-01-01

    The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological...... inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress...... results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications...

  3. DMPD: Complement-mediated phagocytosis--the role of Syk. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16754322 Complement-mediated phagocytosis--the role of Syk. Tohyama Y, Yamamura H. ...IUBMB Life. 2006 May-Jun;58(5-6):304-8. (.png) (.svg) (.html) (.csml) Show Complement-mediated phagocytosis-...-the role of Syk. PubmedID 16754322 Title Complement-mediated phagocytosis--the role of Syk. Authors Tohyama

  4. DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

  5. TDRG1 regulates chemosensitivity of seminoma TCam-2 cells to cisplatin via PI3K/Akt/mTOR signaling pathway and mitochondria-mediated apoptotic pathway.

    Science.gov (United States)

    Gan, Yu; Wang, Yong; Tan, Zhengyu; Zhou, Jun; Kitazawa, Riko; Jiang, Xianzhen; Tang, Yuxin; Yang, Jianfu

    2016-07-02

    We previously identified TDRG1 (testis developmental related gene 1), a novel gene with exclusive expression in testis, promoted the proliferation and progression of cultured human seminoma cells through PI3K/Akt/mTOR signaling. As increasing evidence reveal that aberrant activation of this signaling is involved in cisplatin resistance. Then, in this study, we further explored whether TDRG1 regulated the chemosensitivity of seminoma TCam-2 cells to cisplatin. Our researches showed TDRG1 could regulate the viability of TCam-2 cells following cisplatin treatment in vitro through control of both cell apoptosis and cell cycle. Mechanistically, we observed TDRG1 positively regulated the expression levels of the key elements in PI3K/Akt/mTOR pathway including p-PI3K, p-Akt and p-mTOR and also affected the translocation of nuclear p-Akt in TCam-2 cells during cisplatin treatment. Meanwhile, the levels of Bad, cytochrome c, caspase-9 ratio (activated/total), caspase-3 ratio (activated/total) and cleaved-PARP were negatively modulated by TDRG1, which meant the involvement of mitochondria-mediated apoptotic pathway. Furthermore, we found the effect of TDRG1 knockdown or TDRG1 overexpression could be reversed by IGF-1, a PI3K signaling activator, or LY294002, a inhibitor of this pathway, respectively. Similar effects of TDRG1 on cisplatin chemosensitivity and associated molecular mechanism were also confirmed in vivo by employing xenograft assays. In addition, the positive correlation between TDRG1 and p-PI3K, or p-Akt, was found in tumor tissues from seminoma patients. In conclusion, we uncover that TDRG1 regulates chemosensitivity of TCam-2 cells to cisplatin through PI3K/Akt/mTOR signaling and mitochondria-mediated apoptotic pathway both in vitro and in vivo.

  6. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Yu-Qin Zhang

    Full Text Available The role of Pokemon (POK erythroid myeloid ontogenic actor, a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  7. Ethanol-mediated regulation of cytochrome P450 2A6 expression in monocytes: role of oxidative stress-mediated PKC/MEK/Nrf2 pathway.

    Directory of Open Access Journals (Sweden)

    Mengyao Jin

    Full Text Available Cytochrome P450 2A6 (CYP2A6 is known to metabolize nicotine, the major constituent of tobacco, leading to the production of toxic metabolites and induction of oxidative stress that result in liver damage and lung cancer. Recently, we have shown that CYP2A6 is induced by ethanol and metabolizes nicotine into cotinine and other metabolites leading to generation of reactive oxygen species (ROS in U937 monocytes. However, the mechanism by which CYP2A6 is induced by ethanol is unknown. In this study, we have examined the role of the PKC/Nrf2 pathway (protein kinase C-mediated phosphorylation and translocation of nuclear erythroid 2-related factor 2 to the nucleus in ethanol-mediated CYP2A6 induction. Our results showed that 100 mM ethanol significantly induced CYP2A6 mRNA and protein (~150% and increased ROS formation, and induction of gene expression and ROS were both completely blocked by treatment with either a CYP2E1 inhibitor (diallyl sulfide or an antioxidant (vitamin C. The results suggest the role of oxidative stress in the regulation of CYP2A6 expression. Subsequently, we investigated the role of Nrf2 pathway in oxidative stress-mediated regulation of CYP2A6 expression in U937 monocytes. Our results showed that butylated hydroxyanisole, a stabilizer of nuclear Nrf2, increased CYP2A6 levels >200%. Staurosporine, an inhibitor of PKC, completely abolished ethanol-induced CYP2A6 expression. Furthermore, our results showed that a specific inhibitor of mitogen-activated protein kinase kinase (MEK (U0126 completely abolished ethanol-mediated CYP2A6 induction and Nrf2 translocation. Overall, these results suggest that CYP2E1-mediated oxidative stress produced as a result of ethanol metabolism translocates Nrf2 into the nucleus through PKC/MEK pathway, resulting in the induction of CYP2A6 in monocytes. An increased level of CYP2A6 in monocytes is expected to further increase oxidative stress in smokers through CYP2A6-mediated nicotine metabolism

  8. A novel role for ATM in regulating proteasome-mediated protein degradation through suppression of the ISG15 conjugation pathway.

    Directory of Open Access Journals (Sweden)

    Laurence M Wood

    Full Text Available Ataxia Telangiectasia (A-T is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.

  9. Viral Small T Oncoproteins Transform Cells by Alleviating Hippo-Pathway-Mediated Inhibition of the YAP Proto-oncogene

    Directory of Open Access Journals (Sweden)

    Hung Thanh Nguyen

    2014-08-01

    Full Text Available Primary human cells can be transformed into tumor cells by a defined set of genetic alterations including telomerase, oncogenic RasV12, and the tumor suppressors p53 and pRb. SV40 small T (ST is required for anchorage-independent growth in vitro and in vivo. Here, we identify the Hippo tumor suppressor pathway as a critical target of ST in cellular transformation. We report that ST uncouples YAP from the inhibitory activity of the Hippo pathway through PAK1-mediated inactivation of NF2. Membrane-tethered activated PAK is sufficient to bypass the requirement for ST in anchorage-independent growth. PAK acts via YAP to mediate the transforming effects of ST. Activation of endogenous YAP is required for ST-mediated transformation and is sufficient to bypass ST in anchorage-independent growth and xenograft tumor formation. Our findings uncover the Hippo tumor suppressor pathway as a final gatekeeper to transformation and tumorigenesis of primary cells.

  10. sli-3 negatively regulates the LET-23/epidermal growth factor receptor-mediated vulval induction pathway in Caenorhabditis elegans.

    Science.gov (United States)

    Gupta, Bhagwati P; Liu, Jing; Hwang, Byung J; Moghal, Nadeem; Sternberg, Paul W

    2006-11-01

    The LIN-3-LET-23-mediated inductive signaling pathway plays a major role during vulval development in C. elegans. Studies on the components of this pathway have revealed positive as well as negative regulators that function to modulate the strength and specificity of the signal transduction cascade. We have carried out genetic screens to identify new regulators of this pathway by screening for suppressors of lin-3 vulvaless phenotype. The screens recovered three loci including alleles of gap-1 and a new gene represented by sli-3. Our genetic epistasis experiments suggest that sli-3 functions either downstream or in parallel to nuclear factors lin-1 and sur-2. sli-3 synergistically interacts with the previously identified negative regulators of the let-23 signaling pathway and causes excessive cell proliferation. However, in the absence of any other mutation sli-3 mutant animals display wild-type vulval induction and morphology. We propose that sli-3 functions as a negative regulator of vulval induction and defines a branch of the inductive signaling pathway. We provide evidence that sli-3 interacts with the EGF signaling pathway components during vulval induction but not during viability and ovulation processes. Thus, sli-3 helps define specificity of the EGF signaling to induce the vulva.

  11. Excessive Time on Social Networking Sites and Disordered Eating Behaviors Among Undergraduate Students: Appearance and Weight Esteem as Mediating Pathways.

    Science.gov (United States)

    Murray, Marisa; Maras, Danijela; Goldfield, Gary S

    2016-12-01

    Social networking sites (SNS) are a popular form of communication among undergraduate students. Body image concerns and disordered eating behaviors are also quite prevalent among this population. Maladaptive use of SNS has been associated with disordered eating behaviors; however, the mechanisms remain unclear. The present study examined if body image concerns (e.g., appearance and weight esteem) mediate the relationship between excessive time spent on SNS and disordered eating behaviors (restrained and emotional eating). The sample included 383 (70.2 percent female) undergraduate students (mean age = 23.08 years, standard deviation = 3.09) who completed self-report questionnaires related to SNS engagement, body image, disordered eating behaviors, and demographics. Parallel multiple mediation and moderated mediation analyses revealed that lower weight and appearance esteem mediated the relationship between excessive time on SNS and restrained eating for males and females, whereas appearance esteem mediated the relationship between excessive time on SNS and emotional eating for females only. The study adds to the literature by highlighting mediational pathways and gender differences. Intervention research is needed to determine if teaching undergraduate students more adaptive ways of using SNS or reducing exposure to SNS reduces body dissatisfaction and disordered eating in this high-risk population.

  12. TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål).

    Science.gov (United States)

    Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhou, Qiang

    2016-03-28

    The "target of rapamycin" (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens.

  13. Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway.

    Science.gov (United States)

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-04-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.

  14. Longitudinal pathways from early maternal depression to children's dysregulated representations: a moderated mediation analysis of harsh parenting and gender.

    Science.gov (United States)

    Martoccio, Tiffany L; Brophy-Herb, Holly E; Maupin, Angela N; Robinson, Joann L

    2016-01-01

    There is some evidence linking maternal depression, harsh parenting, and children's internal representations of attachment, yet, longitudinal examinations of these relationships and differences in the developmental pathways between boys and girls are lacking. Moderated mediation growth curves were employed to examine harsh parenting as a mechanism underlying the link between maternal depression and children's dysregulated representations using a nationally-representative, economically-vulnerable sample of mothers and their children (n = 575; 49% boys, 51% girls). Dysregulation representations were measured using the MacArthur Story Stem Battery at five years of age (M = 5.14, SD = 0.29). Harsh parenting mediated the association between early maternal depression and dysregulated representations for girls. Though initial harsh parenting was a significant mediator for boys, a stronger direct effect of maternal depression to dysregulated representations emerged over time. Results are discussed in terms of their implications for intervention efforts aimed at promoting early supportive parenting.

  15. Pathways to fraction learning: Numerical abilities mediate the relation between early cognitive competencies and later fraction knowledge.

    Science.gov (United States)

    Ye, Ai; Resnick, Ilyse; Hansen, Nicole; Rodrigues, Jessica; Rinne, Luke; Jordan, Nancy C

    2016-12-01

    The current study investigated the mediating role of number-related skills in the developmental relationship between early cognitive competencies and later fraction knowledge using structural equation modeling. Fifth-grade numerical skills (i.e., whole number line estimation, non-symbolic proportional reasoning, multiplication, and long division skills) mapped onto two distinct factors: magnitude reasoning and calculation. Controlling for participants' (N=536) demographic characteristics, these two factors fully mediated relationships between third-grade general cognitive competencies (attentive behavior, verbal and nonverbal intellectual abilities, and working memory) and sixth-grade fraction knowledge (concepts and procedures combined). However, specific developmental pathways differed by type of fraction knowledge. Magnitude reasoning ability fully mediated paths from all four cognitive competencies to knowledge of fraction concepts, whereas calculation ability fully mediated paths from attentive behavior and verbal ability to knowledge of fraction procedures (all with medium to large effect sizes). These findings suggest that there are partly overlapping, yet distinct, developmental pathways from cognitive competencies to general fraction knowledge, fraction concepts, and fraction procedures.

  16. Agonist-induced internalisation of the glucagon-like peptide-1 receptor is mediated by the Gαq pathway.

    Science.gov (United States)

    Thompson, Aiysha; Kanamarlapudi, Venkateswarlu

    2015-01-01

    The glucagon-like peptide-1 receptor (GLP-1R) is a G-protein-coupled receptor (GPCR) and an important target in the treatment of type 2 diabetes mellitus (T2DM). Upon stimulation with agonist, the GLP-1R signals through both Gαs and Gαq coupled pathways to stimulate insulin secretion. The agonist-induced GLP-1R internalisation has recently been shown to be important for insulin secretion. However, the molecular mechanisms underlying GLP-1R internalisation remain unknown. The aim of this study was to determine the role of GLP-1R downstream signalling pathways in its internalisation. Agonist-induced human GLP-1R (hGLP-1R) internalisation and activity were examined using a number of techniques including immunoblotting, ELISA, immunofluorescence and luciferase assays to determine cAMP production, intracellular Ca(2+) accumulation and ERK phosphorylation. Agonist-induced hGLP-1R internalisation is dependent on caveolin-1 and dynamin. Inhibition of the Gαq pathway but not the Gαs pathway affected hGLP-1R internalisation. Consistent with this, hGLP-1R mutant T149M and small-molecule agonists (compound 2 and compound B), which activate only the Gαs pathway, failed to induce internalisation of the receptor. Chemical inhibitors of the Gαq pathway, PKC and ERK phosphorylation significantly reduced agonist-induced hGLP-1R internalisation. These inhibitors also suppressed agonist-induced ERK1/2 phosphorylation demonstrating that the phosphorylated ERK acts downstream of the Gαq pathway in the hGLP-1R internalisation. In summary, agonist-induced hGLP-1R internalisation is mediated by the Gαq pathway. The internalised hGLP-1R stimulates insulin secretion from pancreatic β-cells, indicating the importance of GLP-1 internalisation for insulin secretion.

  17. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen Ngoc, Tam Dan [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Son, Young-Ok [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Lim, Shin-Saeng [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Kim, Jong-Ghee [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Heo, Jung Sun [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Choe, Youngji [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Jeon, Young-Mi, E-mail: young@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Lee, Jeong-Chae, E-mail: leejc88@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2012-03-15

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  18. Mast cell mediators: Their differential release and the secretory pathways involved

    Directory of Open Access Journals (Sweden)

    Tae Chul eMoon

    2014-11-01

    Full Text Available Mast cells (MC are widely distributed throughout the body and are common at mucosal surfaces, a major host-environment interface. MC are functionally and phenotypically heterogeneous depending on the microenvironment in which they mature. Although MC have been classically viewed as effector cells of IgE-mediated allergic diseases, they are also recognized as important in host defense, innate and acquired immunity, homeostatic responses, and immunoregulation. MC activation can induce release of preformed mediators such as histamine from their granules, as well as release of de novo synthesized lipid mediators, cytokines and chemokines that play diverse roles, not only in allergic reactions but also in numerous physiological and pathophysiological responses. Indeed, MC release their mediators in a discriminating and chronological manner, depending upon the stimuli involved and their signaling cascades (e.g., IgE-mediated or Toll Like Receptor-mediated. However, the precise mechanisms underlying differential mediator release in response to these stimuli are poorly known. This review summarizes our knowledge of MC mediators and will focus on what is known about the discriminatory release of these mediators dependent upon diverse stimuli, MC phenotypes and species of origin, as well as on the intracellular synthesis, storage and secretory processes involved.

  19. The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

    Science.gov (United States)

    Maymó, Julieta Lorena; Pérez Pérez, Antonio; Maskin, Bernardo; Dueñas, José Luis; Calvo, Juan Carlos; Sánchez Margalet, Víctor; Varone, Cecilia Laura

    2012-01-01

    Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the

  20. Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica.

    Science.gov (United States)

    Dowd, Georgina C; Bhalla, Manmeet; Kean, Bernard; Thomas, Rowan; Ireton, Keith

    2016-06-01

    Many bacterial pathogens subvert mammalian type IA phosphoinositide 3-kinase (PI3K) in order to induce their internalization into host cells. How PI3K promotes internalization is not well understood. Also unclear is whether type IA PI3K affects different pathogens through similar or distinct mechanisms. Here, we performed an RNA interference (RNAi)-based screen to identify components of the type IA PI3K pathway involved in invasin-mediated entry of Yersinia enterocolitica, an enteropathogen that causes enteritis and lymphadenitis. The 69 genes targeted encode known upstream regulators or downstream effectors of PI3K. A similar RNAi screen was previously performed with the food-borne bacterium Listeria monocytogenes The results of the screen with Y. enterocolitica indicate that at least nine members of the PI3K pathway are needed for invasin-mediated entry. Several of these proteins, including centaurin-α1, Dock180, focal adhesion kinase (FAK), Grp1, LL5α, LL5β, and PLD2 (phospholipase D2), were recruited to sites of entry. In addition, centaurin-α1, FAK, PLD2, and mTOR were required for remodeling of the actin cytoskeleton during entry. Six of the human proteins affecting invasin-dependent internalization also promote InlB-mediated entry of L. monocytogenes Our results identify several host proteins that mediate invasin-induced effects on the actin cytoskeleton and indicate that a subset of PI3K pathway components promote internalization of both Y. enterocolitica and L. monocytogenes.

  1. Erythropoietin-mediated protection of insect brain neurons involves JAK and STAT but not PI3K transduction pathways.

    Science.gov (United States)

    Miljus, N; Heibeck, S; Jarrar, M; Micke, M; Ostrowski, D; Ehrenreich, H; Heinrich, R

    2014-01-31

    The cytokine erythropoietin (Epo) initiates adaptive cellular responses to both moderate environmental challenges and tissue damaging insults in various non-hematopoietic mammalian tissues including the nervous system. Neuroprotective and neuroregenerative functions of Epo in mammals are mediated through receptor-associated Janus kinase 2 and intracellular signaling cascades that modify the transcription of Epo-regulated genes. Signal transducers and activators of transcription (STAT) and phosphoinositol-3-kinase (PI3K) represent key components of two important Epo-induced transduction pathways. Our previous study on insects revealed neuroprotective and regenerative functions of recombinant human Epo (rhEpo) similar to those in mammalian nervous tissues. Here we demonstrate that rhEpo effectively rescues primary cultured locust brain neurons from apoptotic cell death induced by hypoxia or the chemical compound H-7. The Janus kinase inhibitor AG-490 and the STAT inhibitor sc-355797 abolished protective effects of rhEpo on locust brain neurons. In contrast, inhibition of PI3K with LY294002 had no effect on rhEpo-mediated neuroprotection. The results indicate that rhEpo mediates the protection of locust brain neurons through interference with apoptotic pathways by the activation of a Janus kinase-associated receptor and STAT transcription factor(s). The involvement of similar transduction pathways in mammals and insects for the mediation of neuroprotection and support of neural regeneration by Epo indicates that an Epo/Epo receptor-like signaling system with high structural and functional similarity exists in both groups of animals. Epo-like signaling involved in tissue protection appears to be an ancient beneficial function shared by vertebrates and invertebrates.

  2. DMPD: Fragments of extracellular matrix as mediators of inflammation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18243041 Fragments of extracellular matrix as mediators of inflammation. Adair-Kirk...l) Show Fragments of extracellular matrix as mediators of inflammation. PubmedID 18243041 Title Fragments of extrace... TL, Senior RM. Int J Biochem Cell Biol. 2008;40(6-7):1101-10. Epub 2007 Dec 24. (.png) (.svg) (.html) (.csm

  3. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Science.gov (United States)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  4. Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures.

    Science.gov (United States)

    Zhao, Jian; Sakai, Kokki

    2003-02-01

    The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.

  5. Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

    Science.gov (United States)

    Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

    2016-07-01

    Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system.

  6. The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Kuksin Dmitry

    2005-04-01

    Full Text Available Abstract Background Simian virus 40 (SV40 enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi. Results We confirmed our earlier finding that SV40 co localizes with the Golgi marker β-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide. Conclusion The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with β-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.

  7. The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum.

    Science.gov (United States)

    Norkin, Leonard C; Kuksin, Dmitry

    2005-04-19

    Simian virus 40 (SV40) enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi. We confirmed our earlier finding that SV40 co localizes with the Golgi marker beta-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide. The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with beta-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.

  8. Dishevelled mediates ephrinB1 signalling in the eye field through the planar cell polarity pathway.

    Science.gov (United States)

    Lee, Hyun-Shik; Bong, Yong-Sik; Moore, Kathryn B; Soria, Kathleen; Moody, Sally A; Daar, Ira O

    2006-01-01

    An important step in retinal development is the positioning of progenitors within the eye field where they receive the local environmental signals that will direct their ultimate fate. Recent evidence indicates that ephrinB1 functions in retinal progenitor movement, but the signalling pathway is unclear. We present evidence that ephrinB1 signals through its intracellular domain to control retinal progenitor movement into the eye field by interacting with Xenopus Dishevelled (Xdsh), and by using the planar cell polarity (PCP) pathway. Blocking Xdsh translation prevents retinal progeny from entering the eye field, similarly to the morpholino-mediated loss of ephrinB1 (ref. 2). Overexpression of Xdsh can rescue the phenotype induced by loss of ephrinB1, and this rescue (as well as a physical association between Xdsh and ephrinB1) is completely dependent on the DEP (Dishevelled, Egl-10, Pleckstrin) domain of Xdsh. Similar gain- and loss-of-function experiments suggest that Xdsh associates with ephrinB1 and mediates ephrinB1 signalling through downstream members of the PCP pathway during eye field formation.

  9. The TEAD/TEF family protein Scalloped mediates transcriptional output of the Hippo growth-regulatory pathway.

    Science.gov (United States)

    Wu, Shian; Liu, Yi; Zheng, Yonggang; Dong, Jixin; Pan, Duojia

    2008-03-01

    The Hippo (Hpo) kinase cascade restricts tissue growth by inactivating the transcriptional coactivator Yorkie (Yki), which regulates the expression of target genes such as the cell death inhibitor diap1 by unknown mechanisms. Here we identify the TEAD/TEF family protein Scalloped (Sd) as a DNA-binding transcription factor that partners with Yki to mediate the transcriptional output of the Hpo growth-regulatory pathway. The diap1 (th) locus harbors a minimal Sd-binding Hpo Responsive Element (HRE) that mediates transcriptional regulation by the Hpo pathway. Sd binds directly to Yki, and a Yki missense mutation that abrogates Sd-Yki binding also inactivates Yki function in vivo. We further demonstrate that sd is required for yki-induced tissue overgrowth and target gene expression, and that sd activity is conserved in its mammalian homolog. Our results uncover a heretofore missing link in the Hpo signaling pathway and provide a glimpse of the molecular events on a Hpo-responsive enhancer element.

  10. Status epilepticus induces vasogenic edema via tumor necrosis factor-α/ endothelin-1-mediated two different pathways.

    Directory of Open Access Journals (Sweden)

    Ji-Eun Kim

    Full Text Available Status epilepticus (SE induces vasogenic edema in the piriform cortex with disruptions of the blood-brain barrier (BBB. However, the mechanisms of vasogenic edema formation following SE are still unknown. Here we investigated the endothelin B (ETB receptor-mediated pathway of SE-induced vasogenic edema. Following SE, the release of tumor necrosis factor-α (TNF-α stimulated endothelin-1 (ET-1 release and expression in neurons and endothelial cells. In addition, TNF-α-induced ET-1 increased BBB permeability via ETB receptor-mediated endothelial nitric oxide synthase (eNOS activation in endothelial cells. ETB receptor activation also increased intracellular reactive oxygen species by NADPH oxidase production in astrocytes. These findings suggest that SE results in BBB dysfunctions via endothelial-astroglial interactions through the TNF-α-ET-1-eNOS/NADPH oxidase pathway, and that these ETB receptor-mediated interactions may be an effective therapeutic strategy for vasogenic edema in various neurological diseases.

  11. Tyrosine-based signal mediates LRP6 receptor endocytosis and desensitization of Wnt/β-catenin pathway signaling.

    Science.gov (United States)

    Liu, Chia-Chen; Kanekiyo, Takahisa; Roth, Barbara; Bu, Guojun

    2014-10-03

    Wnt/β-catenin signaling orchestrates a number of critical events including cell growth, differentiation, and cell survival during development. Misregulation of this pathway leads to various human diseases, specifically cancers. Endocytosis and phosphorylation of the LDL receptor-related protein 6 (LRP6), an essential co-receptor for Wnt/β-catenin signaling, play a vital role in mediating Wnt/β-catenin signal transduction. However, its regulatory mechanism is not fully understood. In this study, we define the mechanisms by which LRP6 endocytic trafficking regulates Wnt/β-catenin signaling activation. We show that LRP6 mutant with defective tyrosine-based signal in its cytoplasmic tail has an increased cell surface distribution and decreased endocytosis rate. These changes in LRP6 endocytosis coincide with an increased distribution to caveolae, increased phosphorylation, and enhanced Wnt/β-catenin signaling. We further demonstrate that treatment of Wnt3a ligands or blocking the clathrin-mediated endocytosis of LRP6 leads to a redistribution of wild-type receptor to lipid rafts. The LRP6 tyrosine mutant also exhibited an increase in signaling activation in response to Wnt3a stimulation when compared with wild-type LRP6, and this activation is suppressed when caveolae-mediated endocytosis is blocked. Our results reveal molecular mechanisms by which LRP6 endocytosis routes regulate its phosphorylation and the strength of Wnt/β-catenin signaling, and have implications on how this pathway can be modulated in human diseases.

  12. Carbon nanotubes enhance intercalated disc assembly in cardiac myocytes via the β1-integrin-mediated signaling pathway.

    Science.gov (United States)

    Sun, Hongyu; Lü, Shuanghong; Jiang, Xiao-Xia; Li, Xia; Li, Hong; Lin, Qiuxia; Mou, Yongchao; Zhao, Yuwei; Han, Yao; Zhou, Jin; Wang, Changyong

    2015-07-01

    Carbon nanotubes (CNTs) offer a new paradigm for constructing functional cardiac patches and repairing myocardial infarction (MI). However, little is known about how CNTs enhance the mechanical integrity and electrophysiological function of cardiac myocytes. To address this issue, we investigated the regularity and precise mechanism of the influence of CNTs on the assembly of intercalated disc (IDs). Here, single walled CNTs incorporated into collagen substrates were utilized as growth supports for neonatal cardiomyocytes, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, western blotting, transmission electron microscopy, and intracellular calcium transient measurement, we discovered that the addition of CNTs remarkably increased ID-related protein expression and enhanced ID assembly and functionality. On that basis, we further explored the underlying mechanism for how CNTs enhanced ID assembly through the use of immunohistochemical staining and western blotting. We found that the β1-integrin-mediated signaling pathway mediated CNT-induced upregulation of electrical and mechanical junction proteins. Notably, CNTs remarkably accelerated gap junction formation via activation of the β1-integrin-mediated FAK/ERK/GATA4 pathway. These findings provide valuable insight into the mechanistic effects that CNTs have on neonatal cardiomyocyte performance and will have a significant impact on the future of nanomedical research.

  13. STAT1 pathway mediates amplification of metastatic potential and resistance to therapy.

    Directory of Open Access Journals (Sweden)

    Nikolai N Khodarev

    Full Text Available BACKGROUND: Traditionally IFN/STAT1 signaling is connected with an anti-viral response and pro-apoptotic tumor-suppressor functions. Emerging functions of a constitutively activated IFN/STAT1 pathway suggest an association with an aggressive tumor phenotype. We hypothesized that tumor clones that constitutively overexpress this pathway are preferentially selected by the host microenvironment due to a resistance to STAT1-dependent cytotoxicity and demonstrate increased metastatic ability combined with increased resistance to genotoxic stress. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that clones of B16F1 tumors grown in the lungs of syngeneic C57BL/6 mice demonstrate variable transcriptional levels of IFN/STAT1 pathway expression. Tumor cells that constitutively overexpress the IFN/STAT1 pathway (STAT1(H genotype are selected by the lung microenvironment. STAT1(H tumor cells also demonstrate resistance to IFN-gamma (IFNgamma, ionizing radiation (IR, and doxorubicin relative to parental B16F1 and low expressors of the IFN/STAT1 pathway (STAT1(L genotype. Stable knockdown of STAT1 reversed the aggressive phenotype and decreased both lung colonization and resistance to genotoxic stress. CONCLUSIONS: Our results identify a pathway activated by tumor-stromal interactions thereby selecting for pro-metastatic and therapy-resistant tumor clones. New therapies targeted against the IFN/STAT1 signaling pathway may provide an effective strategy to treat or sensitize aggressive tumor clones to conventional cancer therapies and potentially prevent distant organ colonization.

  14. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.

    Science.gov (United States)

    Mace, Kimberly A; Pearson, Joseph C; McGinnis, William

    2005-04-15

    We used wounded Drosophila embryos to define an evolutionarily conserved pathway for repairing the epidermal surface barrier. This pathway includes a wound response enhancer from the Ddc gene that requires grainy head (grh) function and binding sites for the Grh transcription factor. At the signaling level, tyrosine kinase and extracellular signal-regulated kinase (ERK) activities are induced in epidermal cells near wounds, and activated ERK is required for a robust wound response. The conservation of this Grh-dependent pathway suggests that the repair of insect cuticle and mammal skin is controlled by an ancient, shared control system for constructing and healing the animal body surface barrier.

  15. Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling

    Directory of Open Access Journals (Sweden)

    Smeets Ruben L

    2012-03-01

    Full Text Available Abstract Background T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells. Results Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells. Conclusions This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell

  16. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    Energy Technology Data Exchange (ETDEWEB)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G., E-mail: deborah.murdock@vanderbilt.edu

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  17. The BDNF/TrkB Signaling Pathway Is Involved in Heat Hyperalgesia Mediated by Cdk5 in Rats

    Science.gov (United States)

    Zhang, Hong-Hai; Zhang, Xiao-Qin; Xue, Qing-Sheng; Yan-Luo; Huang, Jin-Lu; Zhang, Su; Shao, Hai-Jun; Lu, Han; Wang, Wen-Yuan; Yu, Bu-Wei

    2014-01-01

    Background Cyclin-dependent kinase 5 (Cdk5) has been shown to play an important role in mediating inflammation-induced heat hyperalgesia. However, the underlying mechanism remains unclear. The aim of this study was to determine whether roscovitine, an inhibitor of Cdk5, could reverse the heat hyperalgesia induced by peripheral injection of complete Freund's adjuvant (CFA) via the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB) signaling pathway in the dorsal horn of the spinal cord in rats. Results Heat hyperalgesia induced by peripheral injection of CFA was significantly reversed by roscovitine, TrkB-IgG, and the TrkB inhibitor K252a, respectively. Furthermore, BDNF was significantly increased from 0.5 h to 24 h after CFA injection in the spinal cord dorsal horn. Intrathecal adminstration of the Cdk5 inhibitor roscovitine had no obvious effects on BDNF levels. Increased TrkB protein level was significantly reversed by roscovitine between 0.5 h and 6 h after CFA injection. Cdk5 and TrkB co-immunoprecipitation results suggested Cdk5 mediates the heat hyperalgesia induced by CFA injection by binding with TrkB, and the binding between Cdk5 and TrkB was markedly blocked by intrathecal adminstration of roscovitine. Conclusion Our data suggested that the BDNF-TrkB signaling pathway was involved in CFA-induced heat hyperalgesia mediated by Cdk5. Roscovitine reversed the heat hyperalgesia induced by peripheral injection of CFA by blocking BDNF/TrkB signaling pathway, suggesting that severing the close crosstalk between Cdk5 and the BDNF/TrkB signaling cascade may present a potential target for anti-inflammatory pain. PMID:24465591

  18. The BDNF/TrkB signaling pathway is involved in heat hyperalgesia mediated by Cdk5 in rats.

    Directory of Open Access Journals (Sweden)

    Hong-Hai Zhang

    Full Text Available BACKGROUND: Cyclin-dependent kinase 5 (Cdk5 has been shown to play an important role in mediating inflammation-induced heat hyperalgesia. However, the underlying mechanism remains unclear. The aim of this study was to determine whether roscovitine, an inhibitor of Cdk5, could reverse the heat hyperalgesia induced by peripheral injection of complete Freund's adjuvant (CFA via the brain-derived neurotrophic factor (BDNF-tyrosine kinase B (TrkB signaling pathway in the dorsal horn of the spinal cord in rats. RESULTS: Heat hyperalgesia induced by peripheral injection of CFA was significantly reversed by roscovitine, TrkB-IgG, and the TrkB inhibitor K252a, respectively. Furthermore, BDNF was significantly increased from 0.5 h to 24 h after CFA injection in the spinal cord dorsal horn. Intrathecal adminstration of the Cdk5 inhibitor roscovitine had no obvious effects on BDNF levels. Increased TrkB protein level was significantly reversed by roscovitine between 0.5 h and 6 h after CFA injection. Cdk5 and TrkB co-immunoprecipitation results suggested Cdk5 mediates the heat hyperalgesia induced by CFA injection by binding with TrkB, and the binding between Cdk5 and TrkB was markedly blocked by intrathecal adminstration of roscovitine. CONCLUSION: Our data suggested that the BDNF-TrkB signaling pathway was involved in CFA-induced heat hyperalgesia mediated by Cdk5. Roscovitine reversed the heat hyperalgesia induced by peripheral injection of CFA by blocking BDNF/TrkB signaling pathway, suggesting that severing the close crosstalk between Cdk5 and the BDNF/TrkB signaling cascade may present a potential target for anti-inflammatory pain.

  19. Thalidomide decreases gelatinase production by malignant B lymphoid cell lines through disruption of multiple integrin-mediated signaling pathways

    Science.gov (United States)

    Segarra, Marta; Lozano, Ester; Corbera-Bellalta, Marc; Vilardell, Carme; Cibeira, Maria-Teresa; Esparza, Jordi; Izco, Nora; Bladé, Joan; Cid, Maria C.

    2010-01-01

    Background Thalidomide and its analogs are effective agents in the treatment of multiple myeloma. Since gelatinases (matrix metalloproteinases-2 and -9) play a crucial role in tumor progression, we explored the effect of thalidomide on gelatinase production by malignant B lymphoid cell lines. Design and Methods We investigated the effect of therapeutic doses of thalidomide on integrin-mediated production of gelatinases by malignant B lymphoid cell lines by gelatin zymography, western-blot, reverse transcriptase polymerase chain reaction and invasive capacity through Matrigel-coated Boyden chambers. We also explored the effect of thalidomide on the activation status of the main signaling pathways involved in this process. Results Thalidomide strongly inhibited gelatinase production by B-cell lines and primary myeloma cells in response to fibronectin, the most efficient gelatinase inducer identified in lymphoid cells. Thalidomide disrupted integrin-mediated signaling pathways involved in gelatinase induction and release, such as Src and MAP-kinase ERK activation, resulting in decreased cell motility and invasiveness. Unexpectedly, treatment with thalidomide elicited an increase in fibronectin-induced Akt phosphorylation through phosphoinositide 3-kinase-independent pathways since thalidomide decreased fibronectin-induced phosphoinositide 3-kinase phosphorylation and reversed the inhibition of Akt phosphorylation achieved by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Conclusions Disruption of integrin-mediated signaling may be an important mechanism through which thalidomide and its analogs impair tumor cell interactions with the microenvironment. The unexpected effects of thalidomide on Akt activation indicate the need for further studies to elucidate whether the interference with Akt downstream effects would synergize with the anti-tumor activity of thalidomide. PMID:19815837

  20. The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

    Science.gov (United States)

    Linares, Daniel M; del Río, Beatriz; Ladero, Victor; Redruello, Begoña; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2013-07-01

    Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA.

  1. Akt and SHIP modulate Francisella escape from the phagosome and induction of the Fas-mediated death pathway.

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    Murugesan V S Rajaram

    Full Text Available Francisella tularensis infects macrophages and escapes phago-lysosomal fusion to replicate within the host cytosol, resulting in host cell apoptosis. Here we show that the Fas-mediated death pathway is activated in infected cells and correlates with escape of the bacterium from the phagosome and the bacterial burden. Our studies also demonstrate that constitutive activation of Akt, or deletion of SHIP, promotes phago-lysosomal fusion and limits bacterial burden in the host cytosol, and the subsequent induction of Fas expression and cell death. Finally, we show that phagosomal escape/intracellular bacterial burden regulate activation of the transcription factors sp1/sp3, leading to Fas expression and cell death. These data identify for the first time host cell signaling pathways that regulate the phagosomal escape of Francisella, leading to the induction of Fas and subsequent host cell death.

  2. A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway.

    Science.gov (United States)

    Kim, So Yong; Kim, Tae Jin; Lee, Ki-Young

    2008-06-11

    We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.

  3. Distinct Polymer Architecture Mediates Switching of Complement Activation Pathways at the Nanosphere-Serum Interface: Implications for Stealth Nanoparticle Engineering

    DEFF Research Database (Denmark)

    Hamad, I.; Al-Hanbali, O.; Hunter, A.C.

    2010-01-01

    Nanoparticles with surface projected polyethyleneoxide (PEO) chains in 'mushroom-brush' and "brush" configurations display stealth properties in systemic circulation and have numerous applications in site specific targeting for controlled drug delivery and release as well as diagnostic Imaging. We...... reactions in some individuals Conformational states of surface chains, arising from the block copolymer poloxamine 908 adsorption, on polystyrene nanoparticles trigger complement activation differently. Alteration of copolymer architecture on nanospheres from mushroom to brush configuration not only....... Notably, the role properdin mediated activation of alternative pathway was only restricted to particles displaying PEO chains in a transition mushroom-brush configuration Since nanoparticle-mediated complement activation is of clinical concern our findings provide a rational basis for improved surface...

  4. Combination of Cinobufacini and Doxorubicin Increases Apoptosis of Hepatocellular Carcinoma Cells through the Fas- and Mitochondria-Mediated Pathways.

    Science.gov (United States)

    Xia, Jufeng; Inagaki, Yoshinori; Gao, Jianjun; Qi, Fanghua; Song, Peipei; Han, Guohua; Sawakami, Tatsuo; Gao, Bo; Luo, Chuan; Kokudo, Norihiro; Hasegawa, Kiyoshi; Sakamoto, Yoshihiro; Tang, Wei

    2017-09-25

    Cinobufacini, a traditional Chinese medicine, has been used widely for cancer treatment, such as hepatocellular carcinoma (HCC), sarcoma, and leukemia. Previous studies done by our lab indicated that cinobufacini could suppress HCC cells through mitochondria-mediated and Fas-mediated apoptotic pathways. Here, we use a combination of cinobufacini and doxorubicin to inhibit the growth of HCC cells. The combination group induced more significant apoptosis by affecting proteins and RNA of apoptosis-related elements, such as Bcl-2, Bax, Bid, and cytochrome c. Furthermore, cinobufacini, as a mixture of a number of components, had stronger apoptosis-inducing activity than particular individual components or a simple mixture of a few components. Overall, these results suggested that the combination of cinobufacini and doxorubicin may provide a new strategy for inhibiting the proliferation of HCC cells.

  5. Chlamydial Lipoproteins Stimulate Toll-Like Receptors 1/2 Mediated Inflammatory Responses through MyD88-Dependent Pathway

    Science.gov (United States)

    Wang, Yong; Liu, Qiong; Chen, Ding; Guan, Jie; Ma, Linghui; Zhong, Guangming; Shu, Hengping; Wu, Xiang

    2017-01-01

    Chlamydiae are very important pathogens which could cause several types of diseases in human, but little is known about its pathogenic mechanism. In order to elucidate host inflammatory response and the signal pathway induced by Chlamydial lipoproteins, the predicted lipoproteins of Chlamydia trachomatis were tested for their ability to induce the release of proinflammatory cytokines by mouse macrophages or human TLR (Toll-Like Receptor) expressing cell lines. The results showed that recombinant proteins of C. trachomatis D381, D541, D067, and D775 displayed a strong ability to induce the release of IL-8 in TLR expressing cell line. The signal pathways involved TLR1/2 and TLR2/CD14 but not TLR4. Moreover, except D067, the proinflammatory cytokine induction by D381, D541, and D775 required the thioacylation site (cysteine) for lipid modification and the induction was through MyD88-mediated pathway. Our data supported that lipoproteins played a vital role in pathogenesis of C. trachomatis-induced inflammatory responses via TLR pathway. It was the first study to characterize other chlamydial lipoproteins after identifying the role of MIP (D541) on pathogenesis of Chlamydial diseases. PMID:28184217

  6. The Role of MEKK3 Signaling Pathway in the Resistance of Breast Cancer Cells to TNF-(alpha)-Mediated Apoptosis

    Science.gov (United States)

    2005-05-01

    breast cancer cell lines and in primary breast cancer tissues (2-3). Activated signal transduction pathways including the mitogen-activated protein ...TNF-induced NF-kappaB activation. Nat Immunol 2: 620-4. 12. Qin, X.F., D.S. An, I.S. Chen, and D. Baltimore. 2003. Inhibiting HIV -1 infection in...human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 . Proc Natl Acad Sci U S A 100: 183-8. 13. Huang, Q. Jianhua Yang

  7. The Nrde Pathway Mediates Small-RNA-Directed Histone H3 Lysine 27 Trimethylation in Caenorhabditis elegans.

    Science.gov (United States)

    Mao, Hui; Zhu, Chengming; Zong, Dandan; Weng, Chenchun; Yang, Xiangwei; Huang, Hui; Liu, Dun; Feng, Xuezhu; Guang, Shouhong

    2015-09-21

    Small-RNA-mediated chromatin modifications have been widely studied in plants and S. pombe. However, direct evidence of small-RNA-guided sequence-specific chromatin alterations is scarce in animals. In C. elegans, the nuclear RNAi defective (Nrde) pathway functions to transport siRNA from the cytoplasm to the nucleus, modulate transcription elongation, induce histone H3 lysine 9 (H3K9) trimethylation, and mediate transgenerational inheritance of RNAi. Here, we show that both exogenous RNAi and NRDE-bound endogenous 22G RNAs can direct sequence-specific histone H3 lysine 27 (H3K27) trimethylation at targeted loci through the Nrde pathway. The resulting H3K27me3 status can be inherited by progeny for multiple generations. piRNAs and WAGO-1-associated siRNAs induce H3K27 methylation as well. Interestingly, CSR-1-associated endogenous siRNAs fail to trigger H3K27 methylation, whereas exogenous provision of dsRNAs can induce H3K27 methylation at the CSR-1-targeted loci via the Nrde pathway. We further observed distinct genetic requirements of H3K9 and H3K27 trimethylation. Whereas set-25 and met-2 are required for K9 methylation, mes-2 is required for K27 methylation. The depletion of mes-2 leads to a nuclear RNAi defective phenotype. These results indicate that dsRNA-triggered chromatin modification is a sequence-specific response that engages the Nrde pathway in C. elegans. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. p53 and ATF4 mediate distinct and additive pathways to skeletal muscle atrophy during limb immobilization

    Science.gov (United States)

    Fox, Daniel K.; Ebert, Scott M.; Bongers, Kale S.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.; Kunkel, Steven D.

    2014-01-01

    Immobilization causes skeletal muscle atrophy via complex signaling pathways that are not well understood. To better understand these pathways, we investigated the roles of p53 and ATF4, two transcription factors that mediate adaptations to a variety of cellular stresses. Using mouse models, we demonstrate that 3 days of muscle immobilization induces muscle atrophy and increases expression of p53 and ATF4. Furthermore, muscle fibers lacking p53 or ATF4 are partially resistant to immobilization-induced muscle atrophy, and forced expression of p53 or ATF4 induces muscle fiber atrophy in the absence of immobilization. Importantly, however, p53 and ATF4 do not require each other to promote atrophy, and coexpression of p53 and ATF4 induces more atrophy than either transcription factor alone. Moreover, muscle fibers lacking both p53 and ATF4 are more resistant to immobilization-induced atrophy than fibers lacking only p53 or ATF4. Interestingly, the independent and additive nature of the p53 and ATF4 pathways allows for combinatorial control of at least one downstream effector, p21. Using genome-wide mRNA expression arrays, we identified p21 mRNA as a skeletal muscle transcript that is highly induced in immobilized muscle via the combined actions of p53 and ATF4. Additionally, in mouse muscle, p21 induces atrophy in a manner that does not require immobilization, p53 or ATF4, and p21 is required for atrophy induced by immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as essential and complementary mediators of immobilization-induced muscle atrophy and discover p21 as a critical downstream effector of the p53 and ATF4 pathways. PMID:24895282

  9. A pivotal role of the jasmonic acid signal pathway in mediating radiation-induced bystander effects in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Ting; Xu, Wei; Deng, Chenguang; Xu, Shaoxin; Li, Fanghua; Wu, Yuejin; Wu, Lijun; Bian, Po

    Although radiation-induced bystander effects (RIBE) in Arabidopsis thaliana have been well demonstrated in vivo, little is known about their underlying mechanisms, particularly with regard to the participating signaling molecules and signaling pathways. In higher plants, jasmonic acid (JA) and its bioactive derivatives are well accepted as systemic signal transducers that are produced in response to various environmental stresses. It is therefore speculated that the JA signal pathway might play a potential role in mediating radiation-induced bystander signaling of root-to-shoot. In the present study, pretreatment of seedlings with Salicylhydroxamic acid, an inhibitor of lipoxigenase (LOX) in JA biosynthesis, significantly suppressed RIBE-mediated expression of the AtRAD54 gene. After root irradiation, the aerial parts of A. thaliana mutants deficient in JA biosynthesis (aos) and signaling cascades (jar1-1) showed suppressed induction of the AtRAD54 and AtRAD51 genes and TSI and 180-bp repeats, which have been extensively used as endpoints of bystander genetic and epigenetic effects in plants. These results suggest an involvement of the JA signal pathway in the RIBE of plants. Using the root micro-grafting technique, the JA signal pathway was shown to participate in both the generation of bystander signals in irradiated root cells and radiation responses in the bystander aerial parts of plants. The over-accumulation of endogenous JA in mutant fatty acid oxygenation up-regulated 2 (fou2), in which mutation of the Two Pore Channel 1 (TPC1) gene up-regulates expression of the LOX and allene oxide synthase (AOS) genes, inhibited RIBE-mediated expression of the AtRAD54 gene, but up-regulated expression of the AtKU70 and AtLIG4 genes in the non-homologous end joining (NHEJ) pathway. Considering that NHEJ is employed by plants with increased DNA damage, the switch from HR to NHEJ suggests that over-accumulation of endogenous JA might enhance the radiosensitivity of plants

  10. Cre-mediated stress affects sirtuin expression levels, peroxisome biogenesis and metabolism, antioxidant and proinflammatory signaling pathways.

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    Yu Xiao

    Full Text Available Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts, inducing PPARα/γ, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14 as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase. In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2 and a NF-κB activation were noted. TGFβ1 and proinflammatory cytokines like IL1, IL6 and TNFα were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7 in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out-mediated

  11. Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway.

    Science.gov (United States)

    Mo, Jung-Soon; Meng, Zhipeng; Kim, Young Chul; Park, Hyun Woo; Hansen, Carsten Gram; Kim, Soohyun; Lim, Dae-Sik; Guan, Kun-Liang

    2015-04-01

    YAP (Yes-associated protein) is a transcription co-activator in the Hippo tumour suppressor pathway and controls cell growth, tissue homeostasis and organ size. YAP is inhibited by the kinase Lats, which phosphorylates YAP to induce its cytoplasmic localization and proteasomal degradation. YAP induces gene expression by binding to the TEAD family transcription factors. Dysregulation of the Hippo-YAP pathway is frequently observed in human cancers. Here we show that cellular energy stress induces YAP phosphorylation, in part due to AMPK-dependent Lats activation, thereby inhibiting YAP activity. Moreover, AMPK directly phosphorylates YAP Ser 94, a residue essential for the interaction with TEAD, thus disrupting the YAP-TEAD interaction. AMPK-induced YAP inhibition can suppress oncogenic transformation of Lats-null cells with high YAP activity. Our study establishes a molecular mechanism and functional significance of AMPK in linking cellular energy status to the Hippo-YAP pathway.

  12. Two programmed cell death systems in Escherichia coli: an apoptotic-like death is inhibited by the mazEF-mediated death pathway.

    Directory of Open Access Journals (Sweden)

    Ariel Erental

    Full Text Available In eukaryotes, the classical form of programmed cell death (PCD is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxin-antitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF. mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD; ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

  13. SMAD-PI3K-Akt-mTOR pathway mediates BMP-7 polarization of monocytes into M2 macrophages.

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    Crystal Rocher

    Full Text Available Previously we demonstrated that bone morphogenetic protein-7 (BMP-7 treatment polarizes monocytes into M2 macrophages and increases the expression of anti-inflammatory cytokines. Despite these findings, the mechanisms for the observed BMP-7 induced monocyte polarization into M2 macrophages are completely unknown. In this study, we demonstrate the mechanisms involved in the polarization of monocytes into M2 macrophages. Apoptotic conditioned media (ACM was generated to mimic the stressed conditions, inducing monocyte polarization. Monocytes were treated with ACM along with BMP-7 and/or its inhibitor, follistatin, for 48 hours. Furthermore, an inhibitor of the PI3K pathway, LY-294002, was also studied. Our data show that BMP-7 induces polarization of monocytes into M2 macrophages while significantly increasing the expression of anti-inflammatory markers, arginase-1 and IL-10, and significantly (p<0.05 decreasing the expression of pro-inflammatory markers iNOS, IL-6, TNF-α and MCP-1; (p<0.05. Moreover, addition of the PI3K inhibitor, LY-294002, significantly (p<0.05 decreases upregulation of IL-10 and arginase-1, suggesting involvement of the PI3K pathway in M2 macrophage polarization. Next, following BMP-7 treatment, a significant (p<0.05 increase in p-SMAD1/5/8 and p-PI3K expression resulting in downstream activation of p-Akt and p-mTOR was observed. Furthermore, expression of p-PTEN, an inhibitor of the PI3K pathway, was significantly (p<0.05 increased in the ACM group. However, BMP-7 treatment inhibited its expression, suggesting involvement of the PI3K-Akt-mTOR pathway. In conclusion, we demonstrate that BMP-7 polarizes monocytes into M2 macrophages and enhances anti-inflammatory cytokine expression which is mediated by the activated SMAD-PI3K-Akt-mTOR pathway.

  14. Contribution of ATM and ATR kinase pathways to p53-mediated response in etoposide and methyl methanesulfonate induced DNA damage.

    Science.gov (United States)

    Sun, Bin; Ross, Susan M; Rowley, Sean; Adeleye, Yeyejide; Clewell, Rebecca A

    2017-03-01

    p53 is a key integrator of cellular response to DNA damage, supporting post-translational repair and driving transcription-mediated responses including cell cycle arrest, apoptosis, and repair. DNA damage sensing kinases recognize different types of DNA damage and initiate specific responses through various post-translational modifications of p53. This study evaluated chemical specificity of the p53 pathway response by manipulating p53 or its upstream kinases and assessing the effect on DNA damage and cellular responses to prototype chemicals: etoposide (ETP, topoisomerase II inhibitor) and methyl methane sulfonate (MMS, alkylating agent). p53-deficient cells demonstrated reduced accumulation of the p53 target proteins MDM2, p21, and Wip1; reduced apoptotic response; and increased DNA damage (p-H2AX and micronuclei) with both chemicals. However, p53 was not essential for cell cycle arrest in HT1080 or HCT116 cells. The two chemicals induced different patterns of kinase activation, particularly in terms of Chk 1, Chk 2, p38, and ERK 1/2. However, inhibition of the ATM pathway showed a greater effect on p53 activtation, apoptosis, and accumulation of DNA damage than ATR-Chk 1 or the MAP kinases regardless of the chemical used. These results indicate that ATM is the predominant upstream kinase responsible for activation of the p53-mediated DNA damage response for both MMS and ETP, though the downstream kinase response is markedly different. Environ. Mol. Mutagen. 58:72-83, 2017. © 2017 Wiley Periodicals, Inc.

  15. Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety.

    Science.gov (United States)

    Zhao, Xuan; Zhong, Hang; Wang, Rui; Liu, Dan; Waxman, Samuel; Zhao, Linxiang; Jing, Yongkui

    2015-03-20

    Anti-apoptotic protein Mcl-1 plays an important role in protecting cell from death in acute myeloid leukemia (AML). The apoptosis blocking activity of Mcl-1 is inhibited by BH3-only protein Noxa. We found that dihydroartemisinin (DHA) and its derivative X-11 are potent apoptosis inducers in AML cells and act through a Noxa-mediate pathway; X-11 is four-fold more active than DHA. DHA and X-11-induced apoptosis is associated with induction of Noxa; apoptosis is blocked by silencing Noxa. DHA and X-11 induce Noxa expression by upregulating the transcription factor FOXO3a in a reactive oxygen species-mediated pathway. Interfering with the integrity of the endoperoxide moiety of DHA and X-11, as well as chelating intracellular iron with deferoxamine, diminish apoptosis and Noxa induction. AML cells expressing Bcl-xL, or with overexpression of Bcl-2, have decreased sensitivity to DHA and X-11-induced apoptosis which could be overcome by addition of Bcl-2/Bcl-xL inhibitor ABT-737. DHA and X-11 represent a new group of AML cells-apoptosis inducing compounds which work through Noxa up-regulation utilizing the specific endoperoxide moiety and intracellular iron.

  16. A novel cisplatin mediated apoptosis pathway is associated with acid sphingomyelinase and FAS proapoptotic protein activation in ovarian cancer.

    Science.gov (United States)

    Maurmann, L; Belkacemi, L; Adams, N R; Majmudar, P M; Moghaddas, S; Bose, R N

    2015-07-01

    Platinum-based anticancer drugs, including cisplatin and carboplatin, have been cornerstones in the treatment of solid tumors. We report here that these DNA-damaging agents, particularly cisplatin, induce apoptosis through plasma membrane disruption, triggering FAS death receptor via mitochondrial (intrinsic) pathways. Our objectives were to: quantify the composition of membrane metabolites; and determine the potential involvement of acid sphingomyelinase (ASMase) in the FAS-mediated apoptosis in ovarian cancer after cisplatin treatment. The resulting analysis revealed enhanced apoptosis as measured by: increased phosphocholine, and glycerophosphocholine; elevated cellular energetics; and phosphocreatine and nucleoside triphosphate concentrations. The plasma membrane alterations were accompanied by increased ASMase activity, leading to the upregulation of FAS, FASL and related pro-apoptotic BAX and PUMA genes. Moreover FAS, FASL, BAX, PUMA, CASPASE-3 and -9 proteins were upregulated. Our findings implicate ASMase activity and the intrinsic pathways in cisplatin-mediated membrane demise, and contribute to our understanding of the mechanisms by which ovarian tumors may become resistant to cisplatin.

  17. Can the TLR-4-Mediated Signaling Pathway Be “A Key Inflammatory Promoter for Sporadic TAA”?

    Directory of Open Access Journals (Sweden)

    Giovanni Ruvolo

    2014-01-01

    Full Text Available Thoracic aorta shows with advancing age various changes and a progressive deterioration in structure and function. As a result, vascular remodeling (VR and medial degeneration (MD occur as pathological entities responsible principally for the sporadic TAA onset. Little is known about their genetic, molecular, and cellular mechanisms. Recent evidence is proposing the strong role of a chronic immune/inflammatory process in their evocation and progression. Thus, we evaluated the potential role of Toll like receptor- (TLR- 4-mediated signaling pathway and its polymorphisms in sporadic TAA. Genetic, immunohistochemical, and biochemical analyses were assessed. Interestingly, the rs4986790 TLR4 polymorphism confers a higher susceptibility for sporadic TAA (OR=14.4, P=0.0008 and it represents, together with rs1799752 ACE, rs3918242 MMP-9, and rs2285053 MMP-2 SNPs, an independent sporadic TAA risk factor. In consistency with these data, a significant association was observed between their combined risk genotype and sporadic TAA. Cases bearing this risk genotype showed higher systemic inflammatory mediator levels, significant inflammatory/immune infiltrate, a typical MD phenotype, lower telomere length, and positive correlations with histopatological abnormalities, hypertension, smoking, and ageing. Thus, TLR4 pathway should seem to have a key role in sporadic TAA. It might represent a potential useful tool for preventing and monitoring sporadic TAA and developing personalized treatments.

  18. USP21 regulates Hippo pathway activity by mediating MARK protein turnover

    DEFF Research Database (Denmark)

    Nguyen, Thanh Hung; Kugler, Jan-Michael; Loya, Anand Chainsukh

    2017-01-01

    The Hippo pathway, which acts to repress the activity of YAP and TAZ trancriptional co-activators, serve as a barrier for oncogenic transformation. Unlike other oncoproteins, YAP and TAZ are rarely activated by mutations or amplified in cancer. However, elevated YAP/TAZ activity is frequently obs...

  19. The Aryl Hydrocarbon Receptor Pathway: A Key Component of the microRNA-Mediated AML Signalisome

    Directory of Open Access Journals (Sweden)

    Julia E. Rager

    2012-05-01

    Full Text Available Recent research has spotlighted the role of microRNAs (miRNAs as critical epigenetic regulators of hematopoietic stem cell differentiation and leukemia development. Despite the recent advances in knowledge surrounding epigenetics and leukemia, the mechanisms underlying miRNAs’ influence on leukemia development have yet to be clearly elucidated. Our aim was to identify high ranking biological pathways altered at the gene expression level and under epigenetic control. Specifically, we set out to test the hypothesis that miRNAs dysregulated in acute myeloid leukemia (AML converge on a common pathway that can influence signaling related to hematopoiesis and leukemia development. We identified genes altered in AML patients that are under common regulation of seven key miRNAs. By mapping these genes to a global interaction network, we identified the “AML Signalisome”. The AML Signalisome comprises 53 AML-associated molecules, and is enriched for proteins that play a role in the aryl hydrocarbon receptor (AhR pathway, a major regulator of hematopoiesis. Furthermore, we show biological enrichment for hematopoiesis-related proteins within the AML Signalisome. These findings provide important insight into miRNA-regulated pathways in leukemia, and may help to prioritize targets for disease prevention and treatment.

  20. DMPD: Modulation of Toll-interleukin 1 receptor mediated signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15662540 Modulation of Toll-interleukin 1 receptor mediated signaling. Li X, Qin J....iated signaling. PubmedID 15662540 Title Modulation of Toll-interleukin 1 receptor media... J Mol Med. 2005 Apr;83(4):258-66. Epub 2005 Jan 21. (.png) (.svg) (.html) (.csml) Show Modulation of Toll-interleukin 1 receptor med

  1. Two conserved modules of Schizosaccharomyces pombe Mediator regulate distinct cellular pathways

    DEFF Research Database (Denmark)

    Linder, Tomas; Rasmussen, Nina; Samuelsen, Camilla O;

    2008-01-01

    and Deltamed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell...

  2. Endothelin and calciotropic hormones share regulatory pathways in multidrug resistance protein 2-mediated transport

    NARCIS (Netherlands)

    Wever, K.E.; Masereeuw, R.; Miller, D.S.; Hang, X.M.; Flik, G.

    2006-01-01

    The kidney of vertebrates plays a key role in excretion of endogenous waste products and xenobiotics. Active secretion in the proximal nephron is at the basis of this excretion, mediated by carrier proteins including multidrug resistance protein 2 (Mrp2). We previously showed that Mrp2 function is

  3. Viral vector-mediated selective and reversible blockade of the pathway for visual orienting in mice

    Directory of Open Access Journals (Sweden)

    Tadashi eIsa

    2013-10-01

    Full Text Available Recently, by using a combination of two viral vectors, we developed a technique for pathway-selective and reversible synaptic transmission blockade, and successfully induced a behavioral deficit of dexterous hand movements in macaque monkeys by affecting a population of spinal interneurons. To explore the capacity of this technique to work in other pathways and species, and to obtain fundamental methodological information, we tried to block the crossed tecto-reticular pathway, which is known to control orienting responses to visual targets, in mice. A neuron-specific retrograde gene transfer vector with the gene encoding enhanced tetanus neurotoxin (eTeNT tagged with enhanced green fluorescent protein (EGFP under the control of a tetracycline responsive element was injected into the left medial pontine reticular formation. 7–17 days later, an adeno-associated viral vector with a highly efficient Tet-ON sequence, rtTAV16, was injected into the right superior colliculus. 5–9 weeks later, the daily administration of doxycycline (Dox was initiated. Visual orienting responses toward the left side were impaired 1 - 4 days after Dox administration. Anti-GFP immunohistochemistry revealed that a number of neurons in the intermediate and deep layers of the right superior colliculus were positively stained, indicating eTeNT expression. After the termination of Dox administration, the anti-GFP staining returned to the baseline level within 28 days. A second round of Dox administration, starting from 28 days after the termination of the first Dox administration, resulted in the reappearance of the behavioral impairment. These findings showed that pathway-selective and reversible blockade of synaptic transmission causes behavioral effects also in rodents, and that the crossed tecto-reticular pathway surely controls visual orienting behaviors.

  4. Pathway from Delirium to Death: Potential In-Hospital Mediators of Excess Mortality.

    Science.gov (United States)

    Dharmarajan, Kumar; Swami, Sunil; Gou, Ray Y; Jones, Richard N; Inouye, Sharon K

    2017-05-01

    (1) To determine the relationship of incident delirium during hospitalization with 90-day mortality; (2) to identify potential in-hospital mediators through which delirium increases 90-day mortality. Analysis of data from Project Recovery, a controlled clinical trial of a delirium prevention intervention from 1995 to 1998 with follow-up through 2000. Large academic hospital. Patients ≥70 years old without delirium at hospital admission who were at intermediate-to-high risk of developing delirium and received usual care only. (1) Incident delirium; (2) potential mediators of delirium on death including use of restraining devices (physical restraints, urinary catheters), development of hospital acquired conditions (HACs) (falls, pressure ulcers), and exposure to other noxious insults (sleep deprivation, acute malnutrition, dehydration, aspiration pneumonia); (3) death within 90 days of admission. Among 469 patients, 70 (15%) developed incident delirium. These patients were more likely to experience restraining devices (37% vs 16%, P delirium was 4.2 (95% CI = 2.8-6.3) in bivariable analyses, increased in a graded manner with additional exposures to restraining devices, HACs, and other noxious insults, and declined by 10.9% after addition of these potential mediator categories, providing evidence of mediation. Restraining devices, HACs, and additional noxious insults were more frequent among patients with delirium, increased mortality in a graded manner, and were responsible for a significant percentage of the association of delirium with death. Additional efforts to prevent potential downstream mediators through which delirium increases mortality may help to improve outcomes among hospitalized older adults. © 2016, Copyright the Authors Journal compilation © 2016, The American Geriatrics Society.

  5. Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Dipayan Bose

    2016-03-01

    Full Text Available Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

  6. The Arabidopsis mediator complex subunit16 positively regulates salicylate-mediated systemic acquired resistance and jasmonate/ethylene-induced defense pathways.

    Science.gov (United States)

    Zhang, Xudong; Wang, Chenggang; Zhang, Yanping; Sun, Yijun; Mou, Zhonglin

    2012-10-01

    Systemic acquired resistance (SAR) is a long-lasting plant immunity against a broad spectrum of pathogens. Biological induction of SAR requires the signal molecule salicylic acid (SA) and involves profound transcriptional changes that are largely controlled by the transcription coactivator nonexpressor of pathogenesis-related genes1 (NPR1). However, it is unclear how SAR signals are transduced from the NPR1 signaling node to the general transcription machinery. Here, we report that the Arabidopsis thaliana Mediator subunit16 (MED16) is an essential positive regulator of SAR. Mutations in MED16 reduced NPR1 protein levels and completely compromised biological induction of SAR. These mutations also significantly suppressed SA-induced defense responses, altered the transcriptional changes induced by the avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000/avrRpt2, and rendered plants susceptible to both Pst DC3000/avrRpt2 and Pst DC3000. In addition, mutations in MED16 blocked the induction of several jasmonic acid (JA)/ethylene (ET)-responsive genes and compromised resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. The Mediator complex acts as a bridge between specific transcriptional activators and the RNA polymerase II transcription machinery; therefore, our data suggest that MED16 may be a signaling component in the gap between the NPR1 signaling node and the general transcription machinery and may relay signals from both the SA and the JA/ET pathways.

  7. Role of SIRT1-mediated mitochondrial and Akt pathways in glioblastoma cell death induced by Cotinus coggygria flavonoid nanoliposomes

    Directory of Open Access Journals (Sweden)

    Wang G

    2015-08-01

    phosphorylated p53. Together, these results indicated SIRT1/p53-mediated cell death was induced by CCF-NLs, but not by extracellular signal-regulated kinase, in DBTRG-05MG cells. Overall, this study suggested caspase-dependent activation of both the intrinsic and extrinsic signaling pathways, probably through blockade of the SIRT1/p53-mediated mitochondrial and Akt pathways to exert the proapoptotic effect of CCF-NLs in DBTRG-05MG GBM cells. Keywords: Cotinus coggygria flavonoid nanoliposomes, cell death, SIRT1, mitochondrial, PI3K/Akt pathway

  8. TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation.

    Directory of Open Access Journals (Sweden)

    Hannah Greenfeld

    2015-05-01

    Full Text Available The Epstein-Barr virus (EBV encoded oncoprotein Latent Membrane Protein 1 (LMP1 signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC, and stimulated linear (M1-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63

  9. TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation.

    Directory of Open Access Journals (Sweden)

    Hannah Greenfeld

    2015-05-01

    Full Text Available The Epstein-Barr virus (EBV encoded oncoprotein Latent Membrane Protein 1 (LMP1 signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC, and stimulated linear (M1-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63

  10. The mitochondrial 2-oxoglutarate carrier is part of a metabolic pathway that mediates glucose- and glutamine-stimulated insulin secretion.

    Science.gov (United States)

    Odegaard, Matthew L; Joseph, Jamie W; Jensen, Mette V; Lu, Danhong; Ilkayeva, Olga; Ronnebaum, Sarah M; Becker, Thomas C; Newgard, Christopher B

    2010-05-28

    Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

  11. Angiotensin-II-induced Muscle Wasting is Mediated by 25-Hydroxycholesterol via GSK3β Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Congcong Shen

    2017-02-01

    Full Text Available While angiotensin II (ang II has been implicated in the pathogenesis of cardiac cachexia (CC, the molecules that mediate ang II's wasting effect have not been identified. It is known TNF-α level is increased in patients with CC, and TNF-α release is triggered by ang II. We therefore hypothesized that ang II induced muscle wasting is mediated by TNF-α. Ang II infusion led to skeletal muscle wasting in wild type (WT but not in TNF alpha type 1 receptor knockout (TNFR1KO mice, suggesting that ang II induced muscle loss is mediated by TNF-α through its type 1 receptor. Microarray analysis identified cholesterol 25-hydroxylase (Ch25h as the down stream target of TNF-α. Intraperitoneal injection of 25-hydroxycholesterol (25-OHC, the product of Ch25h, resulted in muscle loss in C57BL/6 mice, accompanied by increased expression of atrogin-1, MuRF1 and suppression of IGF-1/Akt signaling pathway. The identification of 25-OHC as an inducer of muscle wasting has implications for the development of specific treatment strategies in preventing muscle loss.

  12. Butyrate induces ROS-mediated apoptosis by modulating miR-22/SIRT-1 pathway in hepatic cancer cells.

    Science.gov (United States)

    Pant, Kishor; Yadav, Ajay K; Gupta, Parul; Islam, Rakibul; Saraya, Anoop; Venugopal, Senthil K

    2017-03-07

    Butyrate is one of the short chain fatty acids, produced by the gut microbiota during anaerobic fermentation of dietary fibres. It has been shown that it can inhibit tumor progression via suppressing histone deacetylase and can induce apoptosis in cancer cells. However, the comprehensive pathway by which butyrate mediates apoptosis and growth arrest in cancer cells still remains unclear. In this study, the role of miR-22 in butyrate-mediated ROS release and induction of apoptosis was determined in hepatic cells. Intracellular expression of miR-22 was increased when the Huh 7 cells were incubated with sodium butyrate. Over-expression of miR-22 or addition of sodium butyrate inhibited SIRT-1 expression and enhanced the ROS production. Incubation of cells with anti-miR-22 reversed the effects of butyrate. Butyrate induced apoptosis via ROS production, cytochrome c release and activation of caspase-3, whereas addition of N-acetyl cysteine or anti-miR-22 reversed these butyrate-induced effects. Furthermore, sodium butyrate inhibited cell growth and proliferation, whereas anti-miR-22 inhibited these butyrate-mediated changes. The expression of PTEN and gsk-3 was found to be increased while p-akt and β-catenin expression was decreased significantly by butyrate. These data showed that butyrate modulated both apoptosis and proliferation via miR-22 expression in hepatic cells.

  13. GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Hidekazu Ishida

    2016-07-01

    Full Text Available A surface marker that distinctly identifies cardiac progenitors (CPs is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2, specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure.

  14. Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy.

    Science.gov (United States)

    Bernard, Amélie; Klionsky, Daniel J

    2015-04-01

    To maintain proper cellular homeostasis, the magnitude of autophagy activity has to be finely tuned in response to environmental changes. Many aspects of autophagy regulation have been extensively studied: pathways integrating signals through the master regulators TORC1 and PKA lead to multiple post-translational modifications affecting the functions, protein-protein interactions, and localization of Atg proteins. The expression of several ATG genes increases sharply upon autophagy induction conditions, and defects in ATG gene expression are associated with various diseases, pointing to the importance of transcriptional regulation of autophagy. Yet, how changes in ATG gene expression affect the rate of autophagy is not well characterized, and transcriptional regulators of the autophagy pathway remain largely unknown. To identify such regulators, we analyzed the expression of several ATG genes in a library of DNA-binding protein mutants. This led to the identification of Rph1 as a master transcriptional regulator of autophagy.

  15. Gi proteins mediate activation of the canonical hedgehog pathway in the myocardium.

    Science.gov (United States)

    Carbe, Christian J; Cheng, Lan; Addya, Sankar; Gold, Jessica I; Gao, Erhe; Koch, Walter J; Riobo, Natalia A

    2014-07-01

    During myocardial ischemia, upregulation of the hedgehog (Hh) pathway promotes neovascularization and increases cardiomyocyte survival. The canonical Hh pathway activates a transcriptional program through the Gli family of transcription factors by derepression of the seven-transmembrane protein smoothened (Smo). The mechanisms linking Smo to Gli are complex and, in some cell types, involve coupling of Smo to Gi proteins. In the present study, we investigated, for the first time, the transcriptional response of cardiomyocytes to sonic hedgehog (Shh) and the role of Gi protein utilization. Our results show that Shh strongly activates Gli1 expression by quantitative PCR in a Smo-dependent manner in neonatal rat ventricular cardiomyocytes. Microarray analysis of gene expression changes elicited by Shh and sensitive to a Smo inhibitor identified a small subset of 37 cardiomyocyte-specific genes regulated by Shh, including some in the PKA and purinergic signaling pathways. In addition, neonatal rat ventricular cardiomyocytes infected with an adenovirus encoding GiCT, a peptide that impairs receptor-Gi protein coupling, showed reduced activation of Hh targets. In vitro data were confirmed in transgenic mice with cardiomyocyte-inducible GiCT expression. Transgenic GiCT mice showed specific reduction of Gli1 expression in the heart under basal conditions and failed to upregulate the Hh pathway upon ischemia and reperfusion injury, unlike their littermate controls. This study characterizes, for the first time, the transcriptional response of cardiomyocytes to Shh and establishes a critical role for Smo coupling to Gi in Hh signaling in the normal and ischemic myocardium. Copyright © 2014 the American Physiological Society.

  16. Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN

    OpenAIRE

    Makoto Nakakido; Zhenzhong Deng; Takehiro Suzuki; Naoshi Dohmae; Yusuke Nakamura; Ryuji Hamamoto

    2015-01-01

    Phosphatase and tensin homologue (PTEN), one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine meth...

  17. Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus.

    Directory of Open Access Journals (Sweden)

    Natapong Jupatanakul

    2017-01-01

    Full Text Available We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg promoter. Transgene expression inhibits infection with several dengue virus (DENV serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV or chikungunya virus (CHIKV infection, thereby indicating a possible specialization of the pathway's antiviral role.

  18. Neuroendocrine pathways mediating nutritional acceleration of puberty: insights from ruminant models

    Directory of Open Access Journals (Sweden)

    Marcel eAmstalden

    2011-12-01

    Full Text Available The pubertal process is characterized by an activation of physiological events within the hypothalamic-adenohypophyseal-gonadal axis which culminate in reproductive competence. Excessive weight gain and adiposity during the juvenile period is associated with accelerated onset of puberty in females. The mechanisms and pathways by which excess energy balance advances puberty are unclear, but appear to involve an early escape from estradiol negative feedback and early initiation of high frequency episodic gonadotropin-releasing hormone (GnRH secretion. Hypothalamic neurons, particularly neuropeptide Y and proopiomelanocortin neurons are likely important components of the pathway sensing and transmitting metabolic information to the control of GnRH secretion. Kisspeptin neurons may also have a role as effector neurons integrating metabolic and gonadal steroid feedback effects on GnRH secretion at the time of puberty. Recent studies indicate that leptin-responsive neurons within the ventral premammillary nucleus play a critical role in pubertal progression and challenge the relevance of kisspeptin neurons in this process. Nevertheless, the nutritional control of puberty is likely to involve an integration of major sensor and effector pathways that interact with modulatory circuitries for a fine control of GnRH neuron function. In this review, observations made in ruminant species are emphasized for a comparative perspective.

  19. Patulin induces apoptosis through ROS-mediated endoplasmic reticulum stress pathway.

    Science.gov (United States)

    Boussabbeh, Manel; Ben Salem, Intidhar; Prola, Alexandre; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe

    2015-04-01

    Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider, and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and unfolded protein response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA, and expression of the proapoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the proapoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetyl cysteine inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Lhx8 mediated Wnt and TGFβ pathways in tooth development and regeneration.

    Science.gov (United States)

    Zhou, Chen; Yang, Guodong; Chen, Mo; Wang, Chenglin; He, Ling; Xiang, Lusai; Chen, Danying; Ling, Junqi; Mao, Jeremy J

    2015-09-01

    LIM homeobox 8 (Lhx8) is a highly conserved transcriptional factor with recently illustrated roles in cholinergic and GABAergic differentiation, and is expressed in neural crest derived craniofacial tissues during development. However, Lhx8 functions and signaling pathways are largely elusive. Here we showed that Lhx8 regulates dental mesenchyme differentiation and function via Wnt and TGFβ pathways. Lhx8 expression was restricted to dental mesenchyme from E11.5 to a peak at E14.5, and absent in dental epithelium. By reconstituting dental epithelium and mesenchyme in an E16.5 tooth organ, Lhx8 knockdown accelerated dental mesenchyme differentiation; conversely, Lhx8 overexpression attenuated dentin formation. Lhx8 overexpressed adult human dental pulp stem/progenitor cells in β-tricalcium phosphate cubes attenuated mineralized matrix production in vivo. Gene profiling revealed that postnatal dental pulp stem/progenitor cells upon Lhx8 overexpression modified matrix related gene expression including Dspp, Cola1 and osteocalcin. Lhx8 transcriptionally activated Wnt and TGFβ pathways, and its attenuation upregulated multiple dentinogenesis genes. Together, Lhx8 regulates dentin development and regeneration by fine-turning Wnt and TGFβ signaling.

  1. Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration.

    Science.gov (United States)

    Xu, Wei; Weissmiller, April M; White, Joseph A; Fang, Fang; Wang, Xinyi; Wu, Yiwen; Pearn, Matthew L; Zhao, Xiaobei; Sawa, Mariko; Chen, Shengdi; Gunawardena, Shermali; Ding, Jianqing; Mobley, William C; Wu, Chengbiao

    2016-05-02

    The endosome/lysosome pathway is disrupted early in the course of both Alzheimer's disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its β-C-terminal fragment (β-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and β-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. β-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or β-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS.

  2. Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus.

    Science.gov (United States)

    Jupatanakul, Natapong; Sim, Shuzhen; Angleró-Rodríguez, Yesseinia I; Souza-Neto, Jayme; Das, Suchismita; Poti, Kristin E; Rossi, Shannan L; Bergren, Nicholas; Vasilakis, Nikos; Dimopoulos, George

    2017-01-01

    We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway's antiviral role.

  3. A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo

    Science.gov (United States)

    Motley, Michael P.; Madsen, Daniel H.; Jürgensen, Henrik J.; Spencer, David E.; Szabo, Roman; Holmbeck, Kenn; Flick, Matthew J.; Lawrence, Daniel A.; Castellino, Francis J.; Weigert, Roberto

    2016-01-01

    Extravascular fibrin deposition accompanies many human diseases and causes chronic inflammation and organ damage, unless removed in a timely manner. Here, we used intravital microscopy to investigate how fibrin is removed from extravascular space. Fibrin placed into the dermis of mice underwent cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin(ogen) receptors, αMβ2 and ICAM-1, the myeloid cell integrin-binding site on fibrin or the endocytic collagen receptor, the mannose receptor. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance and shows that interstitial fibrin and collagen are cleared by different subsets of macrophages employing distinct molecular pathways. PMID:26647393

  4. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  5. 7-O-Geranylquercetin induces apoptosis in gastric cancer cells via ROS-MAPK mediated mitochondrial signaling pathway activation.

    Science.gov (United States)

    Zhu, Yanyan; Jiang, Yameng; Shi, Lei; Du, Linying; Xu, Xiaodong; Wang, Enxia; Sun, Yong; Guo, Xin; Zou, Boyang; Wang, Huaxin; Wang, Changyuan; Sun, Lidan; Zhen, Yuhong

    2017-03-01

    7-O-Geranylquercetin (GQ) is a novel O-alkylated derivate of quercetin. In this study, we evaluated its apoptosis induction effects in human gastric cancer cell lines SGC-7901 and MGC-803 and explored the potential molecular mechanisms. The results demonstrated that GQ lowered viability of SGC-7901 and MGC-803 cells in a dose- and time-dependent manner without apparent cytotoxicity to human gastric epithelial cell line GES-1. GQ could induce apoptosis in SGC-7901 and MGC-803cells, and arrest the gastric cancer cells at G2/M phase. Mechanism study showed that GQ triggered generation of reactive oxygen species (ROS), then activated p38 and JNK signaling pathways, subsequently led to mitochondrial impairment by regulating the expression of Bcl-2, Bcl-xl and Bax, and finally promoted the release of cytochrome c and the activation of caspases to induce apoptosis. In addition, Z-VAD-FMK (caspase inhibitor) could reverse GQ-induced apoptosis. SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) could rescue GQ-induced cell death and attenuate mitochondrial signal pathway activation. Furthermore, NAC (ROS inhibitor) could rescue GQ-induced cell death, reduce ROS generation, decrease the phosphorylation of p38 and JNK, and then attenuate the activation of mitochondrial signal pathway. Taken together, GQ induces caspase-dependent apoptosis in gastric cancer cells through activating ROS-MAPK mediated mitochondrial signal pathway. This study highlights the potential use of GQ as a gastric cancer therapeutic agent. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  6. Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways.

    Science.gov (United States)

    Wang, Jing; Yuan, Li; Xiao, Haifang; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2013-06-01

    Momordin Ic is a natural triterpenoid saponin enriched in various Chinese and Japanese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. So far, there is little scientific evidence for momordin Ic with regard to the anti-tumor activities. The aim of this work was to elucidate the anti-tumor effect of momordin Ic and the signal transduction pathways involved. We found that momordin Ic induced apoptosis in human hepatocellular carcinoma HepG2 cells, which were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, momordin Ic triggered reactive oxygen species (ROS) production together with collapse of mitochondrial membrane potential, cytochrome c release, down-regulation of Bcl-2 and up-regulation of Bax expression. The activation of p38 and JNK, inactivation of Erk1/2 and Akt were also demonstrated. Although ROS production rather than NO was stimulated, the expression of iNOS and HO-1 were altered after momordin Ic treatment for 4 h. Furthermore, the cytochrome c release, caspase-3 activation, Bax/Bcl-2 expression and PARP cleavage were promoted with LY294002 and U0126 intervention but were blocked by SB203580, SP600125, PI3K activator, NAC and 1,400 W pretreatment, demonstrating the mitochondrial disruption. Furthermore, momordin Ic combination with NAC influenced MAPK, PI3K/Akt and HO-1, iNOS pathways, MAPK and PI3K/Akt pathways also regulated the expression of HO-1 and iNOS. These results indicated that momordin Ic induced apoptosis through oxidative stress-regulated mitochondrial dysfunction involving the MAPK and PI3K-mediated iNOS and HO-1 pathways. Thus, momordin Ic might represent a potential source of anticancer candidate.

  7. The Association of CXC Receptor 4 Mediated Signaling Pathway with Oxaliplatin-Resistant Human Colorectal Cancer Cells

    Science.gov (United States)

    Huang, Cheng-Yi; Kuo, Yi-Hung; Tung, Shui-Yi; Shen, Chien-Heng; Hsieh, Yung-Yu; Teng, Chih-Chuan; Lee, Kam-Fai; Chen, Te-Chuan; Lee, Ko-Chao; Kuo, Hsing-Chun

    2016-01-01

    The stromal cell–derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) axis plays an important role in tumor angiogenesis and invasiveness in colorectal cancer (CRC) progression. In addition, metastatic CRC remains one of the most difficult human malignancies to treat because of its chemoresistant behavior. However, the mechanism by which correlation occurs between CXCR4 and the clinical response of CRC to chemotherapy remains unknown. We generated chemoresistant cells with increasing doses of oxaliplatin (OXA) and 5-Fluorouracil (5FU) to develop resistance at a clinical dose. We found that the putative markers did not change in the parental cells, but HCT-116/OxR and HCT-116/5-FUR were more aggressive and had higher tumor growth (demonstrated by wound healing, chemotaxis assay, and a nude mice xenograft model) with the use of oxaliplatin. Apoptosis induced by oxaliplatin treatment was significantly decreased in HCT-116/OxR compared to the parental cells. Moreover, HCT-116/OxR cells displayed increased levels of p-gp, p-Akt p-ERK, p-IKBβ, CXCR4, and Bcl-2, but they also significantly inhibited the apoptotic pathways when compared to the parental strain. We evaluated the molecular mechanism governing the signaling pathway associated with anti-apoptosis activity and the aggressive status of chemoresistant cells. Experiments involving specific inhibitors demonstrated that the activation of the pathways associated with CXCR4, ERK1/2 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt is critical to the functioning of the HCT-116/OxR and HCT-116/5-FUR characteristics of chemosensitivity. These findings elucidate the mechanism of CXCR4/PI3K/Akt downstream signaling and provide strategies to inhibit CXCR4 mediated signaling pathway in order to overcome CRC’s resistance to chemotherapy. PMID:27668882

  8. Hepatic gene expression profiling reveals key pathways involved in leptin-mediated weight loss in ob/ob mice.

    Directory of Open Access Journals (Sweden)

    Ashok Sharma

    Full Text Available BACKGROUND: Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss. METHODOLOGY/PRINCIPAL FINDINGS: WE UTILIZED MICROARRAY TECHNOLOGY TO COMPARE HEPATIC GENE EXPRESSION CHANGES AFTER TWO TYPES OF LEPTIN ADMINISTRATION: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV. We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes, endoplasmic reticulum (22 genes and vacuole (8 genes were significantly over represented. CONCLUSIONS/SIGNIFICANCE: In this study we have identified key molecular pathways and downstream

  9. Hepatic gene expression profiling reveals key pathways involved in leptin-mediated weight loss in ob/ob mice.

    Science.gov (United States)

    Sharma, Ashok; Bartell, Shoshana M; Baile, Clifton A; Chen, Bo; Podolsky, Robert H; McIndoe, Richard A; She, Jin-Xiong

    2010-08-16

    Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss. WE UTILIZED MICROARRAY TECHNOLOGY TO COMPARE HEPATIC GENE EXPRESSION CHANGES AFTER TWO TYPES OF LEPTIN ADMINISTRATION: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ) and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV). We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes), endoplasmic reticulum (22 genes) and vacuole (8 genes) were significantly over represented. In this study we have identified key molecular pathways and downstream genes which respond to leptin treatment and are involved in leptin-mediated weight

  10. BDNF-TrkB pathway mediates neuroprotection of hydrogen sulfide against formaldehyde-induced toxicity to PC12 cells.

    Directory of Open Access Journals (Sweden)

    Jia-Mei Jiang

    Full Text Available Formaldehyde (FA is a common environmental contaminant that has toxic effects on the central nervous system (CNS. Our previous data demonstrated that hydrogen sulfide (H2S, the third endogenous gaseous mediator, has protective effects against FA-induced neurotoxicity. As is known to all, Brain-derived neurotropic factor (BDNF, a member of the neurotrophin gene family, mediates its neuroprotective properties via various intracellular signaling pathways triggered by activating the tyrosine kinase receptor B (TrkB. Intriguingly, our previous data have illustrated the upregulatory role of H2S on BDNF protein expression in the hippocampus of rats. Therefore, in this study, we hypothesized that H2S provides neuroprotection against FA toxicity by regulating BDNF-TrkB pathway. In the present study, we found that NaHS, a donor of H2S, upregulated the level of BDNF protein in PC12 cells, and significantly rescued FA-induced downregulation of BDNF levels. Furthermore, we found that pretreatment of PC12 cells with K252a, an inhibitor of the BDNF receptor TrkB, markedly reversed the inhibition of NaHS on FA-induced cytotoxicity and ablated the protective effects of NaHS on FA-induced oxidative stress, including the accumulation of intracellular reactive oxygen species (ROS, 4-hydroxy-2-trans-nonenal (4-HNE, and malondialdehyde (MDA. We also showed that K252a abolished the inhibition of NaHS on FA-induced apoptosis, as well as the activation of caspase-3 in PC12 cells. In addition, K252a reversed the protection of H2S against FA-induced downregulation of Bcl-2 protein expression and upregulation of Bax protein expression in PC12 cells. These data indicate that the BDNF-TrkB pathway mediates the neuroprotection of H2S against FA-induced cytotoxicity, oxidative stress and apoptosis in PC12 cells. These findings provide a novel mechanism underlying the protection of H2S against FA-induced neurotoxicity.

  11. Trophoblast cell fusion and differentiation are mediated by both the protein kinase C and a pathways.

    Directory of Open Access Journals (Sweden)

    Waka Omata

    Full Text Available The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.

  12. Promotion of Dental Pulp Cell Migration and Pulp Repair by a Bioceramic Putty Involving FGFR-mediated Signaling Pathways.

    Science.gov (United States)

    Zhang, J; Zhu, L X; Cheng, X; Lin, Y; Yan, P; Peng, B

    2015-06-01

    Mineral trioxide aggregate is the currently recommended material of choice for clinical pulp repair despite several disadvantages, including handling inconvenience. Little is known about the signaling mechanisms involved in bioceramic-mediated dental pulp repair-particularly, dental pulp cell (DPC) migration. This study evaluated the effects of iRoot BP Plus, a novel ready-to-use nanoparticulate bioceramic putty, on DPC migration in vitro and pulp repair in vivo, focusing on possible involvement of fibroblast growth factor receptor (FGFR)-related signaling, including mitogen-activated protein kinase and Akt pathways. Treatment with iRoot BP Plus extracts enhanced horizontal and vertical migration of DPCs, which was comparable with the effects induced by mineral trioxide aggregate extracts. The DPCs exposed to iRoot BP Plus extracts demonstrated no evident apoptosis. Importantly, treatment with iRoot BP Plus extracts resulted in rapid activation of FGFR, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), and Akt signaling in DPCs. Confocal immunofluorescence staining revealed that iRoot BP Plus stimulated focal adhesion formation and stress fiber assembly in DPCs, in addition to upregulating the expression of focal adhesion molecules, including p-focal adhesion kinase, p-paxillin, and vinculin. Moreover, activation of FGFR, ERK, JNK, and Akt were found to mediate the upregulated expression of focal adhesion molecules, stress fiber assembly, and enhanced DPC migration induced by iRoot BP Plus. Consistent with the in vitro results, we observed induction of homogeneous dentin bridge formation and expression of p-focal adhesion kinase, p-FGFR, p-ERK 1/2, p-JNK, and p-Akt near injury sites by iRoot BP Plus in an in vivo pulp repair model. These data demonstrate that iRoot BP Plus can promote DPC migration and pulp repair involving the FGFR-mediated ERK 1/2, JNK, and Akt pathways. These findings provide

  13. Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death.

    Science.gov (United States)

    Laborde, E

    2010-09-01

    Glutathione transferases (GSTs) are enzymes that catalyze the conjugation of glutathione (GSH) to a variety of electrophilic substances. Their best known role is as cell housekeepers engaged in the detoxification of xenobiotics. Recently, GSTs have also been shown to act as modulators of signal transduction pathways that control cell proliferation and cell death. Their involvement in cancer cell growth and differentiation, and in the development of resistance to anticancer agents, has made them attractive drug targets. This review is focused on the inhibition of GSTs, in particular GSTP1-1, as a potential therapeutic approach for the treatment of cancer and other diseases associated with aberrant cell proliferation.

  14. Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope.

    Directory of Open Access Journals (Sweden)

    Preetinder K Dhanoa

    Full Text Available BACKGROUND: Tail-anchored (TA proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34 and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9. Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. CONCLUSIONS/SIGNIFICANCE: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie

  15. Neural pathways mediating control of reproductive behaviour in male Japanese quail

    Science.gov (United States)

    Wild, J Martin; Balthazart, Jacques

    2012-01-01

    The sexually dimorphic medial preoptic nucleus (POM) in Japanese quail has for many years been the focus of intensive investigations into its role in reproductive behaviour. The present paper delineates a sequence of descending pathways that finally reach sacral levels of the spinal cord housing motor neurons innervating cloacal muscles involved in reproductive behaviour. We first retrogradely labeled the motor neurons innervating the large cloacal sphincter muscle (mSC) that forms part of the foam gland complex (Seiwert and Adkins-Regan, 1998, Brain Behav Evol 52:61–80) and then putative premotor nuclei in the brainstem, one of which was nucleus retroambigualis (RAm) in the caudal medulla. Anterograde tracing from RAm defined a bulbospinal pathway, terminations of which overlapped the distribution of mSC motor neurons and their extensive dorsally directed dendrites. Descending input to RAm arose from an extensive dorsomedial nucleus of the intercollicular complex (DM-ICo), electrical stimulation of which drove vocalizations. POM neurons were retrogradely labeled by injections of tracer into DM-ICo, but POM projections largely surrounded DM, rather than penetrated it. Thus, although a POM projection to ICo was shown, a POM projection to DM must be inferred. Nevertheless, the sequence of projections in the male quail from POM to cloacal motor neurons strongly resembles that in rats, cats and monkeys for the control of reproductive behaviour, as largely defined by Holstege and co-workers (e.g., Holstege et al., 1997, Neuroscience 80: 587–598). PMID:23225613

  16. Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus

    Science.gov (United States)

    Jupatanakul, Natapong; Sim, Shuzhen; Angleró-Rodríguez, Yesseinia I.; Souza-Neto, Jayme; Das, Suchismita; Poti, Kristin E.; Rossi, Shannan L.; Bergren, Nicholas; Vasilakis, Nikos

    2017-01-01

    We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role. PMID:28081143

  17. DHEA-mediated inhibition of the pentose phosphate pathway alters oocyte lipid metabolism in mice.

    Science.gov (United States)

    Jimenez, Patricia T; Frolova, Antonina I; Chi, Maggie M; Grindler, Natalia M; Willcockson, Alexandra R; Reynolds, Kasey A; Zhao, Quihong; Moley, Kelle H

    2013-12-01

    Women with polycystic ovary syndrome (PCOS) and hyperandrogenism have altered hormone levels and suffer from ovarian dysfunction leading to subfertility. We have attempted to generate a model of hyperandrogenism by feeding mice chow supplemented with dehydroepiandrosterone (DHEA), an androgen precursor that is often elevated in women with PCOS. Treated mice had polycystic ovaries, low ovulation rates, disrupted estrous cycles, and altered hormone levels. Because DHEA is an inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate pathway, we tested the hypothesis that oocytes from DHEA-exposed mice would have metabolic disruptions. Citrate levels, glucose-6-phosphate dehydrogenase activity, and lipid content in denuded oocytes from these mice were significantly lower than controls, suggesting abnormal tricarboxylic acid and pentose phosphate pathway metabolism. The lipid and citrate effects were reversible by supplementation with nicotinic acid, a precursor for reduced nicotinamide adenine dinucleotide phosphate. These findings suggest that elevations in systemic DHEA can have a negative impact on oocyte metabolism and may contribute to poor pregnancy outcomes in women with hyperandrogenism and PCOS.

  18. Mutations in the Lipopolysaccharide biosynthesis pathway interfere with crescentin-mediated cell curvature in Caulobacter crescentus.

    Science.gov (United States)

    Cabeen, Matthew T; Murolo, Michelle A; Briegel, Ariane; Bui, N Khai; Vollmer, Waldemar; Ausmees, Nora; Jensen, Grant J; Jacobs-Wagner, Christine

    2010-07-01

    Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.

  19. Spatial signalling mediated by the transforming growth factor-β signalling pathway during tooth formation.

    Science.gov (United States)

    He, Xin-Yu; Sun, Ke; Xu, Ruo-Shi; Tan, Jia-Li; Pi, Cai-Xia; Wan, Mian; Peng, Yi-Ran; Ye, Ling; Zheng, Li-Wei; Zhou, Xue-Dong

    2016-12-16

    Tooth development relies on sequential and reciprocal interactions between the epithelial and mesenchymal tissues, and it is continuously regulated by a variety of conserved and specific temporal-spatial signalling pathways. It is well known that suspensions of tooth germ cells can form tooth-like structures after losing the positional information provided by the epithelial and mesenchymal tissues. However, the particular stage in which the tooth germ cells start to form tooth-like structures after losing their positional information remains unclear. In this study, we investigated the reassociation of tooth germ cells suspension from different morphological stages during tooth development and the phosphorylation of Smad2/3 in this process. Four tooth morphological stages were designed in this study. The results showed that tooth germ cells formed odontogenic tissue at embryonic day (E) 14.5, which is referred to as the cap stage, and they formed tooth-like structures at E16.5, which is referred to as the early bell stage, and E18.5, which is referred to as the late bell stage. Moreover, the transforming growth factor-β signalling pathway might play a role in this process.

  20. DHEA-Mediated Inhibition of the Pentose Phosphate Pathway Alters Oocyte Lipid Metabolism in Mice

    Science.gov (United States)

    Jimenez, Patricia T.; Frolova, Antonina I.; Chi, Maggie M.; Grindler, Natalia M.; Willcockson, Alexandra R.; Reynolds, Kasey A.; Zhao, Quihong

    2013-01-01

    Women with polycystic ovary syndrome (PCOS) and hyperandrogenism have altered hormone levels and suffer from ovarian dysfunction leading to subfertility. We have attempted to generate a model of hyperandrogenism by feeding mice chow supplemented with dehydroepiandrosterone (DHEA), an androgen precursor that is often elevated in women with PCOS. Treated mice had polycystic ovaries, low ovulation rates, disrupted estrous cycles, and altered hormone levels. Because DHEA is an inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate pathway, we tested the hypothesis that oocytes from DHEA-exposed mice would have metabolic disruptions. Citrate levels, glucose-6-phosphate dehydrogenase activity, and lipid content in denuded oocytes from these mice were significantly lower than controls, suggesting abnormal tricarboxylic acid and pentose phosphate pathway metabolism. The lipid and citrate effects were reversible by supplementation with nicotinic acid, a precursor for reduced nicotinamide adenine dinucleotide phosphate. These findings suggest that elevations in systemic DHEA can have a negative impact on oocyte metabolism and may contribute to poor pregnancy outcomes in women with hyperandrogenism and PCOS. PMID:24036000

  1. Resistance to selective BRAF inhibition can be mediated by modest upstream pathway activation.

    Science.gov (United States)

    Su, Fei; Bradley, William D; Wang, Qiongqing; Yang, Hong; Xu, Lizhong; Higgins, Brian; Kolinsky, Kenneth; Packman, Kathryn; Kim, Min Jung; Trunzer, Kerstin; Lee, Richard J; Schostack, Kathleen; Carter, Jade; Albert, Thomas; Germer, Soren; Rosinski, Jim; Martin, Mitchell; Simcox, Mary Ellen; Lestini, Brian; Heimbrook, David; Bollag, Gideon

    2012-02-15

    A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.

  2. Thyroid Hormone Receptor α1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway*

    Science.gov (United States)

    Grespin, Matthew E.; Bonamy, Ghislain M. C.; Roggero, Vincent R.; Cameron, Nicole G.; Adam, Lindsay E.; Atchison, Andrew P.; Fratto, Victoria M.; Allison, Lizabeth A.

    2008-01-01

    The thyroid hormone receptor α1 (TRα) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T3). Previously, we have shown that TRα, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRα is its ability to exit the nucleus through the nuclear pore complex. TRα export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRα. We show that, in addition to shuttling in heterokaryons, TRα shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRα directly interacts with calreticulin, and point to the intriguing possibility that TRα follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRα from the nucleus to cytoplasm. PMID:18641393

  3. Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

    Science.gov (United States)

    Grespin, Matthew E; Bonamy, Ghislain M C; Roggero, Vincent R; Cameron, Nicole G; Adam, Lindsay E; Atchison, Andrew P; Fratto, Victoria M; Allison, Lizabeth A

    2008-09-12

    The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

  4. Injury of cortical neurons is caused by the advanced glycation end products-mediated pathway

    Institute of Scientific and Technical Information of China (English)

    Ying Xing; Xu Zhang; Xiangfu Song; Zhongwen Lv; Lingling Hou; Fei Li

    2013-01-01

    Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products (50, 100, 200, 400 mg/L), and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.

  5. Identification of miRNAs and miRNA-mediated regulatory pathways in Carica papaya.

    Science.gov (United States)

    Liang, Gang; Li, Yang; He, Hua; Wang, Fang; Yu, Diqiu

    2013-10-01

    Plant microRNAs (miRNAs) post-transcriptionally regulate target gene expression to modulate growth and development and biotic and abiotic stress responses. By analyzing small RNA deep sequencing data in combination with the genome sequence, we identified 75 conserved miRNAs and 11 novel miRNAs. Their target genes were also predicted. For most conserved miRNAs, the miRNA-target pairs were conserved across plant species. In addition to these conserved miRNA-target pairs, we also identified some papaya-specific miRNA-target regulatory pathways. Both miR168 and miR530 target the Argonaute 1 gene, indicating a second autoregulatory mechanism for miRNA regulation. A non-conserved miRNA was mapped within an intron of Dicer-like 1 (DCL1), suggesting a conserved homeostatic autoregulatory mechanism for DCL1 expression. A 21-nt miRNA triggers secondary siRNA production from its target genes, nucleotide-binding site leucine-rich repeat protein genes. Certain phased-miRNAs were processed from their conserved miRNA precursors, indicating a putative miRNA evolution mechanism. In addition, we identified a Carica papaya-specific miRNA that targets an ethylene receptor gene, implying its function in the ethylene signaling pathway. This work will also advance our understanding of miRNA functions and evolution in plants.

  6. Atractylenolide I-mediated Notch pathway inhibition attenuates gastric cancer stem cell traits

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Li; Mao, Rurong; Shen, Ke; Zheng, Yuanhong; Li, Yueqi [State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237 (China); Liu, Jianwen, E-mail: liujian@ecust.edu.cn [State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237 (China); Ni, Lei, E-mail: nilei625@yahoo.com [Department of Respiration, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Road II, Shanghai 200025 (China)

    2014-07-18

    Highlights: • This paper supports the anti-tumor effects of AT-I on gastric cancer in vitro. • AT-I attenuates gastric cancer stem cell traits. • It is the systematic study regarding AT-I suppression of Notch pathway in GC and GCSLCs. - Abstract: Atractylenolide I (AT-I), one of the main naturally occurring compounds of Rhizoma Atractylodis Macrocephalae, has remarkable anti-cancer effects on various cancers. However, its effects on the treatment of gastric cancer remain unclear. Via multiple cellular and molecular approaches, we demonstrated that AT-I could potently inhibit cancer cell proliferation and induce apoptosis through inactivating Notch pathway. AT-I treatment led to the reduction of expressions of Notch1, Jagged1, and its downstream Hes1/ Hey1. Our results showed that AT-I inhibited the self-renewal capacity of gastric stem-like cells (GCSLCs) by suppression of their sphere formation capacity and cell viability. AT-I attenuated gastric cancer stem cell (GCSC) traits partly through inactivating Notch1, leading to reducing the expressions of its downstream target Hes1, Hey1 and CD44 in vitro. Collectively, our results suggest that AT-I might develop as a potential therapeutic drug for the treatment of gastric cancer.

  7. The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth.

    Science.gov (United States)

    Hobecker, Karen Vanesa; Reynoso, Mauricio Alberto; Bustos-Sanmamed, Pilar; Wen, Jiangqi; Mysore, Kirankumar S; Crespi, Martín; Blanco, Flavio Antonio; Zanetti, María Eugenia

    2017-08-01

    Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2 Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development. © 2017 American Society of Plant Biologists. All Rights Reserved.

  8. Curcumin Rescues Diabetic Renal Fibrosis by Targeting Superoxide-Mediated Wnt Signaling Pathways.

    Science.gov (United States)

    Ho, Cheng; Hsu, Yung-Chien; Lei, Chen-Chou; Mau, Shu-Ching; Shih, Ya-Hsueh; Lin, Chun-Liang

    2016-03-01

    The purposes of this study were to investigate whether curcumin can weaken diabetic nephropathy by modulating both oxidative stress and renal injury from Wnt signaling mediation. Wnt5a/β-catenin depression and induction of superoxide synthesis are associated with high glucose (HG) induced transforming growth factor (TGF)-β1 and fibronectin expression in mesangial cells. Curcumin resumes HG depression of Wnt/β-catenin signaling and alleviates HG induction of superoxide, TGF-β1 and fibronectin expression in renal mesangial cell. Exogenous curcumin alleviated urinary total proteinuria and serum superoxide level in diabetic rats. Based on laser-captured microdissection for quantitative real-time polymerase chain reaction, it was found that diabetes significantly increased TGF-β1 and fibronectin expression in line with depressed Wnt5a expression. Curcumin treatment reduced the TGF-β1 and fibronectin activation and the inhibiting effect of diabetes on Wnt5a/β-catenin expression in renal glomeruli. Immunohistochemistry showed that curcumin treatment significantly reduced 8-hydroxy-2'-deoxyguanosine, TGF-β1 and fibronectin, and was in line with the restoration of the suppressed Wnt5a expression immunoreactivities in glomeruli of diabetic rats. Curcumin alleviated extracellular matrix accumulation in diabetic nephropathy by not only preventing the diabetes-mediated superoxide synthesis but also resuming downregulation of Wnt/β-catenin signaling. These findings suggest that regulation of Wnt activity by curcumin is a feasible alternative strategy to rescue diabetic renal injury.

  9. Mitogen-activated protein kinase pathways are required for melatonin-mediated defense responses in plants.

    Science.gov (United States)

    Lee, Hyoung Yool; Back, Kyoungwhan

    2016-04-01

    Melatonin enhances pathogen resistance by inducing the expression of a number of plant defense-related genes. To examine whether the melatonin-mediated pathogen resistance is associated with mitogen-activated protein kinase (MAPK) cascades, Arabidopsis and tobacco leaves were treated with melatonin and investigated for MAPK activation using an antiphospho-p44/42 MAPK (Erk1/2) monoclonal antibody. Two MAPKs, MPK3 and MPK6, were activated rapidly and transiently by 1 μm melatonin treatment in Arabidopsis. Its tobacco ortholog MAPKs were also activated. The activation of MPK3 and MPK6 by 2-hydroxymelatonin and N-acetylserotonin was also observed, albeit to a lesser degree than that by melatonin. Furthermore, MAPK activation by melatonin was uncoupled from G-protein signaling, because melatonin efficiently activated two MAPKs in a G-protein β knockout mutant (agb1). Suppression of both MPK3 and MPK6 in transgenic Arabidopsis exhibited significant decreases in the induction of defense-related gene expression and pathogen resistance relative to wild-type plants. Using an array of MAP kinase kinase (MKK) knockout mutants, we found that four MKKs, namely MKK4, MKK5, MKK7, and MKK9, are responsible for the activation of MPK3 and MPK6 by melatonin, indicating that melatonin-mediated innate immunity is triggered by MAPK signaling through MKK4/5/7/9-MPK3/6 cascades.

  10. Development pathways from abusive parenting to delinquency: the mediating role of depression and aggression.

    Science.gov (United States)

    You, Sukkyung; Lim, Sun Ah

    2015-08-01

    This study investigated the long-term relationship between abusive parenting and adolescent mental health, and the path to delinquent behavior. Longitudinal data from 5th through 7th graders from the Korean Children and Youth Panel Survey (KCYPS) were analyzed to examine if abusive parenting was a predictor of early adolescent delinquency behavior, via aggression and depression as mediating factors. The results were as follows. First, parental abuse (both emotional and physical) was found to have significant effects on children's psychosocial factors (aggression and depression), while parental neglect (both emotional and physical) had significant effects on depression alone and not on aggression. Second, aggression exerted significant effects on both violent and non-violent delinquent behaviors, while depression had a significant effect on only non-violent delinquent behaviors. Third, children's psychosocial factors (aggression and depression) played significant mediating roles between earlier abusive parenting and delinquent behaviors. Fourth, for children living in a family with their grandparents, paths from abusive parenting, psychosocial adaptation, and later delinquent behaviors were not significant, implying that living with grandparents played a protective factor in these relationships.

  11. The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Li Wei; Chen Chunying; Ye Chang; Zhao Yuliang; Chen Zhen; Meng Huan; Gao Yuxi; Yuan Hui; Xing Genmei; Zhao Feng; Chai Zhifang [Laboratory for Bio-Environmental Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Nanotechnology of China and Institute of High Energy Physics, Chinese Academy of Science, Yuquan Road 19B, Beijing 100049 (China); Wei Taotao; Zhang Xujia; Yang Fuyu [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China); Lao Fang; Han Dong [National Center for Nanoscience and Technology of China, No 2, Ist North Street Zhongguancun, Beijing 100080 (China); Tang Xianhua; Zhang Yingge [Chinese Academy of Military Medical Sciences, Beijing 100039 (China)], E-mail: chenchy@nanoctr.cn, E-mail: weitt@moon.ibp.ac.cn, E-mail: zhaoyuliang@ihep.ac.cn

    2008-04-09

    Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles entering cells are mainly via endocytosis with time-, temperature- and energy-dependent manners. The cellular uptake of [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside cells.

  12. The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

    Science.gov (United States)

    Kang, WonKyung; Kurihara, Masaaki; Matsumoto, Shogo

    2006-06-20

    The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

  13. Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3beta pathway.

    Science.gov (United States)

    Chen, Lin; Zhang, Yi; Sun, Xiuli; Li, Hui; LeSage, Gene; Javer, Avani; Zhang, Xiumei; Wei, Xinbing; Jiang, Yulin; Yin, Deling

    2009-07-01

    As resveratrol derivatives, resveratrol aliphatic acids were synthesized in our laboratory. Previously, we reported the improved pharmaceutical properties of the compounds compared to resveratrol, including better solubility in water and much tighter binding with human serum albumin. Here, we investigate the role of resveratrol aliphatic acids in Toll-like receptor 2 (TLR2)-mediated apoptosis. We showed that resveratrol aliphatic acid (R6A) significantly inhibits the expression of TLR2. In addition, overexpression of TLR2 in HEK293 cells caused a significant decrease in apoptosis after R6A treatment. Moreover, inhibition of TLR2 by R6A decreases serum deprivation-reduced the levels of phosphorylated Akt and phosphorylated glycogen synthase kinase 3beta (GSK3beta). Our study thus demonstrates that the resveratrol aliphatic acid inhibits cell apoptosis through TLR2 by the involvement of Akt/GSK3beta pathway.

  14. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  15. Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug

    Energy Technology Data Exchange (ETDEWEB)

    Mosedale, Merrie [Hamner-University of North Carolina Institute for Drug Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Wu, Hong [Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT06340 (United States); Kurtz, C. Lisa [Hamner-University of North Carolina Institute for Drug Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Schmidt, Stephen P. [Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT06340 (United States); Adkins, Karissa, E-mail: Karissa.Adkins@pfizer.com [Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT06340 (United States); Harrill, Alison H. [Hamner-University of North Carolina Institute for Drug Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); University of Arkansas for Medical Sciences, Little Rock, AR72205 (United States)

    2014-10-01

    A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this drug. - Highlights: • Identified susceptible and resistant mouse strains to liver injury induced by a CAD • Liver injury characterized by single cell necrosis, and phospholipidosis

  16. Regulated release of BDNF by cortical oligodendrocytes is mediated through metabotropic glutamate receptors and the PLC pathway

    Directory of Open Access Journals (Sweden)

    Issa P Bagayogo

    2009-04-01

    Full Text Available A number of studies suggest that OLGs (oligodendrocytes), the myelinating cells of the central nervous system, are also a source of trophic molecules, such as neurotrophins that may influence survival of proximate neurons. What is less clear is how the release of these molecules may be regulated. The present study investigated the effects of BDNF (brain-derived neurotrophic factor) derived from cortical OLGs on proximate neurons, as well as regulatory mechanisms mediating BDNF release. Initial work determined that BDNF derived from cortical OLGs increased the numbers of VGLUT1 (vesicular glutamate transporter 1)-positive glutamatergic cortical neurons. Furthermore, glutamate acting through metabotropic, and not AMPA/kainate or NMDA (N-methyl-d-aspartate), receptors increased BDNF release. The PLC (phospholipase C) pathway is a key mediator of metabotropic actions to release BDNF in astrocytes and neurons. Treatment of OLGs with the PLC activator m-3M3FBS [N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide] induced robust release of BDNF. Moreover, release elicited by the metabotropic receptor agonist ACPD [trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] was inhibited by the PLC antagonist U73122, the IP3 (inositol triphosphate 3) receptor inhibitor 2-APB (2-aminoethoxydiphenylborane) and the intracellular calcium chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)]. Taken together, these results suggest that OLG lineage cells release BDNF, a molecule trophic for proximate neurons. BDNF release is regulated by glutamate acting through mGluRs (metabotropic glutamate receptors) and the PLC pathway. Thus glutamate and BDNF may be molecules that support neuron–OLG interactions in the cortex.

  17. The impact of human development on individual health: a causal mediation analysis examining pathways through education and body mass index.

    Science.gov (United States)

    Wang, Aolin; Arah, Onyebuchi A

    2017-01-01

    The macro environment we live in projects what we can achieve and how we behave, and in turn, shapes our health in complex ways. Policymaking will benefit from insights into the mechanisms underlying how national socioeconomic context affects health. This study examined the impact of human development on individual health and the possible mediating roles of education and body mass index (BMI). We analyzed World Health Survey data on 109,448 participants aged 25 or older from 42 low- and middle-income countries with augmented human development index (HDI) in 1990. We used principal components method to create a health score based on measures from eight health state domains, used years of schooling as education indicator and calculated BMI from self-reported height and weight. We used causal mediation analysis technique with random intercepts to account for the multilevel structure. Below a reference HDI level of 0.48, HDI was negatively associated with good health (total effect at HDI of 0.23: b =  - 3.44, 95% CI [-6.39--0.49] for males and b =  - 5.16, 95% CI [-9.24,--1.08] for females) but was positively associated with good health above this reference level (total effect at HDI of 0.75: b = 4.16, 95% CI [-0.33-8.66] for males and b = 6.62, 95% CI [0.85-12.38] for females). We found a small positive effect of HDI on health via education across reference HDI levels (b ranging from 0.24 to 0.29 for males and 0.40 to 0.49 for females) but not via pathways involving BMI only. Human development has a non-linear effect on individual health, but the impact appears to be mainly through pathways other than education and BMI.

  18. Transmitters and pathways mediating inhibition of spinal itch-signaling neurons by scratching and other counterstimuli.

    Directory of Open Access Journals (Sweden)

    Tasuku Akiyama

    Full Text Available Scratching relieves itch, but the underlying neural mechanisms are poorly understood. We presently investigated a role for the inhibitory neurotransmitters GABA and glycine in scratch-evoked inhibition of spinal itch-signaling neurons in a mouse model of chronic dry skin itch. Superficial dorsal horn neurons ipsilateral to hindpaw dry skin treatment exhibited a high level of spontaneous firing that was significantly attenuated by cutaneous scratching, pinch and noxious heat. Scratch-evoked inhibition was nearly abolished by spinal delivery of the glycine antagonist, strychnine, and was markedly attenuated by respective GABA(A and GABA(B antagonists bicuculline and saclofen. Scratch-evoked inhibition was also significantly attenuated (but not abolished by interruption of the upper cervical spinal cord, indicating the involvement of both segmental and suprasegmental circuits that engage glycine- and GABA-mediated inhibition of spinal itch-signaling neurons by noxious counterstimuli.

  19. Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway.

    LENUS (Irish Health Repository)

    Fanning, N F

    2012-02-03

    Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml(-1)) of ionic (diatrizoate 69.6+\\/-2.9%; ioxaglate 58.9+\\/-2.0%) and non-ionic (iohexol 57.3+\\/-2.9%; iotrolan 57.1+\\/-2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7+\\/-1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml(-1)) treated neutrophils to control levels (23.0+\\/-3.5% to 61.2+\\/-5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and

  20. Helicobacter pylori urease activates blood platelets through a lipoxygenase-mediated pathway.

    Science.gov (United States)

    Wassermann, German E; Olivera-Severo, Deiber; Uberti, Augusto F; Carlini, Célia R

    2010-07-01

    The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase-derived eicosanoids. We investigated whether H. pylori urease displays platelet-activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein-labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED(50) 0.28 microM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12-lipoxygenase inhibitor) and enhanced approximately 3-fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12-lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet-activating factor, but required activation of verapamil-inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di- or tri-chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.

  1. Gene expression profiling reveals potential key pathways involved in pyrazinamide-mediated hepatotoxicity in Wistar rats.

    Science.gov (United States)

    Zhang, Yun; Jiang, Zhenzhou; Su, Yijing; Chen, Mi; Li, Fu; Liu, Li; Sun, Lixin; Wang, Yun; Zhang, Shuang; Zhang, Luyong

    2013-08-01

    Pyrazinamide (PZA) is an important sterilizing prodrug that shortens the duration of tuberculosis therapy. However, hepatotoxicity has been reported during clinical trials investigating PZA. To determine the hepatotoxic effects of PZA in vivo and to further investigate the underlying cellular mechanism, we profiled the gene expression patterns of PZA-treated rat livers by microarray analysis. Wistar rats of both sexes were orally administered PZA at doses of 0.5, 1.0 and 2.0 g kg(-1) for 28 days. Body weight, absolute and relative liver weight, biochemical analysis, histopathology, oxidative stress parameters in liver homogenates and changes in global transcriptomic expression were evaluated to study the hepatotoxic effects of PZA. Our results confirm the dose-dependent and sex-related hepatotoxicity of PZA. Female rats were more sensitive to PZA-induced hepatotoxicity than males. Furthermore, changes in the activity of major antioxidant enzymes and nonenzymatic antioxidants (superoxide dismutase, total antioxidant capacity, glutathione and malondialdehyde), indicating the development of oxidative stress, were more significant in the PZA-treated group. PZA-induced gene expression changes were related to pathways involved in drug metabolism, peroxisome proliferator-activated receptor (PPAR) signaling, oxidative stress and apoptosis. Real-time polymerase chain reaction confirmed the regulation of selected genes involved in PZA-hepatotoxicity (Ephx1, Cyp2b1, Gstm1, Gstp1, Fabp7, Acaa1, Cpt-1b, Cyp8b1, Hmox1 and Ntrk1). We observed for the first time that these genes have effects on PZA-induced hepatotoxicity. In addition, drug metabolism and PPAR signaling pathways may play an important role in PZA hepatotoxicity. Taken together, these findings will be useful for future PZA hepatotoxicity studies.

  2. Electroacupuncture Exerts Neuroprotection through Caveolin-1 Mediated Molecular Pathway in Intracerebral Hemorrhage of Rats

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    Hui-Qin Li

    2016-01-01

    Full Text Available Spontaneous intracerebral hemorrhage (ICH is one of the most devastating types of stroke. Here, we aim to demonstrate that electroacupuncture on Baihui (GV20 exerts neuroprotection for acute ICH possibly via the caveolin-1/matrix metalloproteinase/blood-brain barrier permeability pathway. The model of ICH was established by using collagenase VII. Rats were randomly divided into three groups: Sham-operation group, Sham electroacupuncture group, and electroacupuncture group. Each group was further divided into 4 subgroups according to the time points of 6 h, 1 d, 3 d, and 7 d after ICH. The methods were used including examination of neurological deficit scores according to Longa’s scale, measurement of blood-brain barrier permeability through Evans Blue content, in situ immunofluorescent detection of caveolin-1 in brains, western blot analysis of caveolin-1 in brains, and in situ zymography for measuring matrix metalloproteinase-2/9 activity in brains. Compared with Sham electroacupuncture group, electroacupuncture group has resulted in a significant improvement in neurological deficit scores and in a reduction in Evans Blue content, expression of caveolin-1, and activity of matrix metalloproteinase-2/9 at 6 h, 1 d, 3 d, and 7 d after ICH (P<0.05. In conclusion, the present results suggested that electroacupuncture on GV20 can improve neurological deficit scores and reduce blood-brain barrier permeability after ICH, and the mechanism possibly targets caveolin-1/matrix metalloproteinase/blood-brain barrier permeability pathway.

  3. Biochar increases soil N2O emissions produced by nitrification-mediated pathways

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    María eSánchez-García

    2014-07-01

    Full Text Available In spite of the numerous studies reporting a decrease in soil nitrous oxide (N2O emissions after biochar amendment, there is still a lack of understanding of the processes involved. Hence the subject remains controversial, with a number of studies showing no changes or even an increase in N2O emissions after biochar soil application. Unraveling the exact causes of these changes, and in which circumstances biochar decreases or increases emissions, is vital to developing and applying successful mitigation strategies. With this objective, we studied two soils (Haplic Phaeozem (HP and Haplic Calcisol (HC, which showed opposed responses to biochar amendment. Under the same experimental conditions, the addition of biochar to soil HP decreased N2O emissions by 76%; whereas it increased emissions by 54% in soil HC. We combined microcosm experiments adding different nitrogen fertilizers, stable isotope techniques and the use of a nitrification inhibitor (dicyciandiamide with the aim of improving our understanding of the mechanisms involved in the formation of N2O in these two soils. Evidence suggests that denitrification is the main pathway leading to N2O emissions in soil HP, and ammonia oxidation and nitrifier-denitrification being the major processes generating N2O in soil HC. Biochar systematically stimulated nitrification in soil HC, which was probably the cause of the increased N2O emissions. Here we demonstrate that the effectiveness of using biochar for reducing N2O emissions from a particular soil is linked to its dominant N2O formation pathway.

  4. Zinc Prevents Abdominal Aortic Aneurysm Formation by Induction of A20-Mediated Suppression of NF-κB Pathway.

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    Ya-Wei Yan

    Full Text Available Chronic inflammation and degradation of elastin are the main processes in the development of abdominal aortic aneurysm (AAA. Recent studies show that zinc has an anti-inflammatory effect. Based on these, zinc may render effective therapy for the treatment of the AAA. Currently, we want to investigate the effects of zinc on AAA progression and its related molecular mechanism. Rat AAA models were induced by periaortic application of CaCl2. AAA rats were treated by daily intraperitoneal injection of ZnSO4 or vehicle alone. The aorta segments were collected at 4 weeks after surgery. The primary rat aortic vascular smooth muscle cells (VSMCs were stimulated with TNF-α alone or with ZnSO4 for 3 weeks. The results showed that zinc supplementation significantly suppressed the CaCl2-induced expansion of the abdominal aortic diameter, as well as a preservation of medial elastin fibers in the aortas. Zinc supplementation also obviously attenuated infiltration of the macrophages and lymphocytes in the aortas. In addition, zinc reduced MMP-2 and MMP-9 production in the aortas. Most importantly, zinc treatment significantly induced A20 expression, along with inhibition of the NF-κB canonical signaling pathway in vitro in VSMCs and in vivo in rat AAA. This study demonstrated, for the first time, that zinc supplementation could prevent the development of rat experimental AAA by induction of A20-mediated inhibition of the NF-κB canonical signaling pathway.

  5. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

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    Guy-Armel Bounda

    2015-01-01

    Full Text Available Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01. Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

  6. Induction of G2/M arrest by pseudolaric acid B is mediated by activation of the ATM signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Ai-guo MENG; Ling-lingJIANG

    2009-01-01

    Aim: The aim of this study was to investigate the mechanism of pseudolaric acid B (PLAB)-induced cell cycle arrest in human melanoma SK-28 cells. Methods: Cell growth inhibition was detected by MTT assay, the cell cycle was analyzed by flow cytometry, and protein expression was examined by Western blot analysis.Results: PLAB inhibited the growth of human melanoma ceils and induced G2/M arrest in SK-28 cells, accompanied by an up-regulation of Cdc2 phosphorylation and a subsequent down-regulation of Cdc2 expression. Furthermore, PLAB decreased the expression of Cdc25C phosphatase and increased the expression of Wee1 kinase. Meanwhile, a reduction in Cdc2 activity was party due to induction of the expression of p21wsaf1/cip1 in a p53-dependent manner. In addition, PLAB activated the checkpoint kinase, Chk2, and increased the expression of p53, two major targets of ATM kinase. These effects were inhibited by caffeine, an ATM kinase inhibitor. We also found that PLAB significantly enhanced ATM kinase activity. Conclusion: Taken together, these results suggest that PLAB induced G2/M arrest in human melanoma cells via a mechanism involving the activation of ATM, and the effect of PLAB on Cdc2 activity was mediated via interactions with the Chk2-Cdc25C and p53 signalling pathways, two distinct downstream pathways of ATM. PLAB may be a promising chemopreventive agent for treating human melanoma.

  7. Carnosic acid attenuates acute ethanol-induced liver injury via a SIRT1/p66Shc-mediated mitochondrial pathway.

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    Tian, Xinyao; Hu, Yan; Li, Mingzhu; Xia, Kun; Yin, Jiye; Chen, Juan; Liu, Zhaoqian

    2016-04-01

    Ethanol-induced liver injury is associated with oxidative stress and hepatocyte apoptosis. We previously demonstrated that SIRT1/p66Shc pathway activation attenuates hepatocyte apoptosis in liver ischemia/reperfusion. The current study aimed to investigate whether carnosic acid (CA), a natural antioxidant, can inhibit acute ethanol-induced apoptosis of hepatocytes and to determine the effect of SIRT1/p66Shc on this process. Our results showed that CA pretreatment significantly reduced ethanol-induced histologic damage, serum aminotransferase activity, and oxidative stress in rats. Importantly, CA pretreatment increased SIRT1 expression following ethanol exposure. Furthermore, p66Shc expression was negatively correlated with SIRT1 expression. Consistent with the results demonstrating p66Shc inhibition, CA pretreatment inhibited the release of cytochrome C and apoptosis-inducing factor (AIF) from mitochondria. After exposing L02 cells to ethanol, the increased SIRT1 expression induced by CA was abrogated by pharmacologic SIRT1 inhibition or the use of siRNA against SIRT1. Additionally, SIRT1 inhibition significantly abrogated the suppression of p66Shc expression and mitochondrial translocation induced by CA. Accordingly, CA-induced decreases in the release of cytochrome C and AIF and in mitochondrial apoptosis were nearly abolished by SIRT1 knockdown. These data indicated that CA-activated SIRT1 is protective against ethanol treatment. In summary, CA attenuates acute ethanol-induced liver injury via a SIRT1/p66Shc-mediated mitochondrial pathway.

  8. The ROCK/GGTase Pathway Are Essential to the Proliferation and Differentiation of Neural Stem Cells Mediated by Simvastatin.

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    Zhang, Chan; Wu, Jian-Min; Liao, Min; Wang, Jun-Ling; Xu, Chao-Jin

    2016-12-01

    Simvastatin, a lipophilic and fermentation-derived natural statin, is reported to treat neurological disorders, such as traumatic brain injury, Parkinson's disease (PD), Alzheimer disease (AD), etc. Recently, research also indicated that simvastatin could promote regeneration in the dentate gyrus of adult mice by Wnt/β-catenin signaling (Robin et al. in Stem Cell Reports 2:9-17, 2014). However, the effect and mechanisms by which simvastatin may affect the neural stem cells (NSCs; from the embryonic day 14.5 (E14.5) SD rat brain) are not fully understood. Here, we investigated the effects of different doses of simvastatin on the survival, proliferation, differentiation, migration, and cell cycle of NSCs as well as underlying intracellular signaling pathways. The results showed that simvastatin not only inhibits the proliferation of NSCs but also enhances the βIII-tubulin(+) neuron differentiation rate. Additionally, we find that simvastatin could also promote NSC migration and induce cell cycle arrest at M2 phrase. All these effects of simvastatin on NSCs were mimicked with an inhibitor of Rho kinase (ROCK) and a specific inhibitor of geranylgeranyl transferase (GGTase). In conclusion, these data indicate that simvastatin could promote neurogenesis of neural stem cells, and these effects were mediated through the ROCK/GGTase pathway.

  9. Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells.

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    Alaoui-El-Azher, Mounia; Mans, Jeffrey J; Baker, Henry V; Chen, Casey; Progulske-Fox, Ann; Lamont, Richard J; Handfield, Martin

    2010-07-23

    The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

  10. Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells.

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    Mounia Alaoui-El-Azher

    Full Text Available The cytolethal distending toxin (CDT of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK to dissect the functional role of the ataxia telangiectasia mutated (ATM pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2, significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

  11. Zinc Prevents Abdominal Aortic Aneurysm Formation by Induction of A20-Mediated Suppression of NF-κB Pathway.

    Science.gov (United States)

    Yan, Ya-Wei; Fan, Jun; Bai, Shu-Ling; Hou, Wei-Jian; Li, Xiang; Tong, Hao

    2016-01-01

    Chronic inflammation and degradation of elastin are the main processes in the development of abdominal aortic aneurysm (AAA). Recent studies show that zinc has an anti-inflammatory effect. Based on these, zinc may render effective therapy for the treatment of the AAA. Currently, we want to investigate the effects of zinc on AAA progression and its related molecular mechanism. Rat AAA models were induced by periaortic application of CaCl2. AAA rats were treated by daily intraperitoneal injection of ZnSO4 or vehicle alone. The aorta segments were collected at 4 weeks after surgery. The primary rat aortic vascular smooth muscle cells (VSMCs) were stimulated with TNF-α alone or with ZnSO4 for 3 weeks. The results showed that zinc supplementation significantly suppressed the CaCl2-induced expansion of the abdominal aortic diameter, as well as a preservation of medial elastin fibers in the aortas. Zinc supplementation also obviously attenuated infiltration of the macrophages and lymphocytes in the aortas. In addition, zinc reduced MMP-2 and MMP-9 production in the aortas. Most importantly, zinc treatment significantly induced A20 expression, along with inhibition of the NF-κB canonical signaling pathway in vitro in VSMCs and in vivo in rat AAA. This study demonstrated, for the first time, that zinc supplementation could prevent the development of rat experimental AAA by induction of A20-mediated inhibition of the NF-κB canonical signaling pathway.

  12. Activation of mitochondria-mediated apoptotic pathway in tri-ortho-cresyl phosphate-induced delayed neuropathy.

    Science.gov (United States)

    Zou, Chaoshuang; Kou, Ruirui; Gao, Yuan; Xie, Keqin; Song, Fuyong

    2013-06-01

    Previous studies suggest that abnormal neurons death has been implicated in organophosphate-induced delayed neuropathy (OPIDN). However, the precise mechanism of neuronal death in OPIDN remains largely unknown. In this study, adult hens were treated with a dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP) by gavage, and then sacrificed on the time-points of 1, 5, 10, and 21 days after dosing TOCP, respectively. The apoptotic change of spinal cord neurons induced by TOCP was examined, and the role of mitochondria-mediated apoptosis of neurons during OPIDN was investigated. TUNEL assays showed that apoptotic neurons in hen spinal cords began to appear on day 5 following TOCP exposure. Immunohistochemistry and western blot analysis revealed a translocation of cytochrome C from mitochondria to cytoplasm after dosing TOCP. Moreover, the level of Bcl-2, Bcl-xl, Pro-caspase3 and Pro-caspase9 in hen spinal cord was significantly decreased, whereas that of Bax and cleaved-PARP was significantly elevated. Taken together, these findings indicate that the administration of TOCP can induce neuron apoptosis in hen spinal cords, which might be mediated by the activation of mitochondrial apoptotic pathway.

  13. Short-term psychosocial stress protects photoreceptors from damage via corticosterone-mediated activation of the AKT pathway.

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    Forkwa, Tembei K; Neumann, Inga D; Tamm, Ernst R; Ohlmann, Andreas; Reber, Stefan O

    2014-02-01

    Apoptotic death of photoreceptors in hereditary retinal degenerations can be prevented by neuroprotective molecules. Here, we report that adrenal glucocorticoids (GC) released during psychosocial stress protect photoreceptors from apoptosis after light damage. Psychosocial stress is known to be the main type of stressor humans are exposed to and was induced here in mice by 10h of chronic subordinate colony housing (CSC). Photoreceptor damage was generated by subsequent exposure to white light. Short-term psychosocial stress prior to illumination significantly reduced the number of apoptotic photoreceptors, an effect that was absent in adrenalectomized (ADX) mice. The neuroprotective effect was completely restored in ADX mice substituted with GC. Moreover, phosphorylation of retinal AKT increased following CSC or exogenous GC treatment, an effect that was again absent in ADX mice exposed to CSC. Finally, inhibition of AKT signaling with triciribine blocked the stress- and GC-mediated neuroprotective effects on photoreceptors. In summary, we provide evidence that 1) short-term psychosocial stress protects photoreceptors from light-induced damage and 2) the protective effect is most likely mediated by GC-induced activation of the AKT signaling pathway.

  14. Resveratrol inhibits Hexokinases II mediated glycolysis in non-small cell lung cancer via targeting Akt signaling pathway.

    Science.gov (United States)

    Li, Wei; Ma, Xiaoqian; Li, Na; Liu, Huasheng; Dong, Qiong; Zhang, Juan; Yang, Cejun; Liu, Yin; Liang, Qi; Zhang, Shengwang; Xu, Chang; Song, Wei; Tan, Shiming; Rong, Pengfei; Wang, Wei

    2016-12-10

    Deregulation of glycolysis was often observed in human cancer cells. In the present study, we reported resveratrol, a small polyphenol, which has been intensively studied in various tumor models, has a profound anti-tumor effect on human non-small cell lung cancer (NSCLC) via regulation of glycolysis. Resveratrol impaired hexokinase II (HK2)-mediated glycolysis, and markedly inhibited anchorage-dependent and -independent growth of NSCLC cells. Exposure to resveratrol decreased EGFR and downstream kinases Akt and ERK1/2 activation. Moreover, we revealed that resveratrol impaired glucose metabolism by mainly inhibiting expression of HK2 mediated by the Akt signaling pathway, and exogenous overexpression of constitutively activated Akt1 in NSCLC cells substantially rescued resveratrol-induced glycolysis suppression. The in vivo data indicated that resveratrol obviously suppressed tumor growth in a xenograft mouse model. Our results suggest targeting HK2 or metabolic enzymes appears to be a new approach for clinical NSCLC prevention or treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. LKB1 mediates the development of conventional and innate T cells via AMP-dependent kinase autonomous pathways.

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    Marouan Zarrouk

    Full Text Available The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK. The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.

  16. Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway.

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    Abhishek Mandal

    2016-01-01

    Full Text Available The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS, due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis.

  17. Atg7 Knockdown Augments Concanavalin A-Induced Acute Hepatitis through an ROS-Mediated p38/MAPK Pathway.

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    Yan Zhuang

    Full Text Available Concanavalin A (ConA, a T-cell mitogen that induces acute autoimmune hepatitis, is widely used to model pathophysiological processes of human acute autoimmune liver disease. Although autophagy has been extensively studied in the past decade, little is known about its molecular mechanism underlying the regulation of ConA-induced acute hepatitis. In this study, we used a Cre-conditional atg7 KO mouse to investigate the effects of Atg7-associated autophagy on ConA-induced murine hepatitis. Our results demonstrated that atg7 deficiency in mice enhanced macrophage activation and increased pro-inflammatory cytokines upon ConA stimulation. Atg7 silencing resulted in accumulation of dysfunctional mitochondria, disruption of reactive oxygen species (ROS degradation, and increase in pro-inflammatory cytokines in Raw264.7 cells. p38/MAPK and NF-κB levels were increased upon ConA induction due to Atg7 deficiency. Blocking ROS production inhibited ConA-induced p38/IκB phosphorylation and subsequent intracellular inflammatory responses. Hence, this study demonstrated that atg7 knockout in mice or Atg7 knockdown in cell culture augmented ConA-induced acute hepatitis and related cellular malfunction, indicating protective effects of Atg7 on regulating mitochondrial ROS via a p38/MAPK-mediated pathway. Collectively, our findings reveal that autophagy may attenuate macrophage-mediated inflammatory response to ConA and may be the potential therapeutic targets for acute liver injury.

  18. Cholesterol secoaldehyde induces apoptosis in J774 macrophages via mitochondrial pathway but not involving reactive oxygen species as mediators.

    Science.gov (United States)

    Gao, Xueli; Raghavamenon, Achuthan C; D'Auvergne, Oswald; Uppu, Rao M

    2009-11-13

    Cholesterol secoaldehyde (3beta-hydroxy-5-oxo-5,6-secocholestan-6-al or ChSeco) is an oxysterol known to be formed in reactions of ozone with cholesterol and also when cholesterol-5alpha-hydroperoxide undergoes Hock cleavage. In view of its widespread occurrence and atherogenic potential, we examined the effects of ChSeco on mouse J774 macrophage viability and events associated with apoptosis. A dose-dependent decrease in cell viability, disruptions in mitochondrial transmembrane potential (64+/-5.5%; mean+/-SD, n=3), increased levels of cytosolic cytochrome c (8.8+/-0.84 ng/ml; mean+/-SD, n=3), activation of caspase-3 (ca. 3.6-fold) and caspase-9 (ca.1.8-fold), and increased DNA fragmentation (ca. 5-fold), all indicative of apoptosis, were observed in response to exposure to ChSeco. The apoptotic nature of cell death in macrophages was confirmed by dual staining with acridine orange and ethidium bromide. However, unlike the case with cardiomyoblasts and neuronal cells, the apoptotic process in these immune cells was not mediated by increased levels of reactive oxygen species as indicated by a minimal or no increase in 2',7'-dichlorofluorescein fluorescence. It is suggested that the apoptotic process is mediated via the mitochondrial pathway and that ChSeco formed in biological environments contributes to the initiation, progression, and culmination of atherosclerotic plaque formation, as these processes are critically dependent on macrophage apoptosis.

  19. Fungus induces the release of IL- 8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways

    Institute of Scientific and Technical Information of China (English)

    Xu-Dong; Peng; Gui-Qiu; Zhao; Jing; Lin; Nan; Jiang; Qiang; Xu; Cheng-Cheng; Zhu; Jian-Qiu; Qu; Lin; Cong; Hui; Li

    2015-01-01

    AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.

  20. Glucose deprivation induces reticulum stress by the PERK pathway and caspase-7- and calpain-mediated caspase-12 activation.

    Science.gov (United States)

    de la Cadena, Selene García; Hernández-Fonseca, Karla; Camacho-Arroyo, Ignacio; Massieu, Lourdes

    2014-03-01

    Glucose is the main energy source in brain and it is critical for correct brain functioning. Type 1 diabetic patients might suffer from severe hypoglycemia if exceeding insulin administration, which can lead to acute brain injury if not opportunely corrected. The mechanisms leading to hypoglycemic brain damage are not completely understood and the role of endoplasmic reticulum (ER) stress has not been studied. ER stress resulting from the accumulation of unfolded or misfolded proteins in the ER is counteracted by the unfolded protein response (UPR). When the UPR is sustained, apoptotic death might take place. We have examined UPR activation during glucose deprivation (GD) in hippocampal cultured neurons and its role in the induction of apoptosis. Activation of the PERK pathway of the UPR was observed, as increased phosphorylation of eIF2α and elevated levels of the transcription factor ATF4, occurred 30 min after GD and the levels of the chaperone protein, GRP78 and the transcription factor CHOP, increased after 2 h of GD. In addition, we observed an early activation of caspase-7 and 12 during GD, while caspase-3 activity increased only transiently during glucose reintroduction. Inhibition of caspase-3/7 and the calcium-dependent protease, calpain, significantly decreased caspase-12 activity. The ER stress inhibitor, salubrinal prevented neuronal death and caspase-12 activity. Results suggest that the PERK pathway of the UPR is involved in GD-induced apoptotic neuronal death through the activation of caspase-12, rather than the mitochondrial-dependent caspase pathway. In addition, we show that calpain and caspase-7 are soon activated after GD and mediate caspase-12 activation and neuronal death.

  1. Dectin-1-Mediated Pathway Contributes to Fusarium proliferatum-Induced CXCL-8 Release from Human Respiratory Epithelial Cells

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    Chang-Ching Yeh

    2017-03-01

    Full Text Available Fusarium species are causative agents of human respiratory disorders and are distributed widely in our environment. Little is known of their interaction with human respiratory epithelial cells, which may contribute to allergic airway responses. In this study, we report on the release of C–X–C motif chemokine ligand 8 (CXCL-8 from human bronchial epithelial BEAS-2B cells upon stimulation with Fusarium proliferatum extracts. F. proliferatum-induced cytokine release from BEAS-2B cells was determined by cytokine array and CXCL-8 enzyme-linked immunosorbent assay (ELISA kits. Blocking antibodies and signaling pathway inhibitors were employed to delineate cell surface receptors and signaling pathways participating in CXCL-8 release. F. proliferatum extracts induced the release of CXCL-8 in a time-dependent manner. The dectin-1 receptor ligands, curdlan and laminarin, reduced CXCL-8 release. Cells pre-treated with anti-Dectin-1 antibodies (2 µg/mL decreased CXCL-8 release by 24%. Furthermore, F. proliferatum-stimulated CXCL-8 release was reduced by 32%, 53%–81%, 40% and 26% after BEAS-2B cells were pretreated with activation inhibitors of spleen tyrosine kinase (Syk—piceatannol—, mitogen-activated protein kinases (MAPKs—PD98059, U0126, SB202190, SP600125—, phosphatidylinositol-3-kinase (PI3K—LY294002—and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB—BAY117082—, respectively. These results suggest that Dectin-1-mediated activation of the Syk, MAPKs, PI3K and NF-κB signaling pathways contributes to F. proliferatum-stimulated CXCL-8 release from BEAS-2B cells and provides an important basis for developing novel therapeutic strategies in clinical allergy.

  2. Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway.

    Science.gov (United States)

    Zhao, Haosen; Chen, Shurui; Gao, Kai; Zhou, Zipeng; Wang, Chen; Shen, Zhaoliang; Guo, Yue; Li, Zhuo; Wan, Zhanghui; Liu, Chang; Mei, Xifan

    2017-04-21

    on SCI by regulating autophagy and apoptosis mediated by the SIRT1-AMPK signaling pathway. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Anti-inflammatory effect of honokiol is mediated by PI3K/Akt pathway suppression1

    Institute of Scientific and Technical Information of China (English)

    Byung Hun KIM; Jae Youl CHO

    2008-01-01

    Aim: In this study, we investigated the regulatory effects of honokiol on various inflammatory events mediated by monocytes/macrophages (U937/RAW264.7 cells)and lymphocytes (splenic lymphocytes and CTLL-2 cells) and their putative ac-tion mechanism. Methods: In order to investigate the regulatory effects, various cell lines and primary cells (U937, RAW264.7, CTLL-2 cells, and splenic lymphocytes) were employed and various inflammatory events, such as the pro-duction of inflammatory mediators, cell adhesion, cell proliferation, and the early signaling cascade, were chosen. Results: Honokiol strongly inhibited various inflammatory responses, such as: (ⅰ) the upregulation of nitric oxide (NO), pros-taglandin.E2 and TNF-α production and costimulatory molecule CD80 induced by lipopolysaccharide (LPS); (ⅱ) the functional activation of β1-integrin (CD29) as-sessed by U937 cell-cell and cell-fibronectin adhesions; (ⅲ) the enhancement of lymphocytes and CD8+CTLL-2 cell proliferation stimulated by LPS, phytohemaglutinin A (PHA), and concanavalin A or interleukin (IL)-2; and (ⅳ) the transcriptional upregulation of inducible NO synthase, TNF-α, cyclooxygenase-2, IL-12, and monocyte chemoattractant protein (MCP)-1. These anti-inflammatory effects of honokiol seem to be mediated by interrupting the early activated intra-cellular signaling molecule phosphoinositide 3-kinase (PI3K)/Akt, but not Src, the extracellular signal-regulated kinase, and p38, according to pharmacological, biochemical, and functional analyses. Conclusion: These results suggest that honokiol may act as a potent anti-inflammatory agent with multipotential activities due to an inhibitory effect on the PI3K/Akt pathway.

  4. In-cell infection: a novel pathway for Epstein-Barr virus infection mediated by cell-in-cell structures.

    Science.gov (United States)

    Ni, Chao; Chen, Yuhui; Zeng, Musheng; Pei, Rongjuan; Du, Yong; Tang, Linquan; Wang, Mengyi; Hu, Yazhuo; Zhu, Hanyu; He, Meifang; Wei, Xiawei; Wang, Shan; Ning, Xiangkai; Wang, Manna; Wang, Jufang; Ma, Li; Chen, Xinwen; Sun, Qiang; Tang, Hong; Wang, Ying; Wang, Xiaoning

    2015-07-01

    Epstein-Barr virus (EBV) can infect both susceptible B lymphocytes and non-susceptible epithelial cells (ECs). Viral tropism analyses have revealed two intriguing means of EBV infection, either by a receptor-mediated infection of B cells or by a cell-to-cell contact-mediated infection of non-susceptible ECs. Herein, we report a novel "in-cell infection" mechanism for EBV infection of non-susceptible ECs through the formation of cell-in-cell structures. Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro. Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma. Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells. Significantly, EBV generated from in-cell infected ECs displayed altered tropism with higher infection efficacy to both B cells and ECs. In addition to CNE-2 tumor cells, cell-in-cell structure formation could also mediate EBV infection of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified "in-cell infection" as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that "in-cell infection" is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs.

  5. Apocynin inhibits Toll-like receptor-4-mediated activation of NF-κB by suppressing the Akt and mTOR pathways.

    Science.gov (United States)

    Nam, Yoon Jeong; Kim, Arum; Sohn, Dong Suep; Lee, Chung Soo

    2016-12-01

    Microbial product lipopolysaccharide has been shown to be involved in the pathogenesis of inflammatory skin diseases. Apocynin has demonstrated to have an anti-inflammatory effect. However, the effect of apocynin on the Toll-like receptor-4-dependent activation of Akt, mammalian target of rapamycin (mTOR), and nuclear factor (NF)-κB pathway, which is involved in productions of inflammatory mediators in keratinocytes, has not been studied. Using human keratinocytes, we investigated the effect of apocynin on the inflammatory mediator production in relation to the Toll-like receptor-4-mediated-Akt/mTOR and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Apocynin, Akt inhibitor SH-5, Bay 11-7085 and N-acetylcysteine each attenuated the lipopolysaccharide-induced production of cytokines, PGE2, and chemokines, changes in the levels of Toll-like receptor-4, p-Akt, mTOR, and NF-κB, and production of reactive oxygen species in keratinocytes. The results show that apocynin appears to attenuate the lipopolysaccharide-stimulated production of inflammatory mediators in keratinocytes by suppressing the Toll-like receptor-4-mediated activation of the Akt, mTOR, and NF-κB pathways. The effect of apocynin appears to be attributed to its inhibitory effect on the production of reactive oxygen species. Apocynin appears to attenuate the microbial product-mediated inflammatory skin diseases.

  6. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation.

    Science.gov (United States)

    Parl, Angelika; Mitchell, Sabrina L; Clay, Hayley B; Reiss, Sara; Li, Zhen; Murdock, Deborah G

    2013-11-15

    Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  7. Naja nigricollis CMS-9 enhances the mitochondria-mediated death pathway in adaphostin-treated human leukaemia U937 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Wang, Jeh-Jeng; Chang, Long-Sen

    2011-11-01

    1. The aim of the present study was to explore the effect of the Naja nigricollis phospholipase A(2) CMS-9 on adaphostin-induced death of human leukaemia U937 cells. 2. Leukaemia U937 cells (Bcr/Abl-negative cells) were treated with adaphostin (0-10 μmol/L) and CMS-9 (0-1 μmol/L). The effects of CMS-9, adaphostin and their combination on cell viability, the generation reactive oxygen species (ROS), [Ca(2+) ](i) , p38 mitogen-activated protein kinase (MAPK) activation, Akt and extracellular signal-regulated kinase (ERK) inactivation, mitochondrial membrane potential (ΔΨ(m) ) and Bcl-2 family proteins were analysed. 3. Both adaphostin and CMS-9 induced U937 cell apoptosis, characterized by dissipation of ΔΨ(m) and ROS generation. Combined treatment further increased ΔΨ(m) loss and reduced the viability of adaphostin-treated cells. Unlike in CMS-9-treated cells, in adaphostin-treated cells ROS-induced increases in [Ca(2+) ](i) were observed. CMS-9-induced ROS generation resulted in p38 MAPK activation, whereas adaphostin treatment elicited ROS/Ca(2+) -mediated inactivation of Akt and ERK. Moreover, Akt was found to be involved in ERK phosphorylation. Suppression of p38 MAPK activation blocked CMS-9-induced ΔΨ(m) loss and Bcl-xL downregulation. Overexpression of constitutively active Akt and mitogen-activated protein kinase kinase (MEK) 1 rescued adaphostin-induced ΔΨ(m) loss and Bcl-2 downregulation. Similarly, CMS-9 augmented adaphostin toxicity in human leukaemia K562 cells via increased mitochondrial alterations. 4. The results suggest that two distinct pathways mediate adaphostin- and CMS-9-induced mitochondrial damage (i.e. the ROS-Ca(2+) -Akt-ERK and ROS-p38 MAPK pathways, respectively). These distinct pathway explain the augmentation by CMS-9 of ΔΨ(m) loss and apoptosis in adaphostin-treated U937 cells.

  8. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    Energy Technology Data Exchange (ETDEWEB)

    Zaja, Ivan [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bai, Xiaowen, E-mail: xibai@mcw.edu [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bosnjak, Zeljko J. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States)

    2014-10-31

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  9. MicroRNA-24/MODY gene regulatory pathway mediates pancreatic β-cell dysfunction.

    Science.gov (United States)

    Zhu, Yunxia; You, Weiyan; Wang, Hongdong; Li, Yating; Qiao, Nan; Shi, Yuguang; Zhang, Chenyu; Bleich, David; Han, Xiao

    2013-09-01

    Overnutrition and genetics both contribute separately to pancreatic β-cell dysfunction, but how these factors interact is unclear. This study was aimed at determining whether microRNAs (miRNAs) provide a link between these factors. In this study, miRNA-24 (miR-24) was highly expressed in pancreatic β-cells and further upregulated in islets from genetic fatty (db/db) or mice fed a high-fat diet, and islets subject to oxidative stress. Overexpression of miR-24 inhibited insulin secretion and β-cell proliferation, potentially involving 351 downregulated genes. By using bioinformatic analysis combined with luciferase-based promoter activity assays and quantitative real-time PCR assays, we identified two maturity-onset diabetes of the young (MODY) genes as direct targets of miR-24. Silencing either of these MODY genes (Hnf1a and Neurod1) mimicked the cellular phenotype caused by miR-24 overexpression, whereas restoring their expression rescued β-cell function. Our findings functionally link the miR-24/MODY gene regulatory pathway to the onset of type 2 diabetes and create a novel network between nutrient overload and genetic diabetes via miR-24.

  10. Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}-dependent signaling pathways.

    Science.gov (United States)

    Wu, Joy Y; Purton, Louise E; Rodda, Stephen J; Chen, Min; Weinstein, Lee S; McMahon, Andrew P; Scadden, David T; Kronenberg, Henry M

    2008-11-04

    Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein alpha subunit G(s)alpha is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of G(s)alpha early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking G(s)alpha in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in G(s)alpha-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that G(s)alpha-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner.

  11. Osteoblastic regulation of B lymphopoiesis is mediated by Gsα-dependent signaling pathways

    Science.gov (United States)

    Wu, Joy Y.; Purton, Louise E.; Rodda, Stephen J.; Chen, Min; Weinstein, Lee S.; McMahon, Andrew P.; Scadden, David T.; Kronenberg, Henry M.

    2008-01-01

    Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein α subunit Gsα is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of Gsα early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking Gsα in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in Gsα-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that Gsα-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner. PMID:18957542

  12. The new base excision repair pathway in mammals mediated by tyrosyl-DNA-phosphodiesterase 1

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Human tyrosyl-DNA phosphodiesterase 1 (Tdp1 hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety and has been implicated in the repair of Topoisomerase I (TopI-DNA covalent complexes. Tdp1 can also hydrolyze other 3' end DNA alterations including 3' phosphoglycolate and 3' abasic (AP sites, and exhibits the 3' nucleosidase activity indicating that it may function as a general 3' end-processing DNA repair enzyme. Recently we have shown a new Tdp1 activity generating DNA strand break with the 3' phosphate termini from the AP site. AP sites are formed spontaneously and are inevitable intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic, and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair, initiated by DNA glycosylases performing beta, delta-elimination cleavage of the AP sites, has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites that is initiated by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap.

  13. Mechanism of Anti-glioblastoma Effect of Temzolomide Involved in ROS-Mediated SIRT 1 Pathway

    Directory of Open Access Journals (Sweden)

    Yuan Jiang

    2014-03-01

    Full Text Available Objective: To explore the new molecular mechanism of anti-tumor effect of temzolomide (TMZon glioblastoma cell strain. Methods: MTT methods and Hoechst 33342 staining method were applied to determine the effect of TMZ on the proliferation and apoptosis of glioblastoma cell strains U251 and SHG44, while flow cytometry was used to detect the impact of TMZ on cellular cycles. Additionally, DCFH-DA probe was adopted to test intracellular reactive oxygen species (ROS level while Real-time PCR and Western blot tests were applied to determine the influence of TMZ on SIRT1 expression. Results: TMZ in different concentrations added into glioblastoma cell strain for 72 h could concentration-dependently inhibit the proliferation of glioblastoma cells, 100 μmol/L of which could also block cells in phase G2/M and improve cellular apoptosis. In addition, TMZ could evidently increase intracellular ROS level so as to activate SIRT1. Conclusion: The mechanism of anti-tumor effect of TMZ on glioblastoma may be associated with ROS-induced SIRT1 pathway, providing theoretical basis for the clinical efficacy of TMZ.

  14. Mechanism of Anti-glioblastoma Effect of Temzolomide Involved in ROS-Mediated SIRT 1 Pathway

    Institute of Scientific and Technical Information of China (English)

    Jiang Yuan; Sun Yan; Yuan Yuan

    2014-01-01

    Objective:To explore the new molecular mechanism of anti-tumor effect of temzolomide (TMZ) on glioblastoma cell strain. Methods:MTT methods and Hoechst 33342 staining method were applied to determine the effect of TMZ on the proliferation and apoptosis of glioblastoma cell strains U251 and SHG44, while lfow cytometry was used to detect the impact of TMZ on cellular cycles. Additionally, DCFH-DA probe was adopted to test intracellular reactive oxygen species (ROS) level while Real-time PCR and Western blot tests were applied to determine the inlfuence of TMZ on SIRT1 expression. Results: TMZ in different concentrations added into glioblastoma cell strain for 72 h could concentration-dependently inhibit the proliferation of glioblastoma cells, 100 μmol/L of which could also block cells in phase G2/M and improve cellular apoptosis. In addition, TMZ could evidently increase intracellular ROS level so as to activate SIRT1. Conclusion: The mechanism of anti-tumor effect of TMZ on glioblastoma may be associated with ROS-induced SIRT1 pathway, providing theoretical basis for the clinical efifcacy of TMZ.

  15. Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference.

    Science.gov (United States)

    Li, Xiaoxue; Dong, Xiaolong; Zou, Cong; Zhang, Hongyu

    2015-03-03

    RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells.

  16. Elongation factor P mediates a novel post-transcriptional regulatory pathway critical for bacterial virulence

    DEFF Research Database (Denmark)

    Zou, S Betty; Roy, Hervé; Ibba, Michael;

    2012-01-01

    Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability of the path......Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability...... of the pathogen to respond to external cues are typically attenuating. Here we discuss our recent discovery of a novel post-transcriptional regulatory pathway critical for Salmonella virulence and stress resistance. The enzymes PoxA and YjeK coordinately attach a unique beta-amino acid onto a highly conserved...... our laboratory and others now suggests that EF-P, previously thought to be essential, instead plays an ancillary role in translation by regulating the synthesis of a relatively limited subset of proteins. Other observations suggest that the eukaryotic homolog of EF-P, eIF5A, may illicit similar...

  17. Polysaccharide of Dendrobium huoshanense activates macrophages via toll-like receptor 4-mediated signaling pathways.

    Science.gov (United States)

    Xie, Song-Zi; Hao, Ran; Zha, Xue-Qiang; Pan, Li-Hua; Liu, Jian; Luo, Jian-Ping

    2016-08-01

    The present work aimed at investigating the pattern recognition receptor (PRR) and immunostimulatory mechanism of a purified Dendrobium huoshanense polysaccharide (DHP). We found that DHP could bind to the surface of macrophages and stimulate macrophages to secrete NO, TNF-α and IL-1β. To unravel the mechanism for the binding of DHP to macrophages, flow cytometry, confocal laser-scanning microscopy, affinity electrophoresis, SDS-PAGE and western blotting were employed to verify the type of PRR responsible for the recognition of DHP by RAW264.7 macrophages and peritoneal macrophages of C3H/HeN and C3H/HeJ macrophages. Results showed that toll-like receptor 4 (TLR4) was an essential receptor for macrophages to directly bind DHP. Further, the phosphorylation of ERK, JNK, Akt and p38 were observed to be time-dependently promoted by DHP, as well as the nuclear translocation of NF-κB p65. These results suggest that DHP activates macrophages via its direct binding to TLR4 to trigger TLR4 signaling pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Activation and Inactivation of Pseudomonas stutzeri Methylbenzene Catabolism Pathways Mediated by a Transposable Element

    Science.gov (United States)

    Bolognese, Fabrizio; di Lecce, Cinzia; Galli, Enrica; Barbieri, Paola

    1999-01-01

    The arrangement of the genes involved in o-xylene, m-xylene, and p-xylene catabolism was investigated in three Pseudomonas stutzeri strains: the wild-type strain OX1, which is able to grow on o-xylene but not on the meta and para isomers; the mutant M1, which grows on m-xylene and p-xylene but is unable to utilize the ortho isomer; and the revertant R1, which can utilize all the three isomers of xylene. A 3-kb insertion sequence (IS) termed ISPs1, which inactivates the m-xylene and p-xylene catabolic pathway in P. stutzeri OX1 and the o-xylene catabolic genes in P. stutzeri M1, was detected. No IS was detected in the corresponding catabolic regions of the P. stutzeri R1 genome. ISPs1 is present in several copies in the genomes of the three strains. It is flanked by 24-bp imperfect inverted repeats, causes the direct duplication of 8 bp in the target DNA, and seems to be related to the ISL3 family. PMID:10223973

  19. Atractylenolide I-mediated Notch pathway inhibition attenuates gastric cancer stem cell traits.

    Science.gov (United States)

    Ma, Li; Mao, Rurong; Shen, Ke; Zheng, Yuanhong; Li, Yueqi; Liu, Jianwen; Ni, Lei

    2014-07-18

    Atractylenolide I (AT-I), one of the main naturally occurring compounds of Rhizoma Atractylodis Macrocephalae, has remarkable anti-cancer effects on various cancers. However, its effects on the treatment of gastric cancer remain unclear. Via multiple cellular and molecular approaches, we demonstrated that AT-I could potently inhibit cancer cell proliferation and induce apoptosis through inactivating Notch pathway. AT-I treatment led to the reduction of expressions of Notch1, Jagged1, and its downstream Hes1/ Hey1. Our results showed that AT-I inhibited the self-renewal capacity of gastric stem-like cells (GCSLCs) by suppression of their sphere formation capacity and cell viability. AT-I attenuated gastric cancer stem cell (GCSC) traits partly through inactivating Notch1, leading to reducing the expressions of its downstream target Hes1, Hey1 and CD44 in vitro. Collectively, our results suggest that AT-I might develop as a potential therapeutic drug for the treatment of gastric cancer.

  20. Functional identification of gene cluster for the aniline metabolic pathway mediated by transposable element

    Institute of Scientific and Technical Information of China (English)

    LIANG Quanfeng; Takeo Masahiro; LIN Min; CHEN Ming; XU Yuquan; ZHANG Wei; PING Shuzhen; LU Wei; SONG Xianlong; WANG Weiwei; GENG Lizhao

    2005-01-01

    A convenient and widely applicable method has been developed to clone aniline metabolic gene cluster in this study. Three positive recombinant plasmids pDA1, pDB2 and pDB11 were cloned from genomic library of aniline degradation strain AD9. The result of aniline dioxygenase (AD) activity and catechol 2,3-oxygenase (C23O) activity assay showed that pDA1 and pDB11 contain aniline dioxygenase genes and catechol 2,3-dioxygenase genes, respectively. The sequence analysis of the total 24.7-kb region revealed that this region contains 25 ORFs, of which 17 genes involve metabolism of aniline. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR1) were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode meta- cleavage pathway enzymes for catechol degradation. The gene cluster was surrounded by two IS1071 sequences.

  1. Nonadiabatic dissociation dynamics in H2O: Competition between rotationally and nonrotationally mediated pathways

    Science.gov (United States)

    Yuan, Kaijun; Cheng, Yuan; Cheng, Lina; Guo, Qing; Dai, Dongxu; Wang, Xiuyan; Yang, Xueming; Dixon, Richard N.

    2008-01-01

    The photochemistry of H2O in the VUV region is important in interstellar chemistry. Whereas previous studies of the photodissociation used excitation via unbound states, we have used a tunable VUV photolysis source to excite individual levels of the rotationally structured C̃ state near 124 nm. The ensuing OH product state distributions were recorded by using the H-atom Rydberg tagging technique. Experimental results indicate a dramatic variation in the OH product state distributions and its stereodynamics for different resonant states. Photodissociation of H2O(C̃) in rotational states with k′a = 0 occurs exclusively through a newly discovered homogeneous coupling to the à state, leading to OH products that are vibrationally hot (up to v = 13), but rotationally cold. In contrast, for H2O in rotationally excited states with k′a > 0, an additional pathway opens through Coriolis-type coupling to the B̃ state surface. This yields extremely rotationally hot and vibrationally cold ground state OH(X) and electronically excited OH(A) products, through 2 different mechanisms. In the case of excitation via the 110 ← 000 transition the H atoms for these 2 product channels are ejected in completely different directions. Quantum dynamical models for the C̃-state photodissociation clearly support this remarkable dynamical picture, providing a uniquely detailed illustration of nonadiabatic dynamics involving at least 4 electronic surfaces. PMID:19047628

  2. Exosome proteomics reveals transcriptional regulator proteins with potential to mediate downstream pathways.

    Science.gov (United States)

    Ung, Timothy H; Madsen, Helen J; Hellwinkel, Justin E; Lencioni, Alex M; Graner, Michael W

    2014-11-01

    Exosomes are virus-sized, membrane-enclosed vesicles with origins in the cellular endosomal system, but are released extracellularly. As a population, these tiny vesicles carry relatively enormous amounts of information in their protein, lipid and nucleic acid content, and the vesicles can have profound impacts on recipient cells. This review employs publically-available data combined with gene ontology applications to propose a novel concept, that exosomes transport transcriptional and translational machinery that may have direct impacts on gene expression in recipient cells. Here, we examine the previously published proteomic contents of medulloblastoma-derived exosomes, focusing on transcriptional regulators; we found that there are numerous proteins that may have potential roles in transcriptional and translational regulation with putative influence on downstream, cancer-related pathways. We expanded this search to all of the proteins in the Vesiclepedia database; using gene ontology approaches, we see that these regulatory factors are implicated in many of the processes involved in cancer initiation and progression. This information suggests that some of the effects of exosomes on recipient cells may be due to the delivery of protein factors that can directly and fundamentally change the transcriptional landscape of the cells. Within a tumor environment, this has potential to tilt the advantage towards the cancer.

  3. WNK4 enhances the degradation of NCC through a sortilin-mediated lysosomal pathway.

    Science.gov (United States)

    Zhou, Bo; Zhuang, Jieqiu; Gu, Dingying; Wang, Hua; Cebotaru, Liudmila; Guggino, William B; Cai, Hui

    2010-01-01

    WNK kinase is a serine/threonine kinase that plays an important role in electrolyte homeostasis. WNK4 significantly inhibits the surface expression of the sodium chloride co-transporter (NCC) by enhancing the degradation of NCC through a lysosomal pathway, but the mechanisms underlying this trafficking are unknown. Here, we investigated the effect of the lysosomal targeting receptor sortilin on NCC expression and degradation. In Cos-7 cells, we observed that the presence of WNK4 reduced the steady-state amount of NCC by approximately half. Co-transfection with truncated sortilin (a dominant negative mutant) prevented this WNK4-induced reduction in NCC. NCC immunoprecipitated with both wild-type sortilin and, to a lesser extent, truncated sortilin. Immunostaining revealed that WNK4 increased the co-localization of NCC with the lysosomal marker cathepsin D, and NCC co-localized with wild-type sortilin, truncated sortilin, and WNK4 in the perinuclear region. These findings suggest that WNK4 promotes NCC targeting to the lysosome for degradation via a mechanism involving sortilin.

  4. Effects of an invasive plant transcend ecosystem boundaries through a dragonfly-mediated trophic pathway.

    Science.gov (United States)

    Burkle, Laura A; Mihaljevic, Joseph R; Smith, Kevin G

    2012-12-01

    Trophic interactions can strongly influence the structure and function of terrestrial and aquatic communities through top-down and bottom-up processes. Species with life stages in both terrestrial and aquatic systems may be particularly likely to link the effects of trophic interactions across ecosystem boundaries. Using experimental wetlands planted with purple loosestrife (Lythrum salicaria), we tested the degree to which the bottom-up effects of floral density of this invasive plant could trigger a chain of interactions, changing the behavior of terrestrial flying insect prey and predators and ultimately cascading through top-down interactions to alter lower trophic levels in the aquatic community. The results of our experiment support the linkage of terrestrial and aquatic food webs through this hypothesized pathway, with high loosestrife floral density treatments attracting high levels of visiting insect pollinators and predatory adult dragonflies. High floral densities were also associated with increased adult dragonfly oviposition and subsequently high larval dragonfly abundance in the aquatic community. Finally, high-flower treatments were coupled with changes in zooplankton species richness and shifts in the composition of zooplankton communities. Through changes in animal behavior and trophic interactions in terrestrial and aquatic systems, this work illustrates the broad and potentially cryptic effects of invasive species, and provides additional compelling motivation for ecologists to conduct investigations that cross traditional ecosystem boundaries.

  5. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    Science.gov (United States)

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin

    2017-01-25

    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  6. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

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    Luigi Bozzo

    Full Text Available Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM. To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM or switched to different glucose concentrations (0.5 or 10 mM. None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  7. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    Science.gov (United States)

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  8. Genetic depletion of brain 5HT reveals a common molecular pathway mediating compulsivity and impulsivity.

    Science.gov (United States)

    Angoa-Pérez, Mariana; Kane, Michael J; Briggs, Denise I; Sykes, Catherine E; Shah, Mrudang M; Francescutti, Dina M; Rosenberg, David R; Thomas, David M; Kuhn, Donald M

    2012-06-01

    Neuropsychiatric disorders characterized by behavioral disinhibition, including disorders of compulsivity (e.g. obsessive-compulsive disorder; OCD) and impulse-control (e.g. impulsive aggression), are severe, highly prevalent and chronically disabling. Treatment options for these diseases are extremely limited. The pathophysiological bases of disorders of behavioral disinhibition are poorly understood but it has been suggested that serotonin dysfunction may play a role. Mice lacking the gene encoding brain tryptophan hydroxylase 2 (Tph2-/-), the initial and rate-limiting enzyme in the synthesis of serotonin, were tested in numerous behavioral assays that are well known for their utility in modeling human neuropsychiatric diseases. Mice lacking Tph2 (and brain 5HT) show intense compulsive and impulsive behaviors to include extreme aggression. The impulsivity is motor in form and not cognitive because Tph2-/- mice show normal acquisition and reversal learning on a spatial learning task. Restoration of 5HT levels by treatment of Tph2-/- mice with its immediate precursor 5-hydroxytryptophan attenuated compulsive and impulsive-aggressive behaviors. Surprisingly, in Tph2-/- mice, the lack of 5HT was not associated with anxiety-like behaviors. The results indicate that 5HT mediates behavioral disinhibition in the mammalian brain independent of anxiogenesis.

  9. Th17 pathway-mediated immunopathogenesis of schizophrenia: mechanisms and implications.

    Science.gov (United States)

    Debnath, Monojit; Berk, Michael

    2014-11-01

    Schizophrenia is a highly complex and severe neuropsychiatric disorder with an unknown etiopathology. Evidence for a dysregulated immune system in both the risk for and progression of schizophrenia has recently been overwhelming. Importantly, chronic low-grade inflammation both in the periphery and central nervous system has been shown to contribute predominantly to the pathogenesis of schizophrenia in a subset of individuals. Inflammation in the central nervous system is mediated by a range of proinflammatory cytokines, resident immune cells such as microglia, and brain infiltrating peripheral immunocompetent cells, such as T lymphocytes. Recently, Th17 cells, a subset of T helper cells have emerged as crucial players in mucosal defense against infections. It is linked to atopic, inflammatory, and autoimmune disorders. The risk factors/mechanisms leading to low-grade inflammation in schizophrenia are diverse and include infectious agents, stress, trauma, environmental toxins, genetic vulnerability, physical inactivity, obesity, poor diet, and sleep disruption. Herein, we propose that fetal programming of cellular immune components driven by intrauterine adversity can lead to the generation of long-lasting effector/memory Th17 cells. Th17 cells can disrupt the blood-brain barrier, infiltrate the central nervous system, and, along with other cytokines and microglia, lead to neuroprogression through neuroinflammation in schizophrenia. © The Author 2014. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. Rapid α-oligomer formation mediated by the Aβ C terminus initiates an amyloid assembly pathway

    Science.gov (United States)

    Misra, Pinaki; Kodali, Ravindra; Chemuru, Saketh; Kar, Karunakar; Wetzel, Ronald

    2016-01-01

    Since early oligomeric intermediates in amyloid assembly are often transient and difficult to distinguish, characterize and quantify, the mechanistic basis of the initiation of spontaneous amyloid growth is often opaque. We describe here an approach to the analysis of the Aβ aggregation mechanism that uses Aβ-polyglutamine hybrid peptides designed to retard amyloid maturation and an adjusted thioflavin intensity scale that reveals structural features of aggregation intermediates. The results support an aggregation initiation mechanism for Aβ-polyQ hybrids, and by extension for full-length Aβ peptides, in which a modular Aβ C-terminal segment mediates rapid, non-nucleated formation of α-helical oligomers. The resulting high local concentration of tethered amyloidogenic segments within these α-oligomers facilitates transition to a β-oligomer population that, via further remodelling and/or elongation steps, ultimately generates mature amyloid. Consistent with this mechanism, an engineered Aβ C-terminal fragment delays aggregation onset by Aβ-polyglutamine peptides and redirects assembly of Aβ42 fibrils. PMID:27546208

  11. Redundant Vasodilator Pathways Underlying Radial Artery Flow-Mediated Dilation Are Preserved in Healthy Aging

    Directory of Open Access Journals (Sweden)

    Kevin D. Ballard

    2014-01-01

    Full Text Available Background. Blocking nitric oxide (NO and vasodilator prostanoids (PN does not consistently reduce flow-mediated dilation (FMD in young adults. The impact of aging on the contribution of NO and PG to FMD is unknown. Methods. FMD was measured in older adults (n=10, 65±3 y after arterial infusion of saline, N(G-monomethyl-L-arginine (L-NMMA, and ketorolac + L-NMMA. Data were compared to published data in young adults. Results. L-NMMA reduced FMD in older adults (8.9±3.6 to 5.9±3.7% although this was not statistically significant (P=0.08 and did not differ (P=0.74 from the reduction observed in young adults (10.0±3.8 to 7.6±4.7%; P=0.03. Blocking PN did not affect FMD in young or older adults. In older adults, L-NMMA reduced (n=6; range = 36–123% decrease, augmented (n=3; 10–122% increase, or did not change FMD (n=1; 0.4% increase. After PN blockade, FMD responses were reduced (n=2, augmented (n=6, or unaffected (n=1. Conclusions. NO or PN blockade did not consistently reduce FMD in healthy older adults, suggesting the existence of redundant vasodilator phenotypes as observed previously in young adults.

  12. Integrins mediate mechanical compression-induced endothelium-dependent vasodilation through endothelial nitric oxide pathway.

    Science.gov (United States)

    Lu, Xiao; Kassab, Ghassan S

    2015-09-01

    Cardiac and skeletal muscle contraction lead to compression of intramuscular arterioles, which, in turn, leads to their vasodilation (a process that may enhance blood flow during muscle activity). Although endothelium-derived nitric oxide (NO) has been implicated in compression-induced vasodilation, the mechanism whereby arterial compression elicits NO production is unclear. We cannulated isolated swine (n = 39) myocardial (n = 69) and skeletal muscle (n = 60) arteriole segments and exposed them to cyclic transmural pressure generated by either intraluminal or extraluminal pressure pulses to simulate compression in contracting muscle. We found that the vasodilation elicited by internal or external pressure pulses was equivalent; moreover, vasodilation in response to pressure depended on changes in arteriole diameter. Agonist-induced endothelium-dependent and -independent vasodilation was used to verify endothelial and vascular smooth muscle cell viability. Vasodilation in response to cyclic changes in transmural pressure was smaller than that elicited by pharmacological activation of the NO signaling pathway. It was attenuated by inhibition of NO synthase and by mechanical removal of the endothelium. Stemming from previous observations that endothelial integrin is implicated in vasodilation in response to shear stress, we found that function-blocking integrin α5β1 or αvβ3 antibodies attenuated cyclic compression-induced vasodilation and NOx (NO(-)2 and NO(-)3) production, as did an RGD peptide that competitively inhibits ligand binding to some integrins. We therefore conclude that integrin plays a role in cyclic compression-induced endothelial NO production and thereby in the vasodilation of small arteries during cyclic transmural pressure loading.

  13. BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model.

    Science.gov (United States)

    Perna, Daniele; Karreth, Florian A; Rust, Alistair G; Perez-Mancera, Pedro A; Rashid, Mamunur; Iorio, Francesco; Alifrangis, Constantine; Arends, Mark J; Bosenberg, Marcus W; Bollag, Gideon; Tuveson, David A; Adams, David J

    2015-02-10

    BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.

  14. Neuroprotective effect of berberine is mediated by MAPK signaling pathway in experimental diabetic neuropathy in rats.

    Science.gov (United States)

    Zhou, Jiyin; Du, Xiaohuang; Long, Min; Zhang, Zuo; Zhou, Shiwen; Zhou, Jianyun; Qian, Guisheng

    2016-03-05

    The mechanisms leading to diabetic neuropathy are complex. As an active component in several traditional Chinese medicines, berberine has a beneficial effect in the treatment of diabetes with hyperlipidemia. This study evaluated the protective effects of berberine on diabetic neuropathy induced by streptozotocin and a high-carbohydrate/high-fat diet in rats. Diabetic neuropathy was induced in rats by intraperitoneal injection of 35 mg/kg streptozotocin and a high-carbohydrate/high-fat diet. Two weeks after diabetes induction, rats were treated with berberine (100 mg/kg) and rosiglitazone (4 mg/kg) for 24 weeks. Rats were studied using evoked potentials, the Morris water maze, transmission electron microscopy, real-time PCR, and Western blotting. Blood glucose, glycated hemoglobin, lipid profile, body weight, evoked potentials, and memory were altered in diabetic rats, as was the hippocampal expression of neuritin mRNA, p38 mitogen-activated protein kinase mRNA, c-Jun N-terminal kinase (JNK) mRNA, extracellular signal-regulated kinase mRNA and the phospho-proteins of p38, JNK, and extracellular signal-regulated kinase. In diabetic rats, berberine decreased body weight and the blood levels of glucose, glycated hemoglobin, triglyceride, and total cholesterol, improved memory and affected evoked potential by decreasing latency. Berberine decreased the mRNA expression of neuritin, p38, and JNK and the protein expression of neuritin, p-p38, and p-JNK. Slight micropathological changes were observed in the hippocampus of berberine-treated diabetic rats. These findings suggest that berberine has a beneficial effect against diabetic neuropathy by improving micropathology and increasing neuritin expression via the mitogen-activated protein kinase signaling pathway.

  15. Possible Signaling Pathways Mediating Neuronal Calcium Sensor-1-Dependent Spatial Learning and Memory in Mice

    Science.gov (United States)

    Nakamura, Tomoe Y.; Nakao, Shu; Nakajo, Yukako; Takahashi, Jun C.; Wakabayashi, Shigeo; Yanamoto, Hiroji

    2017-01-01

    Intracellular Ca2+ signaling regulates diverse functions of the nervous system. Many of these neuronal functions, including learning and memory, are regulated by neuronal calcium sensor-1 (NCS-1). However, the pathways by which NCS-1 regulates these functions remain poorly understood. Consistent with the findings of previous reports, we revealed that NCS-1 deficient (Ncs1-/-) mice exhibit impaired spatial learning and memory function in the Morris water maze test, although there was little change in their exercise activity, as determined via treadmill-analysis. Expression of brain-derived neurotrophic factor (BDNF; a key regulator of memory function) and dopamine was significantly reduced in the Ncs1-/- mouse brain, without changes in the levels of glial cell-line derived neurotrophic factor or nerve growth factor. Although there were no gross structural abnormalities in the hippocampi of Ncs1-/- mice, electron microscopy analysis revealed that the density of large dense core vesicles in CA1 presynaptic neurons, which release BDNF and dopamine, was decreased. Phosphorylation of Ca2+/calmodulin-dependent protein kinase II-α (CaMKII-α, which is known to trigger long-term potentiation and increase BDNF levels, was significantly reduced in the Ncs1-/- mouse brain. Furthermore, high voltage electric potential stimulation, which increases the levels of BDNF and promotes spatial learning, significantly increased the levels of NCS-1 concomitant with phosphorylated CaMKII-α in the hippocampus; suggesting a close relationship between NCS-1 and CaMKII-α. Our findings indicate that NCS-1 may regulate spatial learning and memory function at least in part through activation of CaMKII-α signaling, which may directly or indirectly increase BDNF production. PMID:28122057

  16. Rereplication in emi1-deficient zebrafish embryos occurs through a Cdh1-mediated pathway.

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    Mara E Robu

    Full Text Available Disruption of early mitotic inhibitor 1 (Emi1 interferes with normal cell cycle progression and results in early embryonic lethality in vertebrates. During S and G2 phases the ubiquitin ligase complex APC/C is inhibited by Emi1 protein, thereby enabling the accumulation of Cyclins A and B so they can regulate replication and promote the transition from G2 phase to mitosis, respectively. Depletion of Emi1 prevents mitotic entry and causes rereplication and an increase in cell size. In this study, we show that the developmental and cell cycle defects caused by inactivation of zebrafish emi1 are due to inappropriate activation of APC/C through its cofactor Cdh1. Inhibiting/slowing progression into S-phase by depleting Cdt1, an essential replication licensing factor, partially rescued emi1 deficiency-induced rereplication and the increased cell size. The cell size effect was enhanced by co-depletion of cell survival regulator p53. These data suggest that the increased size of emi1-deficient cells is either directly or indirectly caused by the rereplication defects. Moreover, enforced expression of Cyclin A partially ablated the rereplicating population in emi1-deficient zebrafish embryos, consistent with the role of Cyclin A in origin licensing. Forced expression of Cyclin B partially restored the G1 population, in agreement with the established role of Cyclin B in mitotic progression and exit. However, expression of Cyclin B also partially inhibited rereplication in emi1-deficient embryos, suggesting a role for Cyclin B in regulating replication in this cellular context. As Cyclin A and B are substrates for APC/C-Cdh1 - mediated degradation, and Cdt1 is under control of Cyclin A, these data indicate that emi1 deficiency-induced defects in vivo are due to the dysregulation of an APC/C-Cdh1 molecular axis.

  17. The signal transduction mediated by erythropoietin and proinflammatory cytokines in the JAK/STAT pathway in the children with cerebral palsy.

    Science.gov (United States)

    Tao, Weiyuan; Wen, Fang; Zhang, Hong; Liu, Guheng

    2009-03-01

    It is well established that erythropoietin (EPO) is a pleiotropic cytokine, which has a brain-derived neuroprotective effect in the central nervous system (CNS). Immune abnormality has a close relationship with cerebral palsy (CP), and may be even involved in the development of CP. There is evidence that the amount of EPO in CP children is lower than in normal children, but the levels of proinflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha, are higher in the CP children. The signal transduction mediated by EPO that has a neuroprotective effect and mediated by proinflammatory cytokines that lead to brain damage shares the common JAK/STAT pathway. Under acute stress, the JAK/STAT pathway is occupied by massive proinflammatory cytokines, and the negative feedback inhibition factors like suppressor of cytokine signaling (SOCS) proteins are simultaneously activated, which exist in reciprocal inhibition to EPO in the JAK/STAT pathway. As a result, the signal transduction mediated by EPO is prevented or reduced, and the neuroprotective effect of EPO is eventually weakened. In this review, a novel approach to CP treatment through neurodevelopmental treatment (NDT) is put forward by analysis of the interrelationship of signal transduction mediated by EPO and proinflammatory cytokines in the JAK/STAT pathway and their roles in the development of CP, and some reasonable ideas for CP treatment are provided.

  18. Flavonoid myricetin inhibits TNF-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR and NF-κB pathways in human keratinocytes.

    Science.gov (United States)

    Lee, Da Hee; Lee, Chung Soo

    2016-08-05

    Flavonoid myricetin has been shown to exhibit anti-inflammatory and anti-oxidant effects. Nevertheless, the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we examined the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in relation to the Akt, mTOR and NF-κB pathways, which regulate the transcription genes involved in immune and inflammatory responses. TNF-α stimulated production of the inflammatory mediators and reactive oxygen species in keratinocytes, and activation of the Akt, mTOR and NF-κB pathways in HaCaT cells and primary keratinocytes. Myricetin, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-κB activation), rapamycin (mTOR inhibitor) and N-acetylcysteine attenuated TNF-α-induced activation of Akt, mTOR and NF-κB. Myricetin and N-acetylcysteine attenuated the TNF-α-stimulated production of cytokines and chemokines, and production of reactive oxygen species in keratinocytes. The results show that myricetin may reduce TNF-α-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR and NF-κB pathways. The effect of myricetin appears to be associated with inhibition of the production of reactive oxygen species. Further, myricetin appears to attenuate the proinflammatory mediator-induced inflammatory skin diseases.

  19. Copper metabolism domain-containing 1 represses the mediators involved in the terminal effector pathways of human labour and delivery.

    Science.gov (United States)

    Lappas, Martha

    2016-04-01

    Does Copper Metabolism MURR1 Domain 1 (COMMD1) play a role in regulating the mediators involved in the terminal processes of human labour and delivery? COMMD1 plays a critical role in the termination of nuclear factor-κB (NF-κB) activity and the control of pro-inflammatory and pro-labour mediators. Inflammation and infection are the biggest aetiological factors associated with preterm birth. NF-κB drives the transcription of pro-inflammatory mediators involved in the terminal effector pathways of human labour and delivery. In non-gestational tissues, COMMD1 is a negative regulator of NF-κB-induced inflammation. The mRNA and/or protein level of COMMD1 was assessed in myometrium (n = 8 per group) and fetal membranes (n = 8 per group) obtained from term non-labouring and labouring women at term, and fetal membranes (n = 8 per group) at preterm with and without histological chorioamnionitis. Primary human myometrial cells were used to determine the effect of pro-inflammatory mediators on COMMD1 level, and the effect of COMMD1 small interfering RNA (siRNA) on pro-labour mediators. Statistical significance was ascribed to a P labour in myometrium; in fetal membranes with histologically confirmed chorioamnionitis and in myometrial cells treated with pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, the bacterial product fibroblast-stimulating lipopeptide and the viral double stranded RNA analogue polyinosinic polycytidilic acid. Loss-of-function studies revealed an increase in inflammation- and infection-induced TNF-α, IL-1α, IL-1β, IL-6, IL-8 and/or monocyte chemoattractant protein-1 mRNA abundance and/or release; and cyclo-oxygenase-2 mRNA level, release of prostaglandin (PG) F2α and mRNA level of the PGF2α receptor FP. In addition, siRNA knockdown of COMMD1 was associated with significantly increased NF-κB activation as evidenced by increased IL-1β-induced IκB-α protein degradation and NF-κB DNA binding activity. The

  20. The impact of human development on individual health: a causal mediation analysis examining pathways through education and body mass index

    Science.gov (United States)

    Arah, Onyebuchi A.

    2017-01-01

    Background The macro environment we live in projects what we can achieve and how we behave, and in turn, shapes our health in complex ways. Policymaking will benefit from insights into the mechanisms underlying how national socioeconomic context affects health. This study examined the impact of human development on individual health and the possible mediating roles of education and body mass index (BMI). Methods We analyzed World Health Survey data on 109,448 participants aged 25 or older from 42 low- and middle-income countries with augmented human development index (HDI) in 1990. We used principal components method to create a health score based on measures from eight health state domains, used years of schooling as education indicator and calculated BMI from self-reported height and weight. We used causal mediation analysis technique with random intercepts to account for the multilevel structure. Results Below a reference HDI level of 0.48, HDI was negatively associated with good health (total effect at HDI of 0.23: b =  − 3.44, 95% CI [−6.39–−0.49] for males and b =  − 5.16, 95% CI [−9.24,–−1.08] for females) but was positively associated with good health above this reference level (total effect at HDI of 0.75: b = 4.16, 95% CI [−0.33–8.66] for males and b = 6.62, 95% CI [0.85–12.38] for females). We found a small positive effect of HDI on health via education across reference HDI levels (b ranging from 0.24 to 0.29 for males and 0.40 to 0.49 for females) but not via pathways involving BMI only. Conclusion Human development has a non-linear effect on individual health, but the impact appears to be mainly through pathways other than education and BMI. PMID:28265517

  1. αν and β1 Integrins mediate Aβ-induced neurotoxicity in hippocampal neurons via the FAK signaling pathway.

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    Hai-Yan Han

    Full Text Available αν and β1 integrins mediate Aβ-induced neurotoxicity in primary hippocampal neurons. We treated hippocampal neurons with 2.5 µg/mL 17E6 and 5 µg/mL ab58524, which are specific αν and β1 integrin antagonists, respectively, for 42 h prior to 10 µM Aβ treatment. Next, we employed small interfering RNA (siRNA to silence focal adhesion kinase (FAK, a downstream target gene of integrins. The siRNAs were designed with a target sequence, an MOI of 10 and the addition of 5 µg/mL polybrene. Under these conditions, the neurons were transfected and the apoptosis of different cell types was detected. Moreover, we used real-time PCR and Western blotting analyses to detect the expression of FAK and ρFAK genes in different cell types and investigated the underlying mechanism and signal pathway by which αν and β1 integrins mediate Aβ-induced neurotoxicity in hippocampal neurons. An MTT assay showed that both 17E6 and ab58524 significantly increased cell viability compared with the Aβ-treated neurons (P<0.01 and P<0.05, respectively. However, this protective effect was markedly attenuated after transfection with silencing FAK (siFAK. Moreover, TUNEL immunostaining and flow cytometry indicated that both 17E6 and ab58524 significantly protected hippocampal neurons against apoptosis induced by Aβ (P<0.05 compared with the Aβ-treated cells. However, this protective effect was reversed with siFAK treatment. Both the gene and protein expression of FAK increased after Aβ treatment. Interestingly, as the gene and protein levels of FAK decreased, the ρFAK protein expression markedly increased. Furthermore, both the gene and protein expression of FAK and ρFAK were significantly diminished. Thus, we concluded that both αν and β1 integrins interfered with Aβ-induced neurotoxicity in hippocampal neurons and that this mechanism partially contributes to the activation of the Integrin-FAK signaling pathway.

  2. 34. Effect and the Possible Mediated Pathway of Cortisol Secretion in Adrenocortical Cells Induced by Lead and Cadmium in Vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To understand the direct effect on the secretion of adreno-cortical cells induced by lead and cadmium and the possible mediated pathway. Methods: The adrenocortical cells of male guinea pigs were dispersed and primarily cultured, then the cells were incubated wich cadmiun chloride and lead acetate in dosage as 0,6.25, 12.5, 25, 50, 100 μmol/L respectively for different periods (30, 60, 120 and 240 minutes). The cortisol levels in culture medium and cellular cAMP concentration were measured with RIA. Results: Under the existence of ACTH, the levels of cortisol secreted from the cultured cells were showed significantly declined in dose-dependent manner when the cells were treated in 6.25-100μmol/L CdCl2 for 30 to 240 minutes. There would be an interaction for cortisol secretion between the dose of CdCl2 and the incubatal period. Nevertheless, it seemed to have no obvious linear relation in the alterations of cortisol secretion after 12.5~100μmol/L PbAc incubated for 30~240 minutes. It appeared to have a tendency of dual-phase response in a manner of inhibiting the cortisol secretion in low dose (lower than 25μmol/L) and stimulating the secretion function in high dose (50 and 100μmol/L). The cAMP level was presented a remarkably decrease after 6.25~100 μmol/L CdCl2 incubated with the cells. It was proved that the cAMP level had does-effect relations with the CdCl2 dose. PbAc appeared not only dual response with the tendency of cAMP inhibition in low dose and activating to raise in high dose but also dose-effect relationship. Conclusion: CdCl2 could directly inhibit the secretion of cortisol. PbAc is also of the toxic effect on the cortisol secretion with the characteristic of dual-response as inhibition in early phase and low dose while induction to raising in high dose. cAMP, as an important second messenger, play a role in synthesis and secretion of adrenocorticoids. The toxic effects on steroids secretion induced by cadmium and lead were

  3. Toll-like receptor 2 induced angiogenesis and invasion is mediated through the Tie2 signalling pathway in rheumatoid arthritis.

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    Tajvur Saber

    Full Text Available BACKGROUND: Angiogenesis is a critical early event in inflammatory arthritis, facilitating leukocyte migration into the synovium resulting in invasion and destruction of articular cartilage and bone. This study investigates the effect of TLR2 on angiogenesis, EC adhesion and invasion using microvascular endothelial cells and RA whole tissue synovial explants ex-vivo. METHODS: Microvascular endothelial cells (HMVEC and RA synovial explants ex vivo were cultured with the TLR2 ligand, Pam3CSK4 (1 µg/ml. Angiopoietin 2 (Ang2, Tie2 and TLR2 expression in RA synovial tissue was assessed by immunohistology. HMVEC tube formation was assessed using Matrigel matrix assays. Ang2 was measured by ELISA. ICAM-1 cell surface expression was assessed by flow cytometry. Cell migration was assessed by wound repair scratch assays. ECM invasion, MMP-2 and -9 expression were assessed using transwell invasion chambers and zymography. To examine if the angiopoietin/Tie2 signalling pathway mediates TLR2 induced EC tube formation, invasion and migration assays were performed in the presence of a specific neutralising anti-Tie2mAb (10 ug/ml and matched IgG isotype control Ab (10 ug/ml. RESULTS: Ang2 and Tie2 were localised to RA synovial blood vessels, and TLR2 was localised to RA synovial blood vessels, sub-lining infiltrates and the lining layer. Pam3CSK4 significantly increased angiogenic tube formation (p<0.05, and upregulated Ang2 production in HMVEC (p<0.05 and RA synovial explants (p<0.05. Pam3CSK4 induced cell surface expression of ICAM-1, from basal level of 149±54 (MFI to 617±103 (p<0.01. TLR-2 activation induced an 8.8±2.8 fold increase in cell invasion compared to control (p<0.05. Pam3CSK4 also induced HMVEC cell migration and induced MMP-2 and -9 from RA synovial explants. Neutralisation of the Ang2 receptor, Tie2 significantly inhibited Pam3CSK4-induced EC tube formation and invasion (p<0.05. CONCLUSION: TLR2 activation promotes angiogenesis, cell

  4. Convergent Signaling Pathways Regulate Parathyroid Hormone and Fibroblast Growth Factor-23 Action on NPT2A-mediated Phosphate Transport.

    Science.gov (United States)

    Sneddon, W Bruce; Ruiz, Giovanni W; Gallo, Luciana I; Xiao, Kunhong; Zhang, Qiangmin; Rbaibi, Youssef; Weisz, Ora A; Apodaca, Gerard L; Friedman, Peter A

    2016-09-02

    Parathyroid hormone (PTH) and FGF23 are the primary hormones regulating acute phosphate homeostasis. Human renal proximal tubule cells (RPTECs) were used to characterize the mechanism and signaling pathways of PTH and FGF23 on phosphate transport and the role of the PDZ protein NHERF1 in mediating PTH and FGF23 effects. RPTECs express the NPT2A phosphate transporter, αKlotho, FGFR1, FGFR3, FGFR4, and the PTH receptor. FGFR1 isoforms are formed from alternate splicing of exon 3 and of exon 8 or 9 in Ir-like loop 3. Exon 3 was absent, but mRNA containing both exons 8 and 9 is present in cytoplasm. Using an FGFR1c-specific antibody together with mass spectrometry analysis, we show that RPTECs express FGFR-β1C. The data are consistent with regulated FGFR1 splicing involving a novel cytoplasmic mechanism. PTH and FGF23 inhibited phosphate transport in a concentration-dependent manner. At maximally effective concentrations, PTH and FGF23 equivalently decreased phosphate uptake and were not additive, suggesting a shared mechanism of action. Protein kinase A or C blockade prevented PTH but not FGF23 actions. Conversely, inhibiting SGK1, blocking FGFR dimerization, or knocking down Klotho expression disrupted FGF23 actions but did not interfere with PTH effects. C-terminal FGF23(180-251) competitively and selectively blocked FGF23 action without disrupting PTH effects. However, both PTH and FGF23-sensitive phosphate transport were abolished by NHERF1 shRNA knockdown. Extended treatment with PTH or FGF23 down-regulated NPT2A without affecting NHERF1. We conclude that FGFR1c and PTHR signaling pathways converge on NHERF1 to inhibit PTH- and FGF23-sensitive phosphate transport and down-regulate NPT2A.

  5. Vasorelaxation Induced by a New Naphthoquinone-Oxime is Mediated by NO-sGC-cGMP Pathway

    Directory of Open Access Journals (Sweden)

    Bruna P. V. Dantas

    2014-07-01

    Full Text Available It has been established that oximes cause endothelium-independent relaxation in blood vessels. In the present study, the cardiovascular effects of the new oxime 3-hydroxy-4–(hydroxyimino-2-(3-methylbut-2-enylnaphtalen-1(4H-one (Oxime S1 derived from lapachol were evaluated. In normotensive rats, administration of Oxime S1 (10, 15, 20 and 30 mg/Kg, i.v. produced dose-dependent reduction in blood pressure. In isolated aorta and superior mesenteric artery rings, Oxime S1 induced endothelium-independent and concentration-dependent relaxations (10−8 M to 10−4 M. In addition, Oxime S1-induced vasorelaxations were attenuated by hydroxocobalamin or methylene blue in aorta and by PTIO or ODQ in mesenteric artery rings, suggesting a role for the nitric oxide (NO pathway. Additionally, Oxime S1 (30 and 100 µM significantly increased NO concentrations (13.9 ± 1.6 nM and 17.9 ± 4.1 nM, respectively measured by nitric oxide microsensors. Furthermore, pre-contraction with KCl (80 mM prevented Oxime S1-derived vasorelaxation in endothelium-denuded aortic rings. Of note, combined treatment with potassium channel inhibitors also reduced Oxime S1-mediated vasorelaxation suggesting a role for potassium channels, more precisely Kir, Kv and KATP channels. We observed the involvement of BKCa channels in Oxime S1-induced relaxation in mesenteric artery rings. In conclusion, these data suggest that the Oxime S1 induces hypotension and vasorelaxation via NO pathway by activating soluble guanylate cyclase (sGC and K+ channels.

  6. An adverse outcome pathway framework for neural tube and axial defects mediated by modulation of retinoic acid homeostasis.

    Science.gov (United States)

    Tonk, Elisa C M; Pennings, Jeroen L A; Piersma, Aldert H

    2015-08-01

    Developmental toxicity can be caused through a multitude of mechanisms and can therefore not be captured through a single simple mechanistic paradigm. However, it may be possible to define a selected group of overarching mechanisms that might allow detection of the vast majority of developmental toxicants. Against this background, we have explored the usefulness of retinoic acid mediated regulation of neural tube and axial patterning as a general mechanism that, when perturbed, may result in manifestations of developmental toxicity that may cover a large part of malformations known to occur in experimental animals and in man. Through a literature survey, we have identified key genes in the regulation of retinoic acid homeostasis, as well as marker genes of neural tube and axial patterning, that may be used to detect developmental toxicants in in vitro systems. A retinoic acid-neural tube/axial patterning adverse outcome pathway (RA-NTA AOP) framework was designed. The framework was tested against existing data of flusilazole exposure in the rat whole embryo culture, the zebrafish embryotoxicity test, and the embryonic stem cell test. Flusilazole is known to interact with retinoic acid homeostasis, and induced common and unique NTA marker gene changes in the three test systems. Flusilazole-induced changes were similar in directionality to gene expression responses after retinoic acid exposure. It is suggested that the RA-NTA framework may provide a general tool to define mechanistic pathways and biomarkers of developmental toxicity that may be used in alternative in vitro assays for the detection of embryotoxic compounds.

  7. Elevated transforming growth factor β and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren's disease fibroblasts

    Directory of Open Access Journals (Sweden)

    Krause Carola

    2011-06-01

    Full Text Available Abstract Background Dupuytren's disease is a fibroproliferative disorder of the palmar fascia. The treatment used to date has mostly been surgery, but there is a high recurrence rate. Transforming growth factor β (TGF-β has been implicated as a key stimulator of myofibroblast activity and fascial contraction in Dupuytren's disease. Results We studied Dupuytren's fibroblasts in tissues ex vivo and in cells cultured in vitro and found increased TGF-β expression compared to control fibroblasts. This correlated not only with elevated expression and activation of downstream Smad effectors but also with overactive extracellular signal-regulated kinase 1/2 (ERK1/2/mitogen-activated protein (MAP kinase signalling. Treatment with the TGF-β type I receptor kinase inhibitor SB-431542 and bone morphogenetic protein 6 (BMP6 led to inhibition of elevated Smad and ERK1/2/MAP kinase signalling as well as to inhibition of the increased contractility of Dupuytren's fibroblasts. BMP6 attenuated TGF-β expression in Dupuytren's fibroblasts, but not in control fibroblasts. Platelet-derived growth factor (PDGF expression was strongly promoted by TGF-β in Dupuytren's fibroblasts and was curbed by SB-431542 or BMP6 treatment. High basal expression of phosphorylated ERK1/2 MAP kinase and fibroproliferative markers was attenuated in Dupuytren's fibroblasts by a selective PDGF receptor kinase inhibitor. Cotreatment of Dupuytren's fibroblasts with SB-431542 and the mitogen-activated protein kinase kinase 1 inhibitor PD98059 was sufficient to abrogate proliferation and contraction of Dupuytren's fibroblasts. Conclusions Both TGF-β and ERK1/2 MAP kinase pathways cooperated in mediating the enhanced proliferation and high spontaneous contraction of Dupuytren's fibroblasts. Our data indicate that both signalling pathways are prime targets for the development of nonsurgical intervention strategies to treat Dupuytren's disease.

  8. Innate immunity mediates myocardial preconditioning through Toll-like receptor 2 and TIRAP-dependent signaling pathways.

    Science.gov (United States)

    Dong, Jian-Wen; Vallejo, Jesus G; Tzeng, Huei-Ping; Thomas, James A; Mann, Douglas L

    2010-03-01

    Recent studies have implicated Toll-like receptor 2 (TLR2) and TLR4 signaling in delimiting liver and brain injury following ischemia-reperfusion (I/R). To determine whether TLR2 and TLR4 conferred cytoprotection in the heart, we subjected hearts of wild-type (WT) mice and mice deficient in TLR2 (TLR2D), TLR4 (TLR4D), and TIR domain-containing adapter protein (TIRAP-D) to ischemic preconditioning (IPC). Langendorff-perfused hearts were subjected to 30 min ischemia and 60 min reperfusion with or without IPC. IPC resulted in a significant increase (P 0.05) in TIRAP-D mouse hearts (43.8 +/- 1.9%) after I/R injury. IPC also resulted in a significant (P < 0.05) decrease in I/R-induced creatine kinase release and Evans blue dye uptake in WT but not TIRAP-D hearts. Interestingly, IPC resulted in a significant (P < 0.05) increase in %LVDP in TLR4-deficient hearts (52.7 +/- 3%) but not in TLR2D hearts (39.3 +/- 1.5%). Pretreatment with a specific TLR2 ligand (Pam3CSK) protected WT hearts against I/R-induced left ventricular dysfunction. The loss of IPC-induced cardioprotection in TIRAP-D mouse hearts was accompanied by a decreased translocation of protein kinase C-epsilon and decreased phosphorylation of GSK-3beta. Taken together, these data suggest that the cardioprotective effect of IPC is mediated, at least in part, through a TLR2-TIRAP-dependent pathway, suggesting that the modulation of this pathway represents a viable target for reducing I/R injury.

  9. Suppressor of cytokine signalling-6 promotes neurite outgrowth via JAK2/STAT5-mediated signalling pathway, involving negative feedback inhibition.

    Directory of Open Access Journals (Sweden)

    Sakshi Gupta

    Full Text Available BACKGROUND: Suppressors of cytokine signalling (SOCS protein family are key regulators of cellular responses to cytokines and play an important role in the nervous system. The SOCS6 protein, a less extensively studied SOCS family member, has been shown to induce insulin resistance in the retina and promote survival of the retinal neurons. But no reports are available about the role of SOCS6 in neuritogenesis. In this study, we examined the role of SOCS6 in neurite outgrowth and neuronal cell signalling. METHODOLOGY/PRINCIPAL FINDINGS: The effect of SOCS6 in neural stem cells differentiation was studied in neural stem cells and PC12 cell line. Highly elevated levels of SOCS6 were found upon neural cell differentiation both at the mRNA and protein level. Furthermore, SOCS6 over-expression lead to increase in neurite outgrowth and degree of branching, whereas SOCS6 knockdown with specific siRNAs, lead to a significant decrease in neurite initiation and extension. Insulin-like growth factor-1 (IGF-1 stimulation which enhanced neurite outgrowth of neural cells resulted in further enhancement of SOCS6 expression. Jak/Stat (Janus Kinase/Signal Transducer And Activator Of Transcription pathway was found to be involved in the SOCS6 mediated neurite outgrowth. Bioinformatics study revealed presence of putative Stat binding sites in the SOCS6 promoter region. Transcription factors Stat5a and Stat5b were involved in SOCS6 gene upregulation leading to neuronal differentiation. Following differentiation, SOCS6 was found to form a ternary complex with IGFR (Insulin Like Growth Factor-1 Receptor and JAK2 which acted in a negative feedback loop to inhibit pStat5 activation. CONCLUSION/SIGNIFICANCE: The current paradigm for the first time states that SOCS6, a SOCS family member, plays an important role in the process of neuronal differentiation. These findings define a novel molecular mechanism for Jak2/Stat5 mediated SOCS6 signalling.

  10. High-fat diet feeding promotes stemness and precancerous changes in murine gastric mucosa mediated by leptin receptor signaling pathway.

    Science.gov (United States)

    Arita, Seiya; Kinoshita, Yuta; Ushida, Kaori; Enomoto, Atsushi; Inagaki-Ohara, Kyoko

    2016-11-15

    Obesity increases the risk for gastric cancers. However, the occurrence and mechanisms of precancerous atrophic gastritis induced by high-fat diet (HFD) remain unclear. Here, we show that HFD-associated lipotoxicity induces precancerous lesions that are accompanied by the disruption of organelle homeostasis, tissue integrity, and deregulated expression of stemness genes in the gastric epithelium mediated by leptin receptor (ObR) signaling. Following HFD feeding, ectopic fat accumulated and expression of LAMP2A in lysosome and COX IV in mitochondria increased in the gastric mucosa. HFD feeding also led to enhanced expression of activated-Notch1 and stem cell markers Lgr5, CD44, and EpCAM. In addition, HFD-fed mice showed intracellular β-catenin accumulation in the gastric mucosa with increased expression of its target genes, Nanog, Oct4, and c-Myc. These observations were abrogated in the leptin-deficient ob/ob mice and ObR-mutated db/db mice, indicating that these HFD-induced changes were responsible for effects downstream of the ObR. Consistent with this, the expression of the Class IA and III PI3Ks was increased following ObR activation in the gastric mucosa of HFD-fed mice. Together, these results suggest that HFD-induced lipotoxicity and deregulated organelle biosynthesis confer cancer stem cell-like properties to the gastric mucosa via signaling pathway mediated by leptin, PI3K and β-catenin. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Does GRK-β arrestin machinery work as a "switch on" for GPR17-mediated activation of intracellular signaling pathways?

    Science.gov (United States)

    Daniele, Simona; Trincavelli, Maria Letizia; Fumagalli, Marta; Zappelli, Elisa; Lecca, Davide; Bonfanti, Elisabetta; Campiglia, Pietro; Abbracchio, Maria P; Martini, Claudia

    2014-06-01

    During oligodendrocyte-precursor cell (OPC) differentiation program, an impairment in the regulatory mechanisms controlling GPR17 spatio-temporal expression and functional activity has been suggested to contribute to defective OPC maturation, a crucial event in the pathogenesis of multiple sclerosis. GRK-β arrestin machinery is the primary actor in the control of G-protein coupled receptor (GPCR) functional responses and changes in these regulatory protein activities have been demonstrated in several immune/inflammatory diseases. Herein, in order to shed light on the molecular mechanisms controlling GPR17 regulatory events during cell differentiation, the role of GRK/β-arrestin machinery in receptor desensitization and signal transduction was investigated, in transfected cells and primary OPC. Following cell treatment with the two classes of purinergic and cysteinyl-leukotriene (cysLT) ligands, different GRK isoforms were recruited to regulate GPR17 functional responses. CysLT-mediated receptor desensitization mainly involved GRK2; this kinase, via a G protein-dependent mechanism, promoted a transient binding of the receptor to β-arrestins, rapid ERK phosphorylation and sustained nuclear CREB activation. Furthermore, GRK2, whose expression parallels that of the receptor during differentiation process, appeared to be crucial to induce cysLT-mediated maturation of OPCs. On the other hand, purinergic ligand exclusively recruited the GRK5 subtype, and induced, via a G protein-independent/β-arrestin-dependent mechanism, a receptor/β-arrestin stable association, slower and sustained ERK stimulation and marginal CREB activation. These results show that purinergic and cysLT ligands, through the recruitment of specific GRK isoforms, address distinct intracellular pathways, most likely reinforcing the same final response. The identification of these mechanisms and players controlling GPR17 responses during OPC differentiation could be useful to identify new targets in

  12. PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma.

    Science.gov (United States)

    Puustinen, Pietri; Junttila, Melissa R; Vanhatupa, Sari; Sablina, Anna A; Hector, Melissa E; Teittinen, Kaisa; Raheem, Olayinka; Ketola, Kirsi; Lin, Shujun; Kast, Juergen; Haapasalo, Hannu; Hahn, William C; Westermarck, Jukka

    2009-04-01

    Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n=222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

  13. Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage

    Directory of Open Access Journals (Sweden)

    Lin Sen

    2012-03-01

    Full Text Available Abstract Background Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4 plays a role in inflammatory damage caused by brain disorders. Methods In this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics. Results Compared to WT mice, TLR4−/− mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4−/−, MyD88−/− and TRIF−/− mice showed attenuated inflammatory damage after ICH. TLR4−/− mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4−/− mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH. Conclusions Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately

  14. Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

    Science.gov (United States)

    Lidder, P; Johnson, M A; Sullivan, M L; Thompson, D M; Pérez-Amador, M A; Howard, C J; Green, P J

    2004-08-01

    mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

  15. Novel perspectives on the PHD-HIF oxygen sensing pathway in cardioprotection mediated by IPC and RIPC

    Directory of Open Access Journals (Sweden)

    Silvia eMartin-Puig

    2015-05-01

    Full Text Available Reperfusion of ischemic cardiac tissue is the standard treatment for improving clinical outcome following myocardial infarction but is inevitably associated with ischemia-reperfusion injury (IRI. Ischemic myocardial injury can be alleviated by exposing the heart to brief episodes of sublethal ischemia-reperfusion prior to the ischemic insult, a phenomenon that has been termed ischemic preconditioning (IPC. Similarly, remote IPC (RIPC is defined as transient episodes of ischemia at a distant site before a subsequent prolonged injury of the target organ. In this setting, adaptive responses to hypoxia/ischemia in peripheral tissues include the release of soluble factors that have the potential to protect cardiomyocytes remotely. Oxygen fluctuation is a hallmark of insufficient tissue perfusion and ischemic episodes. Emerging evidence indicates that prolyl hydroxylase oxygen sensors (PHDs and hypoxia-inducible transcription factors (HIFs are critical regulators of IPC and RIPC. In this minireview, we discuss recent findings concerning the role of the PHD-HIF axis in IPC and RIPC-mediated cardioprotection and examine molecular pathways and cell types that might be involved. We also appraise the therapeutic value of targeting the PHD-HIF axis to enhance cardiac tolerance against IRI.

  16. RTN1-C mediates cerebral ischemia/reperfusion injury via ER stress and mitochondria-associated apoptosis pathways.

    Science.gov (United States)

    Gong, Lingli; Tang, Yuewen; An, Ran; Lin, Muya; Chen, Lijian; Du, Jian

    2017-10-05

    The reticulon family has been found to induce apoptosis, inhibit axon regeneration and regulate protein trafficking. However, little is known about the mechanisms of how reticulon proteins are involved in neuronal death-promoting processes during ischemia. Here, we report that the expression of Reticulon Protein 1-C (RTN1-C) was associated with the progression of cerebral ischemia/reperfusion (I/R) injury. Using a combination of rat middle cerebral artery occlusion (MCAO) stroke and oxygen-glucose deprivation followed by reoxygenation (OGD/R) models, we determined that the expression of RTN1-C was significantly increased during cerebral ischemic/reperfusion. RTN1-C overexpression induced apoptosis and increased the cell vulnerability to ischemic injury, whereas RTN1-C knockdown reversed ischemia-induced apoptosis and attenuated the vulnerability of OGD/R-treated neural cells. Mechanistically, we demonstrated that RTN1-C mediated OGD/R-induced apoptosis through ER stress and mitochondria-associated pathways. RTN1-C interacted with Bcl-xL and increased its localization in the ER, thus reducing the anti-apoptotic activity of Bcl-xL. Most importantly, knockdown of Rtn1-c expression in vivo attenuated apoptosis in MCAO rats and reduced the extent of I/R-induced brain injury, as assessed by infarct volume and neurological score. Collectively, these data support for the first time that RTN1-C may represent a novel candidate for therapies against cerebral ischemia/reperfusion injury.

  17. Cell-cycle-dependent PC-PLC regulation by APC/C(Cdc20)-mediated ubiquitin-proteasome pathway.

    Science.gov (United States)

    Fu, Da; Ma, Yushui; Wu, Wei; Zhu, Xuchao; Jia, Chengyou; Zhao, Qianlei; Zhang, Chunyi; Wu, Xing Zhong

    2009-07-01

    Phosphatidylcholine-specific phospholipase C (PC-PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC-PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC-PLC were co-immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH-7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC-PLC and Cdc20 were co-localized in the perinuclear endoplasmic reticulum region (the "juxtanuclear quality control" compartment, JUNQ). The expression level and activities of PC-PLC changed in a cell-cycle-dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC-PLC, and caused PC-PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC-PLC regulation in cell cycles is controlled by APC/C(Cdc20)-mediated UPP.

  18. DDX3 modulates cell adhesion and motility and cancer cell metastasis via Rac1-mediated signaling pathway.

    Science.gov (United States)

    Chen, H-H; Yu, H-I; Cho, W-C; Tarn, W-Y

    2015-05-21

    The DEAD-box RNA helicase DDX3 is a versatile protein involved in multiple steps of gene expression and various cellular signaling pathways. DDX3 mutations have been implicated in the wingless (Wnt) type of medulloblastoma. We show here that small interfering RNA-mediated DDX3 knockdown in various cell lines increased cell-cell adhesion but decreased cell-extracellular matrix adhesion. Moreover, DDX3 depletion suppressed cell motility and impaired directional migration in the wound-healing assay. Accordingly, DDX3-depleted cells exhibited reduced invasive capacities in vitro as well as reduced metastatic potential in mice. We also examined the mechanism underlying DDX3-regulated cell migration. DDX3 knockdown reduced the levels of both Rac1 and β-catenin proteins, and consequentially downregulated the expression of several β-catenin target genes. Moreover, we demonstrated that DDX3-regulated Rac1 mRNA translation, possibly through an interaction with its 5'-untranslated region, and affected β-catenin protein stability in an Rac1-dependent manner. Taken together, our results indicate the DDX3-Rac1-β-catenin regulatory axis in modulating the expression of Wnt/β-catenin target genes. Therefore, this report provides a mechanistic context for the role of DDX3 in Wnt-type tumors.

  19. Chlorogenic acid attenuates lipopolysaccharide-induced mice mastitis by suppressing TLR4-mediated NF-κB signaling pathway.

    Science.gov (United States)

    Ruifeng, Gao; Yunhe, Fu; Zhengkai, Wei; Ershun, Zhou; Yimeng, Li; Minjun, Yao; Xiaojing, Song; Zhengtao, Yang; Naisheng, Zhang

    2014-04-15

    Chlorogenic acid (CGA), one of the most abundant polyphenols in the diet, has been reported to have potent anti-inflammatory properties. However, the effect of CGA on lipopolysaccharide (LPS)-induced mice mastitis has not been investigated. The purpose of the present study was to elucidate whether CGA could ameliorate the inflammation response in LPS-induced mice mastitis and to clarify the possible mechanism. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. CGA was administered intraperitoneally with the dose of 12.5, 25, and 50mg/kg respectively 1h before and 12h after induction of LPS. In this study, the effect of CGA on LPS-induced mice mastitis was assessed through histopathological examination, ELISA assay, and western blot analysis. The results showed that CGA significantly reduced TNF-α, IL-1β, and IL-6 production compared with LPS group. Besides, western blot analysis showed that CGA could inhibit the expression of TLR4 and the phosphorylation of NF-κB and IκB induced by LPS. These results suggested that anti-inflammatory effects of CGA against LPS-induced mastitis may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathway. Therefore, CGA may be a potent therapeutic reagent for the prevention of the immunopathology encountered during Escherichia coli elicited mastitis. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. The differentiation of skeletal muscle cells involves a protein-tyrosine phosphatase-alpha-mediated C-Src signaling pathway

    DEFF Research Database (Denmark)

    Lu, Huogen; Shah, Poonam; Ennis, David

    2002-01-01

    Protein-tyrosine phosphatase-alpha (PTPalpha) plays an important role in various cellular signaling events, including proliferation and differentiation. In this study, we established L6 cell lines either underexpressing or overexpressing PTPalpha by stable transfection of cells with antisense PTP....... Moreover, enhanced expression of PTPalpha and activation of Src was detected during myogenesis. Together, these data indicate that PTPalpha is involved in the regulation of L6 myoblast growth and skeletal muscle cell differentiation via an Src-mediated signaling pathway....... PTPalpha or with full-length wild-type human or mouse or double catalytic site Cys --> Ala mutant (DM8) PTPalpha cDNA. Expression of PTPalpha in these cell lines was determined by immunoblotting and immunofluorescence. Cells harboring antisense PTPalpha exhibited a significantly reduced growth rate...... myogenesis 2 days earlier than wild-type L6 cells. Overexpression of phosphatase-inactive mutant PTPalpha recapitulated the phenotype of the antisense cells. The different myogenic activities of these cell lines were correlated with the expression of myogenin and creatine kinase activity. Consistent...

  1. Gadolinium induced apoptosis of human embryo liver L02 cell line by ROS-mediated AIF pathway

    Institute of Scientific and Technical Information of China (English)

    YE Lihua; SHI Zhe; LIU Huixue; YANG Xiaoda; WANG Kui

    2011-01-01

    Gd3+ complexes have a variety of medical applications. In order to shed light on the mechanism of hepatotoxicity of Gd3+ compounds, we investigated the effects of GdCl3 on human embryo liver cell strand (L02 cells). The experimental results showed that long-time exposure to GdC13 resulted in L02 cell apoptosis. The incubation of L02 cells with GdCl3 first induced increase in cellular reactive oxygen species (ROS) and decrease in mitochondrial inner membrane potential (△Ψm). It later resulted in the activation of poly (ADP-ribose) polymerase (PARP) and the release of mitochondrial apoptosis-inducing factor (AIF). The activation of caspase 3, however, was not observed.Antioxidants could significantly reduce GdCl3-induced decrease of △Ψm, release of AIF, and cell apoptosis. Although GdCl3 caused a significant increase in cell membrane permeability in L02, the change of cell membrane permeability was unlikely to be involved in GdCl3-induced cell apoptosis. Overall, our experimental results suggested that GdCl3 induced apoptosis of human embryo liver L02 cell line by ROS-mediated AIF pathway.

  2. Novel perspectives on the PHD-HIF oxygen sensing pathway in cardioprotection mediated by IPC and RIPC.

    Science.gov (United States)

    Martin-Puig, Silvia; Tello, Daniel; Aragonés, Julián

    2015-01-01

    Reperfusion of ischemic cardiac tissue is the standard treatment for improving clinical outcome following myocardial infarction but is inevitably associated with ischemia-reperfusion injury (IRI). Ischemic myocardial injury can be alleviated by exposing the heart to brief episodes of sublethal ischemia-reperfusion prior to the ischemic insult, a phenomenon that has been termed ischemic preconditioning (IPC). Similarly, remote IPC (RIPC) is defined as transient episodes of ischemia at a distant site before a subsequent prolonged injury of the target organ. In this setting, adaptive responses to hypoxia/ischemia in peripheral tissues include the release of soluble factors that have the potential to protect cardiomyocytes remotely. Oxygen fluctuations is a hallmark of insufficient tissue perfusion and ischemic episodes. Emerging evidence indicates that prolyl hydroxylase oxygen sensors (PHDs) and hypoxia-inducible transcription factors (HIFs) are critical regulators of IPC and RIPC. In this review, we discuss recent findings concerning the role of the PHD-HIF axis in IPC and RIPC-mediated cardioprotection and examine molecular pathways and cell types that might be involved. We also appraise the therapeutic value of targeting the PHD-HIF axis to enhance cardiac tolerance against IRI.

  3. The cAMP-mediated protein kinase signal transduction pathway is involved in the pyrogenic effect of CRH in rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The primary action of corticotropin releasing hormone (CRH) is stimulation of the synthesis and release of adrenocorticotropic hormone and β-endorphin from the pituitary in response to stress. In addition, a number of studies indicate that CRH exerts other physiological actions within the central nervous system which are independent of the pituitary. These include increased body temperature and thermogenesis. However, the intracellular mechanism responsible for pyrogenic action of CRH is still unclear. The purpose of these studies was to determine whether or not cAMP was involved in the pyrogenic action of CRH in the rat. Intracerebroventricular (icv) microinjection of CRH (2.5 μg, 5.0 μg, 10 μg) caused increases in colonic temperature and hypothalamus cAMP level in conscious rats. The pyrogenic effects of CRH were abolished or markedly inhibited by prior injection (icv) of an adenylate cyclase inhibitor, 2,,3,-dideoxyadenosine (DDA, 30 μg) or an inhibitor of cAMP-dependent protein kinase, adenosine-3,,5,-(cyclic) monophosphorothionate (Rp-cAMPs, 15 μg). This is the first report demonstrating the pyrogenic effcet of centrally administration of CRH on the rat via the cAMP-mediated protein kinase signal transduction pathway.

  4. A novel TLR4-mediated signaling pathway leading to IL-6 responses in human bladder epithelial cells.

    Directory of Open Access Journals (Sweden)

    Jeongmin Song

    2007-04-01

    Full Text Available The vigorous cytokine response of immune cells to Gram-negative bacteria is primarily mediated by a recognition molecule, Toll-like receptor 4 (TLR4, which recognizes lipopolysaccharide (LPS and initiates a series of intracellular NF-kappaB-associated signaling events. Recently, bladder epithelial cells (BECs were reported to express TLR4 and to evoke a vigorous cytokine response upon exposure to LPS. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by Escherichia coli and isolated LPS. We observed that in addition to the classical NF-kappaB-associated pathway, TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca(2+, adenylyl cyclase 3-generated cAMP, and a transcriptional factor, cAMP response element-binding protein. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract.

  5. Fisetin Alleviates Lipopolysaccharide-Induced Acute Lung Injury via TLR4-Mediated NF-κB Signaling Pathway in Rats.

    Science.gov (United States)

    Feng, Guang; Jiang, Ze-Yu; Sun, Bo; Fu, Jie; Li, Tian-Zuo

    2016-02-01

    Acute lung injury (ALI), a common component of systemic inflammatory disease, is a life-threatening condition without many effective treatments. Fisetin, a natural flavonoid from fruits and vegetables, was reported to have wide pharmacological properties such as anti-inflammatory, antioxidant, and anticancer activities. The aim of this study was to detect the effects of fisetin on lipopolysaccharide (LPS)-induced acute lung injury and investigate the potential mechanism. Fisetin was injected (1, 2, and 4 mg/kg, i.v.) 30 min before LPS administration (5 mg/kg, i.v.). Our results showed that fisetin effectively reduced the inflammatory cytokine release and total protein in bronchoalveolar lavage fluids (BALF), decreased the lung wet/dry ratios, and obviously improved the pulmonary histology in LPS-induced ALI. Furthermore, fisetin inhibited LPS-induced increases of neutrophils and macrophage infiltration and attenuated MPO activity in lung tissues. Additionally, fisetin could significantly inhibit the Toll-like receptor 4 (TLR4) expression and the activation of NF-κB in lung tissues. Our data indicates that fisetin has a protective effect against LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways, and fisetin may be a promising candidate for LPS-induced ALI treatment.

  6. The associated regulators and signal pathway in rILl-16/CD4 mediated growth regulation in Jurkat cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    IL-16 is a ligand and chemotactic factor for CD4+ T cells. IL-16 inhibits the CD3 mediated lymphocyteactivation and proliferation. The effects of IL-16 on the target cells are dependent on the cell type, thepresence of co-activators etc. To understand the regulation function and mechanism of IL-16 on targetcells, we used a 130 a.a. recombinant IL-16 to study its effects on the growth of Jurkat T leukemia cellsin vitro. We found that the rIL-16 stimulated the proliferation of Jurkat cells at low dose (10-9M), butinhibited the growth of the cells at higher concentration (10-5M). Results showed that 10-5 M of rIL-16treatment induced an enhanced apoptosis in Jurkat cells. The treatment blocked the expression of FasL, butup-regulated the c-myc and Bid expression in the cells. Pre-treatment of PKC inhibitor or MEK1 inhibitormarkedly increased or decreased the rIL-16 induced growth-inhibiting effects on Jurkat cells, respectively.The results suggested that the rIL-16 might be a regulator for the growth or apoptosis of Jurkat cells ata dose-dependent manner. The growth-inhibiting effects of rIL-16 might be Fas/FasL independent, but,associated with the activation of PKC, up-regulated expression of c-Myc and Bid, and the participation ofthe ERK signal pathway in Jurkat cells.

  7. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway.

    Directory of Open Access Journals (Sweden)

    Wei Yang

    Full Text Available Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA-dependent pathway from an early stage upstream of NDR1 and EDS1.

  8. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway.

    Science.gov (United States)

    Yang, Wei; Xu, Xiaonan; Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.

  9. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  10. Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Two major apoptosis pathways have been defined in mammalian cells, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway. The Bcl-2 family proteins consist of both anti-apoptosis and pro- apoptosis members that regulate apoptosis, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates downstream caspases. Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.

  11. Epstein-Barr virus-encoded LMP1 triggers regulation of the ERK-mediated Op18/stathmin signaling pathway in association with cell cycle.

    Science.gov (United States)

    Lin, Xuechi; Tang, Min; Tao, Yongguang; Li, Lili; Liu, Sufang; Guo, Lili; Li, Zijian; Ma, Xiaoqian; Xu, Juan; Cao, Ya

    2012-06-01

    The MAPKs are activated by a variety of cellular stimuli to participate in a series of signaling cascades and mediate diverse intracellular responses. One potential target of the MAPKs is Op18/stathmin, a molecule that acts as an integrator of diverse cell signaling pathways and regulates the dynamics of microtubules, which are involved in modulating a variety of cellular processes, including cell cycle progression and cell growth. Our study focused on the regulation of the MAPK-mediated Op18/stathmin signaling pathway, which is triggered by the Epstein-Barr virus-encoded latent membrane protein 1 ( LMP1) oncogene in nasopharyngeal carcinoma cells. The results showed that the activity of MAPK, which was induced by LMP1, varied with cell cycle progression; LMP1 upregulated phosphorylation of ERK during the G(1)/S phase, but negatively regulated phosphorylation of ERK during the G(2)/M phase. We found that the regulation of Op18/stathmin signaling by LMP1 was mainly mediated through ERK. The inhibition of LMP1 expression attenuated the interaction of ERK with Op18/stathmin and promoted microtubule depolymerization. These findings indicate the existence of a new cell cycle-associated signaling pathway in which LMP1 regulates ERK-mediated Op18/stathmin signaling.

  12. The gender specific mediational pathways between parenting styles, neuroticism, pathological reasons for drinking, and alcohol-related problems in emerging adulthood.

    Science.gov (United States)

    Patock-Peckham, Julie A; Morgan-Lopez, Antonio A

    2009-03-01

    Mediational links between parenting styles, neuroticism, pathological reasons for drinking, alcohol use and alcohol-related problems were tested. A two-group SEM path model with 441 (216 female, 225 male) college students was examined. In general, pathological reasons for drinking mediated the impact of neuroticism on alcohol use and alcohol-related problems. A different pattern of relationships was found for each of the two genders. Perceptions of having an authoritarian father were positively linked to higher levels of neuroticism among males but this pattern was not found among females. For males, neuroticism mediated the impact of having an authoritarian father on pathological reasons for drinking with pathological reasons for drinking mediating the impact of neuroticism on alcohol-related problems. Perceptions of having a permissive father were linked to lower levels of neuroticism in females (but have been found as a consistent risk factor for other pathways to alcohol use elsewhere). Compared with other work in this area, these findings indicate parental influences regarding vulnerabilities for alcohol use may be specific to parent-child gender matches for some pathways and specific to one parent (irrespective of child gender) for other pathways.

  13. Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways

    Directory of Open Access Journals (Sweden)

    Ying eZhao

    2015-06-01

    Full Text Available Metasequoiaglyptostroboidies is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as5-to-7years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia.

  14. Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes.

    Science.gov (United States)

    Park, Gyu Hwan; Jeon, Se Jin; Ryu, Jae Ryun; Choi, Min Sik; Han, Seol-Heui; Yang, Sung-Il; Ryu, Jong Hoon; Cheong, Jae Hoon; Shin, Chan Young; Ko, Kwang Ho

    2009-09-01

    Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.

  15. mGluR1-mediated excitation of cerebellar GABAergic interneurons requires both G protein-dependent and Src-ERK1/2-dependent signaling pathways.

    Directory of Open Access Journals (Sweden)

    Hideo Kubota

    Full Text Available Stimulation of type I metabotropic glutamate receptors (mGluR1/5 in several neuronal types induces slow excitatory responses through activation of transient receptor potential canonical (TRPC channels. GABAergic cerebellar molecular layer interneurons (MLIs modulate firing patterns of Purkinje cells (PCs, which play a key role in cerebellar information processing. MLIs express mGluR1, and activation of mGluR1 induces an inward current, but its precise intracellular signaling pathways are unknown. We found that mGluR1 activation facilitated spontaneous firing of mouse cerebellar MLIs through an inward current mediated by TRPC1 channels. This mGluR1-mediated inward current depends on both G protein-dependent and -independent pathways. The nonselective protein tyrosine kinase inhibitors genistein and AG490 as well as the selective extracellular signal-regulated kinase 1/2 (ERK1/2 inhibitors PD98059 and SL327 suppressed the mGluR1-mediated current responses. Following G protein blockade, the residual mGluR1-mediated inward current was significantly reduced by the selective Src tyrosine kinase inhibitor PP2. In contrast to cerebellar PCs, GABAB receptor activation in MLIs did not alter the mGluR1-mediated inward current, suggesting that there is no cross-talk between mGluR1 and GABAB receptors in MLIs. Thus, activation of mGluR1 facilitates firing of MLIs through the TRPC1-mediated inward current, which depends on not only G protein-dependent but also Src-ERK1/2-dependent signaling pathways, and consequently depresses the excitability of cerebellar PCs.

  16. Interleukin-1beta can mediate growth arrest and differentiation via the leukemia inhibitory factor/JAK/STAT pathway in medullary thyroid carcinoma cells.

    Science.gov (United States)

    Park, Jong-In; Strock, Christopher J; Ball, Douglas W; Nelkin, Barry D

    2005-02-01

    Interleukin-1beta (IL-1beta) is a pleiotropic cytokine that can induce several cellular signal transduction pathways. Here, we show that IL-1beta can induce cell cycle arrest and differentiation in the human medullary thyroid carcinoma (MTC) cell line, TT. IL-1beta induces cell cycle arrest accompanied by morphological changes and expression of the neuroendocrine marker calcitonin. These changes are blocked by the MEK1/2 specific inhibitor U0126, indicating that MEK1/2 is essential for IL-1beta signaling in TT cells. IL-1beta induces expression of leukemia inhibitory factor (LIF) and activation of STAT3 via the MEK/ERK pathway. This activation of STAT3 could be abrogated by treatment with anti-LIF neutralizing antibody or anti-gp130 blocking antibody, indicating that induction of LIF expression is sufficient and essential for STAT3 activation by IL-1beta. In addition to activation of the LIF/JAK/STAT pathway, IL-1beta also induced an MEK/ERK-mediated intracellular cell-autonomous signaling pathway that is independently sufficient for growth arrest and differentiation. Thus, IL-1beta activates the MEK/ERK pathway to induce growth arrest and differentiation in MTC cells via dual independent signaling mechanisms, the cell-extrinsic LIF/JAK/STAT pathway, and the cell-intrinsic autonomous signaling pathway.

  17. XBP1-Independent UPR Pathways Suppress C/EBP-β Mediated Chondrocyte Differentiation in ER-Stress Related Skeletal Disease.

    Directory of Open Access Journals (Sweden)

    Trevor L Cameron

    2015-09-01

    Full Text Available Schmid metaphyseal chondrodysplasia (MCDS involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2, generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent or pathologically redundant (XBP1-dependent. XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-β, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-β transcriptional co-factors, GADD45-β and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation

  18. Investigation of cyclooxygenase and signaling pathways involved in human platelet aggregation mediated by synergistic interaction of various agonists.

    Science.gov (United States)

    Khan, Nadia; Farooq, Ahsana Dar; Sadek, Bassem

    2015-01-01

    In the present study, the mechanism(s) of synergistic interaction of various platelet mediators such as arachidonic acid (AA) when combined with 5-hydroxytryptamine (5-HT) or adenosine diphosphate (ADP) on human platelet aggregation were examined. The results demonstrated that 5-HT had no or negligible effect on aggregation but it did potentiate the aggregation response of AA. Similarly, the combination of subeffective concentrations of ADP and AA exhibited noticeable rise in platelet aggregation. Moreover, the observed synergistic effect of AA with 5-HT on platelets was inhibited by different cyclooxygenase (COX) inhibitors, namely ibuprofen and celecoxib, with half maximal inhibitory effect (IC50) values of 18.0 ± 1.8 and 15.6 ± 3.4 μmol/L, respectively. Interestingly, the synergistic effect observed for AA with 5-HT was, also, blocked by the 5-HT receptor blockers cyproheptadine (IC50=22.0 ± 7 μmol/L), ketanserin (IC50=152 ± 23 μmol/L), phospholipase C (PLC) inhibitor (U73122; IC50=6.1 ± 0.8 μmol/L), and mitogen activated protein kinase (MAPK) inhibitor (PD98059; IC50=3.8 ± 0.5 μmol/L). Likewise, the synergism of AA and ADP was, also, attenuated by COX inhibitors (ibuprofen; IC50=20 ± 4 μmol/L and celecoxib; IC50=24 ± 7 μmol/L), PLC inhibitor (U73122; IC50=3.7 ± 0.3 μmol/L), and MAPK inhibitor (PD98059; IC50=2.8 ± 1.1 μmol/L). Our observed data demonstrate that the combination of subthreshold concentrations of agonists amplifies platelet aggregation and that these synergistic effects largely depend on activation of COX/thromboxane A2, receptor-operated Ca(2+) channels, Gq/PLC, and MAPK signaling pathways. Moreover, our data revealed that inhibition of COX pathways by using both selective and/or non-selective COX inhibitors blocks not only AA metabolism and thromboxane A2 formation, but also its binding to Gq receptors and activation of receptor-operated Ca(2+) channels in platelets. Overall, our results show that PLC and MAPK inhibitors proved

  19. Secreted cyclophilin A mediates G1/S phase transition of cholangiocarcinoma cells via CD147/ERK1/2 pathway.

    Science.gov (United States)

    Obchoei, Sumalee; Sawanyawisuth, Kanlayanee; Wongkham, Chaisiri; Kasinrerk, Watchara; Yao, Qizhi; Chen, Changyi; Wongkham, Sopit

    2015-02-01

    Cyclophilin A (CypA) was shown to be upregulated in human cholangiocarcinoma (CCA) tissues. Suppression of intracellular CypA (inCypA) significantly reduces cell proliferation in vitro and tumor growth in nude mice. In the present study, the effect and potential mechanism of secreted CypA (sCypA) on cell proliferation of CCA cell lines were further investigated. CCA cells were treated with sCypA-containing conditioned media (CM) or with purified recombinant human CypA (rhCypA). Cell proliferation, cell cycle, ERK1/2, p38 MAPK, NF-κB, and STAT3 activities were examined by MTS assay, flow cytometry, and Western blot. sCypA was detected in CM from MMNK1 (an immortalized human cholangiocyte cell line) and six CCA cell lines. The sCypA levels corresponded to the inCypA levels indicating the intracellular origin of sCypA. Both sCypA-containing CM and rhCypA significantly increased proliferation of CCA cells. CD147 depletion by shRNA-knockdown or neutralizing with a CD147-monoclonal antibody significantly reduced sCypA-, and rhCypA-mediated cell proliferation. Upon rhCypA treatment, ERK1/2 was rapidly phosphorylated; whereas neutralizing CD147 inhibited ERK1/2 phosphorylation. Cell cycle analysis showed a significant increase in S phase and decrease in G1 population in rhCypA-treated cells. The expression levels of cyclin D1 and phosphorylated-retinoblastoma protein in the rhCypA-treated cells were increased compared with those in the non-treated control cells. p38 MAPK pathway was shown to be suppressed in siCypA-treated cells. In summary, CypA is secreted from CCA cells and enhances cell proliferation in an autocrine/paracrine manner, at least via direct binding with CD147, which may activate the ERK1/2 and p38 MAPK signaling pathways.

  20. Paeoniflorin suppresses TGF-β mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway

    Science.gov (United States)

    Ji, Yu; Dou, Yan-nong; Zhao, Qian-wen; Zhang, Ji-zhou; Yang, Yan; Wang, Ting; Xia, Yu-feng; Dai, Yue; Wei, Zhi-feng

    2016-01-01

    Aim: Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-β mediated pulmonary EMT in bleomycin-induced PF mice. Methods: PF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-β1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR. Results: In PF mice, paeoniflorin (50, 100 mg·kg−1·d−1) or prednisone (6 mg·kg−1·d−1) significantly decreased the expression of FSP-1 and α-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-β1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-β1 for 24 h. TGF-β1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1–30 μmol/L) dose-dependently attenuated TGF-β1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-β1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells. Conclusion: Paeoniflorin suppresses the early stages of TGF-β mediated EMT in alveolar

  1. Inhibiting the clathrin-mediated endocytosis pathway rescues K(IR)2.1 downregulation by pentamidine.

    Science.gov (United States)

    Varkevisser, Rosanne; Houtman, Marien J C; Waasdorp, Maaike; Man, Joyce C K; Heukers, Raimond; Takanari, Hiroki; Tieland, Ralph G; van Bergen En Henegouwen, Paul M P; Vos, Marc A; van der Heyden, Marcel A G

    2013-02-01

    Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K(IR)2.x carried I(K1) current and that inhibiting protein degradation in the lysosome increased intracellular K(IR)2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of K(IR)2.1 to further restore normal levels of functional K(IR)2.1 channels. K(IR)2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). K(IR)2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved K(IR)2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased I(K1) (at -60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p Pentamidine (10 μM, 48 h) reduced K(IR)2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in K(IR)2.1 degradation. Pentamidine-induced downregulation of K(IR)2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.

  2. Upregulation of parathyroid VDR expression by extracellular calcium is mediated by ERK1/2-MAPK signaling pathway.

    Science.gov (United States)

    Cañadillas, Sagrario; Canalejo, Rocio; Rodriguez-Ortiz, Maria Encarnacion; Martinez-Moreno, Julio Manuel; Estepa, Jose Carlos; Zafra, Rafael; Perez, Jose; Muñoz-Castañeda, Juan Rafael; Canalejo, Antonio; Rodriguez, Mariano; Almaden, Yolanda

    2010-05-01

    hypocalcemic rats. Our results demonstrate that extracellular calcium stimulates VDR expression in parathyroid glands through the elevation of the cytosolic calcium level and the stimulation of the PLA(2)-AA-dependent ERK1/2-pathway. Furthermore, the transcription factor Sp1 mediates this effect.

  3. Chemokine CXCL13 mediates orofacial neuropathic pain via CXCR5/ERK pathway in the trigeminal ganglion of mice.

    Science.gov (United States)

    Zhang, Qian; Cao, De-Li; Zhang, Zhi-Jun; Jiang, Bao-Chun; Gao, Yong-Jing

    2016-07-11

    Trigeminal nerve damage-induced neuropathic pain is a severely debilitating chronic orofacial pain syndrome. Spinal chemokine CXCL13 and its receptor CXCR5 were recently demonstrated to play a pivotal role in the pathogenesis of spinal nerve ligation-induced neuropathic pain. Whether and how CXCL13/CXCR5 in the trigeminal ganglion (TG) mediates orofacial pain are unknown. The partial infraorbital nerve ligation (pIONL) was used to induce trigeminal neuropathic pain in mice. The expression of ATF3, CXCL13, CXCR5, and phosphorylated extracellular signal-regulated kinase (pERK) in the TG was detected by immunofluorescence staining and western blot. The effect of shRNA targeting on CXCL13 or CXCR5 on pain hypersensitivity was checked by behavioral testing. pIONL induced persistent mechanical allodynia and increased the expression of ATF3, CXCL13, and CXCR5 in the TG. Inhibition of CXCL13 or CXCR5 by shRNA lentivirus attenuated pIONL-induced mechanical allodynia. Additionally, pIONL-induced neuropathic pain and the activation of ERK in the TG were reduced in Cxcr5 (-/-) mice. Furthermore, MEK inhibitor (PD98059) attenuated mechanical allodynia and reduced TNF-α and IL-1β upregulation induced by pIONL. TNF-α inhibitor (Etanercept) and IL-1β inhibitor (Diacerein) attenuated pIONL-induced orofacial pain. Finally, intra-TG injection of CXCL13 induced mechanical allodynia, increased the activation of ERK and the production of TNF-α and IL-1β in the TG of WT mice, but not in Cxcr5 (-/-) mice. Pretreatment with PD98059, Etanercept, or Diacerein partially blocked CXCL13-induced mechanical allodynia, and PD98059 also reduced CXCL13-induced TNF-α and IL-1β upregulation. CXCL13 and CXCR5 contribute to orofacial pain via ERK-mediated proinflammatory cytokines production. Targeting CXCL13/CXCR5/ERK/TNF-α and IL-1β pathway in the trigeminal ganglion may offer effective treatment for orofacial neuropathic pain.

  4. Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells.

    Science.gov (United States)

    Morrissey, Colm; O'Neill, Amanda; Spengler, Barbara; Christoffel, Volker; Fitzpatrick, John M; Watson, R William G

    2005-05-01

    Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro. We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively. Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin. Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells. (c) 2004 Wiley-Liss, Inc.

  5. Modulation of TCR-mediated signaling pathway by thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2).

    Science.gov (United States)

    Saitoh, S; Kosugi, A; Noda, S; Yamamoto, N; Ogata, M; Minami, Y; Miyake, K; Hamaoka, T

    1995-12-15

    Thymic shared antigen-1 (TSA-1) is a glycosyl-phosphatidylinositol (GPI)-anchored differentiation Ag expressed on murine lymphocytes, and is identical to stem cell Ag-2 (Sca-2). Using newly established mAb against TSA-1/Sca-2, we have previously shown that surface TSA-1 expression is induced upon activation in T cells, and that anti-TSA-1 inhibits IL-2 production induced by anti-CD3 stimulation in T cell hybridomas. In the present study, we have analyzed the functional role of TSA-1 during T cell activation using normal T cells, T cell hybridomas, and transfected Jurkat cell lines that expressed either GPI-anchored or transmembrane form of TSA-1. Anti-TSA-1 inhibited IL-2 production from normal T cells stimulated with soluble anti-CD3 plus accessory cells. Anti-TSA-1 exhibited the inhibitory effect on T cells, but not on accessory cells, because anti-TSA-1 inhibited IL-2 production in Jurkat cells transfected with TSA-1 cDNA, but not in control transfectant. A transmembrane form of TSA-1 was expressed in Jurkat cells by fusing the extracellular portion of TSA-1 to the transmembrane and cytoplasmic regions of the class 1 Db. The analysis using this transfectant revealed that anti-TSA-1-mediated inhibition of IL-2 production did not require the GPI anchor of TSA-1. Finally, in addition to the inhibition of IL-2 production, tyrosine phosphorylation of CD3 zeta-chains observed following TCR stimulation, one of the important early activation events, was markedly reduced by anti-TSA-1. These results imply that TSA-1/Sca-2 plays an important regulatory role in the TCR signaling pathway of activated T cells in addition to its role in T cell differentiation.

  6. Knockdown of Rab5a expression decreases cancer cell motility and invasion through integrin-mediated signaling pathway

    Directory of Open Access Journals (Sweden)

    Shi Shu-liang

    2011-08-01

    Full Text Available Abstract Background Rab GTPases function as modulators in intracellular transport. Rab5a, a member of the Rab subfamily of small GTPases, is an important regulator of vesicle traffic from the plasma membrane to early endosomes. Recent findings have reported that Rab5a gene was involved in the progression of cancer. In the present study, we investigated the effect of Rab5a on cervical cancer invasion and metastasis and the molecular mechanism underlying the involvement of Rab5a. Methods Rab5a expression was assessed by immunohistochemical analysis on a cervical cancer tissue microarray. RNA interference (RNAi was performed to knock down the endogenous expression of Rab5a gene in HeLa and SiHa cells. Cell motility was evaluated using invasion assay and wound migration assay in vitro. The expression levels of integrin-associated molecules were detected by Western blot and immunofluorescence. Results We found that Rab5a was expressed at a high level in cervical cancer tissues. Silencing of Rab5a expression significantly decreased cancer cell motility and invasiveness. The down-regulation of integrin-associated focal adhesion signaling molecules was further detected in Rab5a knockdown cells. Meanwhile, active GTP-bound Rac1, Cdc42, and RhoA were also down-regulated, accompanied with the reduction in the number and size of filopodia and lamellipodia. Conclusions Taken together, these data suggest that Rab5a functions in regulating the invasion phenotype, and we propose that this regulation may be via integrin-mediated signaling pathway in cervical cancer cells.

  7. PLC-Mediated Signaling Pathway in Pollen Tubes Regulates the Gametophytic Self-incompatibility of Pyrus Species.

    Science.gov (United States)

    Qu, Haiyong; Guan, Yaqin; Wang, Yongzhang; Zhang, Shaolin

    2017-01-01

    Among the Rosaceae species, the gametophytic self-incompatibility (GSI) is controlled by a single multi-allelic S locus, which is composed of the pistil-S and pollen-S genes. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase), which is essential for identifying self-pollen. However, the S-RNase system has not been fully characterized. In this study, the self-S-RNase inhibited the Ca(2+)-permeable channel activity at pollen tube apices and the selectively decreased phospholipase C (PLC) activity in the plasma membrane of Pyrus pyrifolia pollen tubes. Self-S-RNase decreased the Ca(2+) influx through a PLC-mediated signaling pathway. Phosphatidylinositol-specific PLC has a 26-amino acid insertion in pollen tubes of the 'Jinzhuili' cultivar, which is a spontaneous self-compatible mutant of the 'Yali' cultivar. 'Yali' plants exhibit a typical S-RNase-based GSI. Upon self-pollination, PLC gene expression is significantly higher in 'Jinzhuili' pollen tubes than that in 'Yali' pollen tubes. Moreover, the PLC in pollen tubes can only interact with one of the two types of S-RNase from the style. In the Pyrus x bretschneideri Rehd, the PLC directly interacted with the S7-RNase in the pollen tube, but not with the S34-RNase. Collectively, our results reveal that the effects of S-RNase on PLC activity are required for S-specific pollen rejection, and that PLC-IP3 participates in the self-incompatibility reaction of Pyrus species.

  8. IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells

    Directory of Open Access Journals (Sweden)

    James E Krolopp

    2016-12-01

    Full Text Available Myokines are specialized cytokines that are secreted from skeletal muscle (SKM in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15 is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5 were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.

  9. CD44v6 regulates growth of brain tumor stem cells partially through the AKT-mediated pathway.

    Directory of Open Access Journals (Sweden)

    Mayumi Jijiwa

    Full Text Available Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6 in BTSC of a subset of glioblastoma multiforme (GBM. Patients with CD44(high GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44(high GBM but not from CD44(low GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN, increased expression of phosphorylated AKT in CD44(high GBM, but not in CD44(low GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKT pathway.

  10. DMPD: Protein kinase C epsilon: a new target to control inflammation andimmune-mediated disorders. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14643884 Protein kinase C epsilon: a new target to control inflammation andimmune-mediated disorder...g) (.html) (.csml) Show Protein kinase C epsilon: a new target to control inflammation andimmune-mediated disorder...l inflammation andimmune-mediated disorders. Authors Aksoy E, Goldman M, Willems F. Publication Int J Bioche

  11. Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

    Science.gov (United States)

    Yang, Wei-Na; Ma, Kai-Ge; Qian, Yi-Hua; Zhang, Jian-Shui; Feng, Gai-Feng; Shi, Li-Li; Zhang, Zhi-Chao; Liu, Zhao-Hui

    2015-07-01

    Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aβ). Reuptake of extracellular Aβ is believed to contribute significantly to the intraneuronal Aβ pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aβ1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aβ internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aβ1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aβ internalization in neurons. We found that extracellular Aβ1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aβ1-42 and LRP1 were also found co-localized in neurons during Aβ1-42 internalization, and they could form Aβ1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aβ1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aβ1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aβ1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aβ levels and served a potential therapeutic target for AD.

  12. Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

    Science.gov (United States)

    Kakiuchi-Kiyota, Satoko; Koza-Taylor, Petra H; Mantena, Srinivasa R; Nelms, Linda F; Enayetallah, Ahmed E; Hollingshead, Brett D; Burdick, Andrew D; Reed, Lori A; Warneke, James A; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2014-03-01

    Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.

  13. Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells

    Science.gov (United States)

    Chen, Y.; Hughes-Fulford, M.

    2000-01-01

    Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

  14. Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells

    Science.gov (United States)

    Chen, Y.; Hughes-Fulford, M.

    2000-01-01

    Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

  15. Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure.

    Science.gov (United States)

    Jozic, Ivan; Vukelic, Sasa; Stojadinovic, Olivera; Liang, Liang; Ramirez, Horacio A; Pastar, Irena; Tomic Canic, Marjana

    2017-05-01

    Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, β-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration in vitro and accelerates wound epithelialization of human wounds ex vivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. The NAV2 homolog Sickie regulates F-actin-mediated axonal growth in Drosophila mushroom body neurons via the non-canonical Rac-Cofilin pathway.

    Science.gov (United States)

    Abe, Takashi; Yamazaki, Daisuke; Murakami, Satoshi; Hiroi, Makoto; Nitta, Yohei; Maeyama, Yuko; Tabata, Tetsuya

    2014-12-01

    The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. We demonstrate that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. We observed phenotypic similarities and genetic interactions among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. We propose that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner.

  17. Chlorin e6-Mediated Photodynamic Therapy Suppresses P. acnes-Induced Inflammatory Response via NFκB and MAPKs Signaling Pathway

    Science.gov (United States)

    Wang, Yoon-Young; Ryu, A-Reum; Jin, Solee; Jeon, Yu-Mi; Lee, Mi-Young

    2017-01-01

    Photodynamic therapy (PDT), consisting of photosensitizer, light, and oxygen has been used for the treatment of various diseases including cancers, microbial infections and skin disorders. In this study, we examined the anti-inflammatory effect of chlorin e6-mediated PDT in P. acnes-infected HaCaT cells using photosensitizer chlorin e6 (Ce6) and halogen light. The live and heat-killed P. acnes triggered an upregulation of inflammatory molecules such as iNOS, NO, and inflammatory cytokine in HaCaT cells and mouse model. Ce6-mediated PDT notably downregulated the expression of these inflammatory molecules in vitro and in vivo. Similarly, chlorin e6-mediated PDT was capable of regulating inflammatory response in both live and heat killed S. epidermidis exposed HaCaT cells. Moreover, phosphorylation of p38, JNK, and ERK were reduced by Ce6-mediated PDT. Ce6-mediated PDT also reduced the phosphorylation of IKKα/β, IĸBα and NFκB p65 in P. acnes-stimulated HaCaT cells. In addition, the dramatic increase in the nuclear translocation of NFκB p65 observed upon stimulation with P. acnes was markedly impaired by Ce6-based PDT. This is the first suggestion that Ce6-mediated PDT suppresses P. acnes-induced inflammation through modulating NFκB and MAPKs signaling pathways. PMID:28118375

  18. GDNF-induced leukemia inhibitory factor can mediate differentiation via the MEK/ERK pathway in pheochromocytoma cells derived from nf1-heterozygous knockout mice.

    Science.gov (United States)

    Park, Jong-In; Powers, James F; Tischler, Arthur S; Strock, Christopher J; Ball, Douglas W; Nelkin, Barry D

    2005-02-01

    Glial cell line-derived neurotrophic factor (GDNF) can induce neuron-like differentiation of mouse pheochromocytoma (MPC) cell lines derived from mice with a heterozygous knockout mutation of nf1, the murine counterpart of the human gene mutated in neurofibromatosis type 1 (NF1). Here, we show that GDNF-induced differentiation in the MPC 862L cell line is mediated by the MEK/extracellular signal-regulated kinase (ERK) pathway. Neurite outgrowth, increased expression of growth-associated protein 43, and decreased incorporation of bromodeoxyuridine (BrdU) were induced by treatment with GDNF, H-RasV12, or a constitutively active MEK2. GDNF also induces leukemia inhibitory factor (LIF) via the MEK/ERK pathway, and LIF itself can elicit these differentiative changes via a cell-extrinsic autocrine/paracrine pathway. Treatment with anti-LIF neutralizing antibody depleted the differentiative activity of the conditioned medium from cells stimulated for MEK/ERK signaling, while recombinant LIF could induce differentiation in MPC cells, indicating that LIF is the sole factor with differentiative activity. LIF could activate MEK1/2 and STAT3, but LIF-induced differentiation was blocked only by the MEK1/2-specific inhibitor U0126, indicating that the MEK/ERK pathway is necessary for LIF action in MPC cells. Our findings suggest that LIF may be utilized for signaling mediated by GDNF and may be important in the pathobiology of neuroendocrine tumors.

  19. Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway[OPEN

    Science.gov (United States)

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice. PMID:25841037

  20. Dopamine D1 Receptor-Mediated Transmission Maintains Information Flow Through the Cortico-Striato-Entopeduncular Direct Pathway to Release Movements.

    Science.gov (United States)

    Chiken, Satomi; Sato, Asako; Ohta, Chikara; Kurokawa, Makoto; Arai, Satoshi; Maeshima, Jun; Sunayama-Morita, Tomoko; Sasaoka, Toshikuni; Nambu, Atsushi

    2015-12-01

    In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.

  1. Divergent pathways to influence: Cognition and behavior differentially mediate the effects of optimism on physical and mental quality of life in Chinese university students.

    Science.gov (United States)

    Ramsay, Jonathan E; Yang, Fang; Pang, Joyce S; Lai, Ching-Man; Ho, Roger Cm; Mak, Kwok-Kei

    2015-07-01

    Previous research has indicated that both cognitive and behavioral variables mediate the positive effect of optimism on quality of life; yet few attempts have been made to accommodate these constructs into a single explanatory framework. Adopting Fredrickson's broaden-and-build perspective, we examined the relationships between optimism, self-rated health, resilience, exercise, and quality of life in 365 Chinese university students using path analysis. For physical quality of life, a two-stage model, in which the effects of optimism were sequentially mediated by cognitive and behavioral variables, provided the best fit. A one-stage model, with full mediation by cognitive variables, provided the best fit for mental quality of life. This suggests that optimism influences physical and mental quality of life via different pathways. © The Author(s) 2013.

  2. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway

    DEFF Research Database (Denmark)

    Amit, Sharon; Hatzubai, Ada; Birman, Yaara;

    2002-01-01

    The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin a......, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway. Udgivelsesdato: 2002-May-1...

  3. Oxymatrine improves intestinal epithelial barrier function involving NF-κB-mediated signaling pathway in CCl4-induced cirrhotic rats.

    Directory of Open Access Journals (Sweden)

    Jian-Bo Wen

    CCl4-induced cirrhotic rats. The results indicate that oxymatrine improves intestinal barrier function via NF-κB-mediated signaling pathway and may be used as a new protecting agent for cirrhosis-associated intestinal mucosal damage.

  4. Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth.

    Science.gov (United States)

    Tsouko, E; Khan, A S; White, M A; Han, J J; Shi, Y; Merchant, F A; Sharpe, M A; Xin, L; Frigo, D E

    2014-05-26

    Cancer cells display an increased demand for glucose. Therefore, identifying the specific aspects of glucose metabolism that are involved in the pathogenesis of cancer may uncover novel therapeutic nodes. Recently, there has been a renewed interest in the role of the pentose phosphate pathway in cancer. This metabolic pathway is advantageous for rapidly growing cells because it provides nucleotide precursors and helps regenerate the reducing agent NADPH, which can contribute to reactive oxygen species (ROS) scavenging. Correspondingly, clinical data suggest glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, is upregulated in prostate cancer. We hypothesized that androgen receptor (AR) signaling, which plays an essential role in the disease, mediated prostate cancer cell growth in part by increasing flux through the pentose phosphate pathway. Here, we determined that G6PD, NADPH and ribose synthesis were all increased by AR signaling. Further, this process was necessary to modulate ROS levels. Pharmacological or molecular inhibition of G6PD abolished these effects and blocked androgen-mediated cell growth. Mechanistically, regulation of G6PD via AR in both hormone-sensitive and castration-resistant models of prostate cancer was abolished following rapamycin treatment, indicating that AR increased flux through the pentose phosphate pathway by the mammalian target of rapamycin (mTOR)-mediated upregulation of G6PD. Accordingly, in two separate mouse models of Pten deletion/elevated mTOR signaling, Pb-Cre;Pten(f/f) and K8-CreER(T2);Pten(f/f), G6PD levels correlated with prostate cancer progression in vivo. Importantly, G6PD levels remained high during progression to castration-resistant prostate cancer. Taken together, our data suggest that AR signaling can promote prostate cancer through the upregulation of G6PD and therefore, the flux of sugars through the pentose phosphate pathway. Hence, these findings support a

  5. Glycyrrhizic acid (GA) inhibits reactive oxygen Species mediated photodamage by blocking ER stress and MAPK pathway in UV-B irradiated human skin fibroblasts.

    Science.gov (United States)

    Farrukh, Mufti Rana; Nissar, Ul-Ashraf; Kaiser, Peerzada J; Afnan, Quadri; Sharma, Praduman R; Bhushan, Shashi; Tasduq, Sheikh A

    2015-07-01

    Previously we have reported that generation of reactive oxygen species is the prime event responsible for calcium mediated activation of PERK-eIF2α-CHOP pathway and apoptosis in UV-B irradiated human skin fibroblasts (Hs68). We have also reported that glycyrrhizic acid (GA) mediates potent photoprotective activity against UV-B - irradiation-induced photodamage in human skin fibroblast. In the present study, we aimed to investigate the role of GA in preventing oxidative stress mediated unfolded protein response (UPR) and mitogen activated protein kinases (MAPK) pathway. Human skin fibroblast (Hs68) cells were exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analysed using western-blotting, microscopy and flow cytometry. Our results show that GA has strong photoprotective action against UV-B induced cellular damage. It was observed that: (a) Oxidative disturbances and intracellular Ca(2+) imbalance induced by UV-B irradiation was significantly restored by GA treatment; (b) activation of PERK-eIF2α-CHOP and MAPK pathway induced by UV-B was significantly blocked by GA; (c) Loss of mitochondrial membrane potential and apoptosis induced by UV-B were reduced by GA treatment. Based on the above findings we conclude GA has a highly significant ROS quenching activity, thereby blocking the cascade of events including release of calcium from ER and subsequent ER stress, MAPK pathway and cellular demise. GA offers highly potent anti photodamage effect and can be exploited for cosmetic or therapeutic purposes. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFβ-induced Autophagy.

    Science.gov (United States)

    Korah, Juliana; Canaff, Lucie; Lebrun, Jean-Jacques

    2016-01-29

    TGFβ is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.

  7. Fucoidan extract induces apoptosis in MCF-7 cells via a mechanism involving the ROS-dependent JNK activation and mitochondria-mediated pathways.

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    Zhongyuan Zhang

    Full Text Available BACKGROUND: Fucoidan extract (FE, an enzymatically digested compound with a low molecular weight, is extracted from brown seaweed. As a natural compound with various actions, FE is attractive, especially in Asian countries, for improving the therapeutic efficacy and safety of cancer treatment. The present study was carried out to investigate the anti-tumor properties of FE in human carcinoma cells and further examine the underlying mechanisms of its activities. METHODOLOGY/PRINCIPAL FINDING: FE inhibits the growth of MCF-7, MDA-MB-231, HeLa, and HT1080 cells. FE-mediated apoptosis in MCF-7 cancer cells is accompanied by DNA fragmentation, nuclear condensation, and phosphatidylserine exposure. FE induces mitochondrial membrane permeabilization (MMP through loss of mitochondrial membrane potential (ΔΨm and regulation of the expression of Bcl-2 family members. Release of apoptosis-inducing factor (AIF and cytochrome c precedes MMP. AIF release causes DNA fragmentation, the final stage of apoptosis, via a caspase-independent mitochondrial pathway. Additionally, FE was found to induce phosphorylation of c-Jun N-terminal kinase (JNK, p38, and extracellular signal-regulated kinase (ERK 1/2, and apoptosis was found to be attenuated by inhibition of JNK. Furthermore, FE-mediated apoptosis was found to involve the generation of reactive oxygen species (ROS, which are responsible for the decrease of ΔΨm and phosphorylation of JNK, p38, and ERK1/2 kinases. CONCLUSIONS/SIGNIFICANCE: These data suggest that FE activates a caspase-independent apoptotic pathway in MCF-7 cancer cells through activation of ROS-mediated MAP kinases and regulation of the Bcl-2 family protein-mediated mitochondrial pathway. They also provide evidence that FE deserves further investigation as a natural anticancer and cancer preventive agent.

  8. Role of nuclear factor κB-mediated inflammatory pathways in cancer-related symptoms and their regulation by nutritional agents.

    Science.gov (United States)

    Gupta, Subash C; Kim, Ji Hye; Kannappan, Ramaswamy; Reuter, Simone; Dougherty, Patrick M; Aggarwal, Bharat B

    2011-06-01

    Cancer is a disease characterized by dysregulation of multiple genes and is associated with symptoms such as cachexia, anorexia, fatigue, depression, neuropathic pain, anxiety, cognitive impairment, sleep disorders and delirium (acute confusion state) in medically ill patients. These symptoms are caused by either the cancer itself or the cancer treatment. During the past decade, increasing evidence has shown that the dysregulation of inflammatory pathways contributes to the expression of these symptoms. Cancer patients have been found to have higher levels of proinflammatory cytokines such as interleukin-6. The nuclear factor (NF)-κB is a major mediator of inflammatory pathways. Therefore, anti-inflammatory agents that can modulate the NF-κB activation and inflammatory pathways may have potential in improving cancer-related symptoms in patients. Because of their multitargeting properties, low cost, low toxicity and immediate availability, natural agents have gained considerable attention for prevention and treatment of cancer-related symptoms. How NF-κB and inflammatory pathways contribute to cancer-related symptoms is the focus of this review. We will also discuss how nutritional agents such as curcumin, genistein, resveratrol, epigallocatechin gallate and lycopene can modulate inflammatory pathways and thereby reduce cancer-related symptoms in patients.

  9. DMPD: Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12960231 Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited sign...82. Epub 2003 Jul 22. (.png) (.svg) (.html) (.csml) Show Macrophage activation through CCR5- and CXCR4-media...on through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. Authors Lee C, Liu QH, Tomkowicz B, Yi

  10. JAK/STAT signaling pathway-mediated immune response in silkworm (Bombyx mori) challenged by Beauveria bassiana.

    Science.gov (United States)

    Geng, Tao; Lv, Ding-Ding; Huang, Yu-Xia; Hou, Cheng-Xiang; Qin, Guang-Xing; Guo, Xi-Jie

    2016-12-20

    Innate immunity was critical in insects defensive system and able to be induced by Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway. Currently, it had been identified many JAK/STAT signaling pathway-related genes in silkworm, but little function was known on insect innate immunity. To explore the roles of JAK/STAT pathway in antifungal immune response in silkworm (Bombyx mori) against Beauveria bassiana infection, the expression patterns of B. mori C-type lectin 5 (BmCTL5) and genes encoding 6 components of JAK/STAT signaling pathway in silkworm challenged by B. bassiana were analyzed using quantitative real time PCR. Meanwhile the activation of JAK/STAT signaling pathway by various pathogenic micro-organisms and the affect of JAK/STAT signaling pathway inhibitors on antifungal activity in silkworm hemolymph was also detected. Moreover, RNAi assay of BmCTL5 and the affect on expression levels of signaling factors were also analyzed. We found that JAK/STAT pathway could be obviously activated in silkworm challenged with B. bassiana and had no response to bacteria and B. mori cytoplasmic polyhedrosis virus (BmCPV). However, the temporal expression patterns of JAK/STAT signaling pathway related genes were significantly different. B. mori downstream receptor kinase (BmDRK) might be a positive regulator of JAK/STAT signaling pathway in silkworm against B. bassiana infection. Moreover, antifungal activity assay showed that the suppression of JAK/STAT signaling pathway by inhibitors could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm to B. bassiana infection, indicating that JAK/STAT signaling pathway might be involved in the synthesis and secretion of antifungal substances. The results of RNAi assays suggested that BmCTL5 might be one pattern recognition receptors for JAK/STAT signaling pathway in silkworm. These findings yield insights for better

  11. Pathway markers for pro-resolving lipid mediators in maternal and umbilical cord blood: A Secondary analysis of the Mothers, Omega-3, & Mental Health Study

    Directory of Open Access Journals (Sweden)

    Ellen L Mozurkewich

    2016-09-01

    Full Text Available The omega-3 fatty acids docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA are precursors to immune regulatory and specialized pro-resolving mediators (SPM of inflammation termed resolvins, maresins, and protections. Evidence for lipid mediator formation in vivo can be gained through evaluation of their 5-lipoxygenase (LOX and 15-LOX metabolic pathway precursors and downstream metabolites: We performed a secondary blood sample analysis from 60 participants in the Mothers, Omega-3, and Mental Health study to determine whether SPM and SPM precursors are augmented by dietary EPA- and DHA-rich fish oil supplementation compared to soy oil placebo. We also aimed to study whether SPM and their precursors differ in early and late pregnancy or between maternal and umbilical cord blood. We found that compared to placebo supplementation, EPA- and DHA- rich fish oil supplementation increased SPM precursor 17-HDHA concentrations in maternal and umbilical cord blood (P=0.02 We found that the D-series resolvin pathway marker 17-HDHA increased significantly between enrollment and late pregnancy (P=0.049. Levels of both 14-HDHA, a maresin pathway marker, and 17-HDHA were significantly greater in umbilical cord blood than in maternal blood (P<0.001, both.

  12. Schizandrin A Inhibits Microglia-Mediated Neuroninflammation through Inhibiting TRAF6-NF-κB and Jak2-Stat3 Signaling Pathways.

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    Fangjiao Song

    Full Text Available Microglial-mediated neuroinflammation has been established as playing a vital role in pathogenesis of neurodegenerative disorders. Thus, rational regulation of microglia functions to inhibit inflammation injury may be a logical and promising approach to neurodegenerative disease therapy. The purposes of the present study were to explore the neuroprotective effects and potential molecular mechanism of Schizandrin A (Sch A, a lignin compound isolated from Schisandra chinesnesis. Our observations showed that Sch A could significantly down-regulate the increased production of nitric oxide (NO, tumor necrosis factor (TNF-α and interleukin (IL-6 induced by lipopolysaccharide (LPS both in BV-2 cells and primary microglia cells. Moreover, Sch A exerted obvious neuroprotective effects against inflammatory injury in neurons when exposed to microglia-conditioned medium. Investigations of the mechanism showed the anti-inflammatory effect of Sch A involved the inhibition of inducible nitric oxide synthase (iNOS and cyclooxygenase 2 (COX-2 expression levels and inhibition of the LPS-induced TRAF6-IKKβ-NF-κB pathway. Furthermore, inhibition of Jak2-Stat3 pathway activation and Stat3 nuclear translocation also was observed. In conclusion, SchA can exert anti-inflammatory and neuroprotective effects by alleviating microglia-mediated neuroinflammation injury through inhibiting the TRAF6-IKKβ-NF-κB and Jak2-Stat3 signaling pathways.

  13. Schizandrin A Inhibits Microglia-Mediated Neuroninflammation through Inhibiting TRAF6-NF-κB and Jak2-Stat3 Signaling Pathways

    Science.gov (United States)

    Song, Fangjiao; Zeng, Kewu; Liao, Lixi; Yu, Qian; Tu, Pengfei; Wang, Xuemei

    2016-01-01

    Microglial-mediated neuroinflammation has been established as playing a vital role in pathogenesis of neurodegenerative disorders. Thus, rational regulation of microglia functions to inhibit inflammation injury may be a logical and promising approach to neurodegenerative disease therapy. The purposes of the present study were to explore the neuroprotective effects and potential molecular mechanism of Schizandrin A (Sch A), a lignin compound isolated from Schisandra chinesnesis. Our observations showed that Sch A could significantly down-regulate the increased production of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-6 induced by lipopolysaccharide (LPS) both in BV-2 cells and primary microglia cells. Moreover, Sch A exerted obvious neuroprotective effects against inflammatory injury in neurons when exposed to microglia-conditioned medium. Investigations of the mechanism showed the anti-inflammatory effect of Sch A involved the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression levels and inhibition of the LPS-induced TRAF6-IKKβ-NF-κB pathway. Furthermore, inhibition of Jak2-Stat3 pathway activation and Stat3 nuclear translocation also was observed. In conclusion, SchA can exert anti-inflammatory and neuroprotective effects by alleviating microglia-mediated neuroinflammation injury through inhibiting the TRAF6-IKKβ-NF-κB and Jak2-Stat3 signaling pathways. PMID:26919063

  14. Pathway Markers for Pro-resolving Lipid Mediators in Maternal and Umbilical Cord Blood: A Secondary Analysis of the Mothers, Omega-3, and Mental Health Study.

    Science.gov (United States)

    Mozurkewich, Ellen L; Greenwood, Matthew; Clinton, Chelsea; Berman, Deborah; Romero, Vivian; Djuric, Zora; Qualls, Clifford; Gronert, Karsten

    2016-01-01

    The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are precursors to immune regulatory and specialized pro-resolving mediators (SPM) of inflammation termed resolvins, maresins, and protectins. Evidence for lipid mediator formation in vivo can be gained through evaluation of their 5-lipoxygenase (LOX) and 15-LOX metabolic pathway precursors and downstream metabolites. We performed a secondary blood sample analysis from 60 participants in the Mothers, Omega-3, and Mental Health study to determine whether SPM and SPM precursors are augmented by dietary EPA- and DHA-rich fish oil supplementation compared to soy oil placebo. We also aimed to study whether SPM and their precursors differ in early and late pregnancy or between maternal and umbilical cord blood. We found that compared to placebo supplementation, EPA- and DHA-rich fish oil supplementation increased SPM precursor 17-hydroxy docosahexaenoic acid (17-HDHA) concentrations in maternal and umbilical cord blood (P = 0.02). We found that the D-series resolvin pathway marker 17-HDHA increased significantly between enrollment and late pregnancy (P = 0.049). Levels of both 14-HDHA, a maresin pathway marker, and 17-HDHA were significantly greater in umbilical cord blood than in maternal blood (P < 0.001, both).

  15. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Song, Shasha [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Wang, Shuang [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  16. Cooperative Transcriptional Activation of Antimicrobial Genes by STAT and NF-κB Pathways by Concerted Recruitment of the Mediator Complex

    Directory of Open Access Journals (Sweden)

    Sebastian Wienerroither

    2015-07-01

    Full Text Available The transcriptional response to infection with the bacterium Listeria monocytogenes (Lm requires cooperative signals of the type I interferon (IFN-I-stimulated JAK-STAT and proinflammatory NF-κB pathways. Using ChIP-seq analysis, we define genes induced in Lm-infected macrophages through synergistic transcriptional activation by NF-κB and the IFN-I-activated transcription factor ISGF3. Using the Nos2 and IL6 genes as prime examples of this group, we show that NF-κB functions to recruit enzymes that establish histone marks of transcriptionally active genes. In addition, NF-κB regulates transcriptional elongation by employing the mediator kinase module for the recruitment of the pTEFb complex. ISGF3 has a major role in associating the core mediator with the transcription start as a prerequisite for TFIID and RNA polymerase II (Pol II binding. Our data suggest that the functional cooperation between two major antimicrobial pathways is based on promoter priming by NF-κB and the engagement of the core mediator for Pol II binding by ISGF3.

  17. Desmocollin 3 mediates follicle stimulating hormone-induced ovarian epithelial cancer cell proliferation by activating the EGFR/Akt signaling pathway.

    Science.gov (United States)

    Yang, Xiao; Wang, Jing; Li, Wen-Ping; Jin, Zhi-Jun; Liu, Xiao-Jun

    2015-01-01

    Follicle-stimulating hormone (FSH) is associated with the pathogenesis of ovarian cancer. We sought to explore whether desmocollin 3 (Dsc3) mediates FSH-induced ovarian epithelial cancer cell proliferation and whether the EGFR/Akt signaling pathway may be involved in this process. Dsc3 positivity in ovarian tissue specimens from 72 patients was assessed by immunohistochemistry. The positive expression rates of Dsc3 were similar in ovarian cancer tissues (24/31:77.4%) and borderline ovarian tumor tissues (18/22:81.8%) (P>0.05), but were significantly higher in these cancerous tissues than in benign ovarian cyst tissues (3/19:15.8%) (Pcancer cells (HO8910, Skov3ip, Skov and Hey cells, but not ES-2 and in borderline ovarian MCV152 tumor cells was higher than in the immortalized ovarian epithelial cell line, Moody. FSH up-regulated the expression of Dsc3 and EGFR in a dose- and time-dependent manner. Furthermore, a converse relationship between the expression of Dsc3, EFGR and PI3K/Akt signaling was elucidated using RNA interference and PI3K/Akt inhibitor in the absence and presence of FSH. A role for these proteins in FSH-induced cell proliferation was verified, highlighting their interdependence in mediating ovarian cancer cell function. These results suggest that Dsc3 can mediate FSH-induced ovarian cancer cell proliferation by activating the EGFR/Akt signaling pathway.

  18. Schisandra chinensis regulates drug metabolizing enzymes and drug transporters via activation of Nrf2-mediated signaling pathway

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    He JL

    2014-12-01

    increase in the intracellular level of glutathione and total glutathione S-transferase content. SCE significantly elevated the messenger ribonucleic acid and protein levels of P-glycoprotein and multidrug resistance-associated protein 2 and 4, whereas the expression of organic anion transporting peptide 1A2 and 1B1 was significantly downregulated by SCE. Knockdown of Nrf2 by small interfering ribonucleic acid attenuated the regulatory effect of SCE on these DMEs and drug transporters. SCE significantly upregulated Nrf2 and promoted the translocation of Nrf2 from cytoplasm to the nuclei. Additionally, SCE significantly suppressed the expression of cytosolic Kelch-like ECH-associated protein 1 (the repressor of Nrf2 and remarkably increased Nrf2 stability in HepG2 cells. Taken together, our findings suggest that the hepatoprotective effects of SCE may be partially ascribed to the modulation of DMEs and drug transporters via Nrf2-mediated signaling pathway. SCE may alter the pharmacokinetics of other coadministered drugs that are substrates of these DMEs and transporters and thus cause unfavorable herb–drug interactions. Keywords: Nrf2, Keap1, HepG2 cell, drug metabolizing enzyme, drug transporter, P-gp, MRP, OATP, Schisandra chinensis

  19. Ganoderma atrum polysaccharide evokes antitumor activity via cAMP-PKA mediated apoptotic pathway and down-regulation of Ca(2+)/PKC signal pathway.

    Science.gov (United States)

    Zhang, Shenshen; Nie, Shaoping; Huang, Danfei; Huang, Jianqin; Feng, Yanling; Xie, Mingyong

    2014-06-01

    Ganoderma atrum polysaccharide (PSG-1) has been commonly suggested as a candidate for prevention and therapy of cancer. We investigated the antitumor effect and the underlying molecular mechanisms of PSG-1. The results showed that PSG-1 inhibited tumor growth and resulted in tumor cell apoptosis in vivo. Here, the data revealed that PSG-1 caused a markedly increase in cAMP and PKA activities, rather than cGMP and PKC. Moreover, the treatment of PSG-1 induced a dramatic increase in the protein level of PKA. In contrast, the expression of PKC and intracellular [Ca(2+)]i were inhibited. Our study also revealed that treatment with PSG-1 increased the spleen and thymus weights, lymphocyte proliferation and macrophage phagocytic activity in tumor-bearing mice. Taken together, we conclude that PSG-1 could inhibit the tumor growth, possibly in part by enhancing the induction of apoptosis through cAMP-PKA signaling pathway and down-regulation of Ca(2+)/PKC signal pathway, activating host immune function in S180-bearing mice.

  20. Early activation of FGF and nodal pathways mediates cardiac specification independently of Wnt/beta-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Lee J Samuel

    Full Text Available BACKGROUND: Cardiac induction, the first step in heart development in vertebrate embryos, is thought to be initiated by anterior endoderm during gastrulation, but what the signals are and how they act is unknown. Several signaling pathways, including FGF, Nodal, BMP and Wnt have been implicated in cardiac specification, in both gain- and loss-of-function experiments. However, as these pathways regulate germ layer formation and patterning, their specific roles in cardiac induction have been difficult to define. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanisms of cardiac induction directly we devised an assay based on conjugates of anterior endoderm from early gastrula stage Xenopus embryos as the inducing tissue and pluripotent ectodermal explants as the responding tissue. We show that the anterior endoderm produces a specific signal, as skeletal muscle is not induced. Cardiac inducing signal needs up to two hours of interaction with the responding tissue to produce an effect. While we found that the BMP pathway was not necessary, our results demonstrate that the FGF and Nodal pathways are essential for cardiogenesis. They were required only during the first hour of cardiogenesis, while sustained activation of ERK was required for at least four hours. Our results also show that transient early activation of the Wnt/beta-catenin pathway has no effect on cardiogenesis, while later activation of the pathway antagonizes cardiac differentiation. CONCLUSIONS/SIGNIFICANCE: We have described an assay for investigating the mechanisms of cardiac induction by anterior endoderm. The assay was used to provide evidence for a direct, early and transient requirement of FGF and Nodal pathways. In addition, we demonstrate that Wnt/beta-catenin pathway plays no direct role in vertebrate cardiac specification, but needs to be suppressed just prior to differentiation.

  1. Parallel neural pathways in higher visual centers of the Drosophila brain that mediate wavelength-specific behavior.

    Science.gov (United States)

    Otsuna, Hideo; Shinomiya, Kazunori; Ito, Kei

    2014-01-01

    Compared with connections between the retinae and primary visual centers, relatively less is known in both mammals and insects about the functional segregation of neural pathways connecting primary and higher centers of the visual processing cascade. Here, using the Drosophila visual system as a model, we demonstrate two levels of parallel computation in the pathways that connect primary visual centers of the optic lobe to computational circuits embedded within deeper centers in the central brain. We show that a seemingly simple achromatic behavior, namely phototaxis, is under the control of several independent pathways, each of which is responsible for navigation towards unique wavelengths. Silencing just one pathway is enough to disturb phototaxis towards one characteristic monochromatic source, whereas phototactic behavior towards white light is not affected. The response spectrum of each demonstrable pathway is different from that of individual photoreceptors, suggesting subtractive computations. A choice assay between two colors showed that these pathways are responsible for navigation towards, but not for the detection itself of, the monochromatic light. The present study provides novel insights about how visual information is separated and processed in parallel to achieve robust control of an innate behavior.

  2. Parallel neural pathways in higher visual centers of the Drosophila brain that mediate wavelength-specific behavior

    Directory of Open Access Journals (Sweden)

    Hideo eOtsuna

    2014-02-01

    Full Text Available Compared with connections between the retinae and primary visual centers, relatively less is known in both mammals and insects about the functional segregation of neural pathways connecting primary and higher centers of the visual processing cascade. Here, using the Drosophila visual system as a model, we demonstrate two levels of parallel computation in the pathways that connect primary visual centers of the optic lobe to computational circuits embedded within deeper centers in the central brain. We show that a seemingly simple achromatic behavior, namely phototaxis, is under the control of several independent pathways, each of which is responsible for navigation towards unique wavelengths. Silencing just one pathway is enough to disturb phototaxis towards one characteristic monochromatic source, whereas phototactic behavior towards white light is not affected. The response spectrum of each demonstrable pathway is different from that of individual photoreceptors, suggesting subtractive computations. A choice assay between two colors showed that these pathways are responsible for navigation towards, but not for the detection itself of, the monochromatic light. The present study provides novel insights about how visual information is separated and processed in parallel to achieve robust control of an innate behavior.

  3. Remodeling of global transcription patterns of Cryptococcus neoformans genes mediated by the stress-activated HOG signaling pathways.

    Science.gov (United States)

    Ko, Young-Joon; Yu, Yeong Man; Kim, Gyu-Bum; Lee, Gir-Won; Maeng, Pil Jae; Kim, Sangsoo; Floyd, Anna; Heitman, Joseph; Bahn, Yong-Sun

    2009-08-01

    The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study, we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses but also elucidate novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress defense and HOG-dependent genes, which encode a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system.

  4. Inhibition of the CyclinD1 promoter in response to sonic hedgehog signaling pathway transduction is mediated by Gli1

    Science.gov (United States)

    Lin, Zhongxiao; Sheng, Hansong; You, Chaoguo; Cai, Ming; Zhang, Yiping; Yu, Li Sheng; Yu, Xiaoming; Lin, Jian; Zhang, Nu

    2017-01-01

    Medulloblastoma (MB) is the most common malignant tumor of the central nervous system in children. Accumulating evidence suggests a major role for the activation of the sonic hedgehog (SHH) signaling pathway in the development of MB cells; however, the mechanisms underlying the effect of this pathway on tumor survival and growth remain poorly understood. The Gli family zinc finger 1 (Gli1) transcription factor is considered as a mediator of the SHH signaling pathway in MB cells. Therefore, the present study investigated whether the SHH signaling pathway promotes the apoptosis of MB cells via downregulation of Gli1. GANT61, a novel Gli1 inhibitor, is known to have an in vitro activity against tumors. In the current study, Daoy cells were treated with different concentrations of GANT61 for 24 h, and the effect on cell proliferation was assayed by cell counting kit-8 assay. In addition, the cell cycle progression and apoptosis were assayed by flow cytometry analysis and hematoxylin-eosin (HE) staining. The effects of GANT61 treatment on SHH signaling pathway at the mRNA level were assayed by polymerase chain reaction (PCR). To further elucidate the inhibitory effects of GANT61 on the expression of Gli1 and CyclinD1, their protein levels were examined by western blot and immunofluorescence. The results indicated that GANT61 significantly inhibited the proliferation of Daoy cells in a dose-dependent manner, compared with the control group (PSHH pathway activity in MB, and may be a novel agent for use in combined chemotherapeutic regimens. PMID:28123507

  5. CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway.

    Science.gov (United States)

    Hauck, C R; Meyer, T F; Lang, F; Gulbins, E

    1998-01-15

    The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.

  6. Screening of the Human Kinome Identifies MSK1/2-CREB1 as an Essential Pathway Mediating Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication during Primary Infection

    Science.gov (United States)

    Cheng, Fan; Sawant, Tanvee Vinod; Lan, Ke; Lu, Chun; Jung, Jae U.

    2015-01-01

    ABSTRACT Viruses often hijack cellular pathways to facilitate infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus etiologically associated with Kaposi's sarcoma, a vascular tumor of endothelial cells. Despite intensive studies, cellular pathways mediating KSHV infection and replication are still not well defined. Using an antibody array approach, we examined cellular proteins phosphorylated during primary KSHV infection of primary human umbilical vein endothelial cells. Enrichment analysis identified integrin/mitogen-activated protein kinase (integrin/MAPK), insulin/epidermal growth factor receptor (insulin/EGFR), and JAK/STAT as the activated networks during primary KSHV infection. The transcriptional factor CREB1 (cyclic AMP [cAMP]-responsive element-binding protein 1) had the strongest increase in phosphorylation. While knockdown of CREB1 had no effect on KSHV entry and trafficking, it drastically reduced the expression of lytic transcripts and proteins and the production of infectious virions. Chemical activation of CREB1 significantly enhanced viral lytic replication. In contrast, CREB1 neither influenced the expression of the latent gene LANA nor affected KSHV infectivity. Mechanistically, CREB1 was not activated through the classic cAMP/protein kinase A (cAMP/PKA) pathway or via the AKT, MK2, and RSK pathways. Rather, CREB1 was activated by the mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Consequently, chemical inhibition or knockdown of MSKs significantly inhibited the KSHV lytic replication program; however, it had a minimal effect on LANA expression and KSHV infectivity. Together, these results identify the MSK1/2-CREB1 proteins as novel essential effectors of KSHV lytic replication during primary infection. The differential effect of the MSK1/2-CREB1 pathway on the expression of viral latent and lytic genes might control the robustness of viral lytic replication, and therefore the

  7. Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Smallwood, Heather S.; Lopez Ferrer, Daniel; Eberlein, P. Elis; Watson, David J.; Squier, Thomas C.

    2009-02-05

    Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance metastasis. In this respect, we have identified a dose-dependent increase in the abundance of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose exposures. Increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX protein complexes associated with DNA repair following macrophage irradiation, with no change in the extent of double-stranded DNA damage. In comparison, when NFκB-dependent pathways are inhibited, through the expression of a dominant-negative IκB construct, there is no significant increase in phosphorylated H2AX upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX mediated DNA-repair pathways is not the result of nonspecific NFκB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation to enhance macrophage radioresistance through processes that include the upregulation of DNA repair pathways involving histone protein H2AX phosphorylation.

  8. Advanced glycation end product-induced astrocytic differentiation of cultured neurospheres through inhibition of Notch-Hes1 pathway-mediated neurogenesis.

    Science.gov (United States)

    Guo, Yijing; Wang, Pin; Sun, Haixia; Cai, Rongrong; Xia, Wenqing; Wang, Shaohua

    2014-01-01

    This study aims to investigate the roles of the Notch-Hes1 pathway in the advanced glycation end product (AGE)-mediated differentiation of neural stem cells (NSCs). We prepared pLentiLox3.7 lentiviral vectors that express short hairpin RNA (shRNA) against Notch1 and transfected it into NSCs. Cell differentiation was analyzed under confocal laser-scanning microscopy. The percentage of neurons and astrocytes was quantified by normalizing the total number of TUJ1+ (Neuron-specific class III β-tubulin) and GFAP+ (Glial fibrillary acidic protein) cells to the total number of Hoechst 33342-labeled cell nuclei. The protein and gene expression of Notch-Hes1 pathway components was examined via western blot analysis and real-time PCR. After 1 week of incubation, we found that AGE-bovine serum albumin (BSA) (400 μg/mL) induced the astrocytic differentiation of cultured neurospheres and inhibited neuronal formation. The expression of Notch-Hes1 pathway components was upregulated in the cells in the AGE-BSA culture medium. Immunoblot analysis indicated that shRNA silencing of Notch1 expression in NSCs significantly increases neurogenesis and suppresses astrocytic differentiation in NSCs incubated with AGE-BSA. AGEs promote the astrocytic differentiation of cultured neurospheres by inhibiting neurogenesis through the Notch-Hes1 pathway, providing a potential therapeutic target for hyperglycemia-related cognitive deficits.

  9. The Caenorhabditis elegans Ste20-related kinase and Rac-type small GTPase regulate the c-Jun N-terminal kinase signaling pathway mediating the stress response.

    Science.gov (United States)

    Fujiki, Kota; Mizuno, Tomoaki; Hisamoto, Naoki; Matsumoto, Kunihiro

    2010-02-01

    Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like MAPK signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPKK, and KGB-1 JNK-like MAPK. In this study, we identify the max-2 gene encoding a C. elegans Ste20-related protein kinase as a component functioning upstream of the MLK-1-MEK-1-KGB-1 pathway. The max-2 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. Biochemical analysis reveals that MAX-2 activates MLK-1 through direct phosphorylation of a specific residue in the activation loop of the MLK-1 kinase domain. Our genetic data presented here also show that MIG-2 small GTPase functions upstream of MAX-2 in the KGB-1 pathway. These results suggest that MAX-2 and MIG-2 play a crucial role in mediating the heavy metal stress response regulated by the KGB-1 pathway.

  10. Suppressor of cytokine signalling (SOCS)-3 protects beta cells against IL-1beta-mediated toxicity through inhibition of multiple nuclear factor-kappaB-regulated proapoptotic pathways

    DEFF Research Database (Denmark)

    Karlsen, Allan Ertman; Heding, P E; Frobøse, H;

    2004-01-01

    The proinflammatory cytokine IL-1beta induces apoptosis in pancreatic beta cells via pathways dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein kinase, and protein kinase C. We recently showed suppressor of cytokine signalling (SOCS)-3 to be a natural negative feedback...... regulator of IL-1beta- and IFN-gamma-mediated signalling in rat islets and beta cell lines, preventing their deleterious effects. However, the mechanisms underlying SOCS-3 inhibition of IL-1beta signalling and prevention against apoptosis remain unknown....

  11. Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress

    DEFF Research Database (Denmark)

    Brouwers, O; Niessen, P M; Haenen, G

    2010-01-01

    intracellular methylglyoxal effect. The diabetes-induced impaired potency (pD(2)) in mesenteric arteries of wild-type rats was significantly improved by GLO-I overexpression (p affect NO-dependent vasorelaxation, while under the same conditions the receptor...... for AGE ligand S100b did (p stress marker nitrotyrosine. Antioxidant pre-incubation prevented methylglyoxal......-induced impairment of vasoreactivity. CONCLUSIONS/INTERPRETATION: These data show that hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation is mediated by increased intracellular methylglyoxal levels in a pathway dependent on oxidative stress....

  12. EphA4 promotes cell proliferation and cell adhesion-mediated drug resistance via the AKT pathway in multiple myeloma.

    Science.gov (United States)

    Ding, Linlin; Shen, Yaodong; Ni, Jing; Ou, Yiqing; Ou, Yangyu; Liu, Hong

    2017-03-01

    Eph receptor A4 (EphA4), a member of the erythropoietin-producing hepatocellular (Eph) family, has been reported to upregulate in several tumors. However, the role of EphA4 in multiple myeloma has not been clarified yet. In this study, we found that EphA4 promoted proliferation of multiple myeloma cells via the regulation of cell cycle. Besides, EphA4 was closely related to cell adhesion of multiple myeloma cells and promoted cell adhesion-mediated drug resistance by enhancing the phosphorylation levels of Akt (p-AKT) expression in multiple myeloma. More interestingly, we discovered that EphA4 can interact with cyclin-dependent kinase 5 (CDK5) and regulate its expression in multiple myeloma. CDK5 has been reported to be overexpressed in multiple myeloma which mediated bortezomib resistance and also participated in AKT pathway. And we have also proved the fact. So, we supposed that EphA4 interacted with CDK5 and promoted its expression which in turn enhanced p-AKT expression and promoted cell adhesion-mediated drug resistance in multiple myeloma. Therefore, this study clarifies the molecular mechanism of cell adhesion-mediated drug resistance and may be useful in identifying potential target for treatment of multiple myeloma.

  13. Blocking RhoA/ROCK inhibits the pathogenesis of pemphigus vulgaris by suppressing oxidative stress and apoptosis through TAK1/NOD2-mediated NF-κB pathway.

    Science.gov (United States)

    Liang, Junqin; Zeng, Xuewen; Halifu, Yilinuer; Chen, Wenjing; Hu, Fengxia; Wang, Peng; Zhang, Huan; Kang, Xiaojing

    2017-06-12

    Oxidative stress and apoptosis play critical roles in pemphigus vulgaris (PV). The main aim of the present study was to investigate the effects of RhoA/ROCK signaling on UVB-induced oxidative damage, and to delineate the molecular mechanisms involved in the UVB-mediated inflammatory and apoptotic response. In HaCaT cells, we observed that blockage of RhoA/ROCK signaling with the inhibitor CT04 or Y27632 greatly inhibited the UVB-mediated increase in intracellular reactive oxygen species (ROS). Additionally, inhibition of RhoA/ROCK signaling reduced UVB-induced apoptosis, as exemplified by a reduction in DNA fragmentation, and also elevated anti-apoptotic Bcl-2 protein, concomitant with reduced levels of pro-apoptotic protein Bax, caspase-3 cleavage and decreased PARP-1 protein. The release of inflammatory mediators TNF-α, IL-1β, and IL-6 was also attenuated. Mechanically, we observed that blockage of RhoA/ROCK repressed the TAK1/NOD2-mediated NF-κB pathway in HaCaT cells exposed to UVB. Taken together, these data reveal that RhoA/ROCK signaling is one of the regulators contributing to oxidative damage and apoptosis in human keratinocytes, suggesting that RhoA/ROCK signaling has strong potential to be used as a useful therapeutic target in skin diseases including PV.

  14. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona, E-mail: moroianu@bc.edu

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.

  15. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Yang You

    Full Text Available Increased stromal stiffness is associated with hepatocellular carcinoma (HCC development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

  16. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

    Science.gov (United States)

    You, Yang; Zheng, Qiongdan; Dong, Yinying; Wang, Yaohui; Zhang, Lan; Xue, Tongchun; Xie, Xiaoying; Hu, Chao; Wang, Zhiming; Chen, Rongxin; Wang, Yanhong; Cui, Jiefeng; Ren, Zhenggang

    2015-01-01

    Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

  17. Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes.

    Science.gov (United States)

    Sengenes, Coralie; Bouloumie, Anne; Hauner, Hans; Berlan, Michel; Busse, Rudi; Lafontan, Max; Galitzky, Jean

    2003-12-05

    Our previous studies have demonstrated that natriuretic peptides (NPs), peptide hormones with natriuretic, diuretic, and vasodilating properties, exert a potent control on the lipolysis in human adipocytes via the activation of the type A guanylyl cyclase receptor (1, 2). In the current study we investigated the intracellular mechanisms involved in the NP-stimulated lipolytic effect in human preadipocytes and adipocytes. We demonstrate that the atrial NP (ANP)-induced lipolysis in human adipocytes was associated with an enhanced serine phosphorylation of the hormone-sensitive lipase (HSL). Both ANP-mediated lipolysis and HSL phosphorylation were inhibited in the presence of increasing concentrations of the guanylyl cyclase inhibitor LY-83583. ANP did not modulate the activity of the cAMP-dependent protein kinase (PKA). Moreover, H-89, a PKA inhibitor, did not affect the ANP-induced lipolysis. On primary cultures of human preadipocytes, the ANP-mediated lipolytic effect was dependent on the differentiation process. On differentiated human preadipocytes, ANP-mediated lipolysis, associated with an increased phosphorylation of HSL and of perilipin A, was strongly decreased by treatment with the inhibitor of the cGMP-dependent protein kinase I (cGKI), Rp-8-pCPT-cGMPS. Thus, ANP-induced lipolysis in human adipocytes is a cGMP-dependent pathway that induces the phosphorylation of HSL and perilipin A via the activation of cGKI. The present study shows that lipolysis in human adipocytes can be controlled by an independent cGKI-mediated signaling as well as by the classical cAMP/PKA pathway.

  18. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway.

    Science.gov (United States)

    Said, Hamid M; Wang, Shuling; Ma, Thomas Y

    2005-07-15

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent Km of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+ -H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+ -calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is

  19. Somatic mutational analysis of FAK in breast cancer: A novel gain-of-function mutation due to deletion of exon 33

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Xu-Qian [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China); Liu, Xiang-Fan [Faculty of Medical Laboratory Science, Shanghai JiaoTong University School of Medicine, Shanghai (China); Yao, Ling [Department of Biochemistry and Molecular Biology, Shanghai JiaoTong University School of Medicine, Shanghai (China); Chen, Chang-Qiang; Gu, Zhi-Dong [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China); Ni, Pei-Hua [Faculty of Medical Laboratory Science, Shanghai JiaoTong University School of Medicine, Shanghai (China); Zheng, Xin-Min [Department of Biochemistry and Molecular Biology, Shanghai JiaoTong University School of Medicine, Shanghai (China); Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY (United States); Fan, Qi-Shi, E-mail: qishifan@126.com [Department of Clinical Laboratory, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai (China)

    2014-01-10

    Highlights: •A novel FAK splicing mutation identified in breast tumor. •FAK-Del33 mutation promotes cell migration and invasion. •FAK-Del33 mutation regulates FAK/Src signal pathway. -- Abstract: Focal adhesion kinase (FAK) regulates cell adhesion, migration, proliferation, and survival. We identified a novel splicing mutant, FAK-Del33 (exon 33 deletion, KF437463), in both breast and thyroid cancers through colony sequencing. Considering the low proportion of mutant transcripts in samples, this mutation was detected by TaqMan-MGB probes based qPCR. In total, three in 21 paired breast tissues were identified with the FAK-Del33 mutation, and no mutations were found in the corresponding normal tissues. When introduced into a breast cell line through lentivirus infection, FAK-Del33 regulated cell motility and migration based on a wound healing assay. We demonstrated that the expression of Tyr397 (main auto-phosphorylation of FAK) was strongly increased in FAK-Del33 overexpressed breast tumor cells compared to wild-type following FAK/Src RTK signaling activation. These results suggest a novel and unique role of the FAK-Del33 mutation in FAK/Src signaling in breast cancer with significant implications for metastatic potential.

  20. Dissociation of the pathways mediating ipsilateral and contralateral motor-evoked potentials in human hand and arm muscles

    Science.gov (United States)

    Ziemann, Ulf; Ishii, Kenji; Borgheresi, Alessandra; Yaseen, Zaneb; Battaglia, Fortunato; Hallett, Mark; Cincotta, Massimo; Wassermann, Eric M

    1999-01-01

    Growing evidence points toward involvement of the human motor cortex in the control of the ipsilateral hand. We used focal transcranial magnetic stimulation (TMS) to examine the pathways of these ipsilateral motor effects.Ipsilateral motor-evoked potentials (MEPs) were obtained in hand and arm muscles of all 10 healthy adult subjects tested. They occurred in the finger and wrist extensors and the biceps, but no response or inhibitory responses were observed in the opponens pollicis, finger and wrist flexors and the triceps.The production of ipsilateral MEPs required contraction of the target muscle. The threshold TMS intensity for ipsilateral MEPs was on average 1.8 times higher, and the onset was 5.7 ms later (in the wrist extensor muscles) compared with size-matched contralateral MEPs.The corticofugal pathways of ipsilateral and contralateral MEPs could be dissociated through differences in cortical map location and preferred stimulating current direction.Both ipsi- and contralateral MEPs in the wrist extensors increased with lateral head rotation toward, and decreased with head rotation away from, the side of the TMS, suggesting a privileged input of the asymmetrical tonic neck reflex to the pathway of the ipsilateral MEP.Large ipsilateral MEPs were obtained in a patient with complete agenesis of the corpus callosum.The dissociation of the pathways for ipsilateral and contralateral MEPs indicates that corticofugal motor fibres other than the fast-conducting crossed corticomotoneuronal system can be activated by TMS. Our data suggest an ipsilateral oligosynaptic pathway, such as a corticoreticulospinal or a corticopropriospinal projection as the route for the ipsilateral MEP. Other pathways, such as branching of corticomotoneuronal axons, a transcallosal projection or a slow-conducting monosynaptic ipsilateral pathway are very unlikely or can be excluded. PMID:10420023

  1. Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase.

    Science.gov (United States)

    Lee, L F; Guan, J; Qiu, Y; Kung, H J

    2001-12-01

    The bombesin/gastrin-releasing peptide (GRP) family of neuropeptides has been implicated in various in vitro and in vivo models of human malignancies including prostate cancers. It was previously shown that bombesin and/or neurotensin (NT) acts as a survival and migratory factor(s) for androgen-independent prostate cancers. However, a role in the transition from an androgen-dependent to -refractory state has not been addressed. In this study, we investigate the biological effects and signal pathways of bombesin and NT on LNCaP, a prostate cancer cell line which requires androgen for growth. We show that both neurotrophic factors can induce LNCaP growth in the absence of androgen. Concurrent transactivation of reporter genes driven by the prostate-specific antigen promoter or a promoter carrying an androgen-responsive element (ARE) indicate that growth stimulation is accompanied by androgen receptor (AR) activation. Furthermore, neurotrophic factor-induced gene activation was also present in PC3 cells transfected with the AR but not in the parental line which lacks the AR. Given that bombesin does not directly bind to the AR and is known to engage a G-protein-coupled receptor, we investigated downstream signaling events that could possibly interact with the AR pathway. We found that three nonreceptor tyrosine kinases, focal adhesion kinase (FAK), Src, and Etk/BMX play important parts in this process. Etk/Bmx activation requires FAK and Src and is critical for neurotrophic factor-induced growth, as LNCaP cells transfected with a dominant-negative Etk/BMX fail to respond to bombesin. Etk's activation requires FAK, Src, but not phosphatidylinositol 3-kinase. Likewise, bombesin-induced AR activation is inhibited by the dominant-negative mutant of either Src or FAK. Thus, in addition to defining a new G-protein pathway, this report makes the following points regarding prostate cancer. (i) Neurotrophic factors can activate the AR, thus circumventing the normal growth

  2. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1.

    Directory of Open Access Journals (Sweden)

    Fengjuan Zhang

    2016-01-01

    Full Text Available Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins are essential components of Bacillus thuringiensis (Bt biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1. In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control.

  3. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

    Science.gov (United States)

    Zhang, Fengjuan; Peng, Donghai; Cheng, Chunsheng; Zhou, Wei; Ju, Shouyong; Wan, Danfeng; Yu, Ziquan; Shi, Jianwei; Deng, Yaoyao; Wang, Fenshan; Ye, Xiaobo; Hu, Zhenfei; Lin, Jian; Ruan, Lifang; Sun, Ming

    2016-01-01

    Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control. PMID:26795495

  4. Sulforaphane induces apoptosis in T24 human urinary bladder cancer cells through a reactive oxygen species-mediated mitochondrial pathway: the involvement of endoplasmic reticulum stress and the Nrf2 signaling pathway.

    Science.gov (United States)

    Jo, Guk Heui; Kim, Gi-Young; Kim, Wun-Jae; Park, Kun Young; Choi, Yung Hyun

    2014-10-01

    Sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, has received a great deal of attention because of its ability to inhibit cell proliferation and induce apoptosis in cancer cells. In this study, we investigated the anticancer activity of sulforaphane in the T24 human bladder cancer line, and explored its molecular mechanism of action. Our results showed that treatment with sulforaphane inhibited cell viability and induced apoptosis in T24 cells in a concentration-dependent manner. Sulforaphane-induced apoptosis was associated with mitochondria dysfunction, cytochrome c release and Bcl-2/Bax dysregulation. Furthermore, the increased activity of caspase-9 and -3, but not caspase-8, was accompanied by the cleavage of poly ADP-ribose polymerase, indicating the involvement of the mitochondria-mediated intrinsic apoptotic pathway. Concomitant with these changes, sulforaphane triggered reactive oxygen species (ROS) generation, which, along with the blockage of sulforaphane-induced loss of mitochondrial membrane potential and apoptosis, was strongly attenuated by the ROS scavenger N-acetyl-L-cysteine. Furthermore, sulforaphane was observed to activate endoplasmic reticulum (ER) stress and the nuclear factor-E2-related factor-2 (Nrf2) signaling pathway, as demonstrated by the upregulation of ER stress‑related proteins, including glucose-regulated protein 78 and C/EBP-homologous protein, and the accumulation of phosphorylated Nrf2 proteins in the nucleus and induction of heme oxygenase-1 expression, respectively. Taken together, these results demonstrate that sulforaphane has antitumor effects against bladder cancer cells through an ROS-mediated intrinsic apoptotic pathway, and suggest that ER stress and Nrf2 may represent strategic targets for sulforaphane-induced apoptosis.

  5. Identification of a Wnt/Dvl/beta-Catenin --> Pitx2 pathway mediating cell-type-specific proliferation during development.

    Science.gov (United States)

    Kioussi, Chrissa; Briata, Paola; Baek, Sung Hee; Rose, David W; Hamblet, Natasha S; Herman, Thomas; Ohgi, Kenneth A; Lin, Chijen; Gleiberman, Anatoli; Wang, Jianbo; Brault, Veronique; Ruiz-Lozano, Pilar; Nguyen, H D; Kemler, Rolf; Glass, Christopher K; Wynshaw-Boris, Anthony; Rosenfeld, Michael G

    2002-11-27

    Understanding the cell type-specific molecular mechanisms by which distinct signaling pathways combinatorially control proliferation during organogenesis is a central issue in development and disease. Here, we report that the bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/beta-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Regulated exchange of HDAC1/beta-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for Cyclin D2 gene induction. The molecular strategy underlying interactions between the Wnt and growth factor-dependent signaling pathways in cardiac outflow tract and pituitary proliferation is likely to be prototypic of cell-specific proliferation strategies in other tissues.

  6. CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, C H; Marquart, H V; Prodinger, W M;

    2001-01-01

    Normal human B lymphocytes activate the alternative