WorldWideScience

Sample records for aft2 mediates activation

  1. Convergent Regulation of Candida albicans Aft2 and Czf1 in Invasive and Opaque Filamentation.

    Science.gov (United States)

    Xu, Ning; Dong, Yi-Jie; Yu, Qi-Lin; Zhang, Bing; Zhang, Meng; Jia, Chang; Chen, Yu-Lu; Zhang, Biao; Xing, Lai-Jun; Li, Ming-Chun

    2015-09-01

    Candida albicans is the most common fungal pathogen of mucosal infections and invasive diseases in immuno-compromised humans. The abilities of yeast-hyphal growth and white-opaque switching affect C. albicans physiology and virulence. Here, we showed that C. albicans Aft2 regulator was required for embedded filamentous growth and opaque cell-type formation. Under low-temperature matrix embedded conditions, Aft2 functioned downstream of Czf1-mediated pathway and was required for invasive filamentation. Moreover, deletion of AFT2 significantly reduced opaque cell-type formation under N-acetylglucosamine (GlcNAc) inducing conditions. Ectopic expression of CZF1 slightly increased the white-opaque switching frequency in the aft2Δ/Δ mutant, but did not completely restore to wild-type levels, suggesting that Czf1 at least partially bypassed the essential requirement for Aft2 in response to opaque-inducing cues. In addition, multiple environmental cues altered AFT2 mRNA and protein levels, such as low temperature, physical environment and GlcNAc. Although the absence of Czf1 or Efg1 also increased the expression level of AFT2 gene, deletion of CZF1 remarkably reduced the stability of Aft2 protein. Furthermore, C. albicans Aft2 physically interacted with Czf1 under all tested conditions, whereas the interaction between Aft2 and Efg1 was barely detectable under embedded conditions, supporting the hypothesis that Aft2, together with Czf1, contributed to activate filamentous growth by antagonizing Efg1-mediated repression under matrix-embedded conditions.

  2. Aft2, a novel transcription regulator, is required for iron metabolism, oxidative stress, surface adhesion and hyphal development in Candida albicans.

    Science.gov (United States)

    Xu, Ning; Cheng, Xinxin; Yu, Qilin; Qian, Kefan; Ding, Xiaohui; Liu, Ruming; Zhang, Biao; Xing, Laijun; Li, Mingchun

    2013-01-01

    Morphological transition and iron metabolism are closely relevant to Candida albicans pathogenicity and virulence. In our previous study, we demonstrated that C. albicans Aft2 plays an important role in ferric reductase activity and virulence. Here, we further explored the roles of C. albicans Aft2 in numerous cellular processes. We found that C. albicans Aft2 exhibited an important role in iron metabolism through bi-directional regulation effects on iron-regulon expression. Deletion of AFT2 reduced cellular iron accumulation under iron-deficient conditions. Furthermore, both reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity were remarkably increased in the aft2Δ/Δ mutant, which were thought to be responsible for the defective responses to oxidative stress. However, we found that over-expression of C. albicans AFT2 under the regulation of the strong PGK1 promoter could not effectively rescue Saccharomyces cerevisiae aft1Δ mutant defects in some cellular processes, such as cell-wall assembly, ion homeostasis and alkaline resistance, suggesting a possibility that C. albicans Aft2 weakened its functional role of regulating some cellular metabolism during the evolutionary process. Interestingly, deletion of AFT2 in C. albicans increased cell surface hydrophobicity, cell flocculation and the ability of adhesion to polystyrene surfaces. In addition, our results also revealed that C. albicans Aft2 played a dual role in regulating hypha-specific genes under solid and liquid hyphal inducing conditions. Deletion of AFT2 caused an impaired invasive growth in solid medium, but an increased filamentous aggregation and growth in liquid conditions. Moreover, iron deficiency and environmental cues induced nuclear import of Aft2, providing additional evidence for the roles of Aft2 in transcriptional regulation. PMID:23626810

  3. Mediatized Extreme Right Activism and Discourse

    DEFF Research Database (Denmark)

    Peters, Rikke Alberg

    2015-01-01

    activism. It is a good example of how new contentious action repertoires in which online and street activism intertwine have also spread to extreme right groups. Despite its neo-fascist and extreme right content the ‘Become Immortal’ campaign serves as an illustrative case for the study of mediated...... and mediatized activism. In order to analyse of the protest form, the visual aesthetics and the discourse of ‘The Immortals’, the paper mobilises two concepts from media and communication studies: mediation and mediatization. It will be argued that that the current transformation of the extreme right: that is...

  4. Statins attenuate polymethylmethacrylate-mediated monocyte activation.

    LENUS (Irish Health Repository)

    Laing, Alan J

    2012-02-03

    BACKGROUND: Periprosthetic osteolysis precipitates aseptic loosening of components, increases the risk of periprosthetic fracture and, through massive bone loss, complicates revision surgery and ultimately is the primary cause for failure of joint arthroplasty. The anti-inflammatory properties of HMG-CoA reductase inhibitors belonging to the statin family are well recognized. We investigated a possible role for status in initiating the first stage of the osteolytic cycle, namely monocytic activation. METHODS: We used an in vitro model of the human monocyte\\/macrophage inflammatory response to poly-methylmethacrylate (PMMA) particles after pretreat-ing cells with cerivastatin, a potent member of the statin family. Cell activation based upon production of TNF-alpha and MCP-1 cytokines was analyzed and the intracellular Raf-MEK-ERK signal transduction pathway was evaluated using western blot analysis, to identify its role in cell activation and in any cerivastatin effects observed. RESULTS: We found that pretreatment with cerivastatin significantly abrogates the production of inflammatory cytokines TNF-alpha and MCP-1 by human monocytes in response to polymethylmethacrylate particle activation. This inflammatory activation and attenuation appear to be mediated through the intracellular Raf-MEK-ERK pathway. INTERPRETATION: We propose that by intervening at the upstream activation stage, subsequent osteoclast activation and osteolysis can be suppressed. We believe that the anti-inflammatory properties of statins may potentially play a prophylactic role in the setting of aseptic loosening, and in so doing increase implant longevity.

  5. Evaluating Active U: an internet-mediated physical activity program

    Directory of Open Access Journals (Sweden)

    Goodrich David E

    2009-09-01

    Full Text Available Abstract Background Engaging in regular physical activity can be challenging, particularly during the winter months. To promote physical activity at the University of Michigan during the winter months, an eight-week Internet-mediated program (Active U was developed providing participants with an online physical activity log, goal setting, motivational emails, and optional team participation and competition. Methods This study is a program evaluation of Active U. Approximately 47,000 faculty, staff, and graduate students were invited to participate in the online Active U intervention in the winter of 2007. Participants were assigned a physical activity goal and were asked to record each physical activity episode into the activity log for eight weeks. Statistics for program reach, effectiveness, adoption, and implementation were calculated using the Re-Aim framework. Multilevel regression analyses were used to assess the decline in rates of data entry and goal attainment during the program, to assess the likelihood of joining a team by demographic characteristics, to test the association between various predictors and the number of weeks an individual met his or her goal, and to analyze server load. Results Overall, 7,483 individuals registered with the Active U website (≈16% of eligible, and 79% participated in the program by logging valid data at least once. Staff members, older participants, and those with a BMI P Conclusion Internet-mediated physical activity interventions that focus on physical activity logging and goal setting while incorporating team competition may help a significant percentage of the target population maintain their physical activity during the winter months.

  6. Activism and the Online Mediation Opportunity Structure

    DEFF Research Database (Denmark)

    Uldam, Julie

    2013-01-01

    The annual United Nations (UN) Framework Convention on Climate Change conferences provides a transnational mediation opportunity structure for activist networks to contest policies that favor market-based models for solving the climate crisis. Online technologies, including commercial social media......, have arguably increased possibilities for being involved in protests on a transnational level. However, this article shows how online modes of action privilege lobbying tactics over civil disobedience tactics, arguing that the former is often incommensurate with an anticapitalist climate approach...... to climate change activism. This impedes possibilities for using online media to protest at the radical end of the climate justice movement spectrum. This article explores this interrelationship between activist demands and (online) modes of action through a focus on the mobilization efforts of London...

  7. Protease activated receptors (PARS) mediation in gyroxin biological activity

    International Nuclear Information System (INIS)

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH2, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  8. Fungal mediator tail subunits contain classical transcriptional activation domains.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

  9. Mediated Electrochemical Measurements of Intracellular Catabolic Activities of Yeast Cells

    Institute of Scientific and Technical Information of China (English)

    Jin Sheng ZHAO; Zhen Yu YANG; Yao LU; Zheng Yu YANG

    2005-01-01

    Coupling with the dual mediator system menadione/ferricyanide, microelectrode voltammetric measurements were undertaken to detect the ferrocyanide accumulations arising from the mediated reduction of ferricyanide by yeast cells. The results indicate that the dual mediator system menadione/ferricyanide could be used as a probe to detect cellular catabolic activities in yeast cells and the electrochemical response has a positive relationship with the specific growth rate of yeast cells.

  10. Free radical-mediated activation of hydrazine derivatives.

    OpenAIRE

    Kalyanaraman, B.; Sinha, B. K.

    1985-01-01

    Hydrazines are known to undergo oxidative activation in several enzymatic systems in vitro. Free radicals or carbonium ions have been proposed as active intermediates during such activation. The toxic effects elicited by hydrazines have also been linked to free radical-mediated activation. In this report, we have reviewed the identification of organic free radicals from hydrazines by direct ESR and ESR-spin trapping.

  11. Baker's Yeast Mediated Reduction of Optically Active Diketone

    Institute of Scientific and Technical Information of China (English)

    ZHENG, Guo-Jun(郑国君); GAO, Xiao-Lei(高晓蕾); CHEN, Jin-Chun(陈锦春); LI, Yu-Lin(李裕林)

    2004-01-01

    Baker's yeast mediated reduction of optically active diketone is described. The two keto groups are efficiently differentiated and the ee value of the recovered material is considerably raised. It affords highly optically active key intermediates efficiently for the synthesis of natural polyhydroxylated agarofuran products.

  12. Aldose reductase mediates retinal microglia activation.

    Science.gov (United States)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.

  13. Activation of CFTR by ASBT-mediated bile salt absorption.

    Science.gov (United States)

    Bijvelds, Marcel J C; Jorna, Huub; Verkade, Henkjan J; Bot, Alice G M; Hofmann, Franz; Agellon, Luis B; Sinaasappel, Maarten; de Jonge, Hugo R

    2005-11-01

    In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal ileum might also modulate intestinal fluid secretion. Taurocholate (TC) induced a biphasic rise in the short circuit current across ileal tissue, reflecting transepithelial electrogenic ion transport. This response was sensitive to bumetanide and largely abrogated in Cftr-null mice, indicating that it predominantly reflects CFTR-mediated Cl- secretion. The residual response in Cftr-null mice could be attributed to electrogenic ASBT activity, as it matched the TC-coupled absorptive Na+ flux. TC-evoked Cl- secretion required ASBT-mediated TC uptake, because it was blocked by a selective ASBT inhibitor and was restricted to the distal ileum. Suppression of neurotransmitter or prostaglandin release, blocking of the histamine H1 receptor, or pretreatment with 5-hydroxytryptamine did not abrogate the TC response, suggesting that neurocrine or immune mediators of Cl- secretion are not involved. Responses to TC were retained after carbachol treatment and after permeabilization of the basolateral membrane with nystatin, indicating that BS modulate CFTR channel gating rather than the driving force for Cl- exit. TC-induced Cl- secretion was maintained in cGMP-dependent protein kinase II-deficient mice and only partially inhibited by the cAMP-dependent protein kinase inhibitor H89, suggesting a mechanism of CFTR activation different from cAMP or cGMP signaling. We conclude that active BS absorption in the ileum triggers CFTR activation and, consequently, local salt and water secretion, which may serve to prevent intestinal obstruction in the postprandial state. PMID:16037545

  14. Spillover-mediated feedforward-inhibition functionally segregates interneuron activity

    Science.gov (United States)

    Coddington, Luke T.; Rudolph, Stephanie; Lune, Patrick Vande; Overstreet-Wadiche, Linda; Wadiche, Jacques I.

    2013-01-01

    Summary Neurotransmitter spillover represents a form of neural transmission not restricted to morphologically defined synaptic connections. Communication between climbing fibers (CFs) and molecular layer interneurons (MLIs) in the cerebellum is mediated exclusively by glutamate spillover. Here, we show how CF stimulation functionally segregates MLIs based on their location relative to glutamate release. Excitation of MLIs that reside within the domain of spillover diffusion coordinates inhibition of MLIs outside the diffusion limit. CF excitation of MLIs is dependent on extrasynaptic NMDA receptors that enhance the spatial and temporal spread of CF signaling. Activity mediated by functionally segregated MLIs converges onto neighboring Purkinje cells (PCs) to generate a long-lasting biphasic change in inhibition. These data demonstrate how glutamate release from single CFs modulates excitability of neighboring PCs, thus expanding the influence of CFs on cerebellar cortical activity in a manner not predicted by anatomical connectivity. PMID:23707614

  15. Stress-mediated p38 activation promotes somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Xinxiu Xu; Quan Wang; Yuan Long; Ru Zhang; Xiaoyuan Wei; Mingzhe Xing; Haifeng Gu

    2013-01-01

    Environmental stress-mediated adaptation plays essential roles in the evolution of life.Cellular adaptation mechanisms usually involve the regulation of chromatin structure,transcription,mRNA stability and translation,which eventually lead to efficient changes in gene expression.Global epigenetic change is also involved in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined factors.Here we report that environmental stress such as hyperosmosis not only facilitates four factor-mediated reprogramming,but also enhances two or one factor-induced iPS cell generation.Hyperosmosis-induced p38 activation plays a critical role in this process.Constitutive active p38 mimics the positive effect of hyperosmosis,while dominant negative p38 and p38 inhibitor block the effect of hyperosmosis.Further study indicates stress-mediated p38 activation may promote reprogramming by reducing the global DNA methylation level and enhancing the expression of pluripotency genes.Our results demonstrate how simple environmental stress like hyperosmosis helps to alter the fate of cells via intracellular signaling and epigenetic modulation.

  16. Activation of CFTR-mediated Cl- Transport by Magnolin

    Institute of Scientific and Technical Information of China (English)

    JIN Ling-ling; LIU Xin; SUN Yan; LIN Sen; ZHOU Na; XU Li-na; YU BO; HOU Shu-guang; YANG Hong

    2008-01-01

    Magnolin is a herbal compound from Magnolia biondii Pamp.It possesses numerous biological activities.Cystic fibrosis transmembrane conductance regulator(CFTR)is all epithelial chloride channel that plays a key role in the fluid secretion of various exocrine organs.In the present study,the activation of CFTR-mediated chloride transport by magnolin is indentified and characterized.In CFTR stably trailsfected FRT cells.magnolin increases CFTR Cl- currents in a concentration-dependent manner.The activation of magnolin on CFTR is rapid,reversible,and cAMP-dependent.Magnolin does not elevate cellular cAMP level.indicating that it activates CFTR by direct binding and interaction with CFTR protein.Magnolin selectively activates wildtype CFTR rather than mutant CFTIL Magnolin may present a novel class of therapeutic lead compound for the treatment of diseases associated with reduced CFTR function such as keratoconjunctivitis sicca,idiopathic chronic pancreatiti,and chromc constipation.

  17. 15 CFR 930.44 - Availability of mediation for disputes concerning proposed activities.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Availability of mediation for disputes... PROGRAMS Consistency for Federal Agency Activities § 930.44 Availability of mediation for disputes..., either party may request the Secretarial mediation or OCRM mediation services provided for in subpart G....

  18. 15 CFR 930.45 - Availability of mediation for previously reviewed activities.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Availability of mediation for... PROGRAMS Consistency for Federal Agency Activities § 930.45 Availability of mediation for previously..., either party may request the Secretarial mediation or OCRM mediation services provided for in subpart...

  19. Activation of PERK signaling attenuates Abeta-mediated ER stress.

    Directory of Open Access Journals (Sweden)

    Do Yeon Lee

    Full Text Available Alzheimer's disease (AD is characterized by the deposition of aggregated beta-amyloid (Abeta, which triggers a cellular stress response called the unfolded protein response (UPR. The UPR signaling pathway is a cellular defense system for dealing with the accumulation of misfolded proteins but switches to apoptosis when endoplasmic reticulum (ER stress is prolonged. ER stress is involved in neurodegenerative diseases including AD, but the molecular mechanisms of ER stress-mediated Abeta neurotoxicity still remain unknown. Here, we show that treatment of Abeta triggers the UPR in the SK-N-SH human neuroblastoma cells. Abeta mediated UPR pathway accompanies the activation of protective pathways such as Grp78/Bip and PERK-eIF2alpha pathway, as well as the apoptotic pathways of the UPR such as CHOP and caspase-4. Knockdown of PERK enhances Abeta neurotoxicity through reducing the activation of eIF2alpha and Grp8/Bip in neurons. Salubrinal, an activator of the eIF2alpha pathway, significantly increased the Grp78/Bip ER chaperone resulted in attenuating caspase-4 dependent apoptosis in Abeta treated neurons. These results indicate that PERK-eIF2alpha pathway is a potential target for therapeutic applications in neurodegenerative diseases including AD.

  20. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  1. Functional analysis of Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Hein, Estrid; Honoré, Christian Le Fèvre; Skjoedt, Mikkel-Ole;

    2010-01-01

    assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition...... was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides...

  2. Serum inflammatory mediators as markers of human Lyme disease activity.

    Science.gov (United States)

    Soloski, Mark J; Crowder, Lauren A; Lahey, Lauren J; Wagner, Catriona A; Robinson, William H; Aucott, John N

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005) in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG), CXCL10 (IP-10) and CCL19 (MIP3B) were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively). There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  3. Serum inflammatory mediators as markers of human Lyme disease activity.

    Directory of Open Access Journals (Sweden)

    Mark J Soloski

    Full Text Available Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005 in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG, CXCL10 (IP-10 and CCL19 (MIP3B were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively. There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01 and the decrease correlates with chemokine levels (p = 0.0375. The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  4. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  5. Functional analysis of Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Hein, Estrid; Honoré, Christian; Skjoedt, Mikkel-Ole;

    2010-01-01

    Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation...... assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition...... on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound...

  6. Bactericidal activity of metal-mediated peroxide-ascorbate systems.

    Science.gov (United States)

    Drath, D B; Karnovsky, M L

    1974-11-01

    Model systems containing ascorbate, hydrogen peroxide, and divalent copper or cobalt have been shown to possess marked bactericidal activity. At equivalent concentrations, copper-containing systems were more bactericidal than the corresponding mixtures containing cobalt. Cobalt at concentrations below 10(-4) M did not appreciably augment microbicidal activity, whereas systems containing copper at concentrations as low as 5 x 10(-6) M were still capable of causing some bacterial death. Manganese was inactive. None of these systems was as potent as the well known myeloperoxidase-peroxide-halide system. The mechanisms of action of these systems are not as yet clear. The possibility that they function through the generation of superoxide (O(2) (-)), hydroxyl radical (OH.), or other free radicals was explored through the use of superoxide dismutase and several free radical scavengers. It seems likely at present that the two active metal-mediated systems function via separate mechanisms. The copper system acts with dehydroascorbate, whereas the cobalt system does not. Activity in the cobalt system appears to depend upon the generation of free radicals. PMID:16558093

  7. Dihydrobenzofuran Neolignanamides: Laccase-Mediated Biomimetic Synthesis and Antiproliferative Activity.

    Science.gov (United States)

    Cardullo, Nunzio; Pulvirenti, Luana; Spatafora, Carmela; Musso, Nicolò; Barresi, Vincenza; Condorelli, Daniele Filippo; Tringali, Corrado

    2016-08-26

    The biomimetic synthesis of a small library of dihydrobenzofuran neolignanamides (the natural trans-grossamide (4) and the related compounds 21-28) has been carried out through an eco-friendly oxidative coupling reaction mediated by Trametes versicolor laccase. These products, after complete spectroscopic characterization, were evaluated for their antiproliferative activity against Caco-2 (colon carcinoma), MCF-7 (mammary adenocarcinoma), and PC-3 (prostate cancer) human cells, using an MTT bioassay. The racemic neolignamides (±)-21 and (±)-27, in being the most lipophilic in the series, were potently active, with GI50 values comparable to or even lower than that of the positive control 5-FU. The racemates were resolved through chiral HPLC, and the pure enantiomers were subjected to ECD measurements to establish their absolute configurations at C-2 and C-3. All enantiomers showed potent antiproliferative activity, with, in particular, a GI50 value of 1.1 μM obtained for (2R,3R)-21. The effect of (±)-21 on the Caco-2 cell cycle was evaluated by flow cytometry, and it was demonstrated that (±)-21 exerts its antiproliferative activity by inducing cell cycle arrest and apoptosis. PMID:27504537

  8. FOXO1 mediates RANKL-induced osteoclast formation and activity.

    Science.gov (United States)

    Wang, Yu; Dong, Guangyu; Jeon, Hyeran Helen; Elazizi, Mohamad; La, Lan B; Hameedaldeen, Alhassan; Xiao, E; Tian, Chen; Alsadun, Sarah; Choi, Yongwon; Graves, Dana T

    2015-03-15

    We have previously shown that the transcription factor FOXO1 is elevated in conditions with high levels of bone resorption. To investigate the role of FOXO1 in the formation of osteoclasts, we examined mice with lineage-specific deletion of FOXO1 in osteoclast precursors and by knockdown of FOXO1 with small interfering RNA. The receptor activator for NF-κB ligand (RANKL), a principal bone-resorbing factor, induced FOXO1 expression and nuclear localization 2 d after stimulation in bone marrow macrophages and RAW264.7 osteoclast precursors. RANKL-induced osteoclast formation and osteoclast activity was reduced in half in vivo and in vitro with lineage-specific FOXO1 deletion (LyzM.Cre(+)FOXO1(L/L)) compared with matched controls (LyzM.Cre(-)FOXO1(L/L)). Similar results were obtained by knockdown of FOXO1 in RAW264.7 cells. Moreover, FOXO1-mediated osteoclast formation was linked to regulation of NFATc1 nuclear localization and expression as well as a number of downstream factors, including dendritic cell-specific transmembrane protein, ATP6vod2, cathepsin K, and integrin αv. Lastly, FOXO1 deletion reduced M-CSF-induced RANK expression and migration of osteoclast precursors. In the present study, we provide evidence that FOXO1 plays a direct role in osteoclast formation by mediating the effect of RANKL on NFATc1 and several downstream effectors. This is likely to be significant because FOXO1 and RANKL are elevated in osteolytic conditions.

  9. Wastewater Mediated Activation of Micromotors for Efficient Water Cleaning.

    Science.gov (United States)

    Srivastava, Sarvesh Kumar; Guix, Maria; Schmidt, Oliver G

    2016-01-13

    We present wastewater-mediated activation of catalytic micromotors for the degradation of nitroaromatic pollutants in water. These next-generation hybrid micromotors are fabricated by growing catalytically active Pd particles over thin-metal films (Ti/Fe/Cr), which are then rolled-up into self-propelled tubular microjets. Coupling of catalytically active Pd particles inside the micromotor surface in the presence of a 4-nitrophenol pollutant (with NaBH4 as reductant) results in autonomous motion via the bubble-recoil propulsion mechanism such that the target pollutant mixture (wastewater) is consumed as a fuel, thereby generating nontoxic byproducts. This study also offers several distinct advantages over its predecessors including no pH/temperature manipulation, limited stringent process control and complete destruction of the target pollutant mixture. The improved intermixing ability of the micromotors caused faster degradation ca. 10 times higher as compared to its nonmotile counterpart. The high catalytic efficiency obtained via a wet-lab approach has promising potential in creating hybrid micromotors comprising of multicatalytic systems assembled into one entity for sustainable environmental remediation and theranostics. PMID:26674098

  10. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    Science.gov (United States)

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  11. Impact of leptin-mediated sympatho-activation on cardiovascular function in obese mice

    OpenAIRE

    Belin de Chantemèle, Eric J.; Mintz, James D.; Rainey, William E.; Stepp, David W.

    2011-01-01

    Although the anorexic effects of leptin are lost in obesity, leptin-mediated sympatho-activation is preserved. The cardiovascular consequences of leptin-mediated sympatho-activation in obesity are poorly understood. We tested the hypothesis that 32 weeks of high fat diet (HFD) induces metabolic leptin resistance but preserves leptin-mediated sympatho-activation of the cardiovascular system. HFD in mice significantly increased body weight and plasma leptin concentrations but significantly redu...

  12. Eosinophil recruitment and activation: the role of lipid mediators

    OpenAIRE

    Luna-Gomes, Tatiana; Bozza, Patrícia T.; Bandeira-Melo, Christianne

    2013-01-01

    Eosinophils are effector cells that migrate toward several mediators released at inflammatory sites to perform their multiple functions. The mechanisms driving eosinophil selective accumulation in sites of allergic inflammation are well-established and involve several steps controlled by adhesion molecules, priming agents, chemotactic, and surviving factors. Even though the majority of studies focused on role of protein mediators like IL-5 and eotaxins, lipid mediators also participate in eos...

  13. Lipopolysaccharide (LPS)-mediated macrophage activation: the role of calcium in the generation of tumoricidal activity

    Energy Technology Data Exchange (ETDEWEB)

    Drysdale, B.E.; Shin, H.S.

    1986-03-01

    As the authors reported, calcium ionophore, A23187, activates macrophages (M theta) for tumor cell killing and the activated M theta produce a soluble cytotoxic factor (M theta-CF) that is similar if not identical to tumor necrosis factor. Based on these observations they have investigated whether calcium is involved in the activation mediated by another potent M theta activator, LPS. The authors have shown that A23187 caused uptake of extracellular /sup 45/Ca/sup + +/ but LPS did not. They have examined the effect of depleting extracellular calcium by using medium containing no added calcium containing 1.0 mM EGTA. In no case did depletion result in decreased M theta-CF production by the M theta activated with LPS. Measurements using the fluorescent, intracellular calcium indicator, Quin 2 have also been performed. While ionomycin, caused a rapid change in the Quin-2 signal, LPS at a concentration even in excess of that required to activate the M theta caused no change in the signal. When high doses of Quin 2 or another intracellular chelator, 8-(diethylaminol-octyl-3,4,5-trimethoxybenzoate, were used to treat M theta, M theta-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M theta-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.

  14. Possible Selves and Physical Activity in Retirees: The Mediating Role of Identity.

    Science.gov (United States)

    Perras, Mélanie G M; Strachan, Shaelyn M; Fortier, Michelle S

    2016-11-01

    Many retirees remain insufficiently physically active for health benefits. Self-perceptions can influence physical activity. Possible selves and identity are two self-perceptions that, when examined relative to physical activity, may help explain physical activity levels among retirees. Scholars claim that a focus on possible selves may impact identity, which, in the physical activity domain, is a known physical activity correlate. The aim of this study was to examine the relationship between these variables, and more specifically, to determine whether exercise identity mediates the relationship between physical activity possible selves and physical activity. We examined the proposed mediation relationship in 531 new retirees. Data were collected across three time points (1 month apart). We conducted mediation analyses with bootstrapping. Identity mediated the relationship between possible selves (content and processes) and behavior-all related to physical activity. These findings represent an important first step toward designing relevant physical activity interventions for retirees.

  15. Activation of CFTR by ASBT-mediated bile salt absorption

    NARCIS (Netherlands)

    Bijvelds, MJC; Jorna, H; Verkade, HJ; Bot, AGM; Hofmann, F; Agellon, LB; Sinaasappel, M; de Jonge, HR

    2005-01-01

    In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal

  16. Plasma-activated air mediates plasmid DNA delivery in vivo.

    Science.gov (United States)

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  17. Schisandra polysaccharide evokes immunomodulatory activity through TLR 4-mediated activation of macrophages.

    Science.gov (United States)

    Zhao, Ting; Feng, Yun; Li, Jing; Mao, Riwen; Zou, Ye; Feng, Weiwei; Zheng, Daheng; Wang, Wei; Chen, Yao; Yang, Liuqing; Wu, Xiangyang

    2014-04-01

    Schisandra chinensis (Turcz.) Baill has been used in traditional Chinese medicine for centuries. Previous studies have shown that Schisandra polysaccharide (SCPP11) has robust antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of SCPP11 were investigated further to reveal its mechanism of action against tumors. Results showed that SCPP11 increased the thymus and spleen indices, pinocytic activity of peritoneal macrophages, and hemolysin formation in CTX-induced immunosuppressed mice. Moreover, SCPP11 significantly increased immunoglobulin levels, cytokines levels in vivo and induced RAW264.7 cells to secrete cytokines in vitro. RAW264.7 cells pretreated with SCPP11 significantly inhibited the proliferation of HepG-2 cells. In addition, SCPP11 promoted both the expression of iNOS protein and of iNOS and TNF-α mRNA. TLR-4 is a possible receptor for SCPP11-mediated macrophage activation. Therefore, the data suggest that SCPP11 exerted its antitumor activity by improving immune system functions through TLR-4-mediated up-regulation of NO and TNF-α.

  18. MED16 and MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced transcriptional activators

    OpenAIRE

    Kim, Tae Whan; Kwon, Yong-Jae; Kim, Jung Mo; Song, Young-Hwa; Kim, Se Nyun; Kim, Young-Joon

    2004-01-01

    Transcriptional activators interact with diverse proteins and recruit transcriptional machinery to the activated promoter. Recruitment of the Mediator complex by transcriptional activators is usually the key step in transcriptional activation. However, it is unclear how Mediator recognizes different types of activator proteins. To systematically identify the subunits responsible for the signal- and activator-specific functions of Mediator in Drosophila melanogaster, each Mediator subunit was ...

  19. Mediated Electron Transfer at Redox Active Monolayers. Part 4: Kinetics of Redox Enzymes Coupled With Electron Mediators

    Directory of Open Access Journals (Sweden)

    Michael E.G. Lyons

    2003-01-01

    Full Text Available A detailed kinetic analysis of the pertinent physical processes underlying the operation of enzyme electrodes immobilized within alkane thiol self assembled monolayers is developed. These electrodes utilize a soluble mediator, which partitions into the monolayer, regenerates the active catalytic form of the enzyme and is re-oxidized at the underlying support electrode surface giving rise to a current which reflects kinetic events at the enzyme surface. Both the enzyme/substrate and enzyme mediator kinetics have been quantified fully in terms of a ping-pong mechanism for the former and Michaelis-Menten kinetics for the latter. The effect of substrate and mediator diffusion in solution have also been specifically considered and the latter processes have been shown to result in a complex expression for the reaction flux. Four limiting kinetic cases have been enumerated and simple expressions for the reaction flux in each of these rate limiting situations have been developed. Kinetic case diagrams have been presented as an aid to mechanistic diagnosis. The complicating effects of diffusive loss of reduced mediator from the enzyme layer have also been examined and the relation between the observed flux corresponding to reduced mediator oxidation at the support electrode and the substrate reaction flux in the enzyme layer have been quantified in terms of an efficiency factor. Results extracted from recently published practical realizations of immobilized monolayer enzyme systems have been discussed in the context of the proposed model analysis.

  20. Odor-mediated taste learning requires dorsal hippocampus, but not basolateral amygdala activity

    OpenAIRE

    Wheeler, Daniel S.; Chang, Stephen E.; Holland, Peter C

    2012-01-01

    Mediated learning is a unique cognitive phenomenon in which mental representations of physically absent stimuli enter into associations with directly-activated representations of physically present stimuli. Three experiments investigated the functional physiology of mediated learning involving the use of odor-taste associations. In Experiments 1a and 1b, basolateral amygdala lesions failed to attenuate mediated taste aversion learning. In Experiment 2, dorsal hippocampus inactivation impaired...

  1. Active video games: the mediating effect of aerobic fitness on body composition

    Directory of Open Access Journals (Sweden)

    Maddison Ralph

    2012-05-01

    Full Text Available Abstract Background Increased understanding of why and how physical activity impacts on health outcomes is needed to increase the effectiveness of physical activity interventions. A recent randomized controlled trial of an active video game (PlayStation EyeToy™ intervention showed a statistically significant treatment effect on the primary outcome, change from baseline in body mass index (BMI, which favored the intervention group at 24 weeks. In this short paper we evaluate the mediating effects of the secondary outcomes. Objective To identify mediators of the effect of an active video games intervention on body composition. Methods Data from a two-arm parallel randomized controlled trial of an active video game intervention (n = 322 were analyzed. The primary outcome was change from baseline in BMI. A priori secondary outcomes were considered as potential mediators of the intervention on BMI, including aerobic fitness (VO2Max, time spent in moderate-to-vigorous physical activity (MVPA, and food snacking at 24 weeks. Results Only aerobic fitness at 24 weeks met the conditions for mediation, and was a significant mediator of BMI. Conclusion Playing active video games can have a positive effect on body composition in overweight or obese children and this effect is most likely mediated through improved aerobic fitness. Future trials should examine other potential mediators related to this type of intervention. Trial registration Australian New Zealand Clinical Trials Registry Website: http://www.anzctr.org.au Study ID number: ACTRN12607000632493

  2. Mediation analysis of the relationship between institutional research activity and patient survival

    DEFF Research Database (Denmark)

    Rochon, Justine; du Bois, Andreas; Lange, Theis

    2014-01-01

    mediated through either optimal surgery or chemotherapy. Taken together, about 26% of the beneficial effect of research activity was mediated through the proposed pathways. CONCLUSIONS: Mediation analysis allows proceeding from the question "Does it work?" to the question "How does it work?" In particular......BACKGROUND: Recent studies have suggested that patients treated in research-active institutions have better outcomes than patients treated in research-inactive institutions. However, little attention has been paid to explaining such effects, probably because techniques for mediation analysis...... existing so far have not been applicable to survival data. METHODS: We investigated the underlying mechanisms using a recently developed method for mediation analysis of survival data. Our analysis of the effect of research activity on patient survival was based on 352 patients who had been diagnosed...

  3. Hippocampal activity mediates the relationship between circadian activity rhythms and memory in older adults.

    Science.gov (United States)

    Sherman, Stephanie M; Mumford, Jeanette A; Schnyer, David M

    2015-08-01

    Older adults experience parallel changes in sleep, circadian rhythms, and episodic memory. These processes appear to be linked such that disruptions in sleep contribute to deficits in memory. Although more variability in circadian patterns is a common feature of aging and predicts pathology, little is known about how alterations in circadian activity rhythms within older adults influence new episodic learning. Following 10 days of recording sleep-wake patterns using actigraphy, healthy older adults underwent fMRI while performing an associative memory task. The results revealed better associative memory was related to more consistent circadian activity rhythms, independent of total sleep time, sleep efficiency, and level of physical activity. Moreover, hippocampal activity during successful memory retrieval events was positively correlated with associative memory accuracy and circadian activity rhythm (CAR) consistency. We demonstrated that the link between consistent rhythms and associative memory performance was mediated by hippocampal activity. These findings provide novel insight into how the circadian rhythm of sleep-wake cycles are associated with memory in older adults and encourage further examination of circadian activity rhythms as a biomarker of cognitive functioning. PMID:26205911

  4. Implementation Planning and Progress on Physical Activity Goals: The Mediating Role of Life-Management Strategies

    Science.gov (United States)

    Dugas, Michelle; Gaudreau, Patrick; Carraro, Natasha

    2012-01-01

    This 4-week prospective study examined whether the use of life-management strategies mediates the relationship between implementation planning and short-term progress on physical activity goals. In particular, the strategies of elective selection, compensation, and loss-based selection were disentangled to assess their specific mediating effects.…

  5. Participation in Organized Activities and Conduct Problems in Elementary School: The Mediating Effect of Social Skills

    Science.gov (United States)

    Denault, Anne-Sophie; Déry, Michèle

    2015-01-01

    The goal of this study was to test a mediation model in which social skills mediate the relationship between participation in organized activities and conduct problems among elementary school children. Two moderators of these associations were also examined, namely, gender and reception of special education services. A total of 563 children (45%…

  6. Validity and reliability of instruments to assess potential mediators of children's physical activity: A systematic review

    NARCIS (Netherlands)

    Brown, H.; Hume, C.; Chin A Paw, J.M.M.

    2009-01-01

    This paper aimed to (1) identify potential mediators reported in children's physical activity interventions; and (2) review the psychometric properties of measures of potential mediators included in such interventions. A systematic search of the literature was conducted and studies that reported pot

  7. A systematic review of intervention effects on potential mediators of children’s physical activity

    Directory of Open Access Journals (Sweden)

    Brown Helen

    2013-02-01

    Full Text Available Abstract Background Many interventions aiming to increase children’s physical activity have been developed and implemented in a variety of settings, and these interventions have previously been reviewed; however the focus of these reviews tends to be on the intervention effects on physical activity outcomes without consideration of the reasons and pathways leading to intervention success or otherwise. To systematically review the efficacy of physical activity interventions targeting 5-12 year old children on potential mediators and, where possible, to calculate the size of the intervention effect on the potential mediator. Methods A systematic search identified intervention studies that reported outcomes on potential mediators of physical activity among 5-12 year old children. Original research articles published between 1985 and April 2012 were reviewed. Results Eighteen potential mediators were identified from 31 studies. Positive effects on cognitive/psychological potential mediators were reported in 15 out of 31 studies. Positive effects on social environmental potential mediators were reported in three out of seven studies, and no effects on the physical environment were reported. Although no studies were identified that performed a mediating analysis, 33 positive intervention effects were found on targeted potential mediators (with effect sizes ranging from small to large and 73% of the time a positive effect on the physical activity outcome was reported. Conclusions Many studies have reported null intervention effects on potential mediators of children’s physical activity; however, it is important that intervention studies statistically examine the mediating effects of interventions so the most effective strategies can be implemented in future programs.

  8. Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity.

    Science.gov (United States)

    Wu, Keren; Li, Ning; Sun, Huaqin; Xu, Tao; Jin, Fa; Nie, Jifeng

    2015-10-23

    In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. PMID:26361144

  9. Activation of AhR-mediated toxicity pathway by emerging pollutants polychlorinated diphenyl sulfides

    Science.gov (United States)

    Polychlorinated diphenyl sulfides (PCDPSs) are a group of environmental pollutants for which limited toxicological information is available. This study tested the hypothesis that PCDPSs could activate the mammalian aryl hydrocarbon receptor (AhR) mediated toxicity pathways. Eight...

  10. Cocaine induces astrocytosis through ER stress-mediated activation of autophagy.

    Science.gov (United States)

    Periyasamy, Palsamy; Guo, Ming-Lei; Buch, Shilpa

    2016-08-01

    Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation. PMID:27337297

  11. Hyper-inflammation and skin destruction mediated by rosiglitazone activation of macrophages in IL-6 deficiency

    DEFF Research Database (Denmark)

    Das, Lopa M; Rosenjack, Julie; Au, Liemin;

    2015-01-01

    -antibodies against IL-6, mimicking IL-6 deficiency in human diseases. IL-6 deficiency when combined with Rosi-mediated upregulation of suppressor of cytokine signaling 3 leads to an altered ratio of nuclear signal transducer and activator of transcription 3/NF-κB that allows hyper-induction of inducible nitric oxide...... mediating hyper-inflammatory macrophage activation significant for diseases associated with IL-6 deficiency....

  12. Streetscape greenery and health: stress, social cohesion and physical activity as mediators.

    Science.gov (United States)

    de Vries, Sjerp; van Dillen, Sonja M E; Groenewegen, Peter P; Spreeuwenberg, Peter

    2013-10-01

    Several studies have shown a positive relationship between local greenspace availability and residents' health, which may offer opportunities for health improvement. This study focuses on three mechanisms through which greenery might exert its positive effect on health: stress reduction, stimulating physical activity and facilitating social cohesion. Knowledge on mechanisms helps to identify which type of greenspace is most effective in generating health benefits. In eighty neighbourhoods in four Dutch cities data on quantity and quality of streetscape greenery were collected by observations. Data on self-reported health and proposed mediators were obtained for adults by mail questionnaires (N = 1641). Multilevel regression analyses, controlling for socio-demographic characteristics, revealed that both quantity and quality of streetscape greenery were related to perceived general health, acute health-related complaints, and mental health. Relationships were generally stronger for quality than for quantity. Stress and social cohesion were the strongest mediators. Total physical activity was not a mediator. Physical activity that could be undertaken in the public space (green activity) was, but less so than stress and social cohesion. With all three mediators included in the analysis, complete mediation could statistically be proven in five out of six cases. In these analyses the contribution of green activity was often not significant. The possibility that the effect of green activity is mediated by stress and social cohesion, rather than that it has a direct health effect, is discussed. PMID:23931942

  13. A sociocultural perspective on mediated activity in third grade science

    Science.gov (United States)

    Reveles, John M.; Kelly, Gregory J.; Durán, Richard P.

    2007-02-01

    This ethnographic study of a third grade classroom examined elementary school science learning as a sociocultural accomplishment. The research focused on how a teacher helped his students acquire psychological tools for learning to think and engage in scientific practices as locally defined. Analyses of classroom discourse examined both how the teacher used mediational strategies to frame disciplinary knowledge in science as well as how students internalized and appropriated ways of knowing in science. The study documented and analyzed how students came to appropriate scientific knowledge as their own in an ongoing manner tied to their identities as student scientists. Implications for sociocultural theory in science education research are discussed.

  14. Assessment of DFT methods for computing activation energies of Mo/W-mediated reactions.

    Science.gov (United States)

    Hu, Lianrui; Chen, Hui

    2015-10-13

    Using high level ab initio coupled cluster calculations as reference, the performances of 15 commonly used density functionals (DFs) on activation energy calculations for typical Mo/W-mediated reactions have been systematically assessed for the first time in this work. The selected representative Mo/W-mediated reactions cover a wide range from enzymatic reactions to organometallic reactions, which include Mo-catalyzed aldehyde oxidation (aldehyde oxidoreductase), Mo-catalyzed dimethyl sulfoxide (DMSO) reduction (DMSO reductase), W-catalyzed acetylene hydration (acetylene hydratase), Mo/W-mediated olefin metathesis, Mo/W-mediated olefin epoxidation, W-mediated alkyne metathesis, and W-mediated C-H bond activation. Covering both Mo- and W-mediated reactions, four DFs of B2GP-PLYP, M06, B2-PLYP, and B3LYP are uniformly recommended with and without DFT empirical dispersion correction. Among these four DFs, B3LYP is notably improved in performance by DFT empirical dispersion correction. In addition to the absolute value of calculation error, if the trend of DFT results is also a consideration, B2GP-PLYP, B2-PLYP, and M06 keep better performance than other functionals tested and constitute our final recommendation of DFs for both Mo- and W-mediated reactions. PMID:26574251

  15. Understanding Synchronous Computer-Mediated Classroom Discussion through Cultural-Historical Activity Theory

    Science.gov (United States)

    Park, Yangjoo

    2015-01-01

    This study is about graduate students' discourse practices in classroom text-based synchronous computer mediated discussions (SCMD). Cultural historical activity theory (in short, Activity Theory) is the primary theoretical lens through which the data are analyzed. Engeström's (1987) Activity System model among the various theoretical positions or…

  16. Mediators of physical activity behaviour change among adult non-clinical populations: a review update

    Directory of Open Access Journals (Sweden)

    Pfaeffli Leila A

    2010-05-01

    Full Text Available Abstract Background An understanding of the determinants of physical activity through mediators of behaviour change is important in order to evaluate the efficacy of interventions. Prior reviews on this topic noted that few studies employed mediator analyses in experimental physical activity trials; the purpose of this review is to update these prior reviews in order to evaluate the state of our present understanding of interventions that include proposed mediators of behaviour change. Methods Literature was identified through electronic database (e.g., MEDLINE, psychINFO searching. Studies were eligible if they described a published experimental or quasi-experimental trial examining the effect of an intervention on physical activity behaviour and mediator change in non-clinical adult populations. Quality of included studies was assessed and the analyses examined the symmetry between mediators and behaviour change. Results Twenty seven unique trials passed the eligibility criteria and 22 were included in the analysis with scores of moderate or higher quality. Half of the studies reviewed failed to show an intervention effect on PA. The remaining studies showed evidence that the intervention affected changes in the proposed mediators, but tests of mediated effect were performed in only six of these 11 cases and demonstrated mixed outcomes. Differences by theory were not discernable at this time, but self-regulation constructs had the most evidence for mediation. Conclusion Published literature employing mediators of change analyses in experimental designs is still relatively elusive since the time of prior reviews; however, the general null findings of changes in mediating constructs from these interventions are a more timely concern. Changes in self-regulation constructs may have the most effect on changes in PA while self-efficacy and outcome expectation type constructs have negligible but limited findings. Innovation and increased fidelity of

  17. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    Science.gov (United States)

    Song, Zhen

    Life-threatening cardiac arrhythmias continue to pose a major health problem. Ventricular fibrillation, which is a complex form of electrical wave turbulence in the lower chambers of the heart, stops the heart from pumping and is the largest cause of natural death in the United States. Atrial fibrillation, a related form of wave turbulence in the upper heart chambers, is in turn the most common arrhythmia diagnosed in clinical practice. Despite extensive research to date, mechanisms of cardiac arrhythmias remain poorly understood. It is well established that both spatial disorder of the refractory period of heart cells and triggered activity (TA) jointly contribute to the initiation and maintenance of arrhythmias. TA broadly refers to the abnormal generation of a single or a sequence of abnormal excitation waves from a small submillimeter region of the heart in the interval of time between two normal waves generated by the heart's natural pacemaker (the sinoatrial node). TA has been widely investigated experimentally and occurs in several pathological conditions where the intracellular concentration of free Ca2+ ions in heart cells becomes elevated. Under such conditions, Ca2+ can be spontaneously released from intracellular stores, thereby driving an electrogenic current that exchanges 3Na+ ions for one Ca2+ ion across the cell membrane. This current in turn depolarizes the membrane of heart cells after a normal excitation. If this calcium-mediated "delayed after depolarization'' (DAD) is sufficiently large, it can generate an action potential. While the arrhythmogenic importance of spontaneous Ca2+ release and DADs is well appreciated, the conditions under which they occur in heart pathologies remain poorly understood. Calcium overload is only one factor among several other factors that can promote DADs, including sympathetic nerve stimulation, different expression levels of membrane ion channels and calcium handling proteins, and different mutations of those

  18. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    International Nuclear Information System (INIS)

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

  19. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Teng, E-mail: tengyu33@yahoo.com [Department of Dermatology, Shandong Ji-ning No. 1 People’s Hospital, Shandong Province 272011 (China); Ji, Jiang [Department of Dermatology, The Second Hospital Affiliated of Soochow University, SuZhou, Jiangsu Province 215000 (China); Guo, Yong-li [Department of Oncology, Shandong Ji-ning No. 1 People’s Hospital, Shandong Province 272011 (China)

    2013-11-08

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.

  20. Complement activation by tubular cells is mediated by properdin binding

    NARCIS (Netherlands)

    Gaarkeuken, E.M.; Siezenga, M.A.; Zuidwijk, K.; Kooten, C. van; Rabelink, T.J.; Daha, M.R.; Berger, S.P.

    2008-01-01

    Activation of filtered complement products on the brush border of the tubular epithelium is thought to be a key factor underlying proteinuria-induced tubulointerstitial injury. However, the mechanism of tubular complement activation is still unclear. Recent studies on mechanisms of complement activa

  1. Is the Effect of Reported Physical Activity on Disability Mediated by Cognitive Performance in White and African American Older Adults?

    OpenAIRE

    Popa, Mihaela A.; Reynolds, Sandra L.; Small, Brent J.

    2009-01-01

    This study examined if reported physical activity has beneficial outcomes on disability through cognitive performance–mediated effects and if these mediation effects are comparable for White and African American elders. Longitudinal data from the Assets and Health Dynamics among the Oldest Old study (N = 4,472) are used to test mediation in multilevel models. During the 7-year follow-up, cognitive performance mediated the effects of reported physical activity on disability in the entire sampl...

  2. Receptor-mediated regional sympathetic nerve activation by leptin.

    OpenAIRE

    Haynes, W G; Morgan, D A; Walsh, S A; Mark, A L; Sivitz, W I

    1997-01-01

    Leptin is a peptide hormone produced by adipose tissue which acts centrally to decrease appetite and increase energy expenditure. Although leptin increases norepinephrine turnover in thermogenic tissues, the effects of leptin on directly measured sympathetic nerve activity to thermogenic and other tissues are not known. We examined the effects of intravenous leptin and vehicle on sympathetic nerve activity to brown adipose tissue, kidney, hindlimb, and adrenal gland in anesthetized Sprague-Da...

  3. Minocycline and sulforaphane inhibited lipopolysaccharide-mediated retinal microglial activation

    OpenAIRE

    Li-ping YANG; Zhu, Xiu-an; Tso, Mark O.M.

    2007-01-01

    Purpose To elucidate the inhibitory effect of minocycline and sulforaphane on lipopolysaccharide (LPS)-induced retinal microglial activation and the mechanisms through which they exerted their inhibitory effects. Methods Primary retinal microglial cultures were exposed to LPS with or without minocycline and sulforaphane. The mRNA expression of monocyte chemotactic protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1α, MIP-1β, eotaxin, regulated upon activation normal T-cell express...

  4. Socio-economic status and physical activity among adolescents : The mediating role of self-esteem

    NARCIS (Netherlands)

    Veselska, Z.; Geckova, A. Madarasova; Reijneveld, S. A.; van Dijk, J. P.

    2011-01-01

    Objectives: Physical activity is an essential part of a healthy lifestyle in adolescence. Previous studies have shown physical activity to be associated with socio-economic status and self-esteem; the latter association may mediate the former, but evidence on this is lacking. The aim of this study w

  5. Developmental Benefits of Extracurricular Involvement: Do Peer Characteristics Mediate the Link between Activities and Youth Outcomes?

    Science.gov (United States)

    Fredricks, Jennifer A.; Eccles, Jacquelynn S.

    2005-01-01

    In this article, we test: (a) the relation between school-based extracurricular participation and indicators of positive and negative development across a range of activity contexts, and (b) a mediation model linking activity participation, prosocial peers, and development. Extensive survey information was collected from a predominately White…

  6. Paeonol attenuates inflammation-mediated neurotoxicity and microglial activation

    Institute of Scientific and Technical Information of China (English)

    Kyong Nyon Nam; Byung-Cheol Woo; Sang-Kwan Moon; Seong-Uk Park; Joo-young Park; Jae-Woong Hwang; Hyung-Sup Bae; Chang-Nam Ko; Eunjoo Hwang Lee

    2013-01-01

    Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. The root of Paeonia lactiflora Pall has been considered useful for the treatment of various disorders in traditional oriental medicine. Paeonol, found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, including anti-oxidative, anti-inflammatory and neuroprotective activities. The objective of this study was to examine the efficacy of paeonol in the repression of inflammation-induced neurotoxicity and microglial cell activation. Organotypic hippocampal slice cultures and primary microglial cells from rat brain were stimulated with bacterial lipopolysaccharide. Paeonol pretreatment was performed for 30 minutes prior to lipopolysaccharide addition. Cell viability and nitrite (the production of nitric oxide), tumor necrosis factor-alpha and interleukin-1beta products were measured after lipopolysaccharide treatment. In organotypic hippocampal slice cultures, paeonol blocked lipopolysaccharide-related hippocampal cell death and inhibited the release of nitrite and interleukin-1beta. Paeonol was effective in inhibiting nitric oxide release from primary microglial cells. It also reduced the lipopolysaccharide-stimulated release of tumor necrosis factor-alpha and interleukin-1β from microglial cells. Paeonol possesses neuroprotective activity in a model of inflammation-induced neurotoxicity and reduces the release of neurotoxic and proinflammatory factors in activated microglial cells.

  7. Disorder-mediated crowd control in an active matter system

    Science.gov (United States)

    Pinçe, Erçağ; Velu, Sabareesh K. P.; Callegari, Agnese; Elahi, Parviz; Gigan, Sylvain; Volpe, Giovanni; Volpe, Giorgio

    2016-03-01

    Living active matter systems such as bacterial colonies, schools of fish and human crowds, display a wealth of emerging collective and dynamic behaviours as a result of far-from-equilibrium interactions. The dynamics of these systems are better understood and controlled considering their interaction with the environment, which for realistic systems is often highly heterogeneous and disordered. Here, we demonstrate that the presence of spatial disorder can alter the long-term dynamics in a colloidal active matter system, making it switch between gathering and dispersal of individuals. At equilibrium, colloidal particles always gather at the bottom of any attractive potential; however, under non-equilibrium driving forces in a bacterial bath, the colloids disperse if disorder is added to the potential. The depth of the local roughness in the environment regulates the transition between gathering and dispersal of individuals in the active matter system, thus inspiring novel routes for controlling emerging behaviours far from equilibrium.

  8. [Bone marrow stromal damage mediated by immune response activity].

    Science.gov (United States)

    Vojinović, J; Kamenov, B; Najman, S; Branković, Lj; Dimitrijević, H

    1994-01-01

    The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis.

  9. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    Science.gov (United States)

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  10. CRISPR-mediated Activation of Latent HIV-1 Expression.

    Science.gov (United States)

    Limsirichai, Prajit; Gaj, Thomas; Schaffer, David V

    2016-03-01

    Complete eradication of HIV-1 infection is impeded by the existence of cells that harbor chromosomally integrated but transcriptionally inactive provirus. These cells can persist for years without producing viral progeny, rendering them refractory to immune surveillance and antiretroviral therapy and providing a permanent reservoir for the stochastic reactivation and reseeding of HIV-1. Strategies for purging this latent reservoir are thus needed to eradicate infection. Here, we show that engineered transcriptional activation systems based on CRISPR/Cas9 can be harnessed to activate viral gene expression in cell line models of HIV-1 latency. We further demonstrate that complementing Cas9 activators with latency-reversing compounds can enhance latent HIV-1 transcription and that epigenome modulation using CRISPR-based acetyltransferases can also promote viral gene activation. Collectively, these results demonstrate that CRISPR systems are potentially effective tools for inducing latent HIV-1 expression and that their use, in combination with antiretroviral therapy, could lead to improved therapies for HIV-1 infection. PMID:26607397

  11. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

    Science.gov (United States)

    Naranjo, José R.; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M.; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C.; Arrabal, María D.; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-01-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  12. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

    Science.gov (United States)

    Naranjo, José R; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C; Arrabal, María D; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-02-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  13. H2S-Mediated Thermal and Photochemical Methane Activation

    NARCIS (Netherlands)

    Baltrusaitis, Jonas; Graaf, de Coen; Broer, Ria; Patterson, Eric V.

    2013-01-01

    Sustainable, low-temperature methods for natural gas activation are critical in addressing current and foreseeable energy and hydrocarbon feedstock needs. Large portions of natural gas resources are still too expensive to process due to their high content of hydrogen sulfide gas (H2S) mixed with met

  14. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

    Science.gov (United States)

    Naranjo, José R; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C; Arrabal, María D; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-02-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

  15. Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy

    OpenAIRE

    Putinski, Charis; ABDUL-GHANI, MOHAMMAD; Stiles, Rebecca; Brunette, Steve; Dick, Sarah A.; Fernando, Pasan; Lynn A. Megeney

    2013-01-01

    Cardiac hypertrophy is a pathologic enlargement of the heart, an alteration that leads to contractile dysfunction and eventual organ failure. The hypertrophy phenotype originates from concentric growth of heart muscle cells and shares many biochemical features with programmed cell death, implying a common molecular origin. Here, we show cell-autonomous activation of a mitochondrial cell death pathway during initial stages of muscle cell hypertrophy, a signal that is essential and sufficient t...

  16. Activities, self-referent memory beliefs, and cognitive performance: evidence for direct and mediated relations.

    Science.gov (United States)

    Jopp, Daniela; Hertzog, Christopher

    2007-12-01

    In this study, the authors investigated the role of activities and self-referent memory beliefs for cognitive performance in a life-span sample. A factor analysis identified 8 activity factors, including Developmental Activities, Experiential Activities, Social Activities, Physical Activities, Technology Use, Watching Television, Games, and Crafts. A second-order general activity factor was significantly related to a general factor of cognitive function as defined by ability tests. Structural regression models suggested that prediction of cognition by activity level was partially mediated by memory beliefs, controlling for age, education, health, and depressive affect. Models adding paths from general and specific activities to aspects of crystallized intelligence suggested additional unique predictive effects for some activities. In alternative models, nonsignificant effects of beliefs on activities were detected when cognition predicted both variables, consistent with the hypothesis that beliefs derive from monitoring cognition and have no influence on activity patterns. PMID:18179299

  17. Active video games: the mediating effect of aerobic fitness on body composition

    OpenAIRE

    Maddison Ralph; Mhurchu Cliona; Jull Andrew; Prapavessis Harry; Foley Louise S; Jiang Yannan

    2012-01-01

    Abstract Background Increased understanding of why and how physical activity impacts on health outcomes is needed to increase the effectiveness of physical activity interventions. A recent randomized controlled trial of an active video game (PlayStation EyeToy™) intervention showed a statistically significant treatment effect on the primary outcome, change from baseline in body mass index (BMI), which favored the intervention group at 24 weeks. In this short paper we evaluate the mediating ef...

  18. Socio-economic status and physical activity among adolescents: The mediating role of self-esteem

    OpenAIRE

    Veselska, Z.; Geckova, A. Madarasova; Reijneveld, S.A.; Dijk, J.P. van

    2011-01-01

    Objectives: Physical activity is an essential part of a healthy lifestyle in adolescence. Previous studies have shown physical activity to be associated with socio-economic status and self-esteem; the latter association may mediate the former, but evidence on this is lacking. The aim of this study was to explore the associations of socioeconomic status and the self-esteem of adolescents with physical activity, and their joint effects. Methods: A sample of 3694 elementary-school students from ...

  19. Anti-HBV activity of TRL mediated by recombinant adenovirus

    Institute of Scientific and Technical Information of China (English)

    Wei-Dong Gong; Ya Zhao; Jun Yi; Jin Ding; Jun Liu; Cai-Fang Xue

    2005-01-01

    AIM: To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGlox(delta)E1,3Cre to acquire RAd/TRL, TR, HBVcand hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63±0.14 vs 1.60±0.47, P = 0.0266, <0.05) andother control groups (0.63±0.14 vs 8.50±2.78, 8.25±2.26,8.25±2.29, 8.50±1.51, 8.57±1.63, P<0.01). MTT assaysuggested that there were no significant differences in cell metabolic activity between groups (P>0.05).CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.

  20. Key mediators of intracellular amino acids signaling to mTORC1 activation.

    Science.gov (United States)

    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  1. Rhodium mediated bond activation: from synthesis to catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Ho, Hung-An [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    Recently, our lab has developed monoanionic tridentate ligand, ToR, showing the corresponding coordination chemistry and catalyst reactivity of magnesium, zirconium, zinc and iridium complexes. This thesis details synthetic chemistry, structural study and catalytic reactivity of the ToR-supported rhodium compounds. Tl[ToR] has been proved to be a superior ligand transfer agent for synthesizing rhodium complexes. The salt metathesis route of Tl[ToM] with [Rh(μ-Cl)(CO)]2 and [Rh(μ- Cl)(COE)]2 gives ToMRh(CO)2 (2.2) and ToMRhH(β3-C8H13) (3.1) respectively while Tl[ToM] with [Rh(μ-Cl)(CO)]2 affords ToPRh(CO)2 (2.3). 2.2 reacts with both strong and weak electrophiles, resulting in the oxazoline N-attacked and the metal center-attacked compounds correspondingly. Using one of the metal center-attacked electrophiles, 2.3 was demonstrated to give high diastereoselectivity. Parallel to COE allylic C-H activation complex 3.1, the propene and allylbenzene allylic C-H activation products have also been synthesized. The subsequent functionalization attempts have been examined by treating with Brønsted acids, Lewis acids, electrophiles, nucleophiles, 1,3-dipolar reagents and reagents containing multiple bonds able to be inserted. Various related complexes have been obtained under these conditions, in which one of the azide insertion compounds reductively eliminates to give an allylic functionalization product stoichiometrically. 3.1 reacts with various primary alcohols to give the decarbonylation dihydride complex ToMRh(H)2CO (4.1). 4.1 shows catalytic reactivity for primary alcohol decarbonylation under a photolytic condition. Meanwhile, 2.2 has been found to be more reactive than 4.1 for catalytic alcohol decarbonylation under the same condition. Various complexes and primary

  2. Activation of cortical 5-HT3 receptor-expressing interneurons induces NO mediated vasodilatations and NPY mediated vasoconstrictions

    Directory of Open Access Journals (Sweden)

    Quentin ePerrenoud

    2012-08-01

    Full Text Available GABAergic interneurons are local integrators of cortical activity that have been reported to be involved in the control of cerebral blood flow through their ability to produce vasoactive molecules and their rich innervation of neighboring blood vessels. They form a highly diverse population among which the serotonin 5-hydroxytryptamine 3A receptor (5-HT3A-expressing interneurons share a common developmental origin, in addition to the responsiveness to serotonergic ascending pathway. We have recently shown that these neurons regroup two distinct subpopulations within the somatosensory cortex: Neuropeptide Y (NPY-expressing interneurons, displaying morphological properties similar to those of neurogliaform cells, and Vasoactive Intestinal Peptide (VIP-expressing bipolar/bitufted interneurons. The aim of the present study was to determine the role of these neuronal populations in the control of vascular tone by monitoring blood vessels diameter changes, using infrared videomicroscopy in mouse neocortical slices. Bath applications of 1-(3-Chlorophenylbiguanide hydrochloride (mCPBG, a 5-HT3R agonist, induced both constrictions (30% and dilations (70% of penetrating arterioles within supragranular layers. All vasoconstrictions were abolished in the presence of the NPY receptor antagonist (BIBP 3226, suggesting that they were elicited by NPY release. Vasodilations persisted in the presence of the VIP receptor antagonist VPAC1 (PG-97-269, whereas they were blocked in the presence of the neuronal Nitric Oxide (NO Synthase (nNOS inhibitor, L-NNA. Altogether, these results strongly suggest that activation of neocortical 5-HT3A-expressing interneurons by serotoninergic input could induces NO mediated vasodilatations and NPY mediated vasoconstrictions.

  3. Magnetar activity mediated by plastic deformations of neutron star crust

    CERN Document Server

    Lyutikov, Maxim

    2014-01-01

    We advance a "Solar flare" model of magnetar activity, whereas a slow evolution of the magnetic field in the upper crust, driven by electron MHD (EMHD) flows, twists the external magnetic flux tubes, producing persistent emission, bursts and flares. At the same time the neutron star crust plastically relieves the imposed magnetic field stress, limiting the strain $ \\epsilon_t $ to values well below the critical strain $ \\epsilon_{crit}$ of a brittle fracture, $ \\epsilon_t \\sim 10^{-2}\\epsilon_{crit} $. Magnetar-like behavior, occurring near the magnetic equator, takes place in all neutron stars, but to a different extent. The persistent luminosity is proportional to cubic power of the magnetic field (at a given age), and hence is hardly observable in most rotationally powered neutron stars. Giant flares can occur only if the magnetic field exceeds some threshold value, while smaller bursts and flares may take place in relatively small magnetic fields. Bursts and flares are magnetospheric reconnection events t...

  4. Photobiologic-mediated fabrication of silver nanoparticles with antibacterial activity.

    Science.gov (United States)

    Lee, Jeong-Ho; Lim, Jeong-Muk; Velmurugan, Palanivel; Park, Yool-Jin; Park, Youn-Jong; Bang, Keuk-Soo; Oh, Byung-Taek

    2016-09-01

    We present the simple, eco-friendly synthesis of silver nanoparticles (AgNPs) using sunlight or green, red, blue, or white LED light together with Dryopteris crassirhizoma rhizome extract (DCRE) as the reducing and capping agent. The preliminary indication of AgNP production was a color change from yellowish green to brown after light exposure in the presence of DCRE. Optimization of parameters such as pH, inoculum dose, and metal ion concentration played an important role in achieving nanoparticle production in 30min. The spectroscopic and morphological properties of AgNPs were characterized using UV-Vis spectroscopy through the presence of a characteristic surface plasmon resonance (SPR) band for AgNPs, Fourier transform infrared spectroscopy (FT-IR), high-resolution transmission electron microscopy (HR-TEM), and X-ray diffraction (XRD). The FT-IR results indicated that the phytochemical present in DCRE was the probable reducing/capping agent involved in the synthesis of AgNPs, and light radiation enhanced nanoparticle production. HR-TEM revealed that the AgNPs were almost spherical with an average size of 5-60nm under all light sources. XRD studies confirmed the face cubic center (fcc) unit cell structure of AgNPs. The synthesized AgNPs showed good antimicrobial activity against Bacillus cereus and Pseudomonas aeruginosa. This study will bring a new insight in ecofriendly production of metal nanoparticles. PMID:27348063

  5. Health Worry, Physical Activity Participation, and Walking Difficulty among Older Adults: A Mediation Analysis

    Science.gov (United States)

    Li, Kin-Kit; Cardinal, Bradley J.; Vuchinich, Samuel

    2009-01-01

    This study examined the effect of health worry (i.e., cognitive aspect of anxiety resulting from concern for health) on walking difficulty in a nationally representative sample (N = 7,527) of older adults (M age = 76.83 years). The study further tested whether physical activity mediates the effect of health worry on walking difficulty in a 6-year…

  6. T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex.

    Science.gov (United States)

    Hertweck, Arnulf; Evans, Catherine M; Eskandarpour, Malihe; Lau, Jonathan C H; Oleinika, Kristine; Jackson, Ian; Kelly, Audrey; Ambrose, John; Adamson, Peter; Cousins, David J; Lavender, Paul; Calder, Virginia L; Lord, Graham M; Jenner, Richard G

    2016-06-21

    The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates. PMID:27292648

  7. T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex

    Directory of Open Access Journals (Sweden)

    Arnulf Hertweck

    2016-06-01

    Full Text Available The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC. Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates.

  8. Sympathetic nervous system activation, arterial shear rate, and flow-mediated dilation.

    NARCIS (Netherlands)

    Thijssen, D.H.J.; Atkinson, C.L.; Ono, K.; Sprung, V.S.; Spence, A.L.; Pugh, C.J.; Green, D.J.

    2014-01-01

    The aim of this study was to examine the contribution of arterial shear to changes in flow-mediated dilation (FMD) during sympathetic nervous system (SNS) activation in healthy humans. Ten healthy men reported to our laboratory four times. Bilateral FMD, shear rate (SR), and catecholamines were exam

  9. 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 activity...

  10. Mediated Effects of Perceived Competence on Youth Physical Activity and Sedentary Behavior

    Science.gov (United States)

    Bai, Yang; Chen, Senlin; Vazou, Spyridoula; Welk, Gregory J.; Schaben, Jodee

    2015-01-01

    Purpose: This study evaluates whether physical activity (PA) and sedentary behavior (SB) are influenced by a common mediating relationship. Method: A total of 1,552 participants in 3rd to 12th grade completed an online survey that included assessments of PA at school (PAS), PA at home (PAH), and SB as well as a battery of psychosocial variables…

  11. Investigating Mediations in Student Activities in an English Immersion Context in Mainland China

    Science.gov (United States)

    Liang, Xiaohua

    2009-01-01

    This study aims to investigate the role of mediation in the learning process from a sociocultural perspective, activity theory in particular. This study was carried out in a primary English immersion school within the CCUEI Programs in Mainland China. Data were collected mainly through observations and interviews, which were then supplemented by…

  12. Activity Theory and Technology Mediated Interaction: Cognitive Scaffolding Using Question-Based Consultation on "Facebook"

    Science.gov (United States)

    Rambe, Patient

    2012-01-01

    Studies that employed activity theory as a theoretical lens for exploring computer-mediated interaction have not adopted social media as their object of study. However, social media provides lecturers with personalised learning environments for diagnostic and prognostic assessments of student mastery of content and deep learning. The integration…

  13. Situated Uses of ICT and Mediation of Joint Activity in a Primary Education Instructional Sequence

    Science.gov (United States)

    Coll, Cesar; Rochera, Maria J.; Colomina, Rosa

    2010-01-01

    Introduction: From a socioconstructivist and situated perspective of teaching and learning processes, the authors analyze how one teacher and her group of 19 sixth-grade pupils use ICT. The study focuses on the way these tools mediate their activity, and evaluates the tools' potential for teaching and learning innovation. Method: A case study…

  14. Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

    OpenAIRE

    Jong Hyun Kim; Ji-Man Park; Kyungmoo Yea; Hyun Wook Kim; Pann-Ghill Suh; Sung Ho Ryu

    2010-01-01

    BACKGROUND: Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glu...

  15. Skeletal muscle reflex-mediated changes in sympathetic nerve activity are abnormal in spontaneously hypertensive rats

    OpenAIRE

    Mizuno, Masaki; Murphy, Megan N.; Mitchell, Jere H.; Smith, Scott A.

    2011-01-01

    In hypertension, the blood pressure response to exercise is exaggerated. We demonstrated previously that this heightened pressor response to physical activity is mediated by an overactive skeletal muscle exercise pressor reflex (EPR), with important contributions from its metaboreflex and mechanoreflex components. However, the mechanisms driving the abnormal blood pressure response to EPR activation are largely unknown. Recent evidence in humans suggests that the muscle metaboreflex partially...

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

    KAUST Repository

    Ali, Zahir

    2015-06-03

    © 2015 Taylor and Francis Group, LLC. Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRVmediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.

  17. Efficacy-mediated effects of spirituality and physical activity on quality of life: A path analysis

    Directory of Open Access Journals (Sweden)

    Konopack James F

    2012-05-01

    Full Text Available Abstract Background Physical activity has been established as an important determinant of quality of life, particularly among older adults. Previous research has suggested that physical activity’s influence on quality of life perceptions is mediated by changes in self-efficacy and health status. In the same vein, spirituality may be a salient quality of life determinant for many individuals. Methods In the current study, we used path analysis to test a model in which physical activity, spirituality, and social support were hypothesized to influence global quality of life in paths mediated by self-efficacy and health status. Cross-sectional data were collected from a sample of 215 adults (male, n = 51; female, n = 164 over the age of 50 (M age = 66.55 years. Results The analysis resulted in a model that provided acceptable fit to the data (χ2 = 33.10, df = 16, p  Conclusions These results support previous findings of an efficacy-mediated relationship between physical activity and quality of life, with the exception that self-efficacy in the current study was moderately associated with physical health status (.38 but not mental health status. Our results further suggest that spirituality may influence health and well-being via a similar, efficacy-mediated path, with strongest effects on mental health status. These results suggest that those who are more spiritual and physically active report greater quality of life, and the effects of these factors on quality of life may be partially mediated by perceptions of self-efficacy.

  18. 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

  19. Mediating between practitioner and developer communities: the Learning Activity Design in Education experience

    Directory of Open Access Journals (Sweden)

    Isobel Falconer

    2007-12-01

    Full Text Available The slow uptake by teachers in post-compulsory education of new technological tools and technology- enhanced teaching methods may be symptomatic of a general split in the e-learning community between development of tools, services and standards, and research into how teachers can use these most effectively (i.e. between the teaching practitioner and technical developer communities. This paper reflects on the experience of transferring knowledge and understanding between these two communities during the Learning Activity Design in Education project funded by the UK Joint Information Systems Committee. The discussion is situated within the literature on ‘mediating representations' and ‘mediating artefacts', and shows that the practical operation of mediating representations is far more complex than previously acknowledged. The experience suggests that for effective transfer of concepts between communities, the communities need to overlap to the extent that a single representation is comprehensible to both. This representation may be viewed as a boundary object that is used to negotiate understanding. If the communities do not overlap a chain of intermediate representations and communities may be necessary. Finally, a tentative distinction is drawn between mediating representations and mediating artefacts, based not in the nature of the resources, but in their mode and context of use.

  20. DNMT1-mediated PTEN hypermethylation confers hepatic stellate cell activation and liver fibrogenesis in rats

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Er-Bao; Huang, Cheng; Ma, Tao-Tao; Tao, Hui; Zhang, Hui; Cheng, Chang; Lv, Xiong-Wen; Li, Jun, E-mail: hunkahmu@126.com

    2012-10-01

    Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a negative regulator of this process. PTEN promoter hypermethylation is a major epigenetic silencing mechanism in tumors. The present study aimed to investigate whether PTEN promoter methylation was involved in HSC activation and liver fibrosis. Treatment of activated HSCs with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) decreased aberrant hypermethylation of the PTEN gene promoter and prevented the loss of PTEN expression that occurred during HSC activation. Silencing DNA methyltransferase 1 (DNMT1) gene also decreased the PTEN gene promoter methylation and upregulated the PTEN gene expression in activated HSC-T6 cells. In addition, knockdown of DNMT1 inhibited the activation of both ERK and AKT pathways in HSC-T6 cells. These results suggest that DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the activation of the PI3K/AKT and ERK pathways, resulting in HSC activation. Highlights: ► PTEN methylation status and loss of PTEN expression ► DNMT1 mediated PTEN hypermethylation. ► Hypermethylation of PTEN contributes to the activation of ERK and AKT pathways.

  1. Dexamethasone-induced apoptosis of osteocytic and osteoblastic cells is mediated by TAK1 activation.

    Science.gov (United States)

    Ding, Heyuan; Wang, Tao; Xu, Dongli; Cha, Bingbing; Liu, Jun; Li, Yiming

    2015-05-01

    Increased apoptosis of osteoblasts and osteocytes is the main mechanism of glucocorticoid (GC)-induced osteonecrosis. In the current study, we investigated whether dexamethasone (Dex)-induced osteoblastic and osteocytic cell apoptosis is mediated through activation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), and whether TAK1 inhibition could promote survival opposing the deleterious effects of Dex. We found that TAK1 was activated by Dex in both osteocytic MLO-Y4 and osteoblastic OB-6 cells, which was prevented by two known anti-oxidants N-acetylcysteine (NAC) and ebselen. TAK1 inhibitors, including LYTAK1 and 5Z-7-oxozeaenol (57-OZ), inhibited Dex-induced apoptosis of MLO-Y4 and OB-6 cells. Meanwhile shRNA-mediated knockdown of TAK1 also suppressed Dex-induced damages to MLO-Y4 and OB-6 cells. On the other hand, exogenously over-expressing TAK1 enhanced Dex-induced MLO-Y4 and OB-6 cell apoptosis. At the molecular level, we found that TAK1 mediated Dex-induced pro-apoptotic Pyk2-JNK activation. Inhibition or silencing of TAK1 almost abolished Pyk2-JNK phosphorylations by Dex in MLO-Y4 and OB-6 cells. TAK1 over-expression, on the other hand, increased Dex's activity on Pyk2-JNK phosphorylations in above cells. We conclude that part of the pro-apoptotic actions of Dex on osteoblastic and osteocytic cells are mediated through TAK1 activation, and that inhibition of TAK1 might protect from GC-induced damages to osteoblasts and osteocytes. PMID:25753204

  2. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  3. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ming V. [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Chen, Weiqin [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Harmancey, Romain N. [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Taegtmeyer, Heinrich [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Chan, Lawrence, E-mail: lchan@bcm.tmc.edu [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); St. Luke' s Episcopal Hospital, Houston, TX 77030 (United States)

    2010-05-07

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  4. Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with Apaf-1

    Institute of Scientific and Technical Information of China (English)

    Yong Zuo; Binggang Xiang; Jie Yang; Xuxu Sun; Yumei Wang; Hui Cang; Jing Yi

    2009-01-01

    Intracellular reactive oxygen species (ROS) are known to regulate apoptosis. Activation of caspase-9, the initial caspase in the mitochondrial apoptotic cascade, is closely associated with ROS, but it is unclear whether ROS regulate caspase-9 via direct oxidative modification. The present study aims to elucidate the molecular mechanisms by which ROS mediate caspase-9 activation. Our results show that the cellular oxidative state facilitates caspase-9 activation. Hydrogen peroxide treatment causes the activation of caspase-9 and apoptosis, and promotes an interaction between caspase-9 and apoptotic protease-activating factor 1 (Apaf-1) via disulfide formation. In addition, in an in vitro mitochondria-free system, the thiol-oxidant diamide promotes auto-cleavage of caspase-9 and the caspase-9/ Apaf-1 interaction by facilitating the formation of disulfide-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H202-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulfide formation. The association between cytochrome c and the C403S mutant is significantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modification of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modification of caspase-9 by ROS can mediate its interaction with Apaf-1, and can thus promote its auto-cleavage and activation. This mechanism may facilitate apoptosome formation and caspase-9 activation under oxidative stress.

  5. Phospholipase D1 mediates AMP-activated protein kinase signaling for glucose uptake.

    Directory of Open Access Journals (Sweden)

    Jong Hyun Kim

    Full Text Available BACKGROUND: Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here, we found that AMPK-induced phospholipase D1 (PLD1 activation is required for (14C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT stimulates PLD activity, while AMPK-dominant negative (DN inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in (14C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of (14C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA, which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate (14C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator regulate (14C-glucose uptake and cell surface glucose transport (GLUT 4 through ERK stimulation by AMPK-mediated PLD1 activation. CONCLUSIONS/SIGNIFICANCE: These results

  6. Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin

    Science.gov (United States)

    Foley, Jonathan H.; Walton, Bethany L.; Aleman, Maria M.; O'Byrne, Alice M.; Lei, Victor; Harrasser, Micaela; Foley, Kimberley A.; Wolberg, Alisa S.; Conway, Edward M.

    2016-01-01

    Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution. PMID:27077125

  7. Women, Physical Activity, and Quality of Life: Self-concept as a Mediator.

    Science.gov (United States)

    Gonzalo Silvestre, Tamara; Ubillos Landa, Silvia

    2016-02-22

    The objectives of this research are: (a) analyze the incremental validity of physical activity's (PA) influence on perceived quality of life (PQL); (b) determine if PA's predictive power is mediated by self-concept; and (c) study if results vary according to a unidimensional or multidimensional approach to self-concept measurement. The sample comprised 160 women from Burgos, Spain aged 18 to 45 years old. Non-probability sampling was used. Two three-step hierarchical regression analyses were applied to forecast PQL. The hedonic quality-of-life indicators, self-concept, self-esteem, and PA were included as independent variables. The first regression analysis included global self-concept as predictor variable, while the second included its five dimensions. Two mediation analyses were conducted to see if PA's ability to predict PQL was mediated by global and physical self-concept. Results from the first regression shows that self-concept, satisfaction with life, and PA were significant predictors. PA slightly but significantly increased explained variance in PQL (2.1%). In the second regression, substituting global self-concept with its five constituent factors, only the physical dimension and satisfaction with life predicted PQL, while PA ceased to be a significant predictor. Mediation analysis revealed that only physical self-concept mediates the relationship between PA and PQL (z = 1.97, p concept. Physical self-concept was the strongest predictor and approximately 32.45 % of PA's effect on PQL was mediated by it. This study's findings support a multidimensional view of self-concept, and represent a more accurate image of the relationship between PQL, PA, and self-concept.

  8. Women, Physical Activity, and Quality of Life: Self-concept as a Mediator.

    Science.gov (United States)

    Gonzalo Silvestre, Tamara; Ubillos Landa, Silvia

    2016-01-01

    The objectives of this research are: (a) analyze the incremental validity of physical activity's (PA) influence on perceived quality of life (PQL); (b) determine if PA's predictive power is mediated by self-concept; and (c) study if results vary according to a unidimensional or multidimensional approach to self-concept measurement. The sample comprised 160 women from Burgos, Spain aged 18 to 45 years old. Non-probability sampling was used. Two three-step hierarchical regression analyses were applied to forecast PQL. The hedonic quality-of-life indicators, self-concept, self-esteem, and PA were included as independent variables. The first regression analysis included global self-concept as predictor variable, while the second included its five dimensions. Two mediation analyses were conducted to see if PA's ability to predict PQL was mediated by global and physical self-concept. Results from the first regression shows that self-concept, satisfaction with life, and PA were significant predictors. PA slightly but significantly increased explained variance in PQL (2.1%). In the second regression, substituting global self-concept with its five constituent factors, only the physical dimension and satisfaction with life predicted PQL, while PA ceased to be a significant predictor. Mediation analysis revealed that only physical self-concept mediates the relationship between PA and PQL (z = 1.97, p self-concept. Physical self-concept was the strongest predictor and approximately 32.45 % of PA's effect on PQL was mediated by it. This study's findings support a multidimensional view of self-concept, and represent a more accurate image of the relationship between PQL, PA, and self-concept. PMID:26898406

  9. Clinical development of reovirus for cancer therapy: An oncolytic virus with immune-mediated antitumor activity

    Science.gov (United States)

    Gong, Jun; Sachdev, Esha; Mita, Alain C; Mita, Monica M

    2016-01-01

    Reovirus is a double-stranded RNA virus with demonstrated oncolysis or preferential replication in cancer cells. The oncolytic properties of reovirus appear to be dependent, in part, on activated Ras signaling. In addition, Ras-transformation promotes reovirus oncolysis by affecting several steps of the viral life cycle. Reovirus-mediated immune responses can present barriers to tumor targeting, serve protective functions against reovirus systemic toxicity, and contribute to therapeutic efficacy through antitumor immune-mediated effects via innate and adaptive responses. Preclinical studies have demonstrated the broad anticancer activity of wild-type, unmodified type 3 Dearing strain reovirus (Reolysin®) across a spectrum of malignancies. The development of reovirus as an anticancer agent and available clinical data reported from 22 clinical trials will be reviewed. PMID:27019795

  10. Clinical development of reovirus for cancer therapy: An oncolytic virus with immune-mediated antitumor activity.

    Science.gov (United States)

    Gong, Jun; Sachdev, Esha; Mita, Alain C; Mita, Monica M

    2016-03-26

    Reovirus is a double-stranded RNA virus with demonstrated oncolysis or preferential replication in cancer cells. The oncolytic properties of reovirus appear to be dependent, in part, on activated Ras signaling. In addition, Ras-transformation promotes reovirus oncolysis by affecting several steps of the viral life cycle. Reovirus-mediated immune responses can present barriers to tumor targeting, serve protective functions against reovirus systemic toxicity, and contribute to therapeutic efficacy through antitumor immune-mediated effects via innate and adaptive responses. Preclinical studies have demonstrated the broad anticancer activity of wild-type, unmodified type 3 Dearing strain reovirus (Reolysin(®)) across a spectrum of malignancies. The development of reovirus as an anticancer agent and available clinical data reported from 22 clinical trials will be reviewed.

  11. A transient reversal of miRNA-mediated repression controls macrophage activation.

    Science.gov (United States)

    Mazumder, Anup; Bose, Mainak; Chakraborty, Abhijit; Chakrabarti, Saikat; Bhattacharyya, Suvendra N

    2013-11-01

    In mammalian macrophages, the expression of a number of cytokines is regulated by miRNAs. Upon macrophage activation, proinflammatory cytokine mRNAs are translated, although the expression of miRNAs targeting these mRNAs remains largely unaltered. We show that there is a transient reversal of miRNA-mediated repression during the early phase of the inflammatory response in macrophages, which leads to the protection of cytokine mRNAs from miRNA-mediated repression. This derepression occurs through Ago2 phosphorylation, which results in its impaired binding to miRNAs and to the corresponding target mRNAs. Macrophages expressing a mutant, non-phosphorylatable AGO2--which remains bound to miRNAs during macrophage activation--have a weakened inflammatory response and fail to prevent parasite invasion. These findings highlight the relevance of the transient relief of miRNA repression for macrophage function.

  12. Inhibition of acetyl-CoA carboxylase by cystamine may mediate the hypotriglyceridemic activity of pantethine.

    Science.gov (United States)

    McCarty, M F

    2001-03-01

    Pantethine is a versatile and well-tolerated hypolipidemic agent whose efficacy in this regard appears to be mediated by its catabolic product cystamine, a nucleophile which avidly attacks disulfide groups. An overview of pantethine research suggests that the hypotriglyceridemic activity of pantethine reflects cystamine-mediated inhibition of the hepatic acetyl-CoA carboxylase, which can be expected to activate hepatic fatty acid oxidation. Inhibition of HMG-CoA reductase as well as a more distal enzyme in the cholesterol synthetic pathway may account for pantethine's hypocholesterolemic effects. If pantethine does indeed effectively inhibit hepatic acetyl-CoA carboxylase, it may have adjuvant utility in the hepatothermic therapy of obesity. As a safe and effective compound of natural origin, pantethine merits broader use in the management of hyperlipidemias. PMID:11359352

  13. Two distinct domains of Flo8 activator mediates its role in transcriptional activation and the physical interaction with Mss11.

    Science.gov (United States)

    Kim, Hye Young; Lee, Sung Bae; Kang, Hyen Sam; Oh, Goo Taeg; Kim, TaeSoo

    2014-06-27

    Flo8 is a transcriptional activator essential for the inducible expression of a set of target genes such as STA1, FLO11, and FLO1 encoding an extracellular glucoamylase and two cell surface proteins, respectively. However, the molecular mechanism of Flo8-mediated transcriptional activation remains largely elusive. By generating serial deletion constructs, we revealed here that a novel transcriptional activation domain on its extreme C-terminal region plays a crucial role in activating transcription. On the other hand, the N-terminal LisH motif of Flo8 appears to be required for its physical interaction with another transcriptional activator, Mss11, for their cooperative transcriptional regulation of the shared targets. Additionally, GST pull-down experiments uncovered that Flo8 and Mss11 can directly form either a heterodimer or a homodimer capable of binding to DNA, and we also showed that this formed complex of two activators interacts functionally and physically with the Swi/Snf complex. Collectively, our findings provide valuable clues for understanding the molecular mechanism of Flo8-mediated transcriptional control of multiple targets.

  14. Cytotoxic activities of amentoflavone against human breast and cervical cancers are mediated by increasing of PTEN expression levels due to peroxisomes proliferate-activated receptor {gamma} activation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eunjung; Shin, Soyoung; Lee, Jeeyoung; Lee, So Jung; Kim, Jinkyoung; Yoon, Doyoung; Kim, Yangmee [Konkuk Univ., Seoul (Korea, Republic of); Woo, Eunrhan [Chosun Univ., Gwangju (Korea, Republic of)

    2012-07-15

    Human peroxisomes proliferate-activated receptor gamma (hPPAR{gamma}) has been implicated in numerous pathologies, including obesity, diabetes, and cancer. Previously, we verified that amentoflavone is an activator of hPPAR{gamma} and probed the molecular basis of its action. In this study, we investigated the mechanism of action of amentoflavone in cancer cells and demonstrated that amentoflavone showed strong cytotoxicity against MCF-7 and HeLa cancer cell lines. We showed that hPPAR{gamma} expression in MCF-7 and HeLa cells is specifically stimulated by amentoflavone, and suggested that amentoflavone-induced cytotoxic activities are mediated by activation of hPPAR{gamma} in these two cancer cell lines. Moreover, amentoflavone increased PTEN levels in these two cancer cell lines, indicating that the cytotoxic activities of amentoflavone are mediated by increasing of PTEN expression levels due to hPPAR{gamma} activation.

  15. Activation of the Endothelin System Mediates Pathological Angiogenesis during Ischemic Retinopathy

    OpenAIRE

    Patel, Chintan; Narayanan, S. Priya; Zhang, Wenbo; Xu, Zhimin; Sukumari-Ramesh, Sangeetha; Dhandapani, Krishnan M.; Caldwell, R. William; Caldwell, Ruth B.

    2014-01-01

    Retinopathy of prematurity adversely affects premature infants because of oxygen-induced damage of the immature retinal vasculature, resulting in pathological neovascularization (NV). Our pilot studies using the mouse model of oxygen-induced retinopathy (OIR) showed marked increases in angiogenic mediators, including endothelins and endothelin receptor (EDNR) A. We hypothesized that activation of the endothelin system via EDNRA plays a causal role in pathological angiogenesis and up-regulatio...

  16. PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation.

    OpenAIRE

    Kannan, P; Yu, Y; Wankhade, S; Tainsky, M A

    1999-01-01

    Overexpression of transcription factor AP-2 has been implicated in the tumorigenicity of the human teratocarcinoma cell lines PA-1 that contain an activated ras oncogene. Here we show evidence that overexpression of AP-2 sequesters transcriptional coactivators which results in self-inhibition. We identified AP-2-interacting proteins and determined whether these proteins were coactivators for AP-2-mediated transcription. One such interacting protein is polyADP-ribose polymerase (PARP). PARP su...

  17. Aqueous-mediated Michael Addition of Active Methylene Compounds with Nitroalkenes

    Institute of Scientific and Technical Information of China (English)

    董文凯; 徐东成; 谢建武

    2012-01-01

    A simple, atom economical and highly efficient green protocol has been developed for the synthesis of Michael adducts of nitroalkenes and 2-amino-2-chromene derivatives by Michael addition of active methylene compounds (such as malononitrile and ethyl cyanoacetate) to nitroalkenes under aqueous-mediated conditions. This green approach provided the desired products in high yields and the reaction scope proved to be quite broad.

  18. New perspectives on mannan-binding lectin-mediated complement activation

    DEFF Research Database (Denmark)

    Degn, Søren Egedal; Thiel, Steffen; Jensenius, Jens Christian

    2007-01-01

    The complement system is an important part of the innate immune system, mediating several major effector functions and modulating adaptive immune responses. Three complement activation pathways exist: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The LP...... picture of the complement system is more that of a small "scale-free" network where C3 acts as the main hub, than that of three linear pathways converging in a common terminal pathway....

  19. Leptin‐Induced Endothelial Dysfunction Is Mediated by Sympathetic Nervous System Activity

    OpenAIRE

    Wang, Jintao; Wang, Hui; Luo, Wei; Guo, Chiao; Wang, Julia; Chen, Y.E.; Chang, Lin; Eitzman, Daniel T.

    2013-01-01

    Background The adipocyte‐derived hormone leptin is elevated in obesity and may contribute to vascular risk associated with obesity. The mechanism(s) by which leptin affects vascular disease is unclear, although leptin has been shown to increase sympathetic activity. The aim of this study was to investigate the effect of leptin treatment on endothelial function and the role of the local sympathetic nervous system in mediating these effects. Methods and Results Recombinant leptin was administer...

  20. DNA-PK mediates AKT activation and apoptosis inhibition in clinically acquired platinum resistance.

    Science.gov (United States)

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-11-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  1. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    Science.gov (United States)

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  2. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance

    Directory of Open Access Journals (Sweden)

    Euan A. Stronach

    2011-11-01

    Full Text Available Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinumresistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK, and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Re-sensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage–mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors.

  3. Polycystin-1 promotes PKCα-mediated NF-κB activation in kidney cells

    International Nuclear Information System (INIS)

    Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-κB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293CTT), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-κB nuclear levels and NF-κB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-κB promoter activation was mediated by PKCα because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293CTT cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-κB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCα-mediated NF-κB signalling and cell survival

  4. Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation.

    Science.gov (United States)

    Abdala-Valencia, Hiam; Bryce, Paul J; Schleimer, Robert P; Wechsler, Joshua B; Loffredo, Lucas F; Cook-Mills, Joan M; Hsu, Chia-Lin; Berdnikovs, Sergejs

    2015-08-15

    Mast cells are critical in the pathogenesis of allergic disease due to the release of preformed and newly synthesized mediators, yet the mechanisms controlling mast cell activation are not well understood. Members of the tetraspanin family are recently emerging as modulators of FcεRI-mediated mast cell activation; however, mechanistic understanding of their function is currently lacking. The tetraspanin CD151 is a poorly understood member of this family and is specifically induced on mouse and human mast cells upon FcεRI aggregation but its functional effects are unknown. In this study, we show that CD151 deficiency significantly exacerbates the IgE-mediated late phase inflammation in a murine model of passive cutaneous anaphylaxis. Ex vivo, FcεRI stimulation of bone marrow-derived mast cells from CD151(-/-) mice resulted in significantly enhanced expression of proinflammatory cytokines IL-4, IL-13, and TNF-α compared with wild-type controls. However, FcεRI-induced mast cell degranulation was unaffected. At the molecular signaling level, CD151 selectively regulated IgE-induced activation of ERK1/2 and PI3K, associated with cytokine production, but had no effect on the phospholipase Cγ1 signaling, associated with degranulation. Collectively, our data indicate that CD151 exerts negative regulation over IgE-induced late phase responses and cytokine production in mast cells. PMID:26136426

  5. Brain activation to negative stimuli mediates a relationship between adolescent marijuana use and later emotional functioning

    Directory of Open Access Journals (Sweden)

    Mary M. Heitzeg

    2015-12-01

    Full Text Available This work investigated the impact of heavy marijuana use during adolescence on emotional functioning, as well as the brain functional mediators of this effect. Participants (n = 40 were recruited from the Michigan Longitudinal Study (MLS. Data on marijuana use were collected prospectively beginning in childhood as part of the MLS. Participants were classified as heavy marijuana users (n = 20 or controls with minimal marijuana use. Two facets of emotional functioning—negative emotionality and resiliency (a self-regulatory mechanism—were assessed as part of the MLS at three time points: mean age 13.4, mean age 19.6, and mean age 23.1. Functional neuroimaging data during an emotion-arousal word task were collected at mean age 20.2. Negative emotionality decreased and resiliency increased across the three time points in controls but not heavy marijuana users. Compared with controls, heavy marijuana users had less activation to negative words in temporal, prefrontal, and occipital cortices, insula, and amygdala. Activation of dorsolateral prefrontal cortex to negative words mediated an association between marijuana group and later negative emotionality. Activation of the cuneus/lingual gyrus mediated an association between marijuana group and later resiliency. Results support growing evidence that heavy marijuana use during adolescence affects later emotional outcomes.

  6. Activation of cell-mediated immunity by Morinda citrifolia fruit extract and its constituents.

    Science.gov (United States)

    Murata, Kazuya; Abe, Yumi; Futamura-Masudaa, Megumi; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

    2014-04-01

    Morinda citrifolia, commonly known as noni, is a traditional natural medicine in French Polynesia and Hawaii. Functional foods derived from M. citrifolia fruit have been marketed to help prevent diseases and promote good health. The objective of this study was to assess the effects of M. citrifolia fruit on cell-mediated immunity. In the picryl chloride-induced contact dermatitis test, M. citrifolia fruit extract (Noni-ext) inhibited the suppression of cell-mediated immunity by immunosuppressive substances isolated from freeze-dried ascites of Ehrlich carcinoma-bearing mice (EC-sup). In addition, Noni-ext inhibited reduction of IL-2 production in EC-sup-treated mice and activated natural killer cells in normal mice. These results suggest that Noni-ext has multiple effects on the recovery of cell-mediated immunity. Furthermore, we investigated the active principles of Noni-ext and identified an iridoid glycoside, deacetylasperulosidic acid. Oral administration of deacetylasperulosidic acid inhibited the reduction of ear swelling, and also cancelled the suppression of IL-2 production along with the activation of natural killer cells in the same manner as that of Noni-ext.

  7. Substrate Stiffness Regulates Proinflammatory Mediator Production through TLR4 Activity in Macrophages

    Science.gov (United States)

    Previtera, Michelle L.; Sengupta, Amitabha

    2015-01-01

    Clinical data show that disease adversely affects tissue elasticity or stiffness. While macrophage activity plays a critical role in driving disease pathology, there are limited data available on the effects of tissue stiffness on macrophage activity. In this study, the effects of substrate stiffness on inflammatory mediator production by macrophages were investigated. Bone marrow–derived macrophages were grown on polyacrylamide gels that mimicked the stiffness of a variety of soft biological tissues. Overall, macrophages grown on soft substrates produced less proinflammatory mediators than macrophages grown on stiff substrates when the endotoxin LPS was added to media. In addition, the pathways involved in stiffness–regulated proinflammation were investigated. The TLR4 signaling pathway was examined by evaluating TLR4, p–NF–κB p65, MyD88, and p–IκBα expression as well as p–NF–κB p65 translocation. Expression and translocation of the various signaling molecules were higher in macrophages grown on stiff substrates than on soft substrates. Furthermore, TLR4 knockout experiments showed that TLR4 activity enhanced proinflammation on stiff substrates. In conclusion, these results suggest that proinflammatory mediator production initiated by TLR4 is mechanically regulated in macrophages. PMID:26710072

  8. Integrin αIIb-mediated PI3K/Akt activation in platelets.

    Directory of Open Access Journals (Sweden)

    Haixia Niu

    Full Text Available Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R(724KEFAKFEEER(734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R(724KEFAKFEEER(734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E(724AERKFERKFE(734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

  9. Identification of trans-sialidases as a common mediator of endothelial cell activation by African trypanosomes.

    Directory of Open Access Journals (Sweden)

    Zeinab Ammar

    Full Text Available Understanding African Trypanosomiasis (AT host-pathogen interaction is the key to an "anti-disease vaccine", a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. T. congolense, T. vivax, and T. b. gambiense activated the endothelial NF-κB pathway, but interestingly, not T. b. brucei. The parasitic TS (trans-sialidases mediated this NF-κB activation, remarkably via their lectin-like domain and induced production of pro-inflammatory molecules not only in vitro but also in vivo, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in T. b. gambiense BSF which distinguishes it from the subspecies T. b. brucei. The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.

  10. β2-Glycoprotein I Is a Cofactor for t-PA–Mediated Plasminogen Activation

    Science.gov (United States)

    Bu, Chunya; Gao, Lei; Xie, Weidong; Zhang, Jainwei; He, Yuhong; Cai, Guoping; McCrae, Keith R

    2010-01-01

    Regulation of the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. Here, we report that the abundant plasma glycoprotein, β2-glycoprotein I (β2GPI), stimulates t-PA–dependent plasminogen activation in the fluid phase and within a fibrin gel. The region within β2GPI responsible for stimulating t-PA activity is at least partially contained within β2GPI domain V. β2GPI bound t-PA with high affinity (Kd ~ 20 nM), stimulated t-PA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (kcat/Km) of t-PA–mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of β2GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following β2GPI repletion. Finally, stimulation of t-PA–mediated plasminogen activity by β2GPI was inhibited by monoclonal anti-β2GPI antibodies, as well as by anti-β2GPI antibodies from patients with antiphospholipid syndrome (APS). These findings suggest that β2GPI may be an endogenous regulator of fibrinolysis. Impairment of β2GPI-stimulated fibrinolysis by anti-β2GPI antibodies may contribute to the development of thrombosis in patients with APS. PMID:19180513

  11. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  12. Bam32: a novel mediator of Erk activation in T cells.

    Science.gov (United States)

    Sommers, Connie L; Gurson, Jordan M; Surana, Rishi; Barda-Saad, Mira; Lee, Jan; Kishor, Aparna; Li, Wenmei; Gasser, Adam J; Barr, Valarie A; Miyaji, Michihiko; Love, Paul E; Samelson, Lawrence E

    2008-07-01

    Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.

  13. Peptide-Based Selective Inhibitors of Matrix Metalloproteinase-Mediated Activities

    Directory of Open Access Journals (Sweden)

    Margaret W. Ndinguri

    2012-11-01

    Full Text Available The matrix metalloproteinases (MMPs exhibit a broad array of activities, some catalytic and some non-catalytic in nature. An overall lack of selectivity has rendered small molecule, active site targeted MMP inhibitors problematic in execution. Inhibitors that favor few or individual members of the MMP family often take advantage of interactions outside the enzyme active site. We presently focus on peptide-based MMP inhibitors and probes that do not incorporate conventional Zn2+ binding groups. In some cases, these inhibitors and probes function by binding only secondary binding sites (exosites, while others bind both exosites and the active site. A myriad of MMP mediated-activities beyond selective catalysis can be inhibited by peptides, particularly cell adhesion, proliferation, motility, and invasion. Selective MMP binding peptides comprise highly customizable, unique imaging agents. Areas of needed improvement for MMP targeting peptides include binding affinity and stability.

  14. The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

    Science.gov (United States)

    Li, Hai-Sheng; Shome, Kuntala; Rojas, Raúl; Rizzo, Mark A; Vasudevan, Chandrasekaran; Fluharty, Eric; Santy, Lorraine C; Casanova, James E; Romero, Guillermo

    2003-01-01

    Background Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. Results Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. Conclusions Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor. PMID:12969509

  15. 5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase

    Directory of Open Access Journals (Sweden)

    Lin Yu-Hsin

    2010-04-01

    Full Text Available Abstract Photodynamic therapy (PDT has been developed as an anticancer treatment, which is based on the tumor-specific accumulation of a photosensitizer that induces cell death after irradiation of light with a specific wavelength. Depending on the subcellular localization of the photosensitizer, PDT could trigger various signal transduction cascades and induce cell death such as apoptosis, autophagy, and necrosis. In this study, we report that both AMP-activated protein kinase (AMPK and mitogen-activated protein kinase (MAPK signaling cascades are activated following 5-aminolevulinic acid (ALA-mediated PDT in both PC12 and CL1-0 cells. Although the activities of caspase-9 and -3 are elevated, the caspase inhibitor zVAD-fmk did not protect cells against ALA-PDT-induced cell death. Instead, autophagic cell death was found in PC12 and CL1-0 cells treated with ALA-PDT. Most importantly, we report here for the first time that it is the activation of AMPK, but not MAPKs that plays a crucial role in mediating autophagic cell death induced by ALA-PDT. This novel observation indicates that the AMPK pathway play an important role in ALA-PDT-induced autophagy.

  16. The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

    Directory of Open Access Journals (Sweden)

    Fluharty Eric

    2003-09-01

    Full Text Available Abstract Background Phospholipase D (PLD is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. Results Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. Conclusions Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.

  17. Psychosocial Mediators of a Faith-Based Physical Activity Intervention: Implications and Lessons Learned from Null Findings

    Science.gov (United States)

    Baruth, Meghan; Wilcox, Sara; Blair, Steve; Hooker, Steve; Hussey, Jim; Saunders, Ruth

    2010-01-01

    Mediation analyses in faith-based physical activity (PA) interventions targeting African-American adults are lacking. The purpose of this study was to examine the psychosocial mediators of a faith-based PA intervention with African-American adults. Churches were randomly assigned to receive immediate or delayed (1-year later) training in PA…

  18. Src mediates the mechanical activation of myogenesis by activating TNFα-converting enzyme

    OpenAIRE

    Niu, Airu; Wen, Yefei; Liu, Huijie; Zhan, Mei; Jin, Bingwen; Li, Yi-Ping

    2013-01-01

    Mechanical stimulation affects many biological aspects in living cells through mechanotransduction. In myogenic precursor cells (MPCs), mechanical stimulation activates p38 mitogen-activated protein kinase (MAPK), a key regulator of myogenesis, via activating TNFα-converting enzyme (TACE, also known as ADAM17), to release autocrine TNFα. However, the signaling mechanism of mechanical activation of TACE is unknown. Because TACE possesses the structural features of substrates of the non-recepto...

  19. A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

    Directory of Open Access Journals (Sweden)

    Chang Chao-Chien

    2011-12-01

    Full Text Available Abstract Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM. Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLCγ2 phosphorylation, protein kinase C (PKC activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may

  20. Intelligence in early adulthood and midlife physical performance in men: The mediating roles of education and physical activity

    DEFF Research Database (Denmark)

    Meincke, Rikke Hodal; Hansen, Åse Marie; Mortensen, Laust Hvas;

    2016-01-01

    The objective of this study was to examine whether associations between intelligence in early adulthood and midlife physical performance in men were mediated by education and leisure-time physical activity. Intelligence correlated positively with later education (r = 0.47) and negatively...... with physical activity (r = − 0.04). We found higher early adult intelligence to be associated with better midlife physical performance for five of the seven measures. Education mediated the association between intelligence and chair-rise and jump height with mediated proportions of 32.4% (95% confidence...... performance, but because intelligence in early adulthood was inversely associated with physical activity, the indirect effects through physical activity were negative. Overall, education and leisure-time physical activity were not strong mediators of the association between early adult intelligence...

  1. Cell-mediated cytotoxicity-supporting activity of various human gammaglobulin preparations.

    Directory of Open Access Journals (Sweden)

    Wakiguchi,Hiroshi

    1987-04-01

    Full Text Available Antibody activity, especially that involved in the reaction of antibody-dependent cell-mediated cytotoxicity (ADCC, of five commercially available human gammaglobulin preparations (standard, pepsin-treated, plasmin-treated, polyethylene glycol-fractionated and S-sulfonated gammaglobulin was measured. All these gammaglobulin preparations had high titers of hemagglutination inhibition and neutralizing antibody against measles virus. In ADCC reaction, the pepsin-treated gammaglobulin preparation showed no antibody activity. The standard gammaglobulin preparation showed weak activity only when highly diluted. The remaining three preparations showed high activity. Though the S-sulfonated gammaglobulin preparation showed no activity in ADCC reaction, it showed high activity after reconversion by means of oxidation and reduction in vitro. The plasmin-treated gammaglobulin preparation showed greater activity than the polyethylene glycol-fractionated preparation of the optimal concentration. In ADCC tests using the plasmin-treated gammaglobulin preparation, K cell activity was strongly inhibited by Hg (thimerosal, while, in those using the standard gammaglobulin preparation, the activity was hardly influenced by Hg, suggesting that the low ADCC activity of the standard gammaglobulin preparation of high concentrations was due to the inhibitory effect of aggregated immunoglobulin G molecules.

  2. A cis-acting diversification activator both necessary and sufficient for AID-mediated hypermutation.

    Directory of Open Access Journals (Sweden)

    Artem Blagodatski

    2009-01-01

    Full Text Available Hypermutation of the immunoglobulin (Ig genes requires Activation Induced cytidine Deaminase (AID and transcription, but it remains unclear why other transcribed genes of B cells do not mutate. We describe a reporter transgene crippled by hypermutation when inserted into or near the Ig light chain (IgL locus of the DT40 B cell line yet stably expressed when inserted into other chromosomal positions. Step-wise deletions of the IgL locus revealed that a sequence extending for 9.8 kilobases downstream of the IgL transcription start site confers the hypermutation activity. This sequence, named DIVAC for diversification activator, efficiently activates hypermutation when inserted at non-Ig loci. The results significantly extend previously reported findings on AID-mediated gene diversification. They show by both deletion and insertion analyses that cis-acting sequences predispose neighboring transcription units to hypermutation.

  3. Integrin-mediated adhesion as self-sustained waves of enzymatic activation.

    Science.gov (United States)

    Block, M R; Destaing, O; Petropoulos, C; Planus, E; Albigès-Rizo, C; Fourcade, B

    2015-10-01

    Integrin receptors mediate interaction between the cellular actin-cytoskeleton and extracellular matrix. Based on their activation properties, we propose a reaction-diffusion model where the kinetics of the two-state receptors is modulated by their lipidic environment. This environment serves as an activator variable, while a second variable plays the role of a scaffold protein and controls the self-sustained activation of the receptors. Due to receptor diffusion which couples dynamically the activator and the inhibitor, our model connects major classes of reaction diffusion systems for excitable media. Spot and rosette solutions, characterized by receptor clustering into localized static or dynamic structures, are organized into a phase diagram. It is shown that diffusion and kinetics of receptors determines the dynamics and the stability of these structures. We discuss this model as a precursor model for cell signaling in the context of podosomes forming actoadhesive metastructures, and we study how generic signaling defects influence their organization. PMID:26565269

  4. Stretch-induced hypertrophy activates NFkB-mediated VEGF secretion in adult cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Anna Leychenko

    Full Text Available Hypertension and myocardial infarction are associated with the onset of hypertrophy. Hypertrophy is a compensatory response mechanism to increases in mechanical load due to pressure or volume overload. It is characterized by extracellular matrix remodeling and hypertrophic growth of adult cardiomyocytes. Production of Vascular Endothelial Growth Factor (VEGF, which acts as an angiogenic factor and a modulator of cardiomyocyte function, is regulated by mechanical stretch. Mechanical stretch promotes VEGF secretion in neonatal cardiomyocytes. Whether this effect is retained in adult cells and the molecular mechanism mediating stretch-induced VEGF secretion has not been elucidated. Our objective was to investigate whether cyclic mechanical stretch induces VEGF secretion in adult cardiomyocytes and to identify the molecular mechanism mediating VEGF secretion in these cells. Isolated primary adult rat cardiomyocytes (ARCMs were subjected to cyclic mechanical stretch at an extension level of 10% at 30 cycles/min that induces hypertrophic responses. Cyclic mechanical stretch induced a 3-fold increase in VEGF secretion in ARCMs compared to non-stretch controls. This increase in stretch-induced VEGF secretion correlated with NFkB activation. Cyclic mechanical stretch-mediated VEGF secretion was blocked by an NFkB peptide inhibitor and expression of a dominant negative mutant IkBα, but not by inhibitors of the MAPK/ERK1/2 or PI3K pathways. Chromatin immunoprecipitation assays demonstrated an interaction of NFkB with the VEGF promoter in stretched primary cardiomyocytes. Moreover, VEGF secretion is increased in the stretched myocardium during pressure overload-induced hypertrophy. These findings are the first to demonstrate that NFkB activation plays a role in mediating VEGF secretion upon cyclic mechanical stretch in adult cardiomyocytes. Signaling by NFkB initiated in response to cyclic mechanical stretch may therefore coordinate the hypertrophic

  5. Maturation promoting factor destabilization facilitates postovulatory aging-mediated abortive spontaneous egg activation in rat.

    Science.gov (United States)

    Prasad, Shilpa; Koch, Biplob; Chaube, Shail K

    2016-04-01

    The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging-mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee-1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging-mediated abortive SEA in rats eggs aged in vivo. For this purpose, sexually immature female rats were given a single injection (20 IU IM) of pregnant mare serum gonadotropin for 48 h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21 h post-hCG surge to induce postovulatory aging in vivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr-14/Tyr-15 and dephosphorylation of Thr-161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging-mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging-mediated abortive SEA in rat eggs. PMID:26991553

  6. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

    Science.gov (United States)

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  7. PPAR-γ activation by Tityus serrulatus venom regulates lipid body formation and lipid mediator production.

    Science.gov (United States)

    Zoccal, Karina Furlani; Paula-Silva, Francisco Wanderley Garcia; Bitencourt, Claudia da Silva; Sorgi, Carlos Artério; Bordon, Karla de Castro Figueiredo; Arantes, Eliane Candiani; Faccioli, Lúcia Helena

    2015-01-01

    Tityus serrulatus venom (TsV) consists of numerous peptides with different physiological and pharmacological activities. Studies have shown that scorpion venom increases pro-inflammatory cytokine production, contributing to immunological imbalance, multiple organ dysfunction, and patient death. We have previously demonstrated that TsV is a venom-associated molecular pattern (VAMP) recognized by TLRs inducing intense inflammatory reaction through the production of pro-inflammatory cytokines and arachidonic acid-derived lipid mediators prostaglandin (PG)E2 and leukotriene (LT)B4. Lipid bodies (LBs) are potential sites for eicosanoid production by inflammatory cells. Moreover, recent studies have shown that the peroxisome proliferator-activated receptor gamma (PPAR-γ) is implicated in LB formation and acts as an important modulator of lipid metabolism during inflammation. In this study, we used murine macrophages to evaluate whether the LB formation induced by TsV after TLR recognition correlates with lipid mediator generation by macrophages and if it occurs through PPAR-γ activation. We demonstrate that TsV acts through TLR2 and TLR4 stimulation and PPAR-γ activation to induce LB formation and generation of PGE2 and LTB4. Our data also show that PPAR-γ negatively regulates the pro-inflammatory NF-κB transcription factor. Based on these results, we suggest that during envenomation, LBs constitute functional organelles for lipid mediator production through signaling pathways that depend on cell surface and nuclear receptors. These findings point to the inflammatory mechanisms that might also be triggered during human envenomation by TsV. PMID:25450800

  8. Moms in motion: a group-mediated cognitive-behavioral physical activity intervention

    Directory of Open Access Journals (Sweden)

    Brawley Lawrence R

    2006-08-01

    Full Text Available Abstract Background When examining the prevalence of physical inactivity by gender and age, women over the age of 25 are at an increased risk for sedentary behavior. Childbearing and motherhood have been explored as one possible explanation for this increased risk. Post natal exercise studies to date demonstrate promising physical and psychological outcomes, however few physical activity interventions have been theory-driven and tailored to post natal exercise initiates. The purpose of this study was to compare the effects of a group-mediated cognitive behavioral intervention based upon social-cognitive theory and group dynamics (GMCB to a standard care postnatal exercise program (SE. Method A randomized, two-arm intervention design was used. Fifty-seven post natal women were randomized to one of two conditions: (1 a standard exercise treatment (SE and (2 a standard exercise treatment plus group-mediated cognitive behavioral intervention (GMCB. Participants in both conditions participated in a four-week intensive phase where participants received standard exercise training. In addition, GMCB participants received self-regulatory behavioral skills training via six group-mediated counseling sessions. Following the intensive phase, participants engaged in a four-week home-based phase of self-structured exercise. Measures of physical activity, barrier efficacy, and proximal outcome expectations were administered and data were analyzed using ANCOVA procedures. Results and discussion ANCOVA of change scores for frequency, minutes, and volume of physical activity revealed significant treatment effects over the intensive and home-based phases (p's Conclusion While both exercise programs resulted in improvements to exercise participation, the GMCB intervention produced greater improvement in overall physical activity, barrier efficacy and proximal outcome expectations.

  9. Regulation of ENaC-Mediated Sodium Reabsorption by Peroxisome Proliferator-Activated Receptors

    Directory of Open Access Journals (Sweden)

    Tengis S. Pavlov

    2010-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of a steroid hormone receptor superfamily that responds to changes in lipid and glucose homeostasis. Peroxisomal proliferator-activated receptor subtype γ (PPARγ has received much attention as the target for antidiabetic drugs, as well as its role in responding to endogenous compounds such as prostaglandin J2. However, thiazolidinediones (TZDs, the synthetic agonists of the PPARγ are tightly associated with fluid retention and edema, as potentially serious side effects. The epithelial sodium channel (ENaC represents the rate limiting step for sodium absorption in the renal collecting duct. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. The role of PPARγ agonists on ENaC activity remains controversial. While PPARγ agonists were shown to stimulate ENaC-mediated renal salt absorption, probably via Serum- and Glucocorticoid-Regulated Kinase 1 (SGK1, other studies reported that PPARγ agonist-induced fluid retention is independent of ENaC activity. The current paper provides new insights into the control and function of ENaC and ENaC-mediated sodium transport as well as several other epithelial channels/transporters by PPARs and particularly PPARγ. The potential contribution of arachidonic acid (AA metabolites in PPAR-dependent mechanisms is also discussed.

  10. FGFR2c-mediated ERK-MAPK activity regulates coronal suture development.

    Science.gov (United States)

    Pfaff, Miles J; Xue, Ke; Li, Li; Horowitz, Mark C; Steinbacher, Derek M; Eswarakumar, Jacob V P

    2016-07-15

    Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor's gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis. PMID:27034231

  11. Peroxynitrite Mediates Diabetes-Induced Endothelial Dysfunction: Possible Role of Rho Kinase Activation

    Directory of Open Access Journals (Sweden)

    Azza B. El-Remessy

    2010-01-01

    Full Text Available Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.

  12. The association between physical self-discrepancies and women's physical activity: the mediating role of motivation.

    Science.gov (United States)

    Brunet, Jennifer; Sabiston, Catherine; Castonguay, Andree; Ferguson, Leah; Bessette, Natalia

    2012-02-01

    The objectives of this study were to test the associations between physical self-discrepancies (actual:ideal and actual:ought) and physical activity behavior, and to examine whether motivational regulations mediate these associations using self-discrepancy (Higgins, 1987) and organismic integration (Deci & Ryan, 1985) theories as guiding frameworks. Young women (N = 205; M(age) = 18.87 years, SD = 1.83) completed self-report questionnaires. Main analyses involved path analysis using a polynomial regression approach, estimation of direct and indirect effects, and evaluation of response surface values. Agreement between actual and ideal (or ought) physical self-perceptions was related to physical activity both directly and indirectly as mediated by the motivational regulations (R(2) = .24-.30). Specifically, when actual and ideal self-perceptions scores were similar, physical activity levels increased as actual and ideal scores increased. Furthermore, physical activity levels were lower when the discrepancy was such that ideal or ought self were higher than actual self. These findings provide support for integrating self-discrepancy and organismic integration theories to advance research in this area.

  13. Dimerization of DOCK2 is essential for DOCK2-mediated Rac activation and lymphocyte migration.

    Science.gov (United States)

    Terasawa, Masao; Uruno, Takehito; Mori, Sayako; Kukimoto-Niino, Mutsuko; Nishikimi, Akihiko; Sanematsu, Fumiyuki; Tanaka, Yoshihiko; Yokoyama, Shigeyuki; Fukui, Yoshinori

    2012-01-01

    The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs), DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR)-2 (also known as CZH2 or Docker) domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane. PMID:23050005

  14. Dimerization of DOCK2 is essential for DOCK2-mediated Rac activation and lymphocyte migration.

    Directory of Open Access Journals (Sweden)

    Masao Terasawa

    Full Text Available The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs, DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR-2 (also known as CZH2 or Docker domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane.

  15. Hydrogen peroxide mediates Rac1 activation of S6K1

    International Nuclear Information System (INIS)

    We previously reported that hydrogen peroxide (H2O2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H2O2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H2O2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H2O2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H2O2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H2O2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H2O2 as a mediator in the activation of S6K1 by Rac1. However, H2O2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H2O2 could be produced by arsenite, which has been shown to be a stimulator of H2O2 production. Taken together, these results suggest that H2O2 plays a pivotal role as a mediator in Rac1 activation of S6K1

  16. Does Pedestrian Danger Mediate the Relationship between Local Walkability and Active Travel to Work?

    Directory of Open Access Journals (Sweden)

    Sandy J Slater

    2016-05-01

    Full Text Available Background: Environmental and policy factors play an important role in influencing people’s lifestyles, physical activity (PA, and risks for developing obesity. Research suggests that more walkable communities are needed to sustain lifelong PA behavior, but there is a need to determine what local built environment features facilitate making being active the easy choice.Purpose: This county-level study examined the association between local walkability (walkability and traffic calming scales, pedestrian danger, and the percent of adults who used active transport to work. Methods: Built environment and PA outcome measures were constructed for the 496 most populous counties representing 74 percent of the U.S. population. GIS-based walkability scales were constructed and include a census of roads located within the counties using 2011 Navteq data. The pedestrian danger index (PDI includes data collected from the Fatality Analysis Reporting System 2009-2011, and measures the likelihood of a pedestrian being hit and killed by a vehicle. Four continuous outcome measures were constructed using 2009-2013 American Community Survey county-level 5-year estimates. The measures represent the percentage of workers living in a county who worked away from home and: 1 walked to work; 2 biked to work; 3 took public transit; and 4 used any form of active transport. Linear regression and mediation analyses were conducted to examine the association between walkability, PDI and active transport. Models accounted for clustering within state with robust standard errors, and controlled for median household income, families with children in poverty, race, ethnicity, urbanicity and region.Results: The walkability scale was significantly negatively associated with the PDI (β=-0.06, 95% CI=-0.111, -0.002. In all models, the PDI was significantly negatively associated with all active travel-related outcomes at the p<0.01 level. The walkability scale was positively

  17. Relationships between levels of motor coordination, attention and physical activity in children: The mediation model

    Directory of Open Access Journals (Sweden)

    Jakub Kokštejn

    2012-12-01

    Full Text Available BACKGROUND: Current findings suggest that physical activity of children with developmental difficulties may be limited by low level of motor coordination. Motor difficulties are often connected with children suffering from attention deficit disorder. OBJECTIVE: The aim of the study was to find out the level of physical activity (PA in older school-age children with motor difficulties (MD in comparison with children without MD and to reveal possible mediate impact on attention between the level of motor skills and PA in children of this age. METHODS: Participants were divided into two groups: 15 children with MD (age 13.7 ± 1.6 years and 27 children without MD (age 13.3 ± 1.4 years. Motor functions were assessed by means of test battery MABC-2, weekly physical activity by means of Actigraph accelerometer and attention by both d2 and numeric square tests. To estimate the mediation of the attention level we have used Baron's & Kenny's (1986 analysis. RESULTS: In most of the indicators of PA, children with MD reached lower value than those without MD. The differences of statistical significance were found in the number of steps per week and weekdays (d = 0.50 and 0.64 respectively and in PA of a very high intensity (d =2 .00 in boys with and without MD. In girls with MD we have found out significantly less time spent in vigorous intensity PA (d = 0.86. The study results support the hypothesis of developmental motor deficits to be a risk factor for PA in older school-age children. Significant mediation effect of concentration of attention in the relationship between the level of motor skills and PA was observed in three cases - in the relationship between gross motor skills on the one hand, and energy expenditure per week and weekdays, and vigorous intensity PA per week on the other. The amount of mediation effect of attention concentration ranged between 12-22%. CONCLUSION: The study has indicated that children's participation in PA can be

  18. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

    Energy Technology Data Exchange (ETDEWEB)

    Matt Vaughn Greg Harrington Daniel R Bush

    2002-08-06

    This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose

  19. Oxidative Conversion Mediates Antiproliferative Effects of tert-Butylhydroquinone: Structure and Activity Relationship Study.

    Science.gov (United States)

    Sanidad, Katherine Z; Sukamtoh, Elvira; Wang, Weicang; Du, Zheyuan; Florio, Ellie; He, Lili; Xiao, Hang; Decker, Eric A; Zhang, Guodong

    2016-05-18

    Previous studies have shown that tert-butylhydroquinone (TBHQ), a widely used food antioxidant, has cytotoxic effects at high doses; however, the underlying mechanisms are not well understood. Here, we found that the effects of TBHQ on cell proliferation, cell cycle progression, and apoptosis are mainly mediated by its oxidative conversion to a quinone metabolite tert-butylquinone (TBQ). Co-addition of cupric ion (Cu(2+)) caused accelerated oxidative conversion of TBHQ to TBQ and enhanced the biological activities of TBHQ on cell proliferation, cell cycle progression, and apoptosis in MC38 colon cancer cells. In contrast, co-addition of ethylenediaminetetraacetic acid (EDTA) suppressed TBHQ oxidation and inhibited the biological activities of TBHQ in MC38 cells. For example, after 24 h of treatment in basal medium, low-dose TBHQ (1.88-7.5 μM) had little effect on MC38 cell proliferation, while co-addition of 50 μM Cu(2+) caused 30-70% inhibition of cell proliferation; in contrast, treatment with high-dose TBHQ (15 μM) inhibited 50 ± 4% MC38 proliferation, which was abolished by co-addition of 50 μM EDTA. We further showed that TBQ had more potent actions on cell proliferation and associated cellular responses than TBHQ, supporting a critical role of TBQ formation in the biological activities of TBHQ. Finally, a structure and activity relationship study showed that the fast-oxidized para-hydroquinones had potent antiproliferative effects in MC38 cells, while the slow-oxidized para-hydroquinones had weak or little biological activities. Together, these results suggest that the biological activities of TBHQ and other para-hydroquinones are mainly mediated by their oxidative metabolism to generate more biologically active quinone metabolites. PMID:27111399

  20. Autonomy Mediates the Relationship between Personality and Physical Activity: An Application of Self-Determination Theory

    Directory of Open Access Journals (Sweden)

    Meredith L. Ramsey

    2016-04-01

    Full Text Available This study sought to examine tenets of Self-Determination Theory by testing a mediation model of physical activity and personality via autonomy. A total of 290 adults were recruited to complete a one-time online survey of exercise habits and individual characteristics. Surveys assessed personality, autonomy, and physical activity. A measurement model specifying direct effects between personality dimensions and physical activity and indirect effects operating through autonomy provided an excellent fit to the data (Χ2 = 0.66, df = 3, p = 0.88, RMSEA(90% CI = 0.00 (0.00–0.05, CFI = 0.99, SRMR = 0.01. Results indicated significant (p < 0.05 effects of Extroversion (β = 0.42, Conscientiousness (β = 0.96, and Emotional Stability (β = 0.60 on autonomy, which in turn, was significantly associated with physical activity (β = 0.55. No significant effects were observed for Agreeableness or Intellect. None of the personality constructs were found to be directly associated with physical activity. This model accounted for 27% of the variance in physical activity. The results of this study suggest that autonomy is significantly associated with physical activity. Therefore, attempts to improve autonomy in individuals may be a useful intervention strategy in improving physical activity levels.

  1. CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury.

    Science.gov (United States)

    Kwon, Min Jung; Shin, Hae Young; Cui, Yuexian; Kim, Hyosil; Thi, Anh Hong Le; Choi, Jun Young; Kim, Eun Young; Hwang, Dong Hoon; Kim, Byung Gon

    2015-12-01

    CNS neurons in adult mammals do not spontaneously regenerate axons after spinal cord injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate beyond the injury site by promoting expression of regeneration-associated genes. We have previously shown that peripheral nerve injury increases the number of macrophages in the DRGs and that the activated macrophages are critical to the enhancement of intrinsic regeneration capacity. The present study identifies a novel chemokine signal mediated by CCL2 that links regenerating neurons with proregenerative macrophage activation. Neutralization of CCL2 abolished the neurite outgrowth activity of conditioned medium obtained from neuron-macrophage cocultures treated with cAMP. The neuron-macrophage interactions that produced outgrowth-promoting conditioned medium required CCL2 in neurons and CCR2/CCR4 in macrophages. The conditioning effects were abolished in CCL2-deficient mice at 3 and 7 d after sciatic nerve injury, but CCL2 was dispensable for the initial growth response and upregulation of GAP-43 at the 1 d time point. Intraganglionic injection of CCL2 mimicked conditioning injury by mobilizing M2-like macrophages. Finally, overexpression of CCL2 in DRGs promoted sensory axon regeneration in a rat spinal cord injury model without harmful side effects. Our data suggest that CCL2-mediated neuron-macrophage interaction plays a critical role for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury. Manipulation of chemokine signaling mediating neuron-macrophage interactions may represent a novel therapeutic approach to promote axon regeneration after CNS injury.

  2. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    DEFF Research Database (Denmark)

    Da Roit, F.; Engelberts, P. J.; Taylor, R. P.;

    2015-01-01

    the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre......-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells...

  3. Biochemical activities of berberine, palmatine and sanguinarine mediating chemical defence against microorganisms and herbivores.

    Science.gov (United States)

    Schmeller, T; Latz-Brüning, B; Wink, M

    1997-01-01

    The alkaloids berberine, palmatine and sanguinarine are toxic to insects and vertebrates and inhibit the multiplication of bacteria, fungi and viruses. Biochemical properties which may contribute to these allelochemical activities were analysed. Acetylcholine esterase, butyrylcholinesterase, choline acetyl transferase, alpha 1- and alpha 2-adrenergic, nicotinergic, muscarinergic and serotonin2 receptors were substantially affected. Sanguinarine appears to be the most effective inhibitor of choline acetyl-transferase (IC50 284 nM), while the protoberberines were inactive at this target. Berberine and palmatine were most active at the alpha 2-receptor (binding with IC50 476 and 956 nM, respectively). Furthermore, berberine and sanguinarine intercalate DNA, inhibit DNA synthesis and reverse transcriptase. In addition, sanguinarine (but not berberine) affects membrane permeability and berberine protein biosynthesis. In consequence, these biochemical activities may mediate chemical defence against microorganisms, viruses and herbivores in the plants producing these alkaloids.

  4. Aromatic Cyanoalkylation through Double C-H Activation Mediated by Ni(III).

    Science.gov (United States)

    Zhou, Wen; Zheng, Shuai; Schultz, Jason W; Rath, Nigam P; Mirica, Liviu M

    2016-05-11

    Herein we report an atom- and step-economic aromatic cyanoalkylation reaction that employs nitriles as building blocks and proceeds through Csp(2)-H and Csp(3)-H bond activation steps mediated by Ni(III). In addition to cyanomethylation with MeCN, regioselective α-cyanoalkylation was observed with various nitrile substrates to generate secondary and tertiary nitriles. Importantly, to the best of our knowledge these are the first examples of C-H bond activation reactions occurring at a Ni(III) center, which may exhibit different reactivity and selectivity profiles than those corresponding to analogous Ni(II) centers. These studies provide guiding principles to design catalytic C-H activation and functionalization reactions involving high-valent Ni species. PMID:27120207

  5. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    Science.gov (United States)

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  6. 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.

  7. Platelet and monocyte activity markers and mediators of inflammation in Takotsubo cardiomyopathy.

    Science.gov (United States)

    Pirzer, Rainer; Elmas, Elif; Haghi, Dariusch; Lippert, Christiane; Kralev, Stefan; Lang, Siegfried; Borggrefe, Martin; Kälsch, Thorsten

    2012-03-01

    Patients with Takotsubo cardiomyopathy (TC) often present with symptoms similar to those of myocardial infarction (MI). We analyzed blood concentrations of mediators of inflammation and platelet- and monocyte-activity markers in patients with TC and MI for significant differences. Clinical data of patients with TC (n = 16) and acute MI (n = 16) were obtained. Serial blood samples were taken at the time of hospital admission (t(0)), after 2-4 days (t(1)) and after 4-7 weeks (t(2)), respectively. Plasma concentrations of interleukin (IL)-6, IL-7, soluble CD40 ligand (sCD40L), and monocyte chemotactic protein 1 (MCP-1) were determined with an ELISA. Tissue factor binding on monocytes, platelet-activation marker CD62P, platelet CD40-ligand (CD40L), and platelet-monocyte aggregates were measured using flow cytometry. Expression of CD62P on platelets and IL-6 plasma levels were significantly lower in patients with TC compared to MI at the time of hospital admission. IL-7 plasma levels were significantly elevated in patients with TC compared to patients with MI at 2-4 days after hospital admission. No significant differences were observed concerning sCD40L and MCP-1 plasma levels, tissue factor binding on monocytes, CD40L expression on platelets, and platelet-monocyte aggregates at any point in time. Our results indicate that inflammatory mediators and platelet-activity markers contribute to the differences in the pathogenesis of MI and TC. PMID:21416113

  8. 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.

  9. EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation.

    Science.gov (United States)

    Zhao, Yang; Li, XiaoJun; Sun, XiangFei; Zhang, YunFeng; Ren, Hong

    2012-01-01

    Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis. PMID:22508042

  10. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    Science.gov (United States)

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  11. Activation of band 3 mediates group A Streptococcus streptolysin S-based beta-haemolysis.

    Science.gov (United States)

    Higashi, Dustin L; Biais, Nicolas; Donahue, Deborah L; Mayfield, Jeffrey A; Tessier, Charles R; Rodriguez, Kevin; Ashfeld, Brandon L; Luchetti, Jeffrey; Ploplis, Victoria A; Castellino, Francis J; Lee, Shaun W

    2016-01-01

    Streptococcus pyogenes, or group A Streptococcus (GAS), is a human bacterial pathogen that can manifest as a range of diseases from pharyngitis and impetigo to severe outcomes such as necrotizing fasciitis and toxic shock syndrome. GAS disease remains a global health burden with cases estimated at over 700 million annually and over half a million deaths due to severe infections(1). For over 100 years, a clinical hallmark of diagnosis has been the appearance of complete (beta) haemolysis when grown in the presence of blood. This activity is due to the production of a small peptide toxin by GAS known as streptolysin S. Although it has been widely held that streptolysin S exerts its lytic activity through membrane disruption, its exact mode of action has remained unknown. Here, we show, using high-resolution live cell imaging, that streptolysin S induces a dramatic osmotic change in red blood cells, leading to cell lysis. This osmotic change was characterized by the rapid influx of Cl(-) ions into the red blood cells through SLS-mediated disruption of the major erythrocyte anion exchange protein, band 3. Chemical inhibition of band 3 function significantly reduced the haemolytic activity of streptolysin S, and dramatically reduced the pathology in an in vivo skin model of GAS infection. These results provide key insights into the mechanism of streptolysin S-mediated haemolysis and have implications for the development of treatments against GAS. PMID:27571972

  12. Body size and activity times mediate mammalian responses to climate change.

    Science.gov (United States)

    McCain, Christy M; King, Sarah R B

    2014-06-01

    Model predictions of extinction risks from anthropogenic climate change are dire, but still overly simplistic. To reliably predict at-risk species we need to know which species are currently responding, which are not, and what traits are mediating the responses. For mammals, we have yet to identify overarching physiological, behavioral, or biogeographic traits determining species' responses to climate change, but they must exist. To date, 73 mammal species in North America and eight additional species worldwide have been assessed for responses to climate change, including local extirpations, range contractions and shifts, decreased abundance, phenological shifts, morphological or genetic changes. Only 52% of those species have responded as expected, 7% responded opposite to expectations, and the remaining 41% have not responded. Which mammals are and are not responding to climate change is mediated predominantly by body size and activity times (phylogenetic multivariate logistic regressions, P climate change than a shrew. Obligate diurnal and nocturnal mammals are more than twice as likely to respond as mammals with flexible activity times (P climate change in some analyses, whereas hibernation, heterothermy, burrowing, nesting, and study location did not influence responses. These results indicate that some mammal species can behaviorally escape climate change whereas others cannot, analogous to paleontology's climate sheltering hypothesis. Including body size and activity flexibility traits into future extinction risk forecasts should substantially improve their predictive utility for conservation and management. PMID:24449019

  13. 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

    OpenAIRE

    Thomas Stübig; Anita Badbaran; Tim Luetkens; York Hildebrandt; Djordje Atanackovic; Binder, Thomas M. C.; Boris Fehse; Nicolaus Kröger

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and...

  14. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    Science.gov (United States)

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  15. Characteristics of Gintonin-Mediated Membrane Depolarization of Pacemaker Activity in Cultured Interstitial Cells of Cajal

    Directory of Open Access Journals (Sweden)

    Byung Joo Kim

    2014-08-01

    Full Text Available Background/Aims: Ginseng regulates gastrointestinal (GI motor activity but the underlying components and molecular mechanisms are unknown. We investigated the effect of gintonin, a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA receptor ligand, on the pacemaker activity of the interstitial cells of Cajal (ICC in murine small intestine and GI motility. Materials and Methods: Enzymatic digestion was used to dissociate ICC from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials and currents from cultured ICC in the absence or presence of gintonin. In vivo effects of gintonin on gastrointestinal (GI motility were investigated by measuring the intestinal transit rate (ITR of Evans blue in normal and streptozotocin (STZ-induced diabetic mice. Results: We investigated the effects of gintonin on pacemaker potentials and currents in cultured ICC from mouse small intestine. Gintonin caused membrane depolarization in current clamp mode but this action was blocked by Ki16425, an LPA1/3 receptor antagonist, and by the addition of GDPβS, a GTP-binding protein inhibitor, into the ICC. To study the gintonin signaling pathway, we examined the effects of U-73122, an active PLC inhibitor, and chelerythrine and calphostin, which inhibit PKC. All inhibitors blocked gintonin actions on pacemaker potentials, but not completely. Gintonin-mediated depolarization was lower in Ca2+-free than in Ca2+-containing external solutions and was blocked by thapsigargin. We found that, in ICC, gintonin also activated Ca2+-activated Cl- channels (TMEM16A, ANO1, but not TRPM7 channels. In vivo, gintonin (10-100 mg/kg, p.o. not only significantly increased the ITR in normal mice but also ameliorated STZ-induced diabetic GI motility retardation in a dose-dependent manner. Conclusions: Gintonin-mediated membrane depolarization of pacemaker activity and ANO1 activation are coupled to the stimulation of GI

  16. Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling.

    Science.gov (United States)

    Mihara, Koichiro; Ramachandran, Rithwik; Saifeddine, Mahmoud; Hansen, Kristina K; Renaux, Bernard; Polley, Danny; Gibson, Stacy; Vanderboor, Christina; Hollenberg, Morley D

    2016-05-01

    Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringβ-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.

  17. Development of CpG-oligodeoxynucleotides for effective activation of rabbit TLR9 mediated immune responses.

    Directory of Open Access Journals (Sweden)

    Tsung-Hsien Chuang

    Full Text Available CpG-oligodeoxynucleotides (CpG-ODN are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9 is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18-24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund's adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.

  18. Auditory cortical activity during cochlear implant-mediated perception of spoken language, melody, and rhythm.

    Science.gov (United States)

    Limb, Charles J; Molloy, Anne T; Jiradejvong, Patpong; Braun, Allen R

    2010-03-01

    Despite the significant advances in language perception for cochlear implant (CI) recipients, music perception continues to be a major challenge for implant-mediated listening. Our understanding of the neural mechanisms that underlie successful implant listening remains limited. To our knowledge, this study represents the first neuroimaging investigation of music perception in CI users, with the hypothesis that CI subjects would demonstrate greater auditory cortical activation than normal hearing controls. H(2) (15)O positron emission tomography (PET) was used here to assess auditory cortical activation patterns in ten postlingually deafened CI patients and ten normal hearing control subjects. Subjects were presented with language, melody, and rhythm tasks during scanning. Our results show significant auditory cortical activation in implant subjects in comparison to control subjects for language, melody, and rhythm. The greatest activity in CI users compared to controls was seen for language tasks, which is thought to reflect both implant and neural specializations for language processing. For musical stimuli, PET scanning revealed significantly greater activation during rhythm perception in CI subjects (compared to control subjects), and the least activation during melody perception, which was the most difficult task for CI users. These results may suggest a possible relationship between auditory performance and degree of auditory cortical activation in implant recipients that deserves further study.

  19. Activity deprivation induces neuronal cell death: mediation by tissue-type plasminogen activator.

    Directory of Open Access Journals (Sweden)

    Eldi Schonfeld-Dado

    Full Text Available Spontaneous activity is an essential attribute of neuronal networks and plays a critical role in their development and maintenance. Upon blockade of activity with tetrodotoxin (TTX, neurons degenerate slowly and die in a manner resembling neurodegenerative diseases-induced neuronal cell death. The molecular cascade leading to this type of slow cell death is not entirely clear. Primary post-natal cortical neurons were exposed to TTX for up to two weeks, followed by molecular, biochemical and immunefluorescence analysis. The expression of the neuronal marker, neuron specific enolase (NSE, was down-regulated, as expected, but surprisingly, there was a concomitant and striking elevation in expression of tissue-type plasminogen activator (tPA. Immunofluorescence analysis indicated that tPA was highly elevated inside affected neurons. Transfection of an endogenous tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1, protected the TTX-exposed neurons from dying. These results indicate that tPA is a pivotal player in slowly progressing activity deprivation-induced neurodegeneration.

  20. Hibiscus sabdariffa extractivities on cadmium-mediated alterations of human U937 cell viability and activation

    Institute of Scientific and Technical Information of China (English)

    Tebekeme Okoko; Diepreye Ere

    2012-01-01

    Objective:To investigate the effect of the anthocyanin-rich extract of Hibiscus sabdariffa (H. sabdariffa) calyx on the viability of cadmium-treated U937 cells and cadmium-mediated activation of U937-derived macrophages. Methods:The macrophage cell line U937 was treated with cadmium (0.1μmol/L) and later incubated with the anthocyanin-rich extract and cell viability was assessed via trypan blue staining. In the other experiment, the U937 cells were transformed to the macrophage form by treatment with phorbol 12, myristate 13, and acetate and incubated with cadmium (10μmol/L). The anthocyanin-rich extract was added to the cells later and subsequently, the supernatant of each cell culture was analysed for the production of tumour necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), nitric oxide, and catalase activity as indices for the activation of macrophages. Results:It revealed that the anthocynanin-rich extract significantly (P <0.05) increased the viability of the cells which was suppressed by cadmium when compared to quercetin dihydrate. The extract also reduced the cadmium-mediated production of the markers of macrophage-activation when compared to quercetin dihydrate. In both experiments, the activity of the extract was concentration-dependent (P <0.05). Conclusion:The findings show that H. sabdariffa possesses significant immunoprotective effect. These corroborate the immense reported antioxidant and medicinal potential of the calyces of the plant which could be exploited for pharmacological and neutraceutical advantages.

  1. Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.

    Science.gov (United States)

    Devor, D C; Frizzell, R A

    1993-11-01

    Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells. PMID:7694492

  2. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    International Nuclear Information System (INIS)

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway

  3. Tissue kallikrein mediates pro-inflammatory pathways and activation of protease-activated receptor-4 in proximal tubular epithelial cells.

    Directory of Open Access Journals (Sweden)

    Wai Han Yiu

    Full Text Available Tissue kallikrein (KLK1 expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG in human proximal tubular epithelial cells (PTEC. Since the kallikrein-kinin system (KKS has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs. There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation.

  4. Tissue kallikrein mediates pro-inflammatory pathways and activation of protease-activated receptor-4 in proximal tubular epithelial cells.

    Science.gov (United States)

    Yiu, Wai Han; Wong, Dickson W L; Chan, Loretta Y Y; Leung, Joseph C K; Chan, Kwok Wah; Lan, Hui Yao; Lai, Kar Neng; Tang, Sydney C W

    2014-01-01

    Tissue kallikrein (KLK1) expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG) in human proximal tubular epithelial cells (PTEC). Since the kallikrein-kinin system (KKS) has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE)-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs). There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation. PMID:24586431

  5. LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae

    Energy Technology Data Exchange (ETDEWEB)

    Cleary, S.F.; Marciano-Cabral, F.

    1986-03-01

    Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNA or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.

  6. Physical Activity and Cognitive Function in Older Adults: The Mediating Effect of Depressive Symptoms.

    Science.gov (United States)

    Vance, David E; Marson, Daniel C; Triebel, Kristen L; Ball, Karlene K; Wadley, Virginia G; Cody, Shameka L

    2016-01-01

    Depressive symptoms and social networks may influence the relationship between physical activity and cognition. Using structural equation modeling, depressive symptoms and social networks were examined as mediators between physical activity and cognition in community-dwelling older adults (N = 122), with a range of cognitive abilities (e.g., normal, mild cognitive impairment). The model included age, physical activity, sedentary behavior, sleeping, social networks, depressive symptoms, and cognitive function. A path was observed between physical activity, depressive symptoms, and cognition; specifically, those who were more physically active experienced less depression and better cognitive functioning. No relationship between social networks and cognition was found. This model fits the data well (goodness-of-fit index = .93, adjusted goodness-of-fit index = .90, root mean square error of approximation = .06). Results suggest that physical activity may mitigate depressive symptoms, with beneficial effects on cognitive functioning in both those with and without mild cognitive impairment. Suggestions for managing depression and improving cognitive functioning are provided. PMID:27224681

  7. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor.

    Science.gov (United States)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-20

    Water molecules inside a G-protein coupled receptor (GPCR) have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal water molecules in GPCR activity, we studied the A2A adenosine receptor using microsecond molecular-dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times more slowly than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from ∼O(10(2)) ps to ∼O(10(2)) ns. Especially, water molecules, exhibiting ultraslow relaxation (∼O(10(2)) ns) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow water molecules in the activation of GPCRs. PMID:27653477

  8. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  9. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression.

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  10. CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development

    Institute of Scientific and Technical Information of China (English)

    Yong Chao Lu; Alvin Chun Hang Ma; Anskar Yu Hung Leung; He Feng Huang; Hsiao Chang Chan; Hui Chen; Kin Lam Fok; Lai Ling Tsang; Mei Kuen Yu; Xiao Hu Zhang; Jing Chen; Xiaohua Jiang; Yiu Wa Chung

    2012-01-01

    Although HCO3-is known to be required for early embryo development,its exact role remains elusive.Here we report that HCO3-acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development.The results show that the effect of HCO3-on preimplantation embryo development can be suppressed by interfering the function of a HCO3--conducting channel,CFTR,by a specific inhibitor or gene knockout.Removal of extracellular HCO3-or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos.Knockdown of miR-125b mimics the effect of HCO3-removal and CFTR inhibition,while injection of miR-125b precursor reverses it.Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos.The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-KB.These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO3-to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.

  11. Endogenous fructose production and fructokinase activation mediate renal injury in diabetic nephropathy.

    Science.gov (United States)

    Lanaspa, Miguel A; Ishimoto, Takuji; Cicerchi, Christina; Tamura, Yoshifuru; Roncal-Jimenez, Carlos A; Chen, Wei; Tanabe, Katsuyuki; Andres-Hernando, Ana; Orlicky, David J; Finol, Esteban; Inaba, Shinichiro; Li, Nanxing; Rivard, Christopher J; Kosugi, Tomoki; Sanchez-Lozada, Laura G; Petrash, J Mark; Sautin, Yuri Y; Ejaz, A Ahsan; Kitagawa, Wataru; Garcia, Gabriela E; Bonthron, David T; Asipu, Aruna; Diggle, Christine P; Rodriguez-Iturbe, Bernardo; Nakagawa, Takahiko; Johnson, Richard J

    2014-11-01

    Diabetes is associated with activation of the polyol pathway, in which glucose is converted to sorbitol by aldose reductase. Previous studies focused on the role of sorbitol in mediating diabetic complications. However, in the proximal tubule, sorbitol can be converted to fructose, which is then metabolized largely by fructokinase, also known as ketohexokinase, leading to ATP depletion, proinflammatory cytokine expression, and oxidative stress. We and others recently identified a potential deleterious role of dietary fructose in the generation of tubulointerstitial injury and the acceleration of CKD. In this study, we investigated the potential role of endogenous fructose production, as opposed to dietary fructose, and its metabolism through fructokinase in the development of diabetic nephropathy. Wild-type mice with streptozotocin-induced diabetes developed proteinuria, reduced GFR, and renal glomerular and proximal tubular injury. Increased renal expression of aldose reductase; elevated levels of renal sorbitol, fructose, and uric acid; and low levels of ATP confirmed activation of the fructokinase pathway. Furthermore, renal expression of inflammatory cytokines with macrophage infiltration was prominent. In contrast, diabetic fructokinase-deficient mice demonstrated significantly less proteinuria, renal dysfunction, renal injury, and inflammation. These studies identify fructokinase as a novel mediator of diabetic nephropathy and document a novel role for endogenous fructose production, or fructoneogenesis, in driving renal disease.

  12. Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

    Science.gov (United States)

    Roth, Lise; Prahst, Claudia; Ruckdeschel, Tina; Savant, Soniya; Weström, Simone; Fantin, Alessandro; Riedel, Maria; Héroult, Mélanie; Ruhrberg, Christiana; Augustin, Hellmut G

    2016-04-26

    Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

  13. A Mathematical Model of Neonatal Rat Atrial Monolayers with Constitutively Active Acetylcholine-Mediated K+ Current.

    Science.gov (United States)

    Majumder, Rupamanjari; Jangsangthong, Wanchana; Feola, Iolanda; Ypey, Dirk L; Pijnappels, Daniël A; Panfilov, Alexander V

    2016-06-01

    Atrial fibrillation (AF) is the most frequent form of arrhythmia occurring in the industrialized world. Because of its complex nature, each identified form of AF requires specialized treatment. Thus, an in-depth understanding of the bases of these arrhythmias is essential for therapeutic development. A variety of experimental studies aimed at understanding the mechanisms of AF are performed using primary cultures of neonatal rat atrial cardiomyocytes (NRAMs). Previously, we have shown that the distinct advantage of NRAM cultures is that they allow standardized, systematic, robust re-entry induction in the presence of a constitutively-active acetylcholine-mediated K+ current (IKACh-c). Experimental studies dedicated to mechanistic explorations of AF, using these cultures, often use computer models for detailed electrophysiological investigations. However, currently, no mathematical model for NRAMs is available. Therefore, in the present study we propose the first model for the action potential (AP) of a NRAM with constitutively-active acetylcholine-mediated K+ current (IKACh-c). The descriptions of the ionic currents were based on patch-clamp data obtained from neonatal rats. Our monolayer model closely mimics the action potential duration (APD) restitution and conduction velocity (CV) restitution curves presented in our previous in vitro studies. In addition, the model reproduces the experimentally observed dynamics of spiral wave rotation, in the absence and in the presence of drug interventions, and in the presence of localized myofibroblast heterogeneities. PMID:27332890

  14. A Mathematical Model of Neonatal Rat Atrial Monolayers with Constitutively Active Acetylcholine-Mediated K+ Current.

    Directory of Open Access Journals (Sweden)

    Rupamanjari Majumder

    2016-06-01

    Full Text Available Atrial fibrillation (AF is the most frequent form of arrhythmia occurring in the industrialized world. Because of its complex nature, each identified form of AF requires specialized treatment. Thus, an in-depth understanding of the bases of these arrhythmias is essential for therapeutic development. A variety of experimental studies aimed at understanding the mechanisms of AF are performed using primary cultures of neonatal rat atrial cardiomyocytes (NRAMs. Previously, we have shown that the distinct advantage of NRAM cultures is that they allow standardized, systematic, robust re-entry induction in the presence of a constitutively-active acetylcholine-mediated K+ current (IKACh-c. Experimental studies dedicated to mechanistic explorations of AF, using these cultures, often use computer models for detailed electrophysiological investigations. However, currently, no mathematical model for NRAMs is available. Therefore, in the present study we propose the first model for the action potential (AP of a NRAM with constitutively-active acetylcholine-mediated K+ current (IKACh-c. The descriptions of the ionic currents were based on patch-clamp data obtained from neonatal rats. Our monolayer model closely mimics the action potential duration (APD restitution and conduction velocity (CV restitution curves presented in our previous in vitro studies. In addition, the model reproduces the experimentally observed dynamics of spiral wave rotation, in the absence and in the presence of drug interventions, and in the presence of localized myofibroblast heterogeneities.

  15. Afferent-mediated modulation of the soleus muscle activity during the stance phase of human walking

    DEFF Research Database (Denmark)

    Nazarena, Mazzaro; Grey, Michael James; do Nascimento, Omar Feix;

    2006-01-01

    The aim of this study was to investigate the contribution of proprioceptive feedback to the amplitude modulation of the soleus muscle activity during human walking. We have previously shown that slow-velocity, small-amplitude ankle dorsiflexion enhancements and reductions applied during the stance......-mediated contribution from muscle group II afferents, cutaneous and proprioceptive afferents from the foot, and load-sensitive afferents to the soleus EMG. Slow-velocity, small-amplitude ankle trajectory modifications were combined with the pharmaceutical depression of group II polysynaptic pathways with tizanidine...... that the feedback from group II afferents, and possibly from load-sensitive afferents, contribute to the amplitude modulation of the soleus muscle activity during the stance phase of the step cycle. However, feedback from cutaneous afferents and instrinsic proprioceptive afferents from the foot does not seem...

  16. Guanidinylated neomycin mediates heparan sulfate-dependent transport of active enzymes to lysosomes.

    Science.gov (United States)

    Sarrazin, Stéphane; Wilson, Beth; Sly, William S; Tor, Yitzhak; Esko, Jeffrey D

    2010-07-01

    Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, beta-D-glucuronidase (GUS) and alpha-L-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells.

  17. Structural Waters Define a Functional Channel Mediating Activation of the GPCR, rhodopsin

    Energy Technology Data Exchange (ETDEWEB)

    Angel, T.; Gupta, S; Jastrzebska, B; Palczewski, K; Chance, M

    2009-01-01

    Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H2O18 solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.

  18. The Association between leisure time physical activity and smoking in adolescence: an examination of potential mediating and moderating factors

    DEFF Research Database (Denmark)

    Verkooijen, Kirsten; Nielsen, Gert A; Kremers, Stef P J

    2008-01-01

    , sense of coherence, and physically active self-concept as potential mediating and moderating variables. METHOD: Data were obtained through a postal survey among 3,940 Danes aged 16 to 20. Bivariate and multivariate logistic regressions were performed to identify significant associations as well...... as mediating and moderating effects. RESULTS: In the bivariate model, leisure time physical activity was negatively associated with smoking; adolescents who were active at least one hour per week were up to half as likely to smoke as inactive adolescents. In the adjusted model, however, the association...... disappeared. Further analyses revealed that physically active self-concept acted both as a mediator and as a moderator of the studied relationship. CONCLUSION: The data suggest that participation in leisure time physical activity is indeed inversely associated with adolescent smoking, but only when physical...

  19. DNA methylation mediates persistent epileptiform activity in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Ziv M Machnes

    Full Text Available Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5' regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5' region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5' region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5' region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5' region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.

  20. 1,25 Dihydroxyvitamin D3 Inhibits TGFβ1-Mediated Primary Human Cardiac Myofibroblast Activation.

    Directory of Open Access Journals (Sweden)

    Anna Meredith

    Full Text Available Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av, and investigated the relationship between circulating vitamin D (25(OHD3 and cardiac fibrosis in human myocardial samples.Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OHD3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGFβ1 to induce activation, in the presence or absence of active vitamin D (1,25(OH2D3. Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH2D3. Treatment with 1,25(OH2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.

  1. Mechanistic Variants in Gas-Phase Metal-Oxide Mediated Activation of Methane at Ambient Conditions.

    Science.gov (United States)

    Li, Jilai; Zhou, Shaodong; Zhang, Jun; Schlangen, Maria; Usharani, Dandamudi; Shaik, Sason; Schwarz, Helmut

    2016-09-01

    The C-H bond activation of methane mediated by a prototypical heteronuclear metal-oxide cluster, [Al2Mg2O5](•+), was investigated by using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in conjunction with high-level quantum mechanical calculations. Experimentally, hydrogen-atom abstraction from methane by the cluster ion [Al2Mg2O5](•+) takes place at ambient conditions. As to the mechanism, according to our computational findings, both the proton-coupled electron transfer (PCET) and the conventional hydrogen-atom transfer (HAT) are feasible and compete with each other. This is in distinct contrast to the [XYO2](+) (X, Y = Mg, Al, Si) cluster oxide ions which activate methane exclusively via the PCET route (Li, J.; Zhou, S.; Zhang, J.; Schlangen, M.; Weiske, T.; Usharani, D.; Shaik, S.; Schwarz, H. J. Am. Chem. Soc. 2016, 138, 7973-7981). The electronic origins of the mechanistically rather complex reactivity scenarios of the [Al2Mg2O5](•+)/CH4 couple were elucidated. For the PCET mechanism, in which the Lewis acid-base pair [Al(+)-O(-)] of the cluster acts as the active site, a clear correlation has been established between the nature of the transition state, the corresponding barrier height, the Lewis acidity-basicity of the [M(+)-O(-)] unit, as well as the bond order of the M(+)-O(-) bond. Also addressed is the role of the spin and charge distributions of a terminal oxygen radical site in the direct HAT route. The knowledge of the factors that control the reactivity of PCET and HAT pathways not only deepens our mechanistic understanding of metal-oxide mediated C-H bond activation but may also provide guidance for the rational design of catalysts. PMID:27518766

  2. CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

    Science.gov (United States)

    Scerif, Miski; Füzesi, Tamás; Thomas, Julia D; Kola, Blerina; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-10-01

    AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

  3. The HEART mobile phone trial: The partial mediating effects of self-efficacy on physical activity among cardiac patients

    Directory of Open Access Journals (Sweden)

    Ralph eMaddison

    2014-05-01

    Full Text Available Background: The ubiquitous use of mobile phones provides an ideal opportunity to deliver interventions to increase physical activity levels. Understanding potential mediators of such interventions is needed to increase their effectiveness. A recent randomized controlled trial of a mobile phone and Internet (mHealth intervention was conducted in New Zealand to determine the effectiveness on exercise capacity and physical activity levels in addition to current cardiac rehabilitation (CR services for people (n=171 with ischaemic heart disease (IHD. Significant intervention effect was observed for self-reported leisure time physical activity and walking, but not peak oxygen uptake (PVO2 at 24 weeks. There was also significant improvement in self-efficacy.Objective: To evaluate the mediating effect of self-efficacy on physical activity levels in an mHealth delivered exercise CR programme. Methods: Treatment evaluations were performed on the principle of intention to treat (ITT. Adjusted regression analyses were conducted to evaluate the main treatment effect on leisure time physical activity and walking at 24 weeks, with and without change in self-efficacy as the mediator of interest. Results: Change in self-efficacy at 24 weeks significantly mediated the treatment effect on leisure time physical activity by 13%, but only partially mediated the effect on walking by 4% at 24 weeks. Conclusion: An mHealth intervention involving text messaging and Internet support had a positive treatment effect on leisure time physical activity and walking at 24 weeks, and this effect was likely mediated through changes in self-efficacy. Future trials should examine other potential mediators related to this type of intervention.

  4. γ-Glutamyltranspeptidase is an endogenous activator of Toll-like receptor 4-mediated osteoclastogenesis

    Science.gov (United States)

    Moriwaki, Sawako; Into, Takeshi; Suzuki, Keiko; Miyauchi, Mutsumi; Takata, Takashi; Shibayama, Keigo; Niida, Shumpei

    2016-01-01

    Chronic inflammation-associated bone destruction, which is observed in rheumatoid arthritis (RA) and periodontitis, is mediated by excessive osteoclastogenesis. We showed previously that γ-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, acts as an endogenous activator of such pathological osteoclastogenesis, independent of its enzymatic activity. GGT accumulation is clinically observed in the joints of RA patients, and, in animals, the administration of recombinant GGT to the gingival sulcus as an in vivo periodontitis model induces an increase in the number of osteoclasts. However, the underlying mechanisms of this process remain unclear. Here, we report that Toll-like receptor 4 (TLR4) recognizes GGT to activate inflammation-associated osteoclastogenesis. Unlike lipopolysaccharide, GGT is sensitive to proteinase K treatment and insensitive to polymyxin B treatment. TLR4 deficiency abrogates GGT-induced osteoclastogenesis and activation of NF-κB and MAPK signaling in precursor cells. Additionally, GGT does not induce osteoclastogenesis in cells lacking the signaling adaptor MyD88. The administration of GGT to the gingival sulcus induces increased osteoclastogenesis in wild-type mice, but does not induce it in TLR4-deficient mice. Our findings elucidate a novel mechanism of inflammation-associated osteoclastogenesis, which involves TLR4 recognition of GGT and subsequent activation of MyD88-dependent signaling. PMID:27775020

  5. Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage

    Energy Technology Data Exchange (ETDEWEB)

    Laha, Dipranjan; Pramanik, Arindam [Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032 (India); Laskar, Aparna [CSIR-Indian Institute of Chemical Biology, Kolkata 700032 (India); Jana, Madhurya [Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032 (India); Pramanik, Panchanan [Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India); Karmakar, Parimal, E-mail: pkarmakar_28@yahoo.co.in [Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032 (India)

    2014-11-15

    Highlights: • Spherical and sheet shaped copper oxide nanoparticles were synthesized. • Physical characterizations of these nanoparticles were done by TEM, DLS, XRD, FTIR. • They showed shape dependent antibacterial activity on different bacterial strain. • They induced both membrane damage and ROS mediated DNA damage in bacteria. - Abstract: In this work, we synthesized spherical and sheet shaped copper oxide nanoparticles and their physical characterizations were done by the X-ray diffraction, fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering. The antibacterial activity of these nanoparticles was determined on both gram positive and gram negative bacterial. Spherical shaped copper oxide nanoparticles showed more antibacterial property on gram positive bacteria where as sheet shaped copper oxide nanoparticles are more active on gram negative bacteria. We also demonstrated that copper oxide nanoparticles produced reactive oxygen species in both gram negative and gram positive bacteria. Furthermore, they induced membrane damage as determined by atomic force microscopy and scanning electron microscopy. Thus production of and membrane damage are major mechanisms of the bactericidal activity of these copper oxide nanoparticles. Finally it was concluded that antibacterial activity of nanoparticles depend on physicochemical properties of copper oxide nanoparticles and bacterial strain.

  6. Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Nardou Katya

    2008-06-01

    Full Text Available Abstract Background Histone deacetylase inhibitors (HDACi are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. Results We show here that the HDACi Sodium Butyrate (NaB, suberoylanilide hydroxamic acid (SAHA and Trichostatin A (TSA strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. Conclusion HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

  7. Mediating relationship between body mass index and the direct measures of the Theory of Planned Behaviour on physical activity intention.

    Science.gov (United States)

    Caperchione, Cristina M; Duncan, Mitch J; Mummery, Kerry; Steele, Rebekah; Schofield, Grant

    2008-03-01

    This research examines (a) the interrelationships between body mass index (BMI), the direct measures of the Theory of Planned Behaviour (TPB) and physical activity intention and (b) the potential mediation effects of the direct measures of the TPB in the relationship between BMI and physical activity intention in a sample of Australian adults. A total sample of 1,062 respondents participated in a computer-assisted telephone-interview (CATI) survey comprised of a standardised introduction; questions regarding TPB and physical activity; and standard demographic questions. BMI for each participant was calculated from self-reported height and weight. Separate regression analyses were performed to examine the mediating effects of each of the direct measures of the TPB on the predictive relationship between the BMI and physical activity intention, as proposed by Baron and Kenny (Journal of Personality and Social Psychology, 51(6), 1173 - 1182, 1986). Findings indicated that the direct measure of attitude and perceived behavioural control mediated the relationship between BMI and physical activity intention. However, the direct measure of subjective norm failed to act as a mediating mechanism. To date there has been no research that has examined the mechanism by which body mass may affect physical activity behaviour. Given the current focus for health promotion specialists on promoting physical activity as a strategy for reducing overweight and obesity, a theoretical understanding of weight-related barriers to physical activity may aid in the development of future interventions and community physical activity programs, particularly those targeting overweight and obese populations.

  8. Energy National Mediator activity report 2009; Rapport d'activite du Mediateur National de l'Energie 2009

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2009-07-01

    After some data illustrating the activity of the Energy National Mediator in 2009, and an interview of a representative of this institution who comments its practice, this report proposes the opinions of the different involved actors (communities, consumer associations, providers, and so on) about the mediator. It puts the adopted strategy in perspective from the past year to the coming one. It describes the missions: information, advice, protection. It reports actions, recommendations and facts for 2009 in terms of consumer information, group mediation, poverty management, samples of analysed disputes. It presents the social organisation and gives a financial assessment of the institution

  9. Cytochrome P450-mediated activation of the fragrance compound geraniol forms potent contact allergens

    International Nuclear Information System (INIS)

    Contact sensitization is caused by low molecular weight compounds which penetrate the skin and bind to protein. In many cases, these compounds are activated to reactive species, either by autoxidation on exposure to air or by metabolic activation in the skin. Geraniol, a widely used fragrance chemical, is considered to be a weak allergen, although its chemical structure does not indicate it to be a contact sensitizer. We have shown that geraniol autoxidizes and forms allergenic oxidation products. In the literature, it is suggested but not shown that geraniol could be metabolically activated to geranial. Previously, a skin-like CYP cocktail consisting of cutaneous CYP isoenzymes, was developed as a model system to study cutaneous metabolism. In the present study, we used this system to investigate CYP-mediated activation of geraniol. In incubations with the skin-like CYP cocktail, geranial, neral, 2,3-epoxygeraniol, 6,7-epoxygeraniol and 6,7-epoxygeranial were identified. Geranial was the main metabolite formed followed by 6,7-epoxygeraniol. The allergenic activities of the identified metabolites were determined in the murine local lymph node assay (LLNA). Geranial, neral and 6,7-epoxygeraniol were shown to be moderate sensitizers, and 6,7-epoxygeranial a strong sensitizer. Of the isoenzymes studied, CYP2B6, CYP1A1 and CYP3A5 showed high activities. It is likely that CYP1A1 and CYP3A5 are mainly responsible for the metabolic activation of geraniol in the skin, as they are expressed constitutively at significantly higher levels than CYP2B6. Thus, geraniol is activated through both autoxidation and metabolism. The allergens geranial and neral are formed via both oxidation mechanisms, thereby playing a large role in the sensitization to geraniol

  10. Dynamic Regulation of Activated Leukocyte Cell Adhesion Molecule–mediated Homotypic Cell Adhesion through the Actin CytoskeletonV⃞

    OpenAIRE

    Nelissen, Judith M. D. T.; Peters, Inge M.; de Grooth, Bart G.; Van Kooyk, Yvette; Figdor, Carl G.

    2000-01-01

    Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM–ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM–ALCAM interactions....

  11. Cholinergic-mediated IP3-receptor activation induces long-lasting synaptic enhancement in CA1 pyramidal neurons

    OpenAIRE

    Fernández de Sevilla, D.; Núñez Molina, Ángel; Borde, M.; Malinow, R.; Buño, Washinton

    2008-01-01

    Cholinergic-glutamatergic interactions influence forms of synaptic plasticity that are thought to mediate memory and learning. We tested in vitro the induction of long-lasting synaptic enhancement at Schaffer collaterals by acetylcholine (ACh) at the apical dendrite of CA1 pyramidal neurons and in vivo by stimulation of cholinergic afferents. In vitro ACh induced a Ca2+ wave and synaptic enhancement mediated by insertion of AMPA receptors in spines. Activation of muscarinic ACh receptors (mAC...

  12. Hydrogen adsorption-mediated synthesis of concave Pt nanocubes and their enhanced electrocatalytic activity

    Science.gov (United States)

    Lu, Bang-An; Du, Jia-Huan; Sheng, Tian; Tian, Na; Xiao, Jing; Liu, Li; Xu, Bin-Bin; Zhou, Zhi-You; Sun, Shi-Gang

    2016-06-01

    Concave nanocubes are enclosed by high-index facets and have negative curvature; they are expected to have enhanced reactivity, as compared to nanocubes with flat surfaces. Herein, we propose and demonstrate a new strategy for the synthesis of concave Pt nanocubes with {hk0} high-index facets, by using a hydrogen adsorption-mediated electrochemical square-wave potential method. It was found that Pt atoms prefer to deposit on edge sites rather than terrace sites on Pt surfaces with intensive hydrogen adsorption, resulting in the formation of concave structures. The as-prepared concave Pt nanocubes exhibit enhanced catalytic activity and stability towards oxidation of ethanol and formic acid in acidic solutions, compared to commercial Pt/C catalysts.Concave nanocubes are enclosed by high-index facets and have negative curvature; they are expected to have enhanced reactivity, as compared to nanocubes with flat surfaces. Herein, we propose and demonstrate a new strategy for the synthesis of concave Pt nanocubes with {hk0} high-index facets, by using a hydrogen adsorption-mediated electrochemical square-wave potential method. It was found that Pt atoms prefer to deposit on edge sites rather than terrace sites on Pt surfaces with intensive hydrogen adsorption, resulting in the formation of concave structures. The as-prepared concave Pt nanocubes exhibit enhanced catalytic activity and stability towards oxidation of ethanol and formic acid in acidic solutions, compared to commercial Pt/C catalysts. Electronic supplementary information (ESI) available: Details of DFT calculation, SEM images of concave Pt nanocubes, mass activity and stability characterization of the catalysts. See DOI: 10.1039/c6nr02349e

  13. PLCγ-activated signalling is essential for TrkB mediated sensory neuron structural plasticity

    Directory of Open Access Journals (Sweden)

    Rocha-Sanchez Sonia M

    2010-10-01

    Full Text Available Abstract Background The vestibular system provides the primary input of our sense of balance and spatial orientation. Dysfunction of the vestibular system can severely affect a person's quality of life. Therefore, understanding the molecular basis of vestibular neuron survival, maintenance, and innervation of the target sensory epithelia is fundamental. Results Here we report that a point mutation at the phospholipase Cγ (PLCγ docking site in the mouse neurotrophin tyrosine kinase receptor TrkB (Ntrk2 specifically impairs fiber guidance inside the vestibular sensory epithelia, but has limited effects on the survival of vestibular sensory neurons and growth of afferent processes toward the sensory epithelia. We also show that expression of the TRPC3 cation calcium channel, whose activity is known to be required for nerve-growth cone guidance induced by brain-derived neurotrophic factor (BDNF, is altered in these animals. In addition, we find that absence of the PLCγ mediated TrkB signalling interferes with the transformation of bouton type afferent terminals of vestibular dendrites into calyces (the largest synaptic contact of dendrites known in the mammalian nervous system on type I vestibular hair cells; the latter are normally distributed in these mutants as revealed by an unaltered expression pattern of the potassium channel KCNQ4 in these cells. Conclusions These results demonstrate a crucial involvement of the TrkB/PLCγ-mediated intracellular signalling in structural aspects of sensory neuron plasticity.

  14. Activation of the endothelin system mediates pathological angiogenesis during ischemic retinopathy.

    Science.gov (United States)

    Patel, Chintan; Narayanan, S Priya; Zhang, Wenbo; Xu, Zhimin; Sukumari-Ramesh, Sangeetha; Dhandapani, Krishnan M; Caldwell, R William; Caldwell, Ruth B

    2014-11-01

    Retinopathy of prematurity adversely affects premature infants because of oxygen-induced damage of the immature retinal vasculature, resulting in pathological neovascularization (NV). Our pilot studies using the mouse model of oxygen-induced retinopathy (OIR) showed marked increases in angiogenic mediators, including endothelins and endothelin receptor (EDNR) A. We hypothesized that activation of the endothelin system via EDNRA plays a causal role in pathological angiogenesis and up-regulation of angiogenic mediators, including vascular endothelial growth factor A (VEGFA) in OIR. Mice were exposed to 75% oxygen from post-natal day P7 to P12, treated with either vehicle or EDNRA antagonist BQ-123 or EDNRB antagonist BQ-788 on P12, and kept at room air from P12 to P17 (ischemic phase). RT-PCR analysis revealed increased levels of EDN2 and EDNRA mRNA, and Western blot analysis revealed increased EDN2 expression during the ischemic phase. EDNRA inhibition significantly increased vessel sprouting, resulting in enhanced physiological angiogenesis and decreased pathological NV, whereas EDNRB inhibition modestly improved vascular repair. OIR triggered significant increases in VEGFA protein and mRNA for delta-like ligand 4, apelin, angiopoietin-2, and monocyte chemoattractant protein-1. BQ-123 treatment significantly reduced these alterations. EDN2 expression was localized to retinal glia and pathological NV tufts of the OIR retinas. EDN2 also induced VEGFA protein expression in cultured astrocytes. In conclusion, inhibition of the EDNRA during OIR suppresses pathological NV and promotes physiological angiogenesis. PMID:25203536

  15. Avoidance of activity and disability in patients with osteoarthritis of the knee: the mediating role of muscle strength.

    NARCIS (Netherlands)

    Steultjens, M.P.M.; Dekker, J.; Bijlsma, J.W.J.

    2002-01-01

    OBJECTIVE: Avoidance of activity is hypothesized to lead to muscle weakness and consequently, to physical disability. This study was undertaken to validate the avoidance model by providing evidence for the mediating role of muscle weakness in the relationship between avoidance of activity and physic

  16. Integrating Social Activity Theory and Critical Discourse Analysis: A Multilayered Methodological Model for Examining Knowledge Mediation in Mentoring

    Science.gov (United States)

    Becher, Ayelet; Orland-Barak, Lily

    2016-01-01

    This study suggests an integrative qualitative methodological framework for capturing complexity in mentoring activity. Specifically, the model examines how historical developments of a discipline direct mentors' mediation of professional knowledge through the language that they use. The model integrates social activity theory and a framework of…

  17. Activation of archaeal transcription mediated by recruitment of transcription factor B.

    Science.gov (United States)

    Ochs, Simon M; Thumann, Sybille; Richau, Renate; Weirauch, Matt T; Lowe, Todd M; Thomm, Michael; Hausner, Winfried

    2012-05-25

    Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested. PMID:22496454

  18. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Institute of Scientific and Technical Information of China (English)

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

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

    Science.gov (United States)

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

    2016-06-30

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

  20. The role of CNS TLR2 activation in mediating innate versus adaptive neuroinflammation.

    Science.gov (United States)

    Luz, Avital; Fainstein, Nina; Einstein, Ofira; Ben-Hur, Tamir

    2015-11-01

    Toll-like receptor 2 (TLR2) is expressed on immune cells in the periphery and the CNS and mediates both innate and adaptive immune responses. Recent studies have implicated TLR2 in systemic pathogenesis of adaptive immunity in experimental autoimmune encephalomyelitis (EAE). In addition, TLR2 is expressed on oligodendrocyte progenitor cells and its activation inhibits their differentiation and myelination. We investigated the roles of CNS TLR2 activation in mediating neuro-inflammatory responses in intact versus EAE animals. We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymosan, a TLR2 agonist, on naive versus EAE animals. The neuro-inflammatory response was characterized by immune-fluorescent staining for IBA-1+ microglia/macrophages and CD3+ T cells, and by semi-quantitative real time PCR for TLR2 and immune cytokines. The nature of the immune cells isolated from EAE brain tissue was assessed by their proliferative response to the PLP peptide autoantigen. Survival and clinical scores were monitored; demyelination and axonal loss were quantified by Gold-Black and Bielschowsky stains. Our findings showed that Zymosan injection in naïve mice induced a massive neuro-inflammatory response without any clinical manifestations. In EAE mice, ICV Zymosan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease was not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymosan injection. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide suggested that TLR2 activation induces innate but not adaptive immune response. We conclude that EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. As CNS TLR2 activation

  1. 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.

  2. GPER inhibits diabetes-mediated RhoA activation to prevent vascular endothelial dysfunction.

    Science.gov (United States)

    Li, Zilin; Cheng, Liang; Liang, Hongliang; Duan, Weixun; Hu, Jing; Zhi, Weiwei; Yang, Jinbao; Liu, Zhenhua; Zhao, Minggao; Liu, Jincheng

    2016-02-01

    The effect of estrogen receptors on diabetes-induced vascular dysfunction is critical, but ambiguous. Individuals with diabetic vascular disease may require estrogen receptor-specific targeted therapy in the future. The G protein-coupled estrogen receptor (GPER) has beneficial effects on vascular function. However, its fundamental mechanisms are unclear. The RhoA/Rho-kinase pathway contributes to diabetic vascular complications, whereas estrogen can suppress Rho-kinase function. Thus, we assumed that GPER inhibits diabetes-mediated RhoA activation to prevent vascular dysfunction. We further investigated the underlying mechanisms involved in this process. Vascular endothelial cells and ex vivo cultured ovariectomized (OVX) C57BL/6 mouse aortae were treated with high glucose (HG) alone or in combination with GPER agonist (G1). G1 treatment was also administered to OVX db/db mice for 8 weeks. An ex-vivo isovolumic myograph was used to analyze the endothelium-dependent vasodilation and endothelium-independent contraction of mouse aortae. Apoptosis, oxidative stress, and inflammation were attenuated in G1-pretreated vascular endothelial cells. G1 significantly decreased the phosphorylation of inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495), inhibited RhoA expression, and increased NO production. Additionally, G1 rescued the impaired endothelium-dependent relaxation and inhibited RhoA activation in the thoracic aorta of OVX db/db mice and ex-vivo cultured OVX C57BL/6 mouse aortae treated with HG. Estrogens acting via GPER could protect vascular endothelium, and GPER activation might elicit ERα-independent effect to inhibit RhoA/Rho-kinase pathway. Additionally, GPER activation might reduce vascular smooth muscle contraction by inhibiting RhoA activation. Thus, the results of the present study suggest a new therapeutic paradigm for end-stage vascular dysfunction by inhibiting RhoA/Rho-kinase pathway via GPER activation. PMID:26785611

  3. 2,4,6-Trichlorophenol mediated increases in extracellular peroxidase activity in three species of Lemnaceae.

    Science.gov (United States)

    Biswas, Dilip K; Scannell, Gillian; Akhmetov, Nurlan; Fitzpatrick, Dara; Jansen, Marcel A K

    2010-11-01

    Chlorinated phenols, or chlorophenols, are persistent priority pollutants that are widespread in the environment. Class III peroxidases are well-characterised plant enzymes that can catalyse the oxidative dechlorination of chlorophenols. Expression of these enzymes by plants is commonly associated with plant stress, therefore limiting scope for phytoremediation. In this study, we have quantitatively compared peroxidase activity and phytotoxicity as a function of 2,4,6-trichlorophenol (TCP) concentration in three species of Lemnaceae; Lemna minor, Lemna gibba and Landoltia punctata. Effects of TCP on the growth rates of the three species differed considerably with L. punctata being the most tolerant species. TCP also affected photosynthetic parameters, causing a decrease in open photosystem II reaction centres (qP) and, in L. punctata only, a decrease in non-photochemical quenching (qN). In parallel, TCP exposure resulted in increased peroxidase activity in all three species. Peroxidase activity in L. minor and L. gibba displayed an inverse relationship with biomass accumulation, i.e. the more growth reduction the more peroxidase activity. In contrast, induction of peroxidase activity in L. punctata was bi-phasic, with a TCP-induced activity peak at concentrations that had no major effect on growth, and further induction under phytotoxic concentrations. The mechanism by which L. punctata recognises and responds to low concentrations of an anthropogenic compound, in the absence of wide-ranging stress, remains enigmatic. However, we conclude that this "window" of peroxidase production in the absence of major growth inhibition offers potential for the development of sustainable, peroxidise-mediated phytoremediation systems. PMID:20810175

  4. The Mechanosensory Lateral Line System Mediates Activation of Socially-Relevant Brain Regions during Territorial Interactions.

    Science.gov (United States)

    Butler, Julie M; Maruska, Karen P

    2016-01-01

    Animals use multiple senses during social interactions and must integrate this information in the brain to make context-dependent behavioral decisions. For fishes, the largest group of vertebrates, the mechanosensory lateral line system provides crucial hydrodynamic information for survival behaviors, but little is known about its function in social communication. Our previous work using the African cichlid fish, Astatotilapia burtoni, provided the first empirical evidence that fish use their lateral line system to detect water movements from conspecifics for mutual assessment and behavioral choices. It is unknown, however, where this socially-relevant mechanosensory information is processed in the brain to elicit adaptive behavioral responses. To examine for the first time in any fish species which brain regions receive contextual mechanosensory information, we quantified expression of the immediate early gene cfos as a proxy for neural activation in sensory and socially-relevant brain nuclei from lateral line-intact and -ablated fish following territorial interactions. Our in situ hybridization results indicate that in addition to known lateral line processing regions, socially-relevant mechanosensory information is processed in the ATn (ventromedial hypothalamus homolog), Dl (putative hippocampus homolog), and Vs (putative medial extended amygdala homolog). In addition, we identified a functional network within the conserved social decision-making network (SDMN) whose co-activity corresponds with mutual assessment and behavioral choice. Lateral line-intact and -ablated fight winners had different patterns of co-activity of these function networks and group identity could be determined solely by activation patterns, indicating the importance of mechanoreception to co-activity of the SDMN. These data show for the first time that the mechanosensory lateral line system provides relevant information to conserved decision-making centers of the brain during territorial

  5. Sam68 Mediates the Activation of Insulin and Leptin Signalling in Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Antonio Pérez-Pérez

    Full Text Available Obesity is a well-known risk factor for breast cancer development in postmenopausal women. High insulin and leptin levels seem to have a role modulating the growth of these tumours. Sam68 is an RNA-binding protein with signalling functions that has been found to be overexpressed in breast cancer. Moreover, Sam68 may be recruited to insulin and leptin signalling pathways, mediating its effects on survival, growth and proliferation in different cellular types. We aimed to study the expression of Sam68 and its phosphorylation level upon insulin and leptin stimulation, and the role of Sam68 in the proliferative effect and signalling pathways that are activated by insulin or leptin in human breast adenocarcinoma cells. In the human breast adenocarcinoma cell lines MCF7, MDA-MB-231 and BT-474, Sam68 protein quantity and gene expression were increased upon leptin or insulin stimulation, as it was checked by qPCR and immunoblot. Moreover, both insulin and leptin stimulation promoted an increase in Sam68 tyrosine phosphorylation and negatively regulated its RNA binding capacity. siRNA was used to downregulate Sam68 expression, which resulted in lower proliferative effects of both insulin and leptin, as well as a lower activation of MAPK and PI3K pathways promoted by both hormones. These effects may be partly explained by the decrease in IRS-1 expression by down-regulation of Sam68. These results suggest the participation of Sam68 in both leptin and insulin receptor signaling in human breast cancer cells, mediating the trophic effects of these hormones in proliferation and cellular growth.

  6. Cocaine-mediated microglial activation involves the ER stress-autophagy axis.

    Science.gov (United States)

    Guo, Ming-Lei; Liao, Ke; Periyasamy, Palsamy; Yang, Lu; Cai, Yu; Callen, Shannon E; Buch, Shilpa

    2015-01-01

    Cocaine abuse leads to neuroinflammation, which, in turn, contributes to the pathogenesis of neurodegeneration associated with advanced HIV-1 infection. Autophagy plays important roles in both innate and adaptive immune responses. However, the possible functional link between cocaine and autophagy has not been explored before. Herein, we demonstrate that cocaine exposure induced autophagy in both BV-2 and primary rat microglial cells as demonstrated by a dose- and time-dependent induction of autophagy-signature proteins such as BECN1/Beclin 1, ATG5, and MAP1LC3B. These findings were validated wherein cocaine treatment of BV-2 cells resulted in increased formation of puncta in cells expressing either endogenous MAP1LC3B or overexpressing GFP-MAP1LC3B. Specificity of cocaine-induced autophagy was confirmed by treating cells with inhibitors of autophagy (3-MA and wortmannin). Intriguingly, cocaine-mediated induction of autophagy involved upstream activation of 2 ER stress pathways (EIF2AK3- and ERN1-dependent), as evidenced by the ability of the ER stress inhibitor salubrinal to ameliorate cocaine-induced autophagy. In vivo validation of these findings demonstrated increased expression of BECN1, ATG5, and MAP1LC3B-II proteins in cocaine-treated mouse brains compared to untreated animals. Increased autophagy contributes to cocaine-mediated activation of microglia since pretreatment of cells with wortmannin resulted in decreased expression and release of inflammatory factors (TNF, IL1B, IL6, and CCL2) in microglial cells. Taken together, our findings suggest that cocaine exposure results in induction of autophagy that is closely linked with neuroinflammation. Targeting autophagic proteins could thus be considered as a therapeutic strategy for the treatment of cocaine-related neuroinflammation diseases.

  7. Cocaine-mediated microglial activation involves the ER stress-autophagy axis.

    Science.gov (United States)

    Guo, Ming-Lei; Liao, Ke; Periyasamy, Palsamy; Yang, Lu; Cai, Yu; Callen, Shannon E; Buch, Shilpa

    2015-01-01

    Cocaine abuse leads to neuroinflammation, which, in turn, contributes to the pathogenesis of neurodegeneration associated with advanced HIV-1 infection. Autophagy plays important roles in both innate and adaptive immune responses. However, the possible functional link between cocaine and autophagy has not been explored before. Herein, we demonstrate that cocaine exposure induced autophagy in both BV-2 and primary rat microglial cells as demonstrated by a dose- and time-dependent induction of autophagy-signature proteins such as BECN1/Beclin 1, ATG5, and MAP1LC3B. These findings were validated wherein cocaine treatment of BV-2 cells resulted in increased formation of puncta in cells expressing either endogenous MAP1LC3B or overexpressing GFP-MAP1LC3B. Specificity of cocaine-induced autophagy was confirmed by treating cells with inhibitors of autophagy (3-MA and wortmannin). Intriguingly, cocaine-mediated induction of autophagy involved upstream activation of 2 ER stress pathways (EIF2AK3- and ERN1-dependent), as evidenced by the ability of the ER stress inhibitor salubrinal to ameliorate cocaine-induced autophagy. In vivo validation of these findings demonstrated increased expression of BECN1, ATG5, and MAP1LC3B-II proteins in cocaine-treated mouse brains compared to untreated animals. Increased autophagy contributes to cocaine-mediated activation of microglia since pretreatment of cells with wortmannin resulted in decreased expression and release of inflammatory factors (TNF, IL1B, IL6, and CCL2) in microglial cells. Taken together, our findings suggest that cocaine exposure results in induction of autophagy that is closely linked with neuroinflammation. Targeting autophagic proteins could thus be considered as a therapeutic strategy for the treatment of cocaine-related neuroinflammation diseases. PMID:26043790

  8. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Lushuai [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yanyan; Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Han, Xiao [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Niu, Yuanjie [Chawnshang Chang Sex Hormone Research Center, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300070 (China); Ren, Shancheng, E-mail: renshancheng@gmail.com [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  9. Lamin A Is an Endogenous SIRT6 Activator and Promotes SIRT6-Mediated DNA Repair

    Directory of Open Access Journals (Sweden)

    Shrestha Ghosh

    2015-11-01

    Full Text Available The nuclear lamins are essential for various molecular events in the nucleus, such as chromatin organization, DNA replication, and provision of mechanical support. A specific point mutation in the LMNA gene creates a truncated prelamin A termed progerin, causing Hutchinson-Gilford progeria syndrome (HGPS. SIRT6 deficiency leads to defective genomic maintenance and accelerated aging similar to HGPS, suggesting a potential link between lamin A and SIRT6. Here, we report that lamin A is an endogenous activator of SIRT6 and facilitates chromatin localization of SIRT6 upon DNA damage. Lamin A promotes SIRT6-dependent DNA-PKcs (DNA-PK catalytic subunit recruitment to chromatin, CtIP deacetylation, and PARP1 mono-ADP ribosylation in response to DNA damage. The presence of progerin jeopardizes SIRT6 activation and compromises SIRT6-mediated molecular events in response to DNA damage. These data reveal a critical role for lamin A in regulating SIRT6 activities, suggesting that defects in SIRT6 functions contribute to impaired DNA repair and accelerated aging in HGPS.

  10. VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression.

    Science.gov (United States)

    Pitzschke, Andrea; Djamei, Armin; Teige, Markus; Hirt, Heribert

    2009-10-27

    The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway. PMID:19820165

  11. Brain's reward circuits mediate itch relief. a functional MRI study of active scratching.

    Directory of Open Access Journals (Sweden)

    Alexandru D P Papoiu

    Full Text Available Previous brain imaging studies investigating the brain processing of scratching used an exogenous intervention mimicking scratching, performed not by the subjects themselves, but delivered by an investigator. In real life, scratching is a conscious, voluntary, controlled motor response to itching, which is directed to the perceived site of distress. In this study we aimed to visualize in real-time by brain imaging the core mechanisms of the itch-scratch cycle when scratching was performed by subjects themselves. Secondly, we aimed to assess the correlations between brain patterns of activation and psychophysical ratings of itch relief or pleasurability of scratching. We also compared the patterns of brain activity evoked by self-scratching vs. passive scratching. We used a robust tridimensional Arterial Spin Labeling fMRI technique that is less sensitive to motion artifacts: 3D gradient echo and spin echo (GRASE--Propeller. Active scratching was accompanied by a higher pleasurability and induced a more pronounced deactivation of the anterior cingulate cortex and insula, in comparison with passive scratching. A significant involvement of the reward system including the ventral tegmentum of the midbrain, coupled with a mechanism deactivating the periaqueductal gray matter (PAG, suggests that itch modulation operates in reverse to the mechanism known to suppress pain. Our findings not only confirm a role for the central networks processing reward in the pleasurable aspects of scratching, but also suggest they play a role in mediating itch relief.

  12. Artemisinin induces ROS-mediated caspase3 activation in ASTC-a-1 cells

    Science.gov (United States)

    Xiao, Feng-Lian; Chen, Tong-Sheng; Qu, Jun-Le; Liu, Cheng-Yi

    2010-02-01

    Artemisinin (ART), an antimalarial phytochemical from the sweet wormwood plant or a naturally occurring component of Artemisia annua, has been shown a potential anticancer activity by apoptotic pathways. In our report, cell counting kit (CCK-8) assay showed that treatment of human lung adenocarcinoma (ASTC-a-1) cells with ART effectively increase cell death by inducing apoptosis in a time- and dose-dependent fashion. Hoechst 33258 staining was used to detect apoptosis as well. Reactive oxygen species (ROS) generation was observed in cells exposed to ART at concentrations of 400 μM for 48 h. N-acetyl-L-cysteine (NAC), an oxygen radical scavenger, suppressed the rate of ROS generation and inhibited the ART-induced apoptosis. Moreover, AFC assay (Fluorometric assay for Caspase3 activity) showed that ROS was involved in ART-induced caspase3 acitvation. Taken together, our data indicate that ART induces ROS-mediated caspase3 activation in a time-and dose-dependent way in ASCT-a-1 cells.

  13. Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement.

    Science.gov (United States)

    Tolu, S; Eddine, R; Marti, F; David, V; Graupner, M; Pons, S; Baudonnat, M; Husson, M; Besson, M; Reperant, C; Zemdegs, J; Pagès, C; Hay, Y A H; Lambolez, B; Caboche, J; Gutkin, B; Gardier, A M; Changeux, J-P; Faure, P; Maskos, U

    2013-03-01

    Smoking is the most important preventable cause of mortality and morbidity worldwide. This nicotine addiction is mediated through the nicotinic acetylcholine receptor (nAChR), expressed on most neurons, and also many other organs in the body. Even within the ventral tegmental area (VTA), the key brain area responsible for the reinforcing properties of all drugs of abuse, nicotine acts on several different cell types and afferents. Identifying the precise action of nicotine on this microcircuit, in vivo, is important to understand reinforcement, and finally to develop efficient smoking cessation treatments. We used a novel lentiviral system to re-express exclusively high-affinity nAChRs on either dopaminergic (DAergic) or γ-aminobutyric acid-releasing (GABAergic) neurons, or both, in the VTA. Using in vivo electrophysiology, we show that, contrary to widely accepted models, the activation of GABA neurons in the VTA plays a crucial role in the control of nicotine-elicited DAergic activity. Our results demonstrate that both positive and negative motivational values are transmitted through the dopamine (DA) neuron, but that the concerted activity of DA and GABA systems is necessary for the reinforcing actions of nicotine through burst firing of DA neurons. This work identifies the GABAergic interneuron as a potential target for smoking cessation drug development.

  14. Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids.

    Science.gov (United States)

    Kuramoto, Kenta; Wang, Nan; Fan, Yuying; Zhang, Weiran; Schoenen, Frank J; Frankowski, Kevin J; Marugan, Juan; Zhou, Yifa; Huang, Sui; He, Congcong

    2016-09-01

    Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity. PMID:27305347

  15. HSF1 transcriptional activity mediates alcohol induction of Vamp2 expression and GABA release

    Directory of Open Access Journals (Sweden)

    Florence P. Varodayan

    2013-12-01

    Full Text Available Many central synapses are highly sensitive to alcohol, and it is now accepted that short-term alterations in synaptic function may lead to longer term changes in circuit function. The regulation of postsynaptic receptors by alcohol has been well studied, but the mechanisms underlying the effects of alcohol on the presynaptic terminal are relatively unexplored. To identify a pathway by which alcohol regulates neurotransmitter release, we recently investigated the mechanism by which ethanol induces the Vamp2 gene, but not Vamp1, in mouse primary cortical cultures. These two genes encode isoforms of synaptobrevin, a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE protein required for synaptic vesicle fusion. We found that alcohol activates the transcription factor heat shock factor 1 (HSF1 to induce Vamp2 gene expression, while Vamp1 mRNA levels remain unaffected. As the Vamp2 gene encodes a SNARE protein, we then investigated whether ethanol exposure and HSF1 transcriptional activity alter neurotransmitter release using electrophysiology. We found that alcohol increased the frequency of γ-aminobutyric acid (GABA-mediated miniature IPSCs via HSF1, but had no effect on mEPSCs. Overall, these data indicate that alcohol induces HSF1 transcriptional activity to trigger a specific coordinated adaptation in GABAergic presynaptic terminals. This mechanism could explain some of the changes in synaptic function that occur soon after alcohol exposure, and may underlie some of the more enduring effects of chronic alcohol intake on local circuit function.

  16. CaMKII mediates recruitment and activation of the deubiquitinase CYLD at the postsynaptic density.

    Directory of Open Access Journals (Sweden)

    Soe Thein

    Full Text Available NMDA treatment of cultured hippocampal neurons causes recruitment of CYLD, as well as CaMKII, to the postsynaptic density (PSD, as shown by immunoelectron microscopy. Recruitment of CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is blocked by pre-treatment with tatCN21, a CaMKII inhibitor, at a concentration that inhibits the translocation of CaMKII to the PSD. Furthermore, CaMKII co-immunoprecipitates with CYLD from solubilized PSD fractions, indicating an association between the proteins. Purified CaMKII phosphorylates CYLD on at least three residues (S-362, S-418, and S-772 on the human CYLD protein Q9NQC7-1 and promotes its deubiquitinase activity. Activation of CaMKII in isolated PSDs promotes phosphorylation of CYLD on the same residues and also enhances endogenous deubiquitinase activity specific for K63-linked polyubiquitins. Since K63-linked polyubiquitin conjugation to proteins inhibits their interaction with proteasomes, CaMKII-mediated recruitment and upregulation of CYLD is expected to remove K63-linked polyubiquitins and facilitate proteasomal degradation at the PSD.

  17. Calcineurin signaling mediates activity-dependent relocation of the axon initial segment.

    Science.gov (United States)

    Evans, Mark D; Sammons, Rosanna P; Lebron, Sabrina; Dumitrescu, Adna S; Watkins, Thomas B K; Uebele, Victor N; Renger, John J; Grubb, Matthew S

    2013-04-17

    The axon initial segment (AIS) is a specialized neuronal subcompartment located at the beginning of the axon that is crucially involved in both the generation of action potentials and the regulation of neuronal polarity. We recently showed that prolonged neuronal depolarization produces a distal shift of the entire AIS structure away from the cell body, a change associated with a decrease in neuronal excitability. Here, we used dissociated rat hippocampal cultures, with a major focus on the dentate granule cell (DGC) population, to explore the signaling pathways underlying activity-dependent relocation of the AIS. First, a pharmacological screen of voltage-gated calcium channels (VGCCs) showed that AIS relocation is triggered by activation of L-type Cav1 VGCCs with negligible contribution from any other VGCC subtypes. Additional pharmacological analysis revealed that downstream signaling events are mediated by the calcium-sensitive phosphatase calcineurin; inhibition of calcineurin with either FK506 or cyclosporin A totally abolished both depolarization- and optogenetically-induced activity-dependent AIS relocation. Furthermore, calcineurin activation is sufficient for AIS plasticity, because expression of a constitutively active form of the phosphatase resulted in relocation of the AIS of DGCs without a depolarizing stimulus. Finally, we assessed the role of calcineurin in other forms of depolarization-induced plasticity. Neither membrane resistance changes nor spine density changes were affected by FK506 treatment, suggesting that calcineurin acts via a separate pathway to modulate AIS plasticity. Together, these results emphasize calcineurin as a vital player in the regulation of intrinsic plasticity as governed by the AIS. PMID:23595753

  18. The orexigenic effect of ghrelin is mediated through central activation of the endogenous cannabinoid system.

    Directory of Open Access Journals (Sweden)

    Blerina Kola

    Full Text Available INTRODUCTION: Ghrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK, a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1 antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction. METHODS: The effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors. RESULTS AND CONCLUSIONS: Ghrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK

  19. Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity

    International Nuclear Information System (INIS)

    Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30II, a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30II, a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30II-dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30II-mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30II-mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30II-mediated LTR repression. Collectively, our data indicate that HTLV-1 p30II modulates viral gene expression in a cooperative manner with p300-mediated acetylation

  20. SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression.

    Science.gov (United States)

    Marathe, Himangi G; Mehta, Gaurav; Zhang, Xiaolu; Datar, Ila; Mehrotra, Aanchal; Yeung, Kam C; de la Serna, Ivana L

    2013-01-01

    SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to

  1. SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression.

    Directory of Open Access Journals (Sweden)

    Himangi G Marathe

    Full Text Available SOX10 is a Sry-related high mobility (HMG-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ and the gene that encodes myelin basic protein (MBP. Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate

  2. Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

    Directory of Open Access Journals (Sweden)

    Duane Delimont

    Full Text Available It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

  3. Antimicrobial activity of human prion protein is mediated by its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Mukesh Pasupuleti

    Full Text Available BACKGROUND: Cellular prion-related protein (PrP(c is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

  4. Endogenous Opiates in the Nucleus Tractus Solitarius Mediate Electroacupuncture-Induced Sleep Activities in Rats

    Directory of Open Access Journals (Sweden)

    Chiung-Hsiang Cheng

    2011-01-01

    Full Text Available Electroacupuncture (EA possesses various therapeutic effects, including alleviation of pain, reduction of inflammation and improvement of sleep disturbance. The mechanisms of EA on sleep improvement, however, remain to be determined. It has been stated in ancient Chinese literature that the Anmian (EX17 acupoint is one of the trigger points that alleviates insomnia. We previously demonstrated that EA stimulation of Anmian acupoints in rats during the dark period enhances non-rapid eye movement (NREM sleep, which involves the induction of cholinergic activity in the nucleus tractus solitarius (NTS. In addition to cholinergic activation of the NTS, activation of the endogenous opioidergic system may also be a mechanism by which acupuncture affects sleep. Therefore, this study was designed to investigate the involvement of the NTS opioidergic system in EA-induced alterations in sleep. Our present results indicate that EA of Anmian acupoints increased NREM sleep, but not rapid eye movement sleep, during the dark period in rats. This enhancement in NREM sleep was dose-dependently blocked by microinjection of opioid receptor antagonist, naloxone, and the μ-opioid receptor antagonist, naloxonazine, into the NTS; administrations of δ-receptor antagonist, natrindole, and the κ-receptor antagonist, nor-binaltrophimine, however, did not affect EA-induced alterations in sleep. Furthermore, β-endorphin was significantly increased in both the brainstem and hippocampus after the EA stimuli, an effect blocked by administration of the muscarinic antagonist scopolamine into the NTS. Our findings suggest that mechanisms of EA-induced NREM sleep enhancement may be mediated, in part, by cholinergic activation, stimulation of the opiodergic neurons to increase the concentrations of β-endorphin and the involvement of the μ-opioid receptors.

  5. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.

    Science.gov (United States)

    Chen, Shaoyong; Gulla, Sarah; Cai, Changmeng; Balk, Steven P

    2012-03-01

    Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

  6. covR Mediated Antibiofilm Activity of 3-Furancarboxaldehyde Increases the Virulence of Group A Streptococcus.

    Directory of Open Access Journals (Sweden)

    Ganapathy Ashwinkumar Subramenium

    Full Text Available Group A streptococcus (GAS, Streptococcus pyogenes, a multi-virulent, exclusive human pathogen responsible for various invasive and non-invasive diseases possesses biofilm forming phenomenon as one of its pathogenic armaments. Recently, antibiofilm agents have gained prime importance, since inhibiting the biofilm formation is expected to reduce development of antibiotic resistance and increase their susceptibility to the host immune cells.The current study demonstrates the antibiofilm activity of 3Furancarboxaldehyde (3FCA, a floral honey derived compound, against GAS biofilm, which was divulged using crystal violet assay, light microscopy, and confocal laser scanning microscopy. The report is extended to study its effect on various aspects of GAS (morphology, virulence, aggregation at its minimal biofilm inhibitory concentration (132μg/ml. 3FCA was found to alter the growth pattern of GAS in solid and liquid medium and increased the rate of auto-aggregation. Electron microscopy unveiled the increase in extra polymeric substances around cell. Gene expression studies showed down-regulation of covR gene, which is speculated to be the prime target for the antibiofilm activity. Increased hyaluronic acid production and down regulation of srtB gene is attributed to the enhanced rate of auto-aggregation. The virulence genes (srv, mga, luxS and hasA were also found to be over expressed, which was manifested with the increased susceptibility of the model organism Caenorhabditis elegans to 3FCA treated GAS. The toxicity of 3FCA was ruled out with no adverse effect on C. elegans.Though 3FCA possess antibiofilm activity against GAS, it was also found to increase the virulence of GAS. This study demonstrates that, covR mediated antibiofilm activity may increase the virulence of GAS. This also emphasizes the importance to analyse the acclimatization response and virulence of the pathogen in the presence of antibiofilm compounds prior to their clinical trials.

  7. Somatic modulation of spinal reflex bladder activity mediated by nociceptive bladder afferent nerve fibers in cats.

    Science.gov (United States)

    Xiao, Zhiying; Rogers, Marc J; Shen, Bing; Wang, Jicheng; Schwen, Zeyad; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2014-09-15

    The goal of the present study was to determine if supraspinal pathways are necessary for inhibition of bladder reflex activity induced by activation of somatic afferents in the pudendal or tibial nerve. Cats anesthetized with α-chloralose were studied after acute spinal cord transection at the thoracic T9/T10 level. Dilute (0.25%) acetic acid was used to irritate the bladder, activate nociceptive afferent C-fibers, and trigger spinal reflex bladder contractions (amplitude: 19.3 ± 2.9 cmH2O). Hexamethonium (a ganglionic blocker, intravenously) significantly (P < 0.01) reduced the amplitude of the reflex bladder contractions to 8.5 ± 1.9 cmH2O. Injection of lidocaine (2%, 1-2 ml) into the sacral spinal cord or transection of the sacral spinal roots and spinal cord further reduced the contraction amplitude to 4.2 ± 1.3 cmH2O. Pudendal nerve stimulation (PNS) at frequencies of 0.5-5 Hz and 40 Hz but not at 10-20 Hz inhibited reflex bladder contractions, whereas tibial nerve stimulation (TNS) failed to inhibit bladder contractions at all tested frequencies (0.5-40 Hz). These results indicate that PNS inhibition of nociceptive afferent C-fiber-mediated spinal reflex bladder contractions can occur at the spinal level in the absence of supraspinal pathways, but TNS inhibition requires supraspinal pathways. In addition, this study shows, for the first time, that after acute spinal cord transection reflex bladder contractions can be triggered by activating nociceptive bladder afferent C-fibers using acetic acid irritation. Understanding the sites of action for PNS or TNS inhibition is important for the clinical application of pudendal or tibial neuromodulation to treat bladder dysfunctions. PMID:25056352

  8. Catalyst activation, deactivation, and degradation in palladium-mediated Negishi cross-coupling reactions.

    Science.gov (United States)

    Böck, Katharina; Feil, Julia E; Karaghiosoff, Konstantin; Koszinowski, Konrad

    2015-03-27

    Pd-mediated Negishi cross-coupling reactions were studied by a combination of kinetic measurements, electrospray-ionization (ESI) mass spectrometry, (31)P NMR and UV/Vis spectroscopy. The kinetic measurements point to a rate-determining oxidative addition. Surprisingly, this step seems to involve not only the Pd catalyst and the aryl halide substrate, but also the organozinc reagent. In this context, the ESI-mass spectrometric observation of heterobimetallic Pd-Zn complexes [L2 PdZnR](+) (L=S-PHOS, R=Bu, Ph, Bn) is particularly revealing. The inferred presence of these and related neutral complexes with a direct Pd-Zn interaction in solution explains how the organozinc reagent can modulate the reactivity of the Pd catalyst. Previous theoretical calculations by González-Pérez et al. (Organometallics- 2012, 31, 2053) suggest that the complexation by the organozinc reagent lowers the activity of the Pd catalyst. Presumably, a similar effect also causes the rate decrease observed upon addition of ZnBr2 . In contrast, added LiBr apparently counteracts the formation of Pd-Zn complexes and restores the high activity of the Pd catalyst. At longer reaction times, deactivation processes due to degradation of the S-PHOS ligand and aggregation of the Pd catalyst come into play, thus further contributing to the appreciable complexity of the title reaction. PMID:25709062

  9. Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

    Science.gov (United States)

    Zhang, Jinfang; Xu, Kai; Liu, Pengda; Geng, Yan; Wang, Bin; Gan, Wenjian; Guo, Jianping; Wu, Fei; Chin, Y Rebecca; Berrios, Christian; Lien, Evan C; Toker, Alex; DeCaprio, James A; Sicinski, Piotr; Wei, Wenyi

    2016-06-16

    The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer. PMID:27237051

  10. Ethanol Activation of PKA Mediates Single-Minded 2 Expression in Neuronal Cells.

    Science.gov (United States)

    Wang, Xiaolan; Yang, Zhihua; Sun, Yinan; Zhou, Hanjing; Chu, Guangpin; Zhang, Jing; Meng, Xianfang

    2015-12-01

    Prenatal ethanol exposure can cause extensive apoptotic neurodegeneration throughout the developing central nervous system (CNS), which results in cognitive deficits and memory decline. However, the underlying mechanisms need further study. Single-minded 2 (Sim2), a transcriptional repressor, is reportedly involved in diseases that impair learning and memory, such as Down syndrome (DS) and Alzheimer's disease. It is still unknown whether Sim2 is involved in regulating ethanol-mediated neuronal injury that might ultimately lead to neuronal dysfunction and subsequent learning and memory deficits. To study the effects of ethanol on Sim2 expression and neuronal injury, we used animal models and cell culture experiments. Our results indicated that in SH-SY5Y cells, ethanol exposure increased Sim2 expression and levels of cleaved caspase 3, which is a marker for cells undergoing apoptosis. Silencing Sim2 expression attenuated caspase 3 activation and cellular apoptosis. We also found that protein kinase A (PKA) activation induced Sim2 expression, as did ethanol. Inhibiting the PKA signaling pathway with H-89 decreased Sim2 expression and cleavage of caspase 3 that was induced by ethanol in vivo and in vitro. We further found that PKA regulated Sim2 expression at the transcriptional level. These results demonstrate that ethanol leads to increased Sim2 expression via the PKA pathway, ultimately resulting in apoptotic cell death.

  11. Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

    Science.gov (United States)

    Sukhova, Anna A.; Sagaradze, George D.; Albert, Eugene A.; Ageeva, Ludmila V.; Sharonov, George V.; Tkachuk, Vsevolod A.

    2016-01-01

    Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt. PMID:27110716

  12. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila

    Science.gov (United States)

    Soldano, Alessia; Alpizar, Yeranddy A; Boonen, Brett; Franco, Luis; López-Requena, Alejandro; Liu, Guangda; Mora, Natalia; Yaksi, Emre; Voets, Thomas; Vennekens, Rudi; Hassan, Bassem A; Talavera, Karel

    2016-01-01

    Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism. DOI: http://dx.doi.org/10.7554/eLife.13133.001 PMID:27296646

  13. Self-efficacy as a mediator of the relationship between perceived union barriers and women's participation in union activities.

    Science.gov (United States)

    Bulger, C A; Mellor, S

    1997-12-01

    This study investigated the influence of union self-efficacy (expectations of success in pursuit of union activities) as a mediator of the relationship between perceptions of barriers to union participation and women's participation in union activities (N = 89). Perceived barriers were defined in 4 domains (community, family, union, work), and self-efficacy was operationalized based on C. Lee and P. Bobko's (1994) analysis of self-efficacy measures (self-efficacy magnitude, self-efficacy strength). Union self-efficacy was found to mediate the relationship between the magnitude of perceived union barriers and the magnitude of union participation, although mediation was limited to women with weak union self-efficacy. Implications for designing training and intervention programs to enhance women's participation in the face of perceived barriers are discussed.

  14. Transcriptional activation of transposable elements in mouse zygotes is independent of Tet3-mediated 5-methylcytosine oxidation

    Institute of Scientific and Technical Information of China (English)

    Azusa Inoue; Shogo Matoba; Yi Zhang

    2012-01-01

    The methylation state of the paternal genome is rapidly reprogrammed shortly after fertilization.Recent studies have revealed that loss of 5-methylcytosine(5mC)in zygotes correlates with appearance of 5-hydroxymethylcytosine (5hmC),5-formylcytosine(5fC),and 5-carboxylcytosine(5caC).This process is mediated by Tet3 and the 5mC oxidation products generated in zygotes are gradually lost during preimplantation development through a replicationdependent dilution process.Despite these findings,the biological significance of Tet3-mediated oxidation of 5mC to 5hmC/5fC/5caC in zygotes is unknown.DNA methylation plays an important role in silencing gene expression including the repression of transposable elements(TEs).Given that the activation of TEs during preimplantation development correlates with loss of DNA methylation,it is believed that paternal DNA demethylation may have an important role in TE activation.Here we examined this hypothesis and found that Tet3-mediated 5mC oxidation does not have a significant contribution to TE activation.We show that the expression of LINE-1(long interspersed nucleotide element 1)and ERVL(endogenous retroviruses class Ⅲ)are activated from both paternal and maternal genomes in zygotes.Inhibition of 5mC oxidation by siRNA-mediated depletion of Tet3 affected neither TE activation,nor global transcription in zygotes.Thus,our study provides the first evidence demonstrating that activation of both TEs and global transcription in zygotes are independent of Tet3-mediated 5mC oxidation.

  15. Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia.

    Science.gov (United States)

    Qu, Wen-Sheng; Liu, Jun-Li; Li, Chun-Yu; Li, Xiao; Xie, Min-Jie; Wang, Wei; Tian, Dai-Shi

    2015-11-01

    Previous reports have suggested that epidermal growth factor receptor (EGFR) is involved in microglia activation characterized by cell morphology changes, cytokine production and cell migration; and the biochemical regulation of the microglia migration is a potential therapeutic target following CNS inflammatory damages. However, the role of EGFR in microglia motility after inflammatory stimulation remains unknown. In the present study, lipopolysaccharide (LPS) was found to trigger rapid EGFR phosphorylation within 10 min, which was sustained during long-term stimulation in both primary microglial cells and the cultured BV2 microglial cells, furthermore, blocking EGFR phosphorylation by AG1478 significantly attenuated the LPS-induced chemotactic and chemokinetic migration of microglia. In addition, LPS could initiate calcium oscillation in microglia during live-cell recording, however, an intracellular calcium chelator and a selective inhibitor of calcium/calmodulin-dependent protein kinase II, but not an extracellular calcium chelator, remarkably suppressed the LPS-induced EGFR phosphorylation in BV2 microglia cells. As EGFR is not a traditional receptor for LPS, these findings suggest that the rapid phosphorylation of EGFR is attributed to the LPS-triggered intracellular calcium mobilization. By examining the downstream signals of EGFR, we further proved that extracellular signal-regulated kinase (ERK) is essential for EGFR-mediated microglia migration, because ERK inhibition attenuated the chemotactic and chemokinetic migration of microglia that had been induced by either LPS or EGF. Collectively, these results suggest that LPS could trigger the rapid phosphorylation of EGFR and subsequent ERK activation through mobilizing calcium activity, which underlies the microglia migration in an inflammatory condition.

  16. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Directory of Open Access Journals (Sweden)

    Nicholas D Weber

    Full Text Available Despite an existing effective vaccine, hepatitis B virus (HBV remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB, imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

  17. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Science.gov (United States)

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  18. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling.

    Science.gov (United States)

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling. PMID:25419570

  19. Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

    Science.gov (United States)

    Liang, Hai Po H; Kerschen, Edward J; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; Jia, Shuang; Hessner, Martin J; Toso, Raffaella; Rezaie, Alireza R; Fernández, José A; Camire, Rodney M; Ruf, Wolfram; Griffin, John H; Weiler, Hartmut

    2015-11-19

    The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

  20. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A receptor-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Anna Kakehashi

    Full Text Available Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA A receptor (GABA(AR system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(AR agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN. Formation of glutathione S-transferase placental form positive (GST-P(+ foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(AR alpha 1 subunit was observed in GST-P(+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+ foci by activating GABA(AR-mediated signaling.

  1. p62 regulates CD40-mediated NFκB activation in macrophages through interaction with TRAF6

    Energy Technology Data Exchange (ETDEWEB)

    Seibold, Kristina; Ehrenschwender, Martin, E-mail: martin.ehrenschwender@ukr.de

    2015-08-14

    CD40 is a member of the tumor necrosis factor (TNF) receptor family. Activation-induced recruitment of adapter proteins, so-called TNF-receptor-associated factors (TRAFs) to the cytoplasmic tail of CD40 triggers signaling cascades important in the immune system, but has also been associated with excessive inflammation in diseases such as atherosclerosis and rheumatoid arthritis. Especially, pro-inflammatory nuclear factor κB (NFκB) signaling emanating from CD40-associated TRAF6 appears to be a key pathogenic driving force. Consequently, targeting the CD40-TRAF6 interaction is emerging as a promising therapeutic strategy, but the underlying molecular machinery of this signaling axis is to date poorly understood. Here, we identified the multifunctional adaptor protein p62 as a critical regulator in CD40-mediated NFκB signaling via TRAF6. CD40 activation triggered formation of a TRAF6-p62 complex. Disturbing this interaction tremendously reduced CD40-mediated NFκB signaling in macrophages, while TRAF6-independent signaling pathways remained unaffected. This highlights p62 as a potential target in hyper-inflammatory, CD40-associated pathologies. - Highlights: • CD40 activation triggers interaction of the adapter protein TRAF6 with p62. • TRAF6-p62 interaction regulates CD40-mediated NFκB signaling in macrophages. • Defective TRAF6-p62 interaction reduces CD40-mediated NFκB activation in macrophages.

  2. Antibody-mediated targeting of the urokinase-type plasminogen activator proteolytic function neutralizes fibrinolysis in vivo

    DEFF Research Database (Denmark)

    Lund, Ida K; Jögi, Annika; Rønø, Birgitte;

    2008-01-01

    was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic...

  3. Screen time, adiposity and cardiometabolic markers : mediation by physical activity, not snacking, among 11-year-old children

    NARCIS (Netherlands)

    Berentzen, N. E.; Smit, H. A.; van Rossem, L.; Gehring, U.; Kerkhof, M.; Postma, D. S.; Boshuizen, H. C.; Wijga, A. H.

    2014-01-01

    BACKGROUND: There is evidence for a relation of TV viewing with adiposity and increased cardiometabolic risk factors in children and adolescents. It is unclear to what extent this relation is mediated by snacking and lack of physical activity. We determined whether associations of screen time with a

  4. Screen time, adiposity and cardiometabolic markers: mediation by physical activity, not snacking, among 11-year-old children

    NARCIS (Netherlands)

    Berendtzen, N.E.; Smit, H.A.; Rossem, van L.; Gehring, U.; Kerkhof, van de M.; Postma, D.S.; Boshuizen, H.C.

    2014-01-01

    Background:There is evidence for a relation of TV viewing with adiposity and increased cardiometabolic risk factors in children and adolescents. It is unclear to what extent this relation is mediated by snacking and lack of physical activity. We determined whether associations of screen time with ad

  5. Does HPA-axis activity mediate the relationship between obstetric complications and externalizing behavior problems? The TRAILS study.

    NARCIS (Netherlands)

    Marsman, R.; Rosmalen, J.G.; Oldehinkel, A.J.; Ormel, J.; Buitelaar, J.K.

    2009-01-01

    To examine whether HPA-axis activity mediates the relationship between obstetric complications (OCs) and externalizing behavior problems, and to investigate whether this model is different for boys and girls. In a population-based cohort of 1,768 10- to 12-year-old early adolescents, we assessed the

  6. Fragility, fear of falling, physical activity and falls among older persons: Some theoretical considerations to interpret mediation

    NARCIS (Netherlands)

    Wijlhuizen, G.J.; Chorus, A.M.J.; Hopman-Rock, M.

    2008-01-01

    Background: In their letters to the editor, Lacherez et al. [Lacherez, P.F., Wood, J.M., Kerr, G.K., 2007. Does activity level mediate or suppress the association between fear of falling and falls? Prev. Med. 31; (Electronic publication ahead of print)] and Hafeman and Schwartz [Hafeman, D., Schwart

  7. Remarkable Functional Convergence: Alarmone ppGpp Mediates Persistence by Activating Type I and II Toxin-Antitoxins.

    Science.gov (United States)

    Gerdes, Kenn; Maisonneuve, Etienne

    2015-07-01

    In this issue of Molecular Cell, Verstraeten et al. (2015) demonstrate that the conserved GTPase Obg and the second messenger ppGpp mediate persistence by activation of a type I toxin-antitoxin module (hokB/sokB) in E. coli.

  8. A test of cognitive mediation in a 12-month physical activity workplace intervention: does it explain behaviour change in women?

    Directory of Open Access Journals (Sweden)

    Pickering Michael A

    2010-05-01

    Full Text Available Abstract Background Attempts to demonstrate the efficacy of interventions aimed at increasing physical activity (PA have been mixed. Further, studies are seldom designed in a manner that facilitates the understanding of how or why a treatment is effective or ineffective and PA intervention designs should be guided by a heavier reliance upon behavioral theory. The use of a mediating variable framework offers a systematic methodological approach to testing the role of theory, and could also identify the effectiveness of specific intervention components. The primary purpose of this paper was to test the mediating role that cognitive constructs may have played in regards to the positive effect that a workplace behavioral intervention had on leisure-time PA for women. A subsidiary purpose was to examine the cross-sectional relationships of these cognitive constructs with PA behavior. Methods The Physical Activity Workplace Study was a randomized controlled trial which compared the effects of stage-matched and standard print materials upon self-reported leisure-time PA, within a workplace sample at 6 and 12-months. In this secondary analysis we examined the mediation effects of 14 psychosocial constructs across 3 major social-cognitive theories which were operationalized for the intervention materials and measured at baseline, 6 and 12-months. We examined change in PA and change in the psychological constructs employing a mediation strategy proposed by Baron and Kenny for: (1 the first 6-months (i.e., initial change, (2 the second 6-months (i.e., delayed change, and (3 the entire 12-months (overall change of the study on 323 women (n = 213 control/standard materials group; n = 110 stage-matched materials group. Results Of the 14 constructs and 42 tests (including initial, delayed and overall change two positive results were identified (i.e., overall change in pros, initial change in experiential powerful intervention approaches processes, with very

  9. Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection.

    Science.gov (United States)

    Simpson, A J; Wallace, W A; Marsden, M E; Govan, J R; Porteous, D J; Haslett, C; Sallenave, J M

    2001-08-01

    During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils. PMID:11466403

  10. Walkability parameters, active transportation and objective physical activity: moderating and mediating effects of motor vehicle ownership in a cross-sectional study

    Directory of Open Access Journals (Sweden)

    Eriksson Ulf

    2012-10-01

    Full Text Available Abstract Background Neighborhood walkability has been associated with physical activity in several studies. However, as environmental correlates of physical activity may be context specific, walkability parameters need to be investigated separately in various countries and contexts. Furthermore, the mechanisms by which walkability affects physical activity have been less investigated. Based on previous research, we hypothesized that vehicle ownership is a potential mediator. We investigated the associations between walkability parameters and physical activity, and the mediating and moderating effects of vehicle ownership on these associations in a large sample of Swedish adults. Methods Residential density, street connectivity and land use mix were assessed within polygon-based network buffers (using Geographic Information Systems for 2,178 men and women. Time spent in moderate to vigorous physical activity was assessed by accelerometers, and walking and cycling for transportation were assessed by the International Physical Activity Questionnaire. Associations were examined by linear regression and adjusted for socio-demographic characteristics. The product of coefficients approach was used to investigate the mediating effect of vehicle ownership. Results Residential density and land use mix, but not street connectivity, were significantly associated with time spent in moderate to vigorous physical activity and walking for transportation. Cycling for transportation was not associated with any of the walkability parameters. Vehicle ownership mediated a significant proportion of the association between the walkability parameters and physical activity outcomes. For residential density, vehicle ownership mediated 25% of the association with moderate to vigorous physical activity and 20% of the association with the amount of walking for transportation. For land use mix, the corresponding proportions were 34% and 14%. Vehicle ownership did not

  11. Suppression by Apoptotic Cells Defines Tumor Necrosis Factor-Mediated Induction of Glomerular Mesangial Cell Apoptosis by Activated Macrophages

    OpenAIRE

    Duffield, Jeremy S.; Ware, Carl F.; Ryffel, Bernhardt; Savill, John

    2001-01-01

    Activated macrophages (Mφ) isolated from inflamed glomeruli or generated by interferon-γ and lipopolysaccharide treatment in vitro induce glomerular mesangial cell apoptosis by hitherto incompletely understood mechanisms. In this report we demonstrate that nitric oxide-independent killing of co-cultured mesangial cells by interferon-γ/lipopolysaccharide-activated Mφ is suppressed by binding/ingestion of apoptotic cells and is mediated by tumor necrosis factor (TNF). Thus, soluble TNF receptor...

  12. Troglitazone, a PPAR-γ activator prevents endothelial cell adhesion molecule expression and lymphocyte adhesion mediated by TNF-α

    OpenAIRE

    Itoh Makoto; Joh Takashi; Minagar Alireza; Welbourne Tomas; Jordan Paul; Sasaki Makoto; Elrod John W; Alexander J Steven

    2005-01-01

    Abstract Background Cytokine mediated induction of the mucosal addressin cell adhesion molecule-1(MAdCAM-1) expression is associated with the onset and progression of inflammatory bowel disease (IBD). Results Using western blotting and cell-based ELISA, we show in this study that troglitazone, an activator of the peroxisome proliferator-activated receptor-γ (PPAR-γ), widely used in the treatment of diabetes, has as well recently been highlighted as protective in models of inflammation and can...

  13. Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA.

    Directory of Open Access Journals (Sweden)

    Cristina Antonella Nadalutti

    Full Text Available PURPOSE: To investigate the role of thioredoxin (TRX, a novel regulator of extracellular transglutaminase 2 (TG2, in celiac patients IgA (CD IgA mediated TG2 enzymatic activation. METHODS: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. RESULTS: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. CONCLUSIONS: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.

  14. Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

    International Nuclear Information System (INIS)

    Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN)63−/menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN)63− generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system

  15. Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

    Energy Technology Data Exchange (ETDEWEB)

    Matsumae, Yoshiharu [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Takahashi, Yasufumi [Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan); Ino, Kosuke [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan)

    2014-09-09

    Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN){sub 6}{sup 3−}/menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN){sub 6}{sup 3−} generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.

  16. Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    Chen-Guang Bai; Xiao-Wei Hou; Feng Wang; Cen Qiu; Yan Zhu; Ling Huang; Jing Zhao

    2012-01-01

    AIM:To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth.METHODS:The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry,and the results were correlated with clinicopathological parameters,including the mitotic count,proliferative index (Ki-67 immunohistochemical staining),mitotic index (phospho-histone H3 immunohistochemical staining)and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling).Using primary cultured GIST cells,the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting,methyl thiazolyl tetrazolium (MTT),and apoptosis assays.RESULTS:We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples,respectively,and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis,including larger tumor size (P =0.0118),higher mitotic count (P =0.0058),higher proliferative index (P =0.0012),higher mitotic index (P =0.0282),lower apoptosis index (P =0.0484),and increased National Institutes of Health risk level (P =0.0012).We also found that the introduction of exogenous SCF potently increased KIT kinase activity,stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation,while a KIT immunoblocking antibody suppressed proliferation (P =0.01) and promoted apoptosis (P < 0.01)in cultured GIST cells.CONCLUSION:SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth.The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.

  17. Concurrent inhibition of kit- and FcepsilonRI-mediated signaling: coordinated suppression of mast cell activation

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Beaven, Michael A; Iwaki, Shoko;

    2008-01-01

    characterized Kit inhibitor imatinib mesylate (imatinib). In contrast to imatinib, however, hypothemycin also effectively inhibited FcepsilonRI-mediated degranulation and cytokine production in addition to the potentiation of these responses via Kit. The effect of hypothemycin on Kit-mediated responses could...

  18. Activated Carbon as an Electron Acceptor and Redox Mediator during the Anaerobic Biotransformation of Azo Dyes

    NARCIS (Netherlands)

    Zee, van der F.P.; Bisschops, I.A.E.; Lettinga, G.; Field, J.A.

    2003-01-01

    The role of AC as redox mediator in accelerating the reductive transformation of pollutants as well as a terminal electron acceptor in the biological oxidation of an organic substrate is described. This study explores the use of AC as an immobilized redox mediator for the reduction of a recalcitrant

  19. Sympathetic activity-mediated neuropathic facial pain following simple tooth extraction: a case report.

    Science.gov (United States)

    Kohjitani, Atsushi; Miyawaki, Takuya; Kasuya, Keigo; Shimada, Masahiko

    2002-04-01

    This is a report of a case of sympathetic activity-mediated neuropathic facial pain induced by a traumatic trigeminal nerve injury and by varicella zoster virus infection, following a simple tooth extraction. The patient had undergone extraction of the right lower third molar at a local dental clinic, and soon after the tooth extraction, she became aware of spontaneous pain in the right ear, right temporal region, and in the tooth socket. At our initial examination 30 days after the tooth extraction, the healing of the tooth socket was normal; however, the patient had a tingling and burning sensation (dysesthesia) and spontaneous pain of the right lower lip and the right temporal region, both of which were exacerbated by non-noxious stimuli (allodynia). The patient also showed paralysis of the marginal mandibular branch of the facial nerve, taste dysfunction, and increased varicella zoster serum titers. A diagnostic stellate ganglion block (SGB) 45 days after the tooth extraction using one percent lidocaine markedly alleviated the dysesthesia and allodynia. These symptoms are characteristic of neuropathic pain with sympathetic interaction. The patient was successfully treated with SGB and a tricyclic antidepressant. PMID:12002830

  20. N6-isopentenyladenosine and analogs activate the NRF2-mediated antioxidant response

    Directory of Open Access Journals (Sweden)

    Alice Dassano

    2014-01-01

    Full Text Available N6-isopentenyladenosine (i6A, a naturally occurring modified nucleoside, inhibits the proliferation of human tumor cell lines in vitro, but its mechanism of action remains unclear. Treatment of MCF7 human breast adenocarcinoma cells with i6A or with three synthetic analogs (allyl6A, benzyl6A, and butyl6A inhibited growth and altered gene expression. About 60% of the genes that were differentially expressed in response to i6A treatment were also modulated by the analogs, and pathway enrichment analysis identified the NRF2-mediated oxidative stress response as being significantly modulated by all four compounds. Luciferase reporter gene assays in transfected MCF7 cells confirmed that i6A activates the transcription factor NRF2. Assays for cellular production of reactive oxygen species indicated that i6A and analogs had antioxidant effects, reducing basal levels and inhibiting the H2O2- or 12-O-tetradecanoylphorbol-13-acetate (TPA-induced production in MCF7 or dHL-60 (HL-60 cells induced to differentiate along the neutrophilic lineage cell lines, respectively. In vivo, topical application of i6A or benzyl6A to mouse ears prior to TPA stimulation lessened the inflammatory response and significantly reduced the number of infiltrating neutrophils. These results suggest that i6A and analogs trigger a cellular response against oxidative stress and open the possibility of i6A and benzyl6A being used as topical anti-inflammatory drugs.

  1. Wnt-Signaling-Mediated Antiosteoporotic Activity of Porcine Placenta Hydrolysates in Ovariectomized Rats

    Directory of Open Access Journals (Sweden)

    Byoung-Seob Ko

    2012-01-01

    Full Text Available Anti-osteoporotic effects of two types of porcine placenta hydrolysates (PPH were evaluated in ovariectomized (OVX rats orally administered PPH without (WPPH or with (NPPH ovarian hormones (1 g/kg bw/day. PPH groups were compared with OVX rats with estrogen replacement (0.1 mg/kg bw conjugated estrogen; EST, or dextrose (placebo; OVX-control All rats received high-fat/calcium-deficient diets for 12 weeks. NPPH contained less estrogen and progesterone, but more essential amino acids, whereas the opposite was true for WPPH. NPPH decreased body weight and peri-uterine fat pads, and maintained uterus weight. NPPH rats had higher femur and lumbar spine bone mass density compared to controls; but less than those of EST rats. Serum phosphorus and urinary calcium and phosphorus levels were reduced in NPPH rats compared to OVX-controls. Serum bone-specific alkaline phosphatase, osteocalcin, and bone turnover marker levels were reduced NPPH rats compared to OVX-controls. WPPH produced results similar to those of NPPH, but less significant. Both NPPH and estrogen upregulated low-density lipoprotein receptor-related protein 5 and β-catenin in OVX rats, while the expression of dickkopf-related protein 1 was suppressed. In conclusion, NPPH exerted anti-osteoporotic effects by activating osteogenesis and stimulating Wnt signaling, possibly mediated by the various amino acids and not ovarian hormones.

  2. 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

    Directory of Open Access Journals (Sweden)

    Thomas Stübig

    2014-01-01

    Full Text Available Demethylating agent, 5-Azacytidine (5-Aza, has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1 were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.

  3. 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

    Science.gov (United States)

    Stübig, Thomas; Luetkens, Tim; Hildebrandt, York; Atanackovic, Djordje; Binder, Thomas M. C.; Fehse, Boris; Kröger, Nicolaus

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza. PMID:24757283

  4. 5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity.

    Science.gov (United States)

    Stübig, Thomas; Badbaran, Anita; Luetkens, Tim; Hildebrandt, York; Atanackovic, Djordje; Binder, Thomas M C; Fehse, Boris; Kröger, Nicolaus

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ + T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza. PMID:24757283

  5. Alpha-latrotoxin modulates the secretory machinery via receptor-mediated activation of protein kinase C.

    Science.gov (United States)

    Liu, Jie; Wan, Qunfang; Lin, Xianguang; Zhu, Hongliang; Volynski, Kirill; Ushkaryov, Yuri; Xu, Tao

    2005-09-01

    The hypothesis whether alpha-latrotoxin (LTX) could directly regulate the secretory machinery was tested in pancreatic beta cells using combined techniques of membrane capacitance (Cm) measurement and Ca2+ uncaging. Employing ramp increase in [Ca2+]i to stimulate exocytosis, we found that LTX lowers the Ca2+ threshold required for exocytosis without affecting the size of the readily releasable pool (RRP). The burst component of exocytosis in response to step-like [Ca2+]i increase generated by flash photolysis of caged Ca2+ was also speeded up by LTX treatment. LTX increased the maximum rate of exocytosis compared with control responses with similar postflash [Ca2+]i and shifted the Ca2+ dependence of the exocytotic machinery toward lower Ca2+ concentrations. LTXN4C, a LTX mutant which cannot form membrane pores or penetrate through the plasma membrane but has similar affinity for the receptors as the wild-type LTX, mimicked the effect of LTX. Moreover, the effects of both LTX and LTXN4C) were independent of intracellular or extracellular Ca2+ but required extracellular Mg2+. Our data propose that LTX, by binding to the membrane receptors, sensitizes the fusion machinery to Ca2+ and, hence, may permit release at low [Ca2+]i level. This sensitization is mediated by activation of protein kinase C. PMID:16101679

  6. Thermodynamics of tryptophan-mediated activation of the trp RNA-binding attenuation protein.

    Science.gov (United States)

    McElroy, Craig A; Manfredo, Amanda; Gollnick, Paul; Foster, Mark P

    2006-06-27

    The trp RNA-binding attenuation protein (TRAP) functions in many bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary-structural elements in the 5' leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in protein dynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced-fit conformational change are discussed. PMID:16784236

  7. Repressed PKCδ activation in glycodelin-expressing cells mediates resistance to phorbol ester and TGFβ.

    Science.gov (United States)

    Hautala, Laura C; Koistinen, Riitta; Koistinen, Hannu

    2016-10-01

    Glycodelin is a glycoprotein mainly expressed in well-differentiated epithelial cells in reproductive tissues. In normal secretory endometrium, the expression of glycodelin is abundant and regulated by progesterone. In hormone-related cancers glycodelin expression is associated with well-differentiated tumors. We have previously found that glycodelin drives epithelial differentiation of HEC-1B endometrial adenocarcinoma cells, resulting in reduced tumor growth in a preclinical mouse model. Here we show that glycodelin-transfected HEC-1B cells have repressed protein kinase C delta (PKCδ) activation, likely due to downregulation of PDK1, and are resistant to phenotypic change and enhanced migration induced by phorbol 12-myristate 13-acetate (PMA). In control cells, which do not express glycodelin, the effects of PMA were abolished by using PKCδ and PDK1 inhibitors, and knockdown of PKCδ, MEK1 and 2, or ERK1 and 2 by siRNAs. Similarly, transforming growth factor β (TGFβ)-induced phenotypic change was only seen in control cells, not in glycodelin-producing cells, and it was mediated by PKCδ. Taken together, these results strongly suggest that PKCδ, via MAPK pathway, is involved in the glycodelin-driven cell differentiation rendering the cells resistant to stimulation by PMA and TGFβ. PMID:27373413

  8. Acid Phosphatase Activity May Affect the Tuber Swelling by Partially Regulating Sucrose-mediated Sugar Resorption in Potato

    Institute of Scientific and Technical Information of China (English)

    Da-Yong Wang; Yong Lian; De-Wei Zhu

    2008-01-01

    APase activity is involved in regulating many physiological and developmental events by affecting the resorption process.In this study, we investigate the role of APase activity in tuber development in potato. APase activities were mainly localized in cytoplasm, gaps among cells and stroma of amyloplasts of parenchyma cells at the stage of tuber swelling. AP1, encoding a putative APase, was also highly expressed in swelling tubers and a low level of expression was observed in elongated stolons and matured tubers. Inhibition of APase activity by applying Brefeldin A, an inhibitor of APase production and secretion, significantly suppressed the tuber swelling and moderately affected the stolon elongation and the tuberization frequency. During tuber development, sucrose serves as the main soluble sugar for long-distance transportation and resorption. Moreover, Inhibition of APase activity by Brefeldin A markedly reduced the sucrose content in tubers and further decreased the starch accumulation, suggesting that the function of APase in regulating the tuber swelling might be at least artially mediated by the sugar resorption. Exogenous sucrose treatments further indicate the important role of sucrose-mediated sugar resorption in tuber swelling. These results suggest that the APase activity might affect the tuber swelling by partially regulating the sucrose-mediated sugar resorption.

  9. TRAIL-mediated killing of acute lymphoblastic leukemia by plasmacytoid dendritic cell-activated natural killer cells.

    Science.gov (United States)

    Lelaidier, Martin; Dìaz-Rodriguez, Yildian; Cordeau, Martine; Cordeiro, Paulo; Haddad, Elie; Herblot, Sabine; Duval, Michel

    2015-10-01

    Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT), underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest, but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs, but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement, while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally, adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL.

  10. COUP-TFII mediates progesterone regulation of uterine implantation by controlling ER activity.

    Directory of Open Access Journals (Sweden)

    Isao Kurihara

    2007-06-01

    Full Text Available Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2, a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone-Indian hedgehog-Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.

  11. HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    Directory of Open Access Journals (Sweden)

    James F Curtin

    2009-01-01

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1, an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2 signaling on bone marrow-derived GBM-infiltrating DCs. METHODS AND FINDINGS: Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad expressing Fms-like tyrosine kinase 3 ligand (Flt3L and thymidine kinase (TK delivered into the tumor mass, we demonstrated that CD4(+ and CD8(+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV] treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide

  12. 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...

  13. Biosynthesis and characterization of Acalypha indica mediated copper oxide nanoparticles and evaluation of its antimicrobial and anticancer activity

    Science.gov (United States)

    Sivaraj, Rajeshwari; Rahman, Pattanathu K. S. M.; Rajiv, P.; Narendhran, S.; Venckatesh, R.

    2014-08-01

    Copper oxide nanoparticles were synthesized by biological method using aqueous extract of Acalypha indica leaf and characterized by UV-visible spectroscopy, XRD, FT-IR, SEM TEM and EDX analysis. The synthesised particles were highly stable, spherical and particle size was in the range of 26-30 nm. The antimicrobial activity of A.indica mediated copper oxide nanoparticles was tested against selected pathogens. Copper oxide nanoparticles showed efficient antibacterial and antifungal effect against Escherichia coli, Pseudomonas fluorescens and Candida albicans. The cytotoxicity activity of A.indica mediated copper nanoparticles was evaluated by MTT assay against MCF-7 breast cancer cell lines and confirmed that copper oxide nanoparticles have cytotoxicity activity.

  14. Prostate cancer cell-stromal cell crosstalk via FGFR1 mediates antitumor activity of dovitinib in bone metastases.

    Science.gov (United States)

    Wan, Xinhai; Corn, Paul G; Yang, Jun; Palanisamy, Nallasivam; Starbuck, Michael W; Efstathiou, Eleni; Li Ning Tapia, Elsa M; Tapia, Elsa M Li-Ning; Zurita, Amado J; Aparicio, Ana; Ravoori, Murali K; Vazquez, Elba S; Robinson, Dan R; Wu, Yi-Mi; Cao, Xuhong; Iyer, Matthew K; McKeehan, Wallace; Kundra, Vikas; Wang, Fen; Troncoso, Patricia; Chinnaiyan, Arul M; Logothetis, Christopher J; Navone, Nora M

    2014-09-01

    Bone is the most common site of prostate cancer (PCa) progression to a therapy-resistant, lethal phenotype. We found that blockade of fibroblast growth factor receptors (FGFRs) with the receptor tyrosine kinase inhibitor dovitinib has clinical activity in a subset of men with castration-resistant PCa and bone metastases. Our integrated analyses suggest that FGF signaling mediates a positive feedback loop between PCa cells and bone cells and that blockade of FGFR1 in osteoblasts partially mediates the antitumor activity of dovitinib by improving bone quality and by blocking PCa cell-bone cell interaction. These findings account for clinical observations such as reductions in lesion size and intensity on bone scans, lymph node size, and tumor-specific symptoms without proportional declines in serum prostate-specific antigen concentration. Our findings suggest that targeting FGFR has therapeutic activity in advanced PCa and provide direction for the development of therapies with FGFR inhibitors.

  15. The "Romsas in Motion" Community Intervention: Mediating Effects of Psychosocial Factors on Forward Transition in the Stages of Change in Physical Activity

    Science.gov (United States)

    Lorentzen, Catherine; Ommundsen, Yngvar; Jenum, Anne Karen; Holme, Ingar

    2009-01-01

    This study examines whether a community-based physical activity intervention influenced movement in stages of change in physical activity in an adult population, whether any such effect was mediated by psychosocial influences, and whether any such mediations were moderated by sociodemographic or anthropometric factors. The 3-year-long…

  16. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection.

    Science.gov (United States)

    Zhang, Hua; Li, Fengqi; Pan, Ziye; Wu, Zhijun; Wang, Yanhong; Cui, Yudong

    2014-11-01

    Apoptosis is frequently induced to inhibit virus replication during infection of Enterovirus 71 (EV71). On the contrary, anti-apoptotic pathway, such as PI3K/Akt pathway, is simultaneously exploited by EV71 to accomplish the viral life cycle. The relationship by which EV71-induced apoptosis and PI3K/Akt signaling pathway remains to be elucidated. In this study, we demonstrated that EV71 infection altered Bax conformation and triggered its redistribution from the cytosol to mitochondria in RD cells. Subsequently, cytochrome c was released from mitochondria to cytosol. We also found that c-Jun NH2-terminal kinase (JNK) was activated during EV71 infection. The JNK specific inhibitor significantly inhibited Bax activation and cytochrome c release, suggesting that EV71-induced apoptosis was involved into a JNK-dependent manner. Meanwhile, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Inhibition of the PI3K/Akt pathway enhanced JNK phosphorylation and the JNK-mediated apoptosis upon EV71 infection. Moreover, PI3K/Akt pathway phosphorylated apoptosis signal-regulating kinase 1 (ASK1) and negatively regulated the ASK1 activity. Knockdown of ASK1 significantly decreased JNK phosphorylation, which implied that ASK1 phosphorylation by Akt inhibited ASK1-mediated JNK activation. Collectively, these data reveal that activation of the PI3K/Akt pathway limits JNK-mediated apoptosis by phosphorylating and inactivating ASK1 during EV71 infection.

  17. Perceived sports competence mediates the relationship between childhood motor skill proficiency and adolescent physical activity and fitness: a longitudinal assessment

    Directory of Open Access Journals (Sweden)

    van Beurden Eric

    2008-08-01

    Full Text Available Abstract Background The purpose of this paper was to investigate whether perceived sports competence mediates the relationship between childhood motor skill proficiency and subsequent adolescent physical activity and fitness. Methods In 2000, children's motor skill proficiency was assessed as part of a school-based physical activity intervention. In 2006/07, participants were followed up as part of the Physical Activity and Skills Study and completed assessments for perceived sports competence (Physical Self-Perception Profile, physical activity (Adolescent Physical Activity Recall Questionnaire and cardiorespiratory fitness (Multistage Fitness Test. Structural equation modelling techniques were used to determine whether perceived sports competence mediated between childhood object control skill proficiency (composite score of kick, catch and overhand throw, and subsequent adolescent self-reported time in moderate-to-vigorous physical activity and cardiorespiratory fitness. Results Of 928 original intervention participants, 481 were located in 28 schools and 276 (57% were assessed with at least one follow-up measure. Slightly more than half were female (52.4% with a mean age of 16.4 years (range 14.2 to 18.3 yrs. Relevant assessments were completed by 250 (90.6% students for the Physical Activity Model and 227 (82.3% for the Fitness Model. Both hypothesised mediation models had a good fit to the observed data, with the Physical Activity Model accounting for 18% (R2 = 0.18 of physical activity variance and the Fitness Model accounting for 30% (R2 = 0.30 of fitness variance. Sex did not act as a moderator in either model. Conclusion Developing a high perceived sports competence through object control skill development in childhood is important for both boys and girls in determining adolescent physical activity participation and fitness. Our findings highlight the need for interventions to target and improve the perceived sports competence of youth.

  18. EGFR-mediated carcinoma cell metastasis mediated by integrin αvβ5 depends on activation of c-Src and cleavage of MUC1.

    Directory of Open Access Journals (Sweden)

    Steven K M Lau

    Full Text Available Receptor tyrosine kinases and integrins play an essential role in tumor cell invasion and metastasis. We previously showed that EGF and other growth factors induce human carcinoma cell invasion and metastasis mediated by integrin αvβ5 that is prevented by Src blockade. MUC1, a transmembrane glycoprotein, is expressed in most epithelial tumors as a heterodimer consisting of an extracellular and a transmembrane subunit. The MUC1 cytoplasmic domain of the transmembrane subunit (MUC1.CD translocates to the nucleus where it promotes the transcription of a metastatic gene signature associated with epithelial to mesenchymal transition. Here, we demonstrate a requirement for MUC1 in carcinoma cell metastasis dependent on EGFR and Src without affecting primary tumor growth. EGF stimulates Src-dependent MUC1 cleavage and nuclear localization leading to the expression of genes linked to metastasis. Moreover, expression of MUC1.CD results in its nuclear localization and is sufficient for transcription of the metastatic gene signature and tumor cell metastasis. These results demonstrate that EGFR and Src activity contribute to carcinoma cell invasion and metastasis mediated by integrin αvβ5 in part by promoting proteolytic cleavage of MUC1 and highlight the ability of MUC1.CD to promote metastasis in a context-dependent manner. Our findings may have implications for the use and future design of targeted therapies in cancers known to express EGFR, Src, or MUC1.

  19. Inhibition effect of cypermethrin mediated by co-regulators SRC-1 and SMRT in interleukin-6-induced androgen receptor activation.

    Science.gov (United States)

    Wang, Qi; Zhou, Ji-Long; Wang, Hui; Ju, Qiang; Ding, Zhen; Zhou, Xiao-Long; Ge, Xing; Shi, Qiao-Mei; Pan, Chen; Zhang, Jin-Peng; Zhang, Mei-Rong; Yu, Hong-Min; Xu, Li-Chun

    2016-09-01

    It is hypothesized that the pesticide cypermethrin may induce androgen receptor (AR) antagonism via ligand-independent mechanisms. The Real-Time Cell Analysis (RTCA) iCELLigence system was used to investigate the inhibitory effect of cypermethrin on interleukin-6 (IL-6)-induced ligand-independent LNCaP cell growth. Then, the mammalian two-hybrid assays were applied to clarify whether the mechanism of IL-6-induced AR antagonism of cypermethrin was associated with the interactions of the AR and co-activator steroid receptor co-activator-1 (SRC-1) and co-repressor silencing mediator for retinoid and thyroid hormone receptors (SMRT). Cypermethrin inhibited the LNCaP cell growth induced by IL-6. The interactions of AR-SRC-1 and AR-SMRT mediated by IL-6 were suppressed by cypermethrin. The results indicate that the IL-6-mediated AR antagonism induced by cypermethrin is related to repress the recruitment of co-regulators SRC-1 and SMRT to the AR in a ligand-independent manner. Inhibition of the interactions of AR-SRC-1 and AR-SMRT mediated by IL-6 contributes to the AR antagonism induced by cypermethrin. PMID:27239967

  20. Spinal activity of interleukin 6 mediates myelin basic protein-induced allodynia.

    Science.gov (United States)

    Ko, Justin S; Eddinger, Kelly A; Angert, Mila; Chernov, Andrei V; Dolkas, Jennifer; Strongin, Alex Y; Yaksh, Tony L; Shubayev, Veronica I

    2016-08-01

    Mechanosensory fibers are enveloped by myelin, a unique multilamellar membrane permitting saltatory neuronal conduction. Damage to myelin is thought to contribute to severe pain evoked by innocuous tactile stimulation (i.e., mechanical allodynia). Our earlier (Liu et al., 2012) and present data demonstrate that a single injection of a myelin basic protein-derived peptide (MBP84-104) into an intact sciatic nerve produces a robust and long-lasting (>30days) mechanical allodynia in female rats. The MBP84-104 peptide represents the immunodominant epitope and requires T cells to maintain allodynia. Surprisingly, only systemic gabapentin (a ligand of voltage-gated calcium channel α2δ1), but not ketorolac (COX inhibitor), lidocaine (sodium channel blocker) or MK801 (NMDA antagonist) reverse allodynia induced by the intrasciatic MBP84-104. The genome-wide transcriptional profiling of the sciatic nerve followed by the bioinformatics analyses of the expression changes identified interleukin (IL)-6 as the major cytokine induced by MBP84-104 in both the control and athymic T cell-deficient nude rats. The intrasciatic MBP84-104 injection resulted in both unilateral allodynia and unilateral IL-6 increase the segmental spinal cord (neurons and astrocytes). An intrathecal delivery of a function-blocking IL-6 antibody reduced the allodynia in part by the transcriptional effects in large-diameter primary afferents in DRG. Our data suggest that MBP regulates IL-6 expression in the nervous system and that the spinal IL-6 activity mediates nociceptive processing stimulated by the MBP epitopes released after damage or disease of the somatosensory nervous system. PMID:26970355

  1. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    Science.gov (United States)

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. PMID:26598443

  2. Cardio-respiratory effects of systemic neurotensin injection are mediated through activation of neurotensin NTS₁ receptors.

    Science.gov (United States)

    Kaczyńska, Katarzyna; Szereda-Przestaszewska, Małgorzata

    2012-09-15

    The purpose of our study was to determine the cardio-respiratory pattern exerted by the systemic injection of neurotensin, contribution of neurotensin NTS(1) receptors and the neural pathways mediating the responses. The effects of an intravenous injection (i.v.) of neurotensin were investigated in anaesthetized, spontaneously breathing rats in following experimental schemes: (i) control animals before and after midcervical vagotomy; (ii) in three separate subgroups of rats: neurally intact, vagotomized at supranodosal level and initially midcervically vagotomized exposed to section of the carotid sinus nerves (CSNs); (iii) in the intact rats 2 minutes after blockade of neurotensin NTS(1) receptors with SR 142948. Intravenous injection of 10 μg/kg of neurotensin in the intact rats evoked prompt increase in the respiratory rate followed by a prolonged slowing down coupled with augmented tidal volume. Midcervical vagotomy precluded the effects of neurotensin on the frequency of breathing, while CSNs section reduced the increase in tidal volume. In all the neural states neurotensin caused significant fall in mean arterial blood pressure preceded by prompt hypertensive response. The cardio-respiratory effects of neurotensin were blocked by pre-treatment with NTS(1) receptor antagonist. The results of this study showed that neurotensin acting through NTS(1) receptors augments the tidal component of the breathing pattern in a large portion via carotid body afferentation whereas the respiratory timing response to neurotensin depends entirely on the intact midcervical vagi. Blood pressure effects evoked by an intravenous neurotensin occur outside vagal and CSNs pathways and might result from activation of the peripheral vascular NTS(1) receptors.

  3. Relations Between Autonomous Motivation and Leisure-Time Physical Activity Participation: The Mediating Role of Self-Regulation Techniques.

    Science.gov (United States)

    Nurmi, Johanna; Hagger, Martin S; Haukkala, Ari; Araújo-Soares, Vera; Hankonen, Nelli

    2016-04-01

    This study tested the predictive validity of a multitheory process model in which the effect of autonomous motivation from self-determination theory on physical activity participation is mediated by the adoption of self-regulatory techniques based on control theory. Finnish adolescents (N = 411, aged 17-19) completed a prospective survey including validated measures of the predictors and physical activity, at baseline and after one month (N = 177). A subsample used an accelerometer to objectively measure physical activity and further validate the physical activity self-report assessment tool (n = 44). Autonomous motivation statistically significantly predicted action planning, coping planning, and self-monitoring. Coping planning and self-monitoring mediated the effect of autonomous motivation on physical activity, although self-monitoring was the most prominent. Controlled motivation had no effect on self-regulation techniques or physical activity. Developing interventions that support autonomous motivation for physical activity may foster increased engagement in self-regulation techniques and positively affect physical activity behavior. PMID:27390147

  4. Stat3 activation of NF-κB p100 processing involves CBP/p300-mediated acetylation

    OpenAIRE

    Nadiminty, Nagalakshmi; Lou, Wei; Lee, Soo Ok; Lin, Xin; Trump, Donald L.; Gao, Allen C.

    2006-01-01

    Activation of the noncanonical NF-κB signaling pathway involved in the proteolytic processing of NF-κB p100 to p52 is tightly regulated, and overproduction of p52 leads to lymphocyte hyperplasia and transformation. We have demonstrated that active but not latent Stat3, expressed in many types of human cancers involved in cell proliferation and survival, induces p100 processing to p52 by activation of IKKα and subsequent phosphorylation of p100. The Stat3-mediated p100 processing to p52 requir...

  5. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2015-01-30

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  6. Physical activity as a mediator of the impact of chronic conditions on quality of life in older adults

    Directory of Open Access Journals (Sweden)

    Miller William C

    2007-12-01

    Full Text Available Abstract Background Chronic conditions could negatively affect the quality of life of older adults. This may be partially due to a relative lack of physical activity. We examined whether physical activity mediates the relationship between different chronic conditions and several health outcomes that are important to the quality of life of older adults. Methods The data were taken from the Canadian Community Health Survey (cycle 1.1, a cross-section survey completed in 2001. Only respondents who were 65 years or older were included in our study (N = 22,432. The Health Utilities Index Mark 3 (HUI3 was used to measure overall quality of life, and to measure selected health outcomes (dexterity, mobility, pain, cognition, and emotional wellbeing that are considered to be of importance to the quality of life of older adults. Leisure-time physical activity was assessed by determining weekly energy expenditure (Kcal per week based on the metabolic equivalents of self-reported leisure activities. Linear and logistic regression models were used to determine the mediating effect of leisure-time physical activity while controlling for demographic variables (age and sex, substance use (tobacco use and alcohol consumption, and obesity. Results Having a chronic condition was associated with a relative decrease in health utility scores and a relative increase in mobility limitations, dexterity problems, pain, emotional problems (i.e., decreased happiness, and cognitive limitations. These negative consequences could be partially attributed to a relative lack of physical activity in older adults with a chronic condition (14% mediation for the HUI3 score. The corresponding degree of mediation was 18% for mobility limitations, 5% for pain, and 13% for emotional wellbeing (statistically significant mediation was not observed for the other health attributes. These values varied with respect to the different chronic conditions examined in our study. Conclusion Older

  7. Procarcinogenic effects of cyclosporine A are mediated through the activation of TAK1/TAB1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Jianmin; Walsh, Stephanie B.; Verney, Zoe M. [Department of Dermatology, University of Alabama at Birmingham, AL 35294 (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, Bethesda, MD (United States); Elmets, Craig A. [Department of Dermatology, University of Alabama at Birmingham, AL 35294 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, AL 35294 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology, University of Alabama at Birmingham, AL 35294 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, AL 35294 (United States)

    2011-05-13

    Research highlights: {yields} Organ transplant recipients are highly susceptible to early skin cancer development. {yields} CsA-mediated TGFB1-dependent TAK1/TAB1 signaling augments invasive tumor growth. {yields} CsA enhances accumulation of upstream kinases, ZMP, AMPK and IRAK to activate TAK1. {yields} TAK1 mediates enhanced proliferation and reduced apoptosis via CsA-dependent NF{kappa}B. -- Abstract: Cyclosporine A (CsA) is an immunosuppressive drug commonly used for maintaining chronic immune suppression in organ transplant recipients. It is known that patients receiving CsA manifest increased growth of aggressive non-melanoma skin cancers. However, the underlying mechanism by which CsA augments tumor growth is not fully understood. Here, we show that CsA augments the growth of A431 epidermoid carcinoma xenograft tumors by activating tumor growth factor {beta}-activated kinase1 (TAK1). The activation of TAK1 by CsA occurs at multiple levels by kinases ZMP, AMPK and IRAK. TAK1 forms heterodimeric complexes with TAK binding protein 1 and 2 (TAB1/TAB2) which in term activate nuclear factor {kappa}B (NF{kappa}B) and p38 MAP kinase. Transcriptional activation of NF{kappa}B is evidenced by IKK{beta}-mediated phosphorylation-dependent degradation of I{kappa}B and consequent nuclear translocation of p65. This also leads to enhancement in the expression of its transcriptional target genes cyclin D1, Bcl2 and COX-2. Similarly, activation of p38 leads to enhanced inflammation-related signaling shown by increased phosphorylation of MAPKAPK2 and which in turn phosphorylates its substrate HSP27. Activation of both NF{kappa}B and p38 MAP kinase provide mitogenic stimuli to augment the growth of SCCs.

  8. MicroRNA-122 down-regulation is involved in phenobarbital-mediated activation of the constitutive androstane receptor.

    Directory of Open Access Journals (Sweden)

    Ryota Shizu

    Full Text Available Constitutive androstane receptor (CAR is a nuclear receptor that regulates the transcription of target genes, including CYP2B and 3A. Phenobarbital activates CAR, at least in part, in an AMP-activated protein kinase (AMPK-dependent manner. However, the precise mechanisms underlying phenobarbital activation of AMPK are still unclear. In the present study, it was demonstrated that phenobarbital administration to mice decreases hepatic miR-122, a liver-enriched microRNA involved in both hepatic differentiation and function. The time-course change in the phenobarbital-mediated down-regulation of miR-122 was inversely correlated with AMPK activation. Phenobarbital decreased primary miR-122 to approximately 25% of the basal level as early as 1 h and suppressed transactivity of mir-122 promoter in HuH-7 cells, suggesting that the down-regulation occurred at the transcriptional level. AMPK activation by metformin or 5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside had no evident effect on miR-122 levels. An inhibitory RNA specific for miR-122 increased activated AMPK and CAR-mediated trancactivation of the phenobarbital-responsive enhancer module in HepG2 cells. Conversely, the reporter activity induced by the ectopic CAR was almost completely suppressed by co-transfection with the miR-122 mimic RNA. GFP-tagged CAR was expressed in the cytoplasm in addition to the nucleus in the majority of HuH-7 cells in which miR-122 was highly expressed. Co-transfection of the mimic or the inhibitor RNA for miR-122 further increased or decreased, respectively, the number of cells that expressed GFP-CAR in the cytoplasm. Taken together, these results suggest that phenobarbital-mediated down-regulation of miR-122 is an early and important event in the AMPK-dependent CAR activation and transactivation of its target genes.

  9. MicroRNA-122 down-regulation is involved in phenobarbital-mediated activation of the constitutive androstane receptor.

    Science.gov (United States)

    Shizu, Ryota; Shindo, Sawako; Yoshida, Takemi; Numazawa, Satoshi

    2012-01-01

    Constitutive androstane receptor (CAR) is a nuclear receptor that regulates the transcription of target genes, including CYP2B and 3A. Phenobarbital activates CAR, at least in part, in an AMP-activated protein kinase (AMPK)-dependent manner. However, the precise mechanisms underlying phenobarbital activation of AMPK are still unclear. In the present study, it was demonstrated that phenobarbital administration to mice decreases hepatic miR-122, a liver-enriched microRNA involved in both hepatic differentiation and function. The time-course change in the phenobarbital-mediated down-regulation of miR-122 was inversely correlated with AMPK activation. Phenobarbital decreased primary miR-122 to approximately 25% of the basal level as early as 1 h and suppressed transactivity of mir-122 promoter in HuH-7 cells, suggesting that the down-regulation occurred at the transcriptional level. AMPK activation by metformin or 5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside had no evident effect on miR-122 levels. An inhibitory RNA specific for miR-122 increased activated AMPK and CAR-mediated trancactivation of the phenobarbital-responsive enhancer module in HepG2 cells. Conversely, the reporter activity induced by the ectopic CAR was almost completely suppressed by co-transfection with the miR-122 mimic RNA. GFP-tagged CAR was expressed in the cytoplasm in addition to the nucleus in the majority of HuH-7 cells in which miR-122 was highly expressed. Co-transfection of the mimic or the inhibitor RNA for miR-122 further increased or decreased, respectively, the number of cells that expressed GFP-CAR in the cytoplasm. Taken together, these results suggest that phenobarbital-mediated down-regulation of miR-122 is an early and important event in the AMPK-dependent CAR activation and transactivation of its target genes. PMID:22815988

  10. Procarcinogenic effects of cyclosporine A are mediated through the activation of TAK1/TAB1 signaling pathway

    International Nuclear Information System (INIS)

    Research highlights: → Organ transplant recipients are highly susceptible to early skin cancer development. → CsA-mediated TGFB1-dependent TAK1/TAB1 signaling augments invasive tumor growth. → CsA enhances accumulation of upstream kinases, ZMP, AMPK and IRAK to activate TAK1. → TAK1 mediates enhanced proliferation and reduced apoptosis via CsA-dependent NFκB. -- Abstract: Cyclosporine A (CsA) is an immunosuppressive drug commonly used for maintaining chronic immune suppression in organ transplant recipients. It is known that patients receiving CsA manifest increased growth of aggressive non-melanoma skin cancers. However, the underlying mechanism by which CsA augments tumor growth is not fully understood. Here, we show that CsA augments the growth of A431 epidermoid carcinoma xenograft tumors by activating tumor growth factor β-activated kinase1 (TAK1). The activation of TAK1 by CsA occurs at multiple levels by kinases ZMP, AMPK and IRAK. TAK1 forms heterodimeric complexes with TAK binding protein 1 and 2 (TAB1/TAB2) which in term activate nuclear factor κB (NFκB) and p38 MAP kinase. Transcriptional activation of NFκB is evidenced by IKKβ-mediated phosphorylation-dependent degradation of IκB and consequent nuclear translocation of p65. This also leads to enhancement in the expression of its transcriptional target genes cyclin D1, Bcl2 and COX-2. Similarly, activation of p38 leads to enhanced inflammation-related signaling shown by increased phosphorylation of MAPKAPK2 and which in turn phosphorylates its substrate HSP27. Activation of both NFκB and p38 MAP kinase provide mitogenic stimuli to augment the growth of SCCs.

  11. The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Xiong, Lingxin; Xie, Jingshu; Song, Chenxue; Liu, Jinping; Zheng, Jingtong; Liu, Chuangui; Zhang, Xiaotian; Li, Pingya; Wang, Fang

    2015-01-01

    Epidemiological studies have verified the critical role that antioxidative stress plays in protecting vascular endothelial cells. The aims of the present study were to investigate the antioxidative activities and differential regulation of nuclear erythroid-related factor 2- (Nrf2-) mediated gene expression by Xueshuan Xinmaining Tablet (XXT), a traditional Chinese medicine with the effect of treating cardiovascular diseases. The antioxidative activities of XXT were investigated using quantitative real-time PCR (qPCR), a PCR array, and western blotting. Our results indicated that XXT exhibited potent antioxidative activities by suppressing the levels of hydrogen peroxide- (H2O2-) induced reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs). We were also conscious of strong Nrf2-mediated antioxidant induction. XXT enhanced the expressions of Keap1, Nrf2, and Nrf2-mediated genes, such as glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H: quinine oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), and glutathione peroxidase (GPX) in HUVECs. In summary, XXT strongly activated Nrf2 and its downstream regulated genes, which may contribute to the antioxidative and vascular endothelial cell protective activities of XXT. PMID:26681964

  12. EGF AND TGF-α motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms

    International Nuclear Information System (INIS)

    EGF and TGF-α induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70S6K participating in EGF signalling and phospholipase Cγ in TGF-α signalling. We additionally demonstrate that EGF and TGF-α motogenic activities may be resolved into two stages: (a) cell 'activation' by a transient exposure to either cytokine, and (b) the subsequent 'manifestation' of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-α requires EGFR and integrin αvβ3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-α. In contrast, the mitogenic activities of EGF and TGF-α are independent of CD44 and αvβ3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-α pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity

  13. ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells.

    Science.gov (United States)

    Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R

    2009-11-30

    Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

  14. Role of ROS in Aβ42 Mediated Activation of Cerebral Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Andrey Tsoy

    2014-12-01

    Full Text Available Introduction. There is substantial evidence that the deposition of aggregated amyloid-beta peptide (Aβ in brain parenchyma and brain vessels is the main cause of neuronal dysfunction and death in Alzheimer’s disease (AD. Aβ exhibits multiple cytotoxic effects on neurons and glial cells and causes dysfunction of the blood brain barrier (BBB. In AD brains, an increased deposition of Aβ in the cerebral vasculature has been found to be correlated with increased transmigration of blood-borne inflammatory cells and neurovascular inflammation. However, regulatory mediators of these processes remain to be elucidated. In this study, we examined the role of ROS in actin polymerization and expression of adhesion molecules (P-selectin on the surface of the cerebral endothelial cells (CECs that are activated by Aβ42.Materials and methods. Mouse BEnd3 line (ATCC was used in this research. BEnd3 cells respond to Aβ treatment similarly to human primary CECs and are a common model to investigate CECs’ function. We used immortalized bEnd3 cells as the following: controls; cells incubated with Aβ42 for 10, 30, and 60 minutes; cells incubated with 30 mM of antioxidant N-acetylcysteine (NAC for 1 hr; and, cells pre-treated with NAC followed by Aβ42 exposure. We measured DHE fluorescence to investigate intracellular ROS production. Immunofluorescent microscopy of anti-P-selectin and oregon green phalloidin was used to quantify the surface P-selectin expression and actin polymerization, and Western blot analysis was used to analyze total P-selectin expression.Results. The results of this study have demonstrated a significant time-dependent ROS accumulation after 10 minutes, 30 minutes, and 60 minutes of Aβ42 treatment, while Aβ42 stimulated ROS production in CECs was attenuated by pre-treatment with the NAC antioxidant. We also found that Aβ42 increased P-selectin fluorescence at the surface of bEnd3 cells in a time dependent manner in parallel to ROS

  15. Inhibition of bleomycin-induced pulmonary fibrosis by nordihydroguaiaretic acid. The role of alveolar macrophage activation and mediator production.

    OpenAIRE

    Phan, S. H.; Kunkel, S L

    1986-01-01

    The role of alveolar macrophage activation and release of mediators remains unclear. In this study, this role is examined with respect to the effects of relatively selective inhibitors of arachidonate metabolism on the pathogenesis of pulmonary fibrosis. CBA/J mice were administered bleomycin (0.037 units) endotracheally to induce pulmonary fibrosis. Daily intraperitoneal injections of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA) inhibited pulmonary fibrosis in a dose-dependent ...

  16. TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4

    OpenAIRE

    Malik, Sohail; Wallberg, Annika E.; Kang, Yun Kyoung; Roeder, Robert G.

    2002-01-01

    The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regu...

  17. Cryptic tetracycline resistance determinant (class F) from Bacteroides fragilis mediates resistance in Escherichia coli by actively reducing tetracycline accumulation.

    OpenAIRE

    Park, B. H.; Hendricks, M; Malamy, M H; Tally, F P; Levy, S. B.

    1987-01-01

    Escherichia coli bearing a cryptic tetracycline resistance determinant from Bacteroides fragilis expressed low-level constitutive resistance to tetracycline under aerobic, but not anaerobic, growth conditions and accumulated less tetracycline aerobically than did isogenic susceptible cells. This decreased uptake was energy dependent and reversible by increased concentrations of tetracycline, suggesting a saturable carrier-mediated active efflux mechanism. Decreased uptake was not seen when th...

  18. Impact of aspirin dose on adenosine diphosphate-mediated platelet activities. Results of an in vitro pilot investigation.

    Science.gov (United States)

    Tello-Montoliu, Antonio; Thano, Estela; Rollini, Fabiana; Patel, Ronakkumar; Wilson, Ryan E; Muñiz-Lozano, Ana; Franchi, Francesco; Darlington, Andrew; Desai, Bhaloo; Guzman, Luis A; Bass, Theodore A; Angiolillo, Dominick J

    2013-10-01

    Different aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel's active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (paspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 μM: p=0.888; 3 μM: p=0.524; 10 μM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (paspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators. PMID:23884248

  19. Skullcap (Scutellaria baicalensis) Extract and Its Active Compound, Wogonin, Inhibit Ovalbumin-Induced Th2-Mediated Response

    OpenAIRE

    Hee Soon Shin; Min-Jung Bae; Dae Woon Choi; Dong-Hwa Shon

    2014-01-01

    Skullcap (Scutellaria baicalensis) has been widely used as a dietary ingredient and traditional herbal medicine owing to its anti-inflammatory and anticancer properties. In this study, we investigated the anti-allergic effects of skullcap and its active compounds, focusing on T cell-mediated responses ex vivo and in vivo. Splenocytes from mice sensitized with ovalbumin (OVA) were isolated for analyses of cytokine production and cell viability. Mice sensitized with OVA were orally administere...

  20. Carbon nanotube-mediated delivery of nucleic acids does not result in non-specific activation of B lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Cai Dong [Department of Biology, Boston College, Chestnut Hill, MA 02467 (United States); Doughty, Cheryl A [Department of Biology, Boston College, Chestnut Hill, MA 02467 (United States); Potocky, Terra B [Department of Biology, Boston College, Chestnut Hill, MA 02467 (United States); Dufort, Fay J [Department of Biology, Boston College, Chestnut Hill, MA 02467 (United States); Huang Zhongping [NanoLab, Incorporated, Newton, MA 02458 (United States); Blair, Derek [Department of Biology, Boston College, Chestnut Hill, MA 02467 (United States); Kempa, Krzysztof [Department of Physics, Boston College, Chestnut Hill, MA 02467 (United States); Ren, Z F [Department of Physics, Boston College, Chestnut Hill, MA 02467 (United States); Chiles, Thomas C [Department of Biology, Boston College, Chestnut Hill, MA 02467 (United States)

    2007-09-12

    The efficient delivery of genes and proteins into primary mammalian cells and tissues has represented a formidable challenge. Recent advances in the research of carbon nanotubes (CNTs) offer much promise for their use as delivery platforms into mammalian cells. Ideally, CNT-mediated applications should not result in cellular toxicity nor perturb cellular homeostasis (e.g., result in non-specific activation of primary cells). It is therefore critical to evaluate the impact of CNT exposure on the cellular metabolism, proliferation and survival of primary mammalian cells. We investigated the compatibility of a recently developed CNT-mediated delivery method, termed nanospearing, with primary ex vivo cultures of B lymphocytes. Several parameters were evaluated to assess the impact of CNTs on naive B lymphocytes, including cell survival, activation, proliferation and intracellular signal transduction. Our results indicate that nanospearing does not result in the activation of naive primary B lymphocytes nor alter survival in ex vivo cultures. Herein, B cells exposed to CNTs were capable of responding to extrinsic pro-survival signals such as interleukin-4 and signaling by the B-cell antigen receptor in a manner similar to that of B cells cultured in the absence of CNTs. Our study demonstrates the biocompatibility of the CNT-mediated nanospearing procedure with respect to primary B lymphocytes.

  1. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    Energy Technology Data Exchange (ETDEWEB)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun, E-mail: hirayama.dbio@mri.tmd.ac.jp; Nishina, Hiroshi, E-mail: nishina.dbio@mri.tmd.ac.jp

    2014-01-17

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.

  2. Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi.

    Science.gov (United States)

    Wong, Tania A; Fairn, Gregory D; Poon, Pak P; Shmulevitz, Maya; McMaster, Christopher R; Singer, Richard A; Johnston, Gerald C

    2005-09-01

    The budding yeast Saccharomyces cerevisiae contains a family of Arf (ADP-ribosylation factor) GTPase activating protein (GAP) proteins with the Gcs1 + Age2 ArfGAP pair providing essential overlapping function for the movement of transport vesicles from the trans-Golgi network. We have generated a temperature-sensitive but stable version of the Gcs1 protein that is impaired only for trans-Golgi transport and find that deleterious effects of this enfeebled Gcs1-4 mutant protein are relieved by increased gene dosage of the gcs1-4 mutant gene itself or by the SFH2 gene (also called CSR1), encoding a phosphatidylinositol transfer protein (PITP). This effect was not seen for the SEC14 gene, encoding the founding member of the yeast PITP protein family, even though the Gcs1 and Age2 ArfGAPs are known to be downstream effectors of Sec14-mediated activity for trans-Golgi transport. Sfh2-mediated suppression of inadequate Gcs1-4 function depended on phospholipase D, whereas inadequate Gcs1-4 activity was relieved by increasing levels of diacylglycerol (DAG). Recombinant Gcs1 protein was found to bind certain phospholipids but not DAG. Our findings favor a model of Gcs1 localization through binding to specific phospholipids and activation of ArfGAP activity by DAG-mediated membrane curvature as the transport vesicle is formed. Thus, ArfGAPs are subject to both temporal and spatial regulation that is facilitated by Sfh2-mediated modulation of the lipid environment. PMID:16126894

  3. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1

    Directory of Open Access Journals (Sweden)

    Matthew J. Randall

    2013-01-01

    Full Text Available Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal. Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1, a critical enzyme involved in regulation of thioredoxin (Trx-mediated redox signaling, by alkylation at its selenocysteine (Sec residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases

  4. Hot-electron-mediated surface chemistry: toward electronic control of catalytic activity.

    Science.gov (United States)

    Park, Jeong Young; Kim, Sun Mi; Lee, Hyosun; Nedrygailov, Ievgen I

    2015-08-18

    Energy dissipation at surfaces and interfaces is mediated by excitation of elementary processes, including phonons and electronic excitation, once external energy is deposited to the surface during exothermic chemical processes. Nonadiabatic electronic excitation in exothermic catalytic reactions results in the flow of energetic electrons with an energy of 1-3 eV when chemical energy is converted to electron flow on a short (femtosecond) time scale before atomic vibration adiabatically dissipates the energy (in picoseconds). These energetic electrons that are not in thermal equilibrium with the metal atoms are called "hot electrons". The detection of hot electron flow under atomic or molecular processes and understanding its role in chemical reactions have been major topics in surface chemistry. Recent studies have demonstrated electronic excitation produced during atomic or molecular processes on surfaces, and the influence of hot electrons on atomic and molecular processes. We outline research efforts aimed at identification of the intrinsic relation between the flow of hot electrons and catalytic reactions. We show various strategies for detection and use of hot electrons generated by the energy dissipation processes in surface chemical reactions and photon absorption. A Schottky barrier localized at the metal-oxide interface of either catalytic nanodiodes or hybrid nanocatalysts allows hot electrons to irreversibly transport through the interface. We show that the chemicurrent, composed of hot electrons excited by the surface reaction of CO oxidation or hydrogen oxidation, correlates well with the turnover rate measured separately by gas chromatography. Furthermore, we show that hot electron flows generated on a gold thin film by photon absorption (or internal photoemission) can be amplified by localized surface plasmon resonance. The influence of hot charge carriers on the chemistry at the metal-oxide interface are discussed for the cases of Au, Ag, and Pt

  5. Epinephrine-induced activation of LW-mediated sickle cell adhesion and vaso-occlusion in vivo

    OpenAIRE

    Zennadi, Rahima; Moeller, Benjamin J; Whalen, Erin J.; Batchvarova, Milena; Xu, Ke; Shan, Siqing; Delahunty, Martha; Dewhirst, Mark W.; Telen, Marilyn J.

    2007-01-01

    Sickle red cell (SS RBC) adhesion is believed to contribute to the process of vaso-occlusion in sickle cell disease (SCD). We previously found that the LW RBC adhesion receptor can be activated by epinephrine to mediate SS RBC adhesion to endothelial αvβ3 integrin. To determine the contribution of LW activation to vaso-occlusive events in vivo, we investigated whether in vitro treatment of SS RBCs by epinephrine resulted in vaso-occlusion in intact microvasculature after RBC infusion into nud...

  6. Receptor-mediated activation of gastric vagal afferents by glucagon-like peptide-1 in the rat

    DEFF Research Database (Denmark)

    Bucinskaite, V; Tolessa, T; Pedersen, J;

    2009-01-01

    The vagus nerve plays a role in mediating effects of the two glucagon-like peptides GLP-1 and GLP-2 on gastrointestinal growth, functions and eating behaviour. To obtain electrophysiological and molecular evidence for the contribution of afferent pathways in chemoreception from the gastrointestinal...... tract, afferent mass activity in the ventral gastric branch of the vagus nerve and gene expression of GLP-1 receptors and GLP-2 receptors in the nodose ganglion were examined in Sprague-Dawley rats. Intravenous administration of GLP-1 (30-1000 pmol kg(-1)), reaching high physiological plasma...... concentrations, increased vagal afferent mass activity peaking (13-52% above basal level, P

  7. Sucrose-fueled, energy dissipative, transient formation of molecular hydrogels mediated by yeast activity

    OpenAIRE

    Angulo Pachón, César Augusto; Miravet Celades, Juan Felipe

    2016-01-01

    A biologically mediated, energy dissipative, reversible formation of fibrillar networks is reported. The process of gelation is linked to sucrose-fueled production of CO2 by baker’s yeast (Saccharomyces cerevisiae). Continuous fueling of the system is required to maintain the self-assembled fibrillar network.

  8. Using a Peer-Mediated Intervention To Facilitate Children's Participation in Inclusive Childcare Activities.

    Science.gov (United States)

    Robertson, Jo; Green, Kim; Alper, Sandra; Schloss, Patrick J.; Kohler, Frank

    2003-01-01

    A study examined the impact of a peer-mediated intervention consisting of songs and finger plays, attention to photographs of desired behavior, and peer verbal cues, on two preschoolers with developmental delays. The intervention resulted in increases in engaged and on-task behavior, interactive play, and appropriate participation in circle/story…

  9. Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids

    Science.gov (United States)

    Limbourg, Florian P.; Huang, Zhihong; Plumier, Jean-Christophe; Simoncini, Tommaso; Fujioka, Masayuki; Tuckermann, Jan; Schütz, Günther; Moskowitz, Michael A.; Liao, James K.

    2002-01-01

    Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS–/– mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF. PMID:12464678

  10. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  11. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    Science.gov (United States)

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation.

  12. HBXIP and LSD1 Scaffolded by lncRNA Hotair Mediate Transcriptional Activation by c-Myc.

    Science.gov (United States)

    Li, Yinghui; Wang, Zhen; Shi, Hui; Li, Hang; Li, Leilei; Fang, Runping; Cai, Xiaoli; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2016-01-15

    c-Myc is regarded as a transcription factor, but the basis for its function remains unclear. Here, we define a long noncoding RNA (lncRNA)/protein complex that mediates the transcriptional activation by c-Myc in breast cancer cells. Among 388 c-Myc target genes in human MCF-7 breast cancer cells, we found that their promoters could be occupied by the oncoprotein HBXIP. We confirmed that the HBXIP expression correlated with expression of the c-Myc target genes cyclin A, eIF4E, and LDHA. RNAi-mediated silencing of HBXIP abolished c-Myc-mediated upregulation of these target genes. Mechanistically, HBXIP interacted directly with c-Myc through the leucine zippers and recruited the lncRNA Hotair along with the histone demethylase LSD1, for which Hotair serves as a scaffold. Silencing of HBXIP, Hotair, or LSD1 was sufficient to block c-Myc-enhanced cancer cell growth in vitro and in vivo. Taken together, our results support a model in which the HBXIP/Hotair/LSD1 complex serves as a critical effector of c-Myc in activating transcription of its target genes, illuminating long-standing questions on how c-Myc drives carcinogenesis.

  13. Suppression of Ultraviolet B Exposure-Mediated Activation of NF-κB in Normal Human Keratinocytes by Resveratrol

    Directory of Open Access Journals (Sweden)

    Vaqar Mustafa Adhami

    2003-01-01

    Full Text Available Chemoprevention by naturally occurring agents is a newer dimension in the management of neoplasia, including skin cancer. Solar ultraviolet (UV radiation is the major cause of skin cancer. We recently demonstrated that resveratrol (3,5,4'-trihydroxystilbene, a polyphenolic antioxidant found in grapes and red wine, imparts protection from UVB-mediated cutaneous damages in SKH-1 hairless mice. The mechanism of action of resveratrol is not clearly understood. Here, we investigated the involvement of nuclear factor kappa B (NF-κB, which is known to play a critical role in skin biology and the development of skin cancer, as the mechanism of chemoprevention of UV damage by resveratrol. In the normal human epidermal keratinocytes, resveratrol blocked UVB-mediated (40 mJ/cm2 activation of NF-κB in a dose-dependent (5, 10, and 25μM resveratrol for 24 hours as well as time-dependent (5μ/M resveratrol for 12, 24, and 48 hours fashion. Resveratrol treatment of keratinocytes also inhibited UVB-mediated 1 phosphorylation and degradation of IκBα, and 2 activation of IKKα. We suggest that NF-κB pathway plays a critical role in the chemopreventive effects of resveratrol against the adverse effects of UV radiation including photocarcinogenesis.

  14. Natural Environments and Childhood Experiences Promoting Physical Activity, Examining the Mediational Effects of Feelings about Nature and Social Networks

    Science.gov (United States)

    Calogiuri, Giovanna

    2016-01-01

    The importance of natural environments (NEs) for physical activity (PA) has been studied extensively. However, there is scant evidence to explain the motivational processes underlying the NE-PA relation. The aim of this study was to investigate the NE-PA relation using an ecological framework, focusing on perception of NEs, childhood experiences and possible intra- and inter-individual mediators. Data were retrieved from a cross-sectional survey among 2168 adults from all over Norway. In addition, the coverage of NEs by municipalities was retrieved from national registers. Logistic regression showed that, unlike the self-reported proximity to NEs, higher ratings of perceived supportiveness of NEs for PA predicted participation in NE-based PA for at least 60 min/week or 150 min/week, before and after controlling for socio-demographic characteristics. Reporting frequent experiences in nature during childhood was also an important predictor of higher levels of NE-based PA. Furthermore, a mediational analysis showed that the effect of both predictors was mediated by “feelings about nature” and “social networks”. These findings indicate that to encourage the use of local NE for PA, not only should environmental perceptions be taken into account, positive feelings towards nature alongside opportunities to share activity in nature with others should also be promoted. PMID:27110802

  15. HBXIP and LSD1 Scaffolded by lncRNA Hotair Mediate Transcriptional Activation by c-Myc.

    Science.gov (United States)

    Li, Yinghui; Wang, Zhen; Shi, Hui; Li, Hang; Li, Leilei; Fang, Runping; Cai, Xiaoli; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2016-01-15

    c-Myc is regarded as a transcription factor, but the basis for its function remains unclear. Here, we define a long noncoding RNA (lncRNA)/protein complex that mediates the transcriptional activation by c-Myc in breast cancer cells. Among 388 c-Myc target genes in human MCF-7 breast cancer cells, we found that their promoters could be occupied by the oncoprotein HBXIP. We confirmed that the HBXIP expression correlated with expression of the c-Myc target genes cyclin A, eIF4E, and LDHA. RNAi-mediated silencing of HBXIP abolished c-Myc-mediated upregulation of these target genes. Mechanistically, HBXIP interacted directly with c-Myc through the leucine zippers and recruited the lncRNA Hotair along with the histone demethylase LSD1, for which Hotair serves as a scaffold. Silencing of HBXIP, Hotair, or LSD1 was sufficient to block c-Myc-enhanced cancer cell growth in vitro and in vivo. Taken together, our results support a model in which the HBXIP/Hotair/LSD1 complex serves as a critical effector of c-Myc in activating transcription of its target genes, illuminating long-standing questions on how c-Myc drives carcinogenesis. PMID:26719542

  16. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination

    Science.gov (United States)

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P.; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M.; Reddy, Janardan K.; Borggrefe, Tilman; Skok, Jane A.

    2016-01-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. PMID:26903242

  17. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    Science.gov (United States)

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. PMID:26903242

  18. Sirtuin 1 (SIRT1 activation mediates sildenafil induced delayed cardioprotection against ischemia-reperfusion injury in mice.

    Directory of Open Access Journals (Sweden)

    Mona Shalwala

    Full Text Available BACKGROUND: It has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL protects against myocardial ischemia/reperfusion (I-R injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury. OBJECTIVE/HYPOTHESIS: We tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1. METHODS: Adult male ICR mice were treated with SIL (0.7 mg/kg, i.p., Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control, or saline (0.2 mL. The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis. RESULTS: Both SIL- and RSV-treated mice had increased cardiac SIRT1 activity (P<0.001 as compared to the saline-treated controls 24 hours after drug treatment. In isolated ventricular cardiomyocytes, pretreatment with SIL (1 µM or RSV (1 µM for one hour in vitro also upregulated SIRT1 activity (P<0.05. We further examined the causative relationship between SIRT1 activation and SIL-induced late cardioprotection. Pretreatment with SIL (or RSV 24 hours prior to 30 min ischemia and 24 hours of reperfusion significantly reduced infarct size, which was associated with a significant increase in SIRT1 activity (P<0.05. Moreover, sirtinol (a SIRT1 inhibitor, 5 mg/kg, i.p. given 30 min before I-R blunted the infarct-limiting effect of SIL and RSV (P<0.001. CONCLUSION: Our study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications.

  19. Effectiveness of group-mediated lifestyle physical activity (glpa) program for health benefit in physical activity among elderly people at rural thailand

    International Nuclear Information System (INIS)

    Elderly population is considered as a vulnerable group and prone to develop multiple medical problems. This aging population is rapidly increasing in developing countries especially in Thailand. Methods: This study was a quasi-experimental study to evaluate the effectiveness of Group-Mediated Lifestyle Physical Activity program on change health benefit in physical activity among elderly people by using validated and reliable Global Physical Activity Questionnaire-GPAQv2. The study was conducted in Phranakhonsiayutthaya district, Ayutthaya province due to its population being the second highest elderly in the Central Region of Thailand. A total of 102 persons of age 60 and over who could read and write Thai language were selected purposively. However, 52 elderly were enrolled in the intervention group and 50 were enrolled for the control group. General Linear Model repeated-measures ANOVA was used to evaluate the effects of Group-Mediated Lifestyle Physical Activity (GLPA) program on change health benefit in physical activity among elderly. Results: Overall health benefit at baseline were similar between intervention and control group and found statistically non-significant with p-value 0.638 (>0.05). However, the mean score of health benefit was 23.21 ± 29.23 in intervention group and 20.74 ± 23.18 in control group. One third of participants of intervention group had not found health benefit due to physical activity while in control group this number was more than half. After elderly received Group-Mediated Lifestyle Physical Activity program intervention for 6 month found significant statistical differences as compared with mean score at baseline (health benefit 6 month, intervention group =40.7 ± 34.28 and control group =4.56 ± 8.79). Conclusion: The effect of Group-Mediated Lifestyle Physical Activity program change intervention was statistically significant in health benefit after intervention program between intervention and control group. Our study

  20. Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

    Directory of Open Access Journals (Sweden)

    Bost Kenneth L

    2003-04-01

    Full Text Available Abstract Background Staphylococcus aureus infection of normal osteoblasts induces expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL. Results Normal osteoblasts were incubated in the presence of purified bacterial products over a range of concentrations. Results demonstrate that purified surface structures and a selected superantigen present in the extracellular environment are not capable of inducing TRAIL expression by osteoblasts. Osteoblasts were co-cultured with S. aureus at various multiplicities of infection utilizing cell culture chamber inserts. Results of those experiments suggest that direct contact between bacteria and osteoblasts is necessary for optimal TRAIL induction. Finally, S. aureus infection of osteoblasts in the presence of anti-TRAIL antibody demonstrates that TRAIL mediates caspase-8 activation and apoptosis of infected cells. Conclusions Collectively, these findings suggest a mechanism whereby S. aureus mediates bone destruction via induction of osteoblast apoptosis.

  1. Synthesis and characterization of Eichhornia-mediated copper oxide nanoparticles and assessing their antifungal activity against plant pathogens

    Indian Academy of Sciences (India)

    P VANATHI; P RAJIV; RAJESHWARI SIVARAJ

    2016-09-01

    In this paper, we report the biosynthesis and characterization of copper oxide nanoparticles from an aquatic noxious weed, Eichhornia crassipes by green chemistry approach. The aim of this work is to synthesize copper oxide nanoparticles by simple, cost-effective and ecofriendly method as an alternative to other available techniques. The synthesized copper oxide nanoparticles were characterized by UV–visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscopy (FESEM) and Energy dispersive X-ray spectroscopy (EDX) analyses. The synthesized particles were highly stable, spherical in shape with an average diameter of $28\\pm 4$ nm. The synthesized nanoparticles were then explored to antifungal activity against plant pathogens. Highest zone of inhibition were observed in 100 $\\mu$g ml$^{−1}$ of Eichhornia-mediated copper oxide nanoparticle against Fusarium culmorum and Aspergillus niger. This Eichhornia-mediated copper oxide nanoparticles wereproved to be good antifungal agents against plant fungal pathogens.

  2. The relationship between transformational teaching and adolescent physical activity: the mediating roles of personal and relational efficacy beliefs.

    Science.gov (United States)

    Bourne, Jessica; Liu, Yan; Shields, Christopher A; Jackson, Ben; Zumbo, Bruno D; Beauchamp, Mark R

    2015-02-01

    The purpose of this study was to examine the extent to which transformational teaching, exhibited by secondary school physical education teachers, predicts within-class physical activity and leisure-time physical activity among adolescents. The study used a prospective observational design and involved data collected from 874 Grade 10 adolescents (M age = 15.41, (SD) = .61). Through use of structural equation modeling, the results revealed that adolescents' perceptions of transformational teaching were positively related to within-class physical activity and leisure-time physical activity, and these effects were mediated by adolescents' estimation of their teacher's confidence in their abilities (i.e. relation-inferred self-efficacy) and self-efficacy beliefs.

  3. Activation of estrogen response elements is mediated both via estrogen and muscle contractions in rat skeletal muscle myotubes

    DEFF Research Database (Denmark)

    Wiik, A.; Hellsten, Ylva; Berthelson, P.;

    2009-01-01

    The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction...... then differentiated into myotubes and subjected to either estrogen or electrical stimulation. Activation of the ERE sequence was determined by measurement of luciferase activity. The results show that both ERalpha and ERbeta are expressed in myotubes from rats. Both estrogen stimulation and muscle contraction...... increased (P muscle contraction. Use of ER antagonists showed that, whereas the estrogen-induced transactivation is mediated via ERs, the effect of muscle contraction...

  4. p53 Transcriptional activity is mediated through the SRC1-interacting domain of CBP/p300.

    Science.gov (United States)

    Livengood, Jill A; Scoggin, Kirsten E S; Van Orden, Karen; McBryant, Steven J; Edayathumangalam, Rajeswari S; Laybourn, Paul J; Nyborg, Jennifer K

    2002-03-15

    The tumor suppressor p53 recruits the cellular coactivator CBP/p300 to mediate the transcriptional activation of target genes. In this study, we identify a novel p53-interacting region in CBP/p300, which we call CR2, located near the carboxyl terminus. The 95-amino acid CR2 region (amino acids 2055--2150) is located adjacent to the C/H3 domain and corresponds precisely with the minimal steroid receptor coactivator 1 (SRC1)-interacting domain of CBP (also called IBiD). We show that the region of p53 that participates in the CR2 interaction resides within the first 107 amino acids of the protein. p53 binds strongly to the CR2 domain of both CBP and the highly homologous coactivator p300. Importantly, an in-frame deletion of CR2 within the full-length p300 protein strongly compromises p300-mediated p53 transcriptional activation from a chromatin template in vitro. The identification of the p53-interacting CR2 domain in CBP/p300 prompted us to ask if the human T-cell leukemia virus (HTLV-I) Tax protein, which also interacts with CR2, competes with p53 for binding to this domain. We show that p53 and Tax exhibit mutually exclusive binding to the CR2 region, possibly contributing to the previously reported Tax repression of p53 function. Together, these studies identify and molecularly characterize a new p53 binding site on CBP/p300 that participates in coactivator-mediated p53 transcription function. The identity of the p53.CR2 interaction indicates that at least three distinct sites on CBP/p300 may participate in mediating p53 transactivation. PMID:11782467

  5. TGFβ1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

    Science.gov (United States)

    Zhou, Xiaolai; Zöller, Tanja; Krieglstein, Kerstin; Spittau, Björn

    2015-07-01

    Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor β1 (TGFβ1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFβ1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFβ1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFβ1 treatment. Finally, we demonstrate that TGFβ1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFβ1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor β1 (TGFβ1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFβ1

  6. In Vivo Mechanical Loading Rapidly Activates β–catenin Signaling in Osteocytes through a Prostaglandin Mediated Mechanism

    Science.gov (United States)

    Lara-Castillo, N; Kim-Weroha, NA; Kamel, MA; Javaheri, B; Ellies, DL; Krumlauf, RE; Thiagarajan, G; Johnson, ML

    2015-01-01

    The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in the in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1 hour post loading, spreading to additional osteocytes and finally to cells on the bone surface by 24 hrs. This early activation at 1 hour appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly down regulated in osteocytes in vivo at 24 hours post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo. PMID:25836764

  7. In vivo mechanical loading rapidly activates β-catenin signaling in osteocytes through a prostaglandin mediated mechanism.

    Science.gov (United States)

    Lara-Castillo, N; Kim-Weroha, N A; Kamel, M A; Javaheri, B; Ellies, D L; Krumlauf, R E; Thiagarajan, G; Johnson, M L

    2015-07-01

    The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1h post-loading, spreading to additional osteocytes and finally to cells on the bone surface by 24h. This early activation at 1h appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly downregulated in osteocytes in vivo at 24h post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo. PMID:25836764

  8. Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy.

    Directory of Open Access Journals (Sweden)

    Sung Won Lee

    Full Text Available Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence

  9. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

  10. The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release.

    Science.gov (United States)

    Kola, Blerina; Wittman, Gábor; Bodnár, Ibolya; Amin, Faisal; Lim, Chung Thong; Oláh, Márk; Christ-Crain, Mirjam; Lolli, Francesca; van Thuijl, Hinke; Leontiou, Chrysanthia A; Füzesi, Tamás; Dalino, Paolo; Isidori, Andrea M; Harvey-White, Judith; Kunos, George; Nagy, György M; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-12-01

    This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

  11. Positive reinforcement mediated by midbrain dopamine neurons requires D1 and D2 receptor activation in the nucleus accumbens.

    Directory of Open Access Journals (Sweden)

    Elizabeth E Steinberg

    Full Text Available The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS, a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc, a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.

  12. Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS.

    Science.gov (United States)

    Huang, William Y C; Yan, Qingrong; Lin, Wan-Chen; Chung, Jean K; Hansen, Scott D; Christensen, Sune M; Tu, Hsiung-Lin; Kuriyan, John; Groves, Jay T

    2016-07-19

    The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

  13. Antitumor activities and on-target toxicities mediated by a TRAIL receptor agonist following cotreatment with panobinostat.

    Science.gov (United States)

    Martin, Ben P; Frew, Ailsa J; Bots, Michael; Fox, Stephen; Long, Fenella; Takeda, Kazuyoshi; Yagita, Hideo; Atadja, Peter; Smyth, Mark J; Johnstone, Ricky W

    2011-06-01

    The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti-TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti-mouse TRAIL receptor antibody MD5-1. We demonstrate that the combination of panobinostat and MD5-1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on-target MD5-1-mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5-1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimens.

  14. Differential sensitivity of myosin-heavy-chain-typed fibers to distinct aggregates of nerve-mediated activation.

    Science.gov (United States)

    Dunn, S E; Michel, R N

    1999-02-01

    We studied the regulatory effects of nerve-mediated activity on the early expression of embryonic and adult myosin heavy chains (MHC) within inactive though still innervated rat plantaris and soleus muscle fibers. To this end, we stimulated motor nerves that were quiescent following treatment with tetrodotoxin (TTX) with paradigms designed to partition the influence of neural activation frequency and assessed the selective expression and accumulation of MHCs within muscle fibers using an array of specific antibodies. We show rapid de novo expression of IIx MHC within select soleus fibers in response to high-frequency activation for more than 0.01% of daily time. High-frequency aggregates were also the most effective in preventing the TTX-induced reexpression of embryonic MHCs within specific fibers. Only configurations that included high-frequency trains for more than 0.01% of daily time or combined with 10 Hz stimulation preserved the size of select fibers, used as a measure of the net cellular content of MHC. The effectiveness of this preservation varied according to the muscle type and MHC expressed, and, in a subset of fibers, was influenced by contractile loading status. Our results demonstrate that distinct subsets of MHC-typed fibers are differentially sensitive to the neural activation cues mediating the cellular expression of these proteins.

  15. Positive reinforcement mediated by midbrain dopamine neurons requires D1 and D2 receptor activation in the nucleus accumbens.

    Science.gov (United States)

    Steinberg, Elizabeth E; Boivin, Josiah R; Saunders, Benjamin T; Witten, Ilana B; Deisseroth, Karl; Janak, Patricia H

    2014-01-01

    The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.

  16. Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Spegel, Christer; Kostesha, Natalie;

    2009-01-01

    ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing...... either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in...... vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric...

  17. A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

    OpenAIRE

    Leonard, Joshua N.; Hung, Michelle E.

    2016-01-01

    Extracellular vesicles (EVs) mediate intercellular communication through transfer of RNA and protein between cells. Thus, understanding how cargo molecules are loaded and delivered by EVs is of central importance for elucidating the biological roles of EVs and developing EV-based therapeutics. While some motifs modulating the loading of biomolecular cargo into EVs have been elucidated, the general rules governing cargo loading and delivery remain poorly understood. To investigate how general ...

  18. Labelling of endogenous target protein via N-S acyl transfer-mediated activation of N-sulfanylethylanilide.

    Science.gov (United States)

    Denda, Masaya; Morisaki, Takuya; Kohiki, Taiki; Yamamoto, Jun; Sato, Kohei; Sagawa, Ikuko; Inokuma, Tsubasa; Sato, Youichi; Yamauchi, Aiko; Shigenaga, Akira; Otaka, Akira

    2016-07-14

    The ligand-dependent incorporation of a reporter molecule (e.g., fluorescence dye or biotin) onto a endogenous target protein has emerged as an important strategy for elucidating protein function using various affinity-based labelling reagents consisting of reporter, ligand and reactive units. Conventional labelling reagents generally use a weakly activated reactive unit, which can result in the non-specific labelling of proteins in a ligand-independent manner. In this context, the activation of a labelling reagent through a targeted protein-ligand interaction could potentially overcome the problems associated with conventional affinity-based labelling reagents. We hypothesized that this type of protein-ligand-interaction-mediated activation could be accomplished using N-sulfanylethylanilide (SEAlide) as the reactive unit in the labelling reagent. Electrophilically unreactive amide-type SEAlide can be activated by its conversion to the corresponding active thioester in the presence of a phosphate salt, which can act as an acid-base catalyst. It has been suggested that protein surfaces consisting of hydrophilic residues such as amino, carboxyl and imidazole groups could function as acid-base catalysts. We therefore envisioned that a SEAlide-based labelling reagent (SEAL) bearing SEAlide as a reactive unit could be activated through the binding of the SEAL with a target protein. Several SEALs were readily prepared in this study using standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase protocols. These SEAL systems were subsequently applied to the ligand-dependent labelling of human carbonic anhydrase (hCA) and cyclooxyganese 1. Although we have not yet obtained any direct evidence for the target protein-mediated activation of the SEAlide unit, our results for the reaction of these SEALs with hCA1 or butylamine indirectly support our hypothesis. The SEALs reported in this study represent valuable new entries to the field of affinity-based labelling reagents

  19. Use of serum C-reactive protein as an early marker of inflammatory activity in canine type II immune-mediated polyarthritis: case report

    OpenAIRE

    Kristensen Annemarie T; Jessen Lisbeth; Houser Geoffrey A; Jensen Asger; Kjelgaard-Hansen Mads

    2006-01-01

    Abstract Background Monitoring systemic inflammatory activity during steroid therapy of canine immune-mediated polyarthritis (IMPA) is difficult and mainly relies on clinical signs. Case presentation Canine serum C-reactive protein (CRP) was measured serially and blinded during a 27-week follow-up period of a case of Anaplasma phagocytophilia induced type II immune-mediated polyarthritis. Conclusion WBC was, as expected, observed not to reflect the inflammatory activity during steroid treatme...

  20. mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration.

    Science.gov (United States)

    Deng, Zhili; Lei, Xiaohua; Zhang, Xudong; Zhang, Huishan; Liu, Shuang; Chen, Qi; Hu, Huimin; Wang, Xinyue; Ning, Lina; Cao, Yujing; Zhao, Tongbiao; Zhou, Jiaxi; Chen, Ting; Duan, Enkui

    2015-02-01

    Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration.

  1. mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration.

    Science.gov (United States)

    Deng, Zhili; Lei, Xiaohua; Zhang, Xudong; Zhang, Huishan; Liu, Shuang; Chen, Qi; Hu, Huimin; Wang, Xinyue; Ning, Lina; Cao, Yujing; Zhao, Tongbiao; Zhou, Jiaxi; Chen, Ting; Duan, Enkui

    2015-02-01

    Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration. PMID:25609845

  2. Sri Lankan black tea (Camellia sinensis L.) inhibits the methylglyoxal mediated protein glycation and potentiates its reversing activity in vitro

    Institute of Scientific and Technical Information of China (English)

    Wanigasekara Daya Ratnasooriya

    2016-01-01

    Objective: To evaluate inhibitory activity of methylglyoxal (MGO) mediated protein glycation and ability to potentiate its reversing activity and range of antioxidant properties of Sri Lankan low grown orthodox orange pekoe grade black tea. Methods: Freeze dried black tea brew (BTB) was used as the sample in this study. Anti-glycation and glycation reversing activity was studied in bovine serum albumin (BSA)-MGO model. Antioxidant properties were studied using total polyphenolic content, total flavonoid content, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, 1,1-diphenyl-2-picrylhydrazine and ferric reducing antioxidant power in vitro antioxidant assays. Results: The results demonstrated significant (P Conclusions: The novel properties observed for Sri Lankan orange pekoe grade black tea indicate its usefulness as a supplementary beverage in managing MGO and advanced glycation end products related diseases and ailments.

  3. Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway.

    Science.gov (United States)

    Zhang, Qian-Qian; Zhou, Da-Lei; Lei, Yan; Zheng, Li; Chen, Sheng-Xia; Gou, Hong-Ju; Gu, Qu-Liang; He, Xiao-Dong; Lan, Tian; Qi, Cui-Ling; Li, Jiang-Chao; Ding, Yan-Qing; Qiao, Liang; Wang, Li-Jing

    2015-02-20

    Slit2 is often overexpressed in cancers. Slit2 is a secreted protein that binds to Roundabout (Robo) receptors to regulate cell growth and migration. Here, we employed several complementary mouse models of intestinal cancers, including the Slit2 transgenic mice, the ApcMin/+ spontaneous intestinal adenoma mouse model, and the DMH/DSS-induced colorectal carcinoma model to clarify function of Slit2/Robo1 signaling in intestinal tumorigenesis. We showed that Slit2 and Robo1 are overexpressed in intestinal tumors and may contribute to tumor generation. The Slit2/Robo1 signaling can induce precancerous lesions of the intestine and tumor progression. Ectopic expression of Slit2 activated Slit2/Robo1 signaling and promoted tumorigenesis and tumor growth. This was mediated in part through activation of the Src signaling, which then down-regulated E-cadherin, thereby activating Wnt/β-catenin signaling. Thus, Slit2/Robo1 signaling is oncogenic in intestinal tumorigenesis.

  4. Anticancer Activity of γ-Bisabolene in Human Neuroblastoma Cells via Induction of p53-Mediated Mitochondrial Apoptosis

    Directory of Open Access Journals (Sweden)

    Yu-Jen Jou

    2016-05-01

    Full Text Available γ-Bisabolene has demonstrated antiproliferative activities against several human cancer cell lines. This study first discloses the antiproliferative and apoptosis induction activities of γ-bisabolene to human neuroblastoma TE671 cells. A CC50 value of γ-bisabolene was 8.2 μM to TE671 cells. Cell cycle analysis with PI staining showed γ-bisabolene elevating the sub-G1 fractions in a time-dependent manner. In addition, annexin V-FITC/PI staining showed γ-bisabolene significantly triggering early (annexin-V positive/PI negative and late (annexin-V positive/PI positive apoptosis in dose-dependent manners. γ-Bisabolene induced caspase 3/8/9 activation, intracellular ROS increase, and mitochondrial membrane potential decrease in apoptosis of human neuro-blastoma cells. Moreover, γ-bisabolene increased p53 phosphorylation and up-regulated p53-mediated apoptotic genes Bim and PUMA, as well as decreased the mRNA and protein levels of CK2α. Notably, the results indicated the involvement of CK2α-p53 pathways in mitochondria-mediated apoptosis of human neuroblastoma cells treated with γ-bisabolene. This study elucidated the apoptosis induction pathways of γ-bisabolene-treated neuroblastoma cells, in which could be useful for developing anti-neuroblastoma drugs.

  5. Ex Vivo Antioxidant Activity of Selected Medicinal Plants against Fenton Reaction-Mediated Oxidation of Biological Lipid Substrates

    Directory of Open Access Journals (Sweden)

    Namratha Pai Kotebagilu

    2015-01-01

    Full Text Available Free radical-mediated oxidation is often linked to various degenerative diseases. Biological substrates with lipids as major components are susceptible to oxygen-derived lipid peroxidation due to their composition. Lipid peroxide products act as biomarkers in evaluating the antioxidant potential of various plants and functional foods. The study focused on evaluation of the antioxidant potential of two extracts (methanol and 80% methanol of four medicinal plants, Andrographis paniculata, Costus speciosus, Canthium parviflorum, and Abrus precatorius, against Fenton reaction-mediated oxidation of three biological lipid substrates; cholesterol, low-density lipoprotein, and brain homogenate. The antioxidant activity of the extracts was measured by thiobarbituric acid reactive substances method. Also, the correlation between the polyphenol, flavonoid content, and the antioxidant activity in biological substrates was analyzed. Results indicated highest antioxidant potential by 80% methanol extract of Canthium parviflorum (97.55%, methanol extract of Andrographis paniculata (72.15%, and methanol extract of Canthium parviflorum (49.55% in cholesterol, low-density lipoprotein, and brain, respectively. The polyphenol and flavonoid contents of methanol extract of Andrographis paniculata in cholesterol (r=0.816 and low-density lipoprotein (r=0.948 and Costus speciosus in brain (r=0.977, polyphenols, and r=0.949, flavonoids correlated well with the antioxidant activity. The findings prove the antioxidant potential of the selected medicinal plants against Fenton reaction in biological lipid substrates.

  6. Ex Vivo Antioxidant Activity of Selected Medicinal Plants against Fenton Reaction-Mediated Oxidation of Biological Lipid Substrates.

    Science.gov (United States)

    Pai Kotebagilu, Namratha; Reddy Palvai, Vanitha; Urooj, Asna

    2015-01-01

    Free radical-mediated oxidation is often linked to various degenerative diseases. Biological substrates with lipids as major components are susceptible to oxygen-derived lipid peroxidation due to their composition. Lipid peroxide products act as biomarkers in evaluating the antioxidant potential of various plants and functional foods. The study focused on evaluation of the antioxidant potential of two extracts (methanol and 80% methanol) of four medicinal plants, Andrographis paniculata, Costus speciosus, Canthium parviflorum, and Abrus precatorius, against Fenton reaction-mediated oxidation of three biological lipid substrates; cholesterol, low-density lipoprotein, and brain homogenate. The antioxidant activity of the extracts was measured by thiobarbituric acid reactive substances method. Also, the correlation between the polyphenol, flavonoid content, and the antioxidant activity in biological substrates was analyzed. Results indicated highest antioxidant potential by 80% methanol extract of Canthium parviflorum (97.55%), methanol extract of Andrographis paniculata (72.15%), and methanol extract of Canthium parviflorum (49.55%) in cholesterol, low-density lipoprotein, and brain, respectively. The polyphenol and flavonoid contents of methanol extract of Andrographis paniculata in cholesterol (r = 0.816) and low-density lipoprotein (r = 0.948) and Costus speciosus in brain (r = 0.977, polyphenols, and r = 0.949, flavonoids) correlated well with the antioxidant activity. The findings prove the antioxidant potential of the selected medicinal plants against Fenton reaction in biological lipid substrates. PMID:26933511

  7. Mitochondrial reactive oxygen species-mediated NLRP3 inflammasome activation contributes to aldosterone-induced renal tubular cells injury.

    Science.gov (United States)

    Ding, Wei; Guo, Honglei; Xu, Chengyan; Wang, Bin; Zhang, Minmin; Ding, Feng

    2016-04-01

    Aldosterone (Aldo) is an independent risk factor for chronic kidney disease (CKD), and although Aldo directly induces renal tubular cell injury, the underlying mechanisms remain unclear. NLRP3 inflammasome and mitochondrial reactive oxygen species (ROS) have recently been implicated in various kinds of CKD. The present study hypothesized that mitochondrial ROS and NLRP3 inflammasome mediated Aldo-induced tubular cell injury. The NLRP3 inflammasome is induced by Aldo in a dose- and time-dependent manner, as evidenced by increased NLRP3, ASC, caspase-1, and downstream cytokines, such as interleukin (IL)-1β and IL-18. The activation of the NLRP3 inflammasome was significantly prevented by the selective mineralocorticoid receptor (MR) antagonist eplerenone (EPL) (P < 0.01). Mice harboring genetic knock-out of NLRP3 (NLRP3(-/-)) showed decreased maturation of renal IL-1β and IL-18, reduced renal tubular apoptosis, and improved renal epithelial cell phenotypic alternation, and attenuated renal function in response to Aldo-infusion. In addition, mitochondrial ROS was also increased in Aldo-stimulated HK-2 cells, as assessed by MitoSOXTM red reagent. Mito-Tempo, the mitochondria-targeted antioxidant, significantly decreased HK-2 cell apoptosis, oxidative stress, and the activation of NLRP3 inflammasome. We conclude that Aldo induces renal tubular cell injury via MR dependent, mitochondrial ROS-mediated NLRP3 inflammasome activation. PMID:27014913

  8. Mitochondrial reactive oxygen species-mediated NLRP3 inflammasome activation contributes to aldosterone-induced renal tubular cells injury

    Science.gov (United States)

    Ding, Wei; Guo, Honglei; Xu, Chengyan; Wang, Bin; Zhang, Minmin; Ding, Feng

    2016-01-01

    Aldosterone (Aldo) is an independent risk factor for chronic kidney disease (CKD), and although Aldo directly induces renal tubular cell injury, the underlying mechanisms remain unclear. NLRP3 inflammasome and mitochondrial reactive oxygen species (ROS) have recently been implicated in various kinds of CKD. The present study hypothesized that mitochondrial ROS and NLRP3 inflammasome mediated Aldo–induced tubular cell injury. The NLRP3 inflammasome is induced by Aldo in a dose- and time-dependent manner, as evidenced by increased NLRP3, ASC, caspase-1, and downstream cytokines, such as interleukin (IL)-1β and IL-18. The activation of the NLRP3 inflammasome was significantly prevented by the selective mineralocorticoid receptor (MR) antagonist eplerenone (EPL) (P < 0.01). Mice harboring genetic knock-out of NLRP3 (NLRP3−/−) showed decreased maturation of renal IL-1β and IL-18, reduced renal tubular apoptosis, and improved renal epithelial cell phenotypic alternation, and attenuated renal function in response to Aldo-infusion. In addition, mitochondrial ROS was also increased in Aldo-stimulated HK-2 cells, as assessed by MitoSOXTM red reagent. Mito-Tempo, the mitochondria-targeted antioxidant, significantly decreased HK-2 cell apoptosis, oxidative stress, and the activation of NLRP3 inflammasome. We conclude that Aldo induces renal tubular cell injury via MR dependent, mitochondrial ROS-mediated NLRP3 inflammasome activation. PMID:27014913

  9. Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria.

    Directory of Open Access Journals (Sweden)

    Kyler B Pallister

    Full Text Available In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.

  10. Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome

    Science.gov (United States)

    Cao, Zhongying; Zhou, Yaqin; Zhu, Shengli; Feng, Jian; Chen, Xueyuan; Liu, Shi; Peng, Nanfang; Yang, Xiaodan; Xu, Gang; Zhu, Ying

    2016-01-01

    When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling. PMID:26906558

  11. Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

    Institute of Scientific and Technical Information of China (English)

    SugaT

    2002-01-01

    PPAR(peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.

  12. Effects of Pioglitazone Mediated Activation of PPAR-γ on CIDEC and Obesity Related Changes in Mice

    OpenAIRE

    Shamsi, Bilal Haider; Ma, Chaofeng; Naqvi, Saima; Xiao, Yanfeng

    2014-01-01

    Objective Obesity is a metabolic disorder that can lead to high blood pressure, increased blood cholesterol and triglycerides, insulin resistance, and diabetes mellitus. The aim was to study the effects of pioglitazone mediated sensitization of peroxisome proliferator-activated receptor gamma (PPAR-γ) on the relationship of Cell death-inducing DFFA-like effector C (CIDEC) with obesity related changes in mice. Methods Sixty C57B/L6 mice weighing 10–12g at 3 weeks of age were randomly divided i...

  13. In Vivo Mechanical Loading Rapidly Activates β–catenin Signaling in Osteocytes through a Prostaglandin Mediated Mechanism

    OpenAIRE

    Lara-Castillo, N; Kim-Weroha, NA; Kamel, MA; Javaheri, B; Ellies, DL; Krumlauf, RE; Thiagarajan, G.; Johnson, ML

    2015-01-01

    The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in the in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1 hour post loading, spreading to addition...

  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. Tanshinone IIA Prevents Leu27IGF-II-Induced Cardiomyocyte Hypertrophy Mediated by Estrogen Receptor and Subsequent Akt Activation.

    Science.gov (United States)

    Weng, Yueh-Shan; Wang, Hsueh-Fang; Pai, Pei-Ying; Jong, Gwo-Ping; Lai, Chao-Hung; Chung, Li-Chin; Hsieh, Dennis Jine-Yuan; HsuanDay, Cecilia; Kuo, Wei-Wen; Huang, Chih-Yang

    2015-01-01

    IGF-IIR plays important roles as a key regulator in myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound in Danshen and is structurally similar to 17[Formula: see text]-estradiol (E[Formula: see text]. However, whether tanshinone IIA improves cardiomyocyte survival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Akt phosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequent NFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced the cell size and suppressed ANP and BNP, inhibiting antihypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed by ICI. Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3K inhibitor), and PI3K siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activating the PI3K/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATC3. Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective ER modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy. PMID:26621443

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

    International Nuclear Information System (INIS)

    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

  17. Amino acid 1-209 is essential for PDX-1-mediated repression of human CMV IE promoter activity

    Institute of Scientific and Technical Information of China (English)

    Jing CHEN; Lei CHEN; Ge LI; Lu CHENG; Yin HUANG; Jia-xin ZHANG; Wei-wei FAN; Da-ru LU

    2006-01-01

    Aim: To explore the different roles of pancreatic duodenal homeobox factors-1 (PDX-1) domains in PDX-1 mediated repression of human cytomegalovirus immediately early (CMV IE) promoter. Methods: A series of truncated PDX-1 mutants were constructed. The binding of PDX-1 and CMV IE promoter was identified by electrophoretic mobility shift assay (EMSA). The dual-reporter assay was applied to examine the repression activities of PDX-1 mutants on CMV IE promoter. In addition, RNAi technology was used to specifically knock down the endogenous PDX-1 expression. Results: The reporter assay indicated that compared to the mock controls (pEGFP-N2), overexpression of PDX-1 resulted in a 41% decrease of CMV IE promoter activity in the 293 cells (P<0.05) and 43% decrease in HeLa cells (P<0.05), and the repression levels of various truncated mutants played on CMV IE promoter were different. Specific knock down of the endogenous PDX-1 expression significantly restored the activity of CMV IE promoter. EMS A demonstrated that domain 3 is necessary for nuclear localization and DNA binding activity of PDX-1. However, binding of PDX-1 alone to CMV IE promoter was not sufficient to inhibit its transcriptional activity, and other domains of PDX-1 presented were also required. Conclusion: Our data suggested that the DNA binding activity of PDX-1 domain 3 and the cooperative binding of PDX-1 domain 1/2 with other proteins were required for PDX-1 mediated repression of CMV IE promoter.

  18. Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages.

    Science.gov (United States)

    Varì, Rosaria; D'Archivio, Massimo; Filesi, Carmelina; Carotenuto, Simona; Scazzocchio, Beatrice; Santangelo, Carmela; Giovannini, Claudio; Masella, Roberta

    2011-05-01

    Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH(2)-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 μM) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences. PMID:20621462

  19. Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Reavey, Caitlin T; Hickman, Mark J; Dobi, Krista C; Botstein, David; Winston, Fred

    2015-10-01

    Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.

  20. Physical Activity and Health Perception in Aging: Do Body Mass and Satisfaction Matter? A Three-Path Mediated Link

    Science.gov (United States)

    Capranica, Laura; Stager, Joel; Forte, Roberta; Falbo, Simone; Di Baldassarre, Angela; Segura-Garcia, Cristina; Pesce, Caterina

    2016-01-01

    Although ageing people could benefit from healthy diet and physical activity to maintain health and quality of life, further understandings of the diet- and physical activity-related mechanisms that may cause changes in health and quality of life perception are necessary. The purpose of the study was to investigate the effect of eating attitudes, body mass and image satisfaction, and exercise dependence in the relationship between physical activity and health and quality of life perception in older individuals. Hundred and seventy-nine late middle-aged, (55–64 yrs), young-old (65–74 yrs), and old (75–84 yrs) senior athletes (n = 56), physically active (n = 58) or sedentary adults (n = 65) were submitted to anthropometric evaluations (body mass, height) and self-reported questionnaires: Body Image Dimensional Assessment, Exercise Dependence Scale, Eating Attitude Test, and Short Form Health Survey (Physical Component Summary [PCS] and Mental Component Summary [MCS] of and health and quality of life perception). Senior athletes, physically active, and sedentary participants subgroups differed (Pathletes showed, compared to their sedentary counterparts, further differences (Pmental health. The novelty of the three-path mediated link between physical activity level and mental health perception suggests that the beneficial effect of a physically active lifestyle on weight control can positively impinge on the cognitive-emotional dimension of mental health by ensuring the maintenance, also at older age, of a satisfactory body image. PMID:27611689

  1. Notch activation is dispensable for D, L-sulforaphane-mediated inhibition of human prostate cancer cell migration.

    Directory of Open Access Journals (Sweden)

    Eun-Ryeong Hahm

    Full Text Available D, L-Sulforaphane (SFN, a synthetic racemic analog of broccoli constituent L-sulforaphane, is a highly promising cancer chemopreventive agent with in vivo efficacy against chemically-induced as well as oncogene-driven cancer in preclinical rodent models. Cancer chemopreventive effect of SFN is characterized by G(2/M phase cell cycle arrest, apoptosis induction, and inhibition of cell migration and invasion. Moreover, SFN inhibits multiple oncogenic signaling pathways often hyperactive in human cancers, including nuclear factor-κB, Akt, signal transducer and activator of transcription 3, and androgen receptor. The present study was designed to determine the role of Notch signaling, which is constitutively active in many human cancers, in anticancer effects of SFN using prostate cancer cells as a model. Exposure of human prostate cancer cells (PC-3, LNCaP, and/or LNCaP-C4-2B to SFN as well as its naturally-occurring thio-, sulfinyl-, and sulfonyl-analogs resulted in cleavage (activation of Notch1, Notch2, and Notch4, which was accompanied by a decrease in levels of full-length Notch forms especially at the 16- and 24-hour time points. The SFN-mediated cleavage of Notch isoforms was associated with its transcriptional activation as evidenced by RBP-Jk-, HES-1A/B- and HEY-1 luciferase reporter assays. Migration of PC-3 and LNCaP cells was decreased significantly by RNA interference of Notch1 and Notch2, but not Notch4. Furthermore, SFN-mediated inhibition of PC-3 and LNCaP cell migration was only marginally affected by knockdown of Notch1 and Notch2. Strikingly, SFN administration to Transgenic Adenocarcinoma of Mouse Prostate transgenic mice failed to increase levels of cleaved Notch1, cleaved Notch2, and HES-1 proteins in vivo in prostatic intraepithelial neoplasia, well-differentiated carcinoma or poorly-differentiated prostate cancer lesions. These results indicate that Notch activation is largely dispensable for SFN-mediated inhibition of cell

  2. APPL1-mediated activation of STAT3 contributes to inhibitory effect of adiponectin on hepatic gluconeogenesis.

    Science.gov (United States)

    Ding, Youming; Zhang, Deling; Wang, Bin; Zhang, Yemin; Wang, Lei; Chen, Xiaoyan; Li, Mingxin; Tang, Zhao; Wang, Changhua

    2016-09-15

    Adiponectin has been shown to suppress hepatic gluconeogenesis. However, the signaling pathways underlying its action remain ill-defined. The purpose of this study was to examine the potential role of APPL1 in mediating anti-gluconeogenic ability of adiponectin. Primary hepatocytes were isolated from male C57BL/6 mice. Western blot and RT-PCR were performed to detect protein expression and mRNA level, respectively. The protein-protein association was determined by immunoprecipitation and GST pull-down assay. We found that APPL1 protein levels were negatively associated with expressions of proteins and mRNAs of gluconeogenesis enzymes under stimulation with adiponectin. In addition, adiponectin-stimulated STAT3 phosphorylation and acetylation were positively regulated by APPL1 and negative regulated by SirT1. Pharmacological and genetic inhibition of STAT3 mitigated impact of adiponectin on hepatic gluconeogenesis. Furthermore, adiponectin administration facilitated the binding of APPL1 to SirT1 and suppressed the association of SirT1 with STAT3. Taken together, our study showed that APPL1-SirT1-STAT3 pathway mediated adiponectin signaling in primary hepatocytes. This new finding provides a novel mechanism by which adiponectin suppresses hepatic gluconeogenesis. PMID:27246173

  3. 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 level and would prove helpful in understanding and incorporating mechanisms associated with nitrosative stress responses in plants. PMID:27446194

  4. Mechanisms mediating renal sympathetic nerve activation in obesity-related hypertension.

    Science.gov (United States)

    Chen, W; Leo, S; Weng, C; Yang, X; Wu, Y; Tang, X

    2015-04-01

    Excessive renal sympathetic nerve activation may be one of the mechanisms underlying obesity-related hypertension. Impaired baroreflex sensitivity, adipokine disorders-such as leptin, adiponectin, and resistin-activation of the renin-angiotensin system, hyperinsulinemia, insulin resistance, and renal sodium retention present in obesity increase renal sympathetic nerve activity, thus contributing to the development of hypertension. Renal sympathetic denervation reduces both renal sympathetic activity and blood pressure in patients with obesity-related hypertension. PMID:24609799

  5. Emotion Recognition with Eigen Features of Frequency Band Activities Embedded in Induced Brain Oscillations Mediated by Affective Pictures.

    Science.gov (United States)

    Aydin, Serap; Demirtaş, Serdar; Ateş, Kahraman; Tunga, M Alper

    2016-05-01

    In this study, singular spectrum analysis (SSA) has been used for the first time in order to extract emotional features from well-defined electroencephalography (EEG) frequency band activities (BAs) so-called delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-16 Hz), beta (16-32 Hz), gamma (32-64 Hz). These five BAs were estimated by applying sixth-level multi-resolution wavelet decomposition (MRWD) with Daubechies wavelets (db-8) to single channel nonaveraged emotional EEG oscillations of 6 s for each scalp location over 16 recording sites (Fp1, Fp2, F3, F4, F7, F8, C3, C4, P3, P4, T3, T4, T5, T6, O1, O2). Every trial was mediated by different emotional stimuli which were selected from international affective picture system (IAPS) to induce emotional states such as pleasant (P), neutral (N), and unpleasant (UP). Largest principal components (PCs) of BAs were considered as emotional features and data mining approaches were used for the first time in order to classify both three different (P, N, UP) and two contrasting (P and UP) emotional states for 30 healthy controls. Emotional features extracted from gamma BAs (GBAs) for 16 recording sites provided the high classification accuracies of 87.1% and 100% for classification of three emotional states and two contrasting emotional states, respectively. In conclusion, we found the followings: (1) Eigenspectra of high frequency BAs in EEG are highly sensitive to emotional hemispheric activations, (2) emotional states are mostly mediated by GBA, (3) pleasant pictures induce the higher cortical activation in contrast to unpleasant pictures, (4) contrasting emotions induce opposite cortical activations, (5) cognitive activities are necessary for an emotion to occur. PMID:26971786

  6. Interleukin-6 modulation of intestinal epithelial tight junction permeability is mediated by JNK pathway activation of claudin-2 gene.

    Directory of Open Access Journals (Sweden)

    Rana Al-Sadi

    Full Text Available Defective intestinal epithelial tight junction (TJ barrier has been shown to be a pathogenic factor in the development of intestinal inflammation. Interleukin-6 (IL-6 is a pleiotropic, pro-inflammatory cytokine which plays an important role in promoting inflammatory response in the gut and in the systemic circulation. Despite its key role in mediating variety inflammatory response, the effect of IL-6 on intestinal epithelial barrier remains unclear. The purpose of this study was to investigate the effect of IL-6 on intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved using in-vitro (filter-grown Caco-2 monolayers and in-vivo model (mouse intestinal perfusion systems. Our results indicated that IL-6 causes a site-selective increase in Caco-2 intestinal epithelia TJ permeability, causing an increase in flux of small-sized molecules having molecular radius <4 Å. The size-selective increase in Caco-2 TJ permeability was regulated by protein-specific increase in claudin-2 expression. The IL-6 increase in TJ permeability required activation of JNK signaling cascade. The JNK pathway activation of AP-1 resulted in AP-1 binding to its binding sequence on the claudin-2 promoter region, leading to promoter activation and subsequent increase in claudin-2 gene transcription and protein synthesis and TJ permeability. Our in-vivo mouse perfusion showed that IL-6 modulation of mouse intestinal permeability was also mediated by AP-1 dependent increase in claudin-2 expression. In conclusion, our studies show for the first time that the IL-6 modulation of intestinal TJ permeability was regulated by JNK activation of AP-1 and AP-1 activation of claudin-2 gene.

  7. Positive alcohol expectancies mediate the influence of the behavioral activation system on alcohol use: a prospective path analysis.

    Science.gov (United States)

    Wardell, Jeffrey D; Read, Jennifer P; Colder, Craig R; Merrill, Jennifer E

    2012-04-01

    Gray's (1975, 1987) behavioral activation (BAS) and behavioral inhibition systems (BIS) are thought to underlie sensitivity to reinforcement and punishment, respectively. Consistent with Gray's theory and the Acquired Preparedness model, BAS may facilitate the learning of positive alcohol expectancies (PAEs) over time, leading to increases in drinking. Yet, no prospective tests of this pathway have been reported. The present study investigated whether BAS prospectively predicted PAEs and whether PAEs mediated the association between BAS and subsequent alcohol use. We hypothesized that BAS would influence drinking specifically via enhancement-related PAEs. We also explored the role of BIS in PAEs and drinking. College students (N=557) completed online BAS, PAE, and alcohol use measures in September of their first (T1), second (T2), and third (T3) years of college. We conducted autoregressive path analyses with three BAS subscales and BIS (T1) as predictors, four PAE types (T2) as mediators, and quantity and frequency of drinking (T3) as outcomes. The BAS Fun-Seeking scale was prospectively associated with PAEs, and there was a significant indirect path from Fun-Seeking to alcohol use mediated specifically through activity enhancement PAEs. BIS was positively associated with some PAE types, but did not have indirect effects on drinking. Findings are consistent with both the theory of the BAS and the Acquired Preparedness model, as individuals high on BAS Fun-Seeking may find the rewarding properties of alcohol more reinforcing, leading to stronger enhancement PAEs and increased drinking over time. The prospective design helps establish the temporal association between BAS and alcohol-related learning, and points to the need for prevention efforts that target these at-risk students. PMID:22209025

  8. Role of hypoxia-inducible factor-α in hepatitis-B-virus X protein-mediated MDR1 activation

    International Nuclear Information System (INIS)

    The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1α (HIF-1α) and induced the nuclear translocation of C/EBPβ. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1α siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1α activation, and suggest HIF-1α for the therapeutic target of HBV-mediated chemoresistance

  9. P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

    Directory of Open Access Journals (Sweden)

    Ji Yang eKim

    2015-09-01

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP1 plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE in the distinct brain regions. In addition, P2X7 receptor (P2X7R, an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death. Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths.

  10. GEF-H1 mediated control of NOD1 dependent NF-kappaB activation by Shigella effectors.

    Directory of Open Access Journals (Sweden)

    Atsuko Fukazawa

    2008-11-01

    Full Text Available Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1 mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-kappaB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-kappaB activation by H-Ala-D-gammaGlu-DAP (gammaTriDAP. GEF-H1 is essential for NF-kappaB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens.

  11. Rupestonic acid derivative YZH-106 suppresses influenza virus replication by activation of heme oxygenase-1-mediated interferon response.

    Science.gov (United States)

    Ma, Lin-Lin; Wang, Hui-Qiang; Wu, Ping; Hu, Jin; Yin, Jin-Qiu; Wu, Shuo; Ge, Miao; Sun, Wen-Fang; Zhao, Jiang-Yu; Aisa, Haji Akber; Li, Yu-Huan; Jiang, Jian-Dong

    2016-07-01

    Given the limitation of available antiviral drugs and vaccines, there remains to be a pressing need for novel anti-influenza drugs. Rupestonic acid derivatives were reported to have an anti-influenza virus activity, but their mechanism remains to be elucidated. Herein, we aim to evaluate the antiviral activity of YZH-106, a rupestonic acid derivative, against a broad-spectrum of influenza viruses and to dissect its antiviral mechanisms. Our results demonstrated that YZH-106 exhibited a broad-spectrum antiviral activity against influenza viruses, including drug-resistant strains in vitro. Furthermore, YZH-106 provided partial protection of the mice to Influenza A virus (IAV) infection, as judged by decreased viral load in lungs, improved lung pathology, reduced body weight loss and partial survival benefits. Mechanistically, YZH-106 induced p38 MAPK and ERK1/2 phosphorylation, which led to the activation of erythroid 2-related factor 2 (Nrf2) that up-regulated heme oxygenase-1 (HO-1) expression in addition to other genes. HO-1 inhibited IAV replication by activation of type I IFN expression and subsequent induction of IFN-stimulated genes (ISGs), possibly in a HO-1 enzymatic activity-independent manner. These results suggest that YZH-106 inhibits IAV by up-regulating HO-1-mediated IFN response. HO-1 is thus a promising host target for antiviral therapeutics against influenza and other viral infectious diseases. PMID:27107768

  12. Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity.

    Directory of Open Access Journals (Sweden)

    Sophiya Karki

    Full Text Available The zinc finger antiviral protein (ZAP is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV, the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.

  13. The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis

    Science.gov (United States)

    Pekovic-Vaughan, Vanja; Gibbs, Julie; Yoshitane, Hikari; Yang, Nan; Pathiranage, Dharshika; Guo, Baoqiang; Sagami, Aya; Taguchi, Keiko; Bechtold, David; Loudon, Andrew; Yamamoto, Masayuki; Chan, Jefferson; van der Horst, Gijsbertus T.J.; Fukada, Yoshitaka; Meng, Qing-Jun

    2014-01-01

    The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock “gated” pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic ClockΔ19 mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis. PMID:24637114

  14. Troglitazone, a PPAR-γ activator prevents endothelial cell adhesion molecule expression and lymphocyte adhesion mediated by TNF-α

    Directory of Open Access Journals (Sweden)

    Itoh Makoto

    2005-02-01

    Full Text Available Abstract Background Cytokine mediated induction of the mucosal addressin cell adhesion molecule-1(MAdCAM-1 expression is associated with the onset and progression of inflammatory bowel disease (IBD. Results Using western blotting and cell-based ELISA, we show in this study that troglitazone, an activator of the peroxisome proliferator-activated receptor-γ (PPAR-γ, widely used in the treatment of diabetes, has as well recently been highlighted as protective in models of inflammation and cancer. We found that troglitazone (10–40 μM, significantly reduced the TNF-α (1 ng/ml mediated induction of endothelial MAdCAM-1 in a dose-dependent manner, achieving a 34.7% to 98.4% reduction in induced MAdCAM-1. Trogliazone (20μM reduced TNF-α induced VCAM-1, ICAM-1 and E-selectin expression. Moreover, troglitazone significantly reduced α4β7-integrin dependent lymphocyte adhesion to TNF-α cultured endothelial cells. Conclusions These results suggest that PPAR-γ agonists like troglitazone may be useful in the clinical treatment of IBD.

  15. Parthenolide induces apoptosis by activating the mitochondrial and death receptor pathways and inhibits FAK-mediated cell invasion.

    Science.gov (United States)

    Kwak, Sang Won; Park, Eon Sub; Lee, Chung Soo

    2014-01-01

    The natural product parthenolide induces apoptosis in cancer cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to parthenolide is not clear. In addition, it is unclear whether parthenolide-induced apoptosis is mediated by the formation of reactive oxygen species and the depletion of GSH contents, and the effect of parthenolide on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of parthenolide exposure on apoptosis, cell adhesion, and migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that parthenolide may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of parthenolide appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Parthenolide inhibited fetal bovine serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase-dependent activation of cytoskeletal-associated components. Therefore, parthenolide might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy. PMID:24065392

  16. Palladium- and copper-mediated N-aryl bond formation reactions for the synthesis of biological active compounds

    Directory of Open Access Journals (Sweden)

    Burkhard Koenig

    2011-01-01

    Full Text Available N-Arylated aliphatic and aromatic amines are important substituents in many biologically active compounds. In the last few years, transition-metal-mediated N-aryl bond formation has become a standard procedure for the introduction of amines into aromatic systems. While N-arylation of simple aromatic halides by simple amines works with many of the described methods in high yield, the reactions may require detailed optimization if applied to the synthesis of complex molecules with additional functional groups, such as natural products or drugs. We discuss and compare in this review the three main N-arylation methods in their application to the synthesis of biologically active compounds: Palladium-catalysed Buchwald–Hartwig-type reactions, copper-mediated Ullmann-type and Chan–Lam-type N-arylation reactions. The discussed examples show that palladium-catalysed reactions are favoured for large-scale applications and tolerate sterically demanding substituents on the coupling partners better than Chan–Lam reactions. Chan–Lam N-arylations are particularly mild and do not require additional ligands, which facilitates the work-up. However, reaction times can be very long. Ullmann- and Buchwald–Hartwig-type methods have been used in intramolecular reactions, giving access to complex ring structures. All three N-arylation methods have specific advantages and disadvantages that should be considered when selecting the reaction conditions for a desired C–N bond formation in the course of a total synthesis or drug synthesis.

  17. Skullcap (Scutellaria baicalensis Extract and Its Active Compound, Wogonin, Inhibit Ovalbumin-Induced Th2-Mediated Response

    Directory of Open Access Journals (Sweden)

    Hee Soon Shin

    2014-02-01

    Full Text Available Skullcap (Scutellaria baicalensis has been widely used as a dietary ingredient and traditional herbal medicine owing to its anti-inflammatory and anticancer properties. In this study, we investigated the anti-allergic effects of skullcap and its active compounds, focusing on T cell-mediated responses ex vivo and in vivo. Splenocytes from mice sensitized with ovalbumin (OVA were isolated for analyses of cytokine production and cell viability. Mice sensitized with OVA were orally administered skullcap or wogonin for 16 days, and then immunoglobulin (Ig and cytokine levels were measured by enzyme-linked immunosorbent assays. Treatment with skullcap significantly inhibited interleukin (IL-4 production without reduction of cell viability. Moreover, wogonin, but not baicalin and baicalein, suppressed IL-4 and interferon-gamma production. In vivo, skullcap and wogonin downregulated OVA-induced Th2 immune responses, especially IgE and IL-5 prediction. Wogonin as an active component of skullcap may be applied as a therapeutic agent for IgE- and IL-5-mediated allergic disorders.

  18. Tissue plasminogen activator and plasminogen mediate stress-induced decline of neuronal and cognitive functions in the mouse hippocampus.

    Science.gov (United States)

    Pawlak, Robert; Rao, B S Shankaranarayana; Melchor, Jerry P; Chattarji, Sumantra; McEwen, Bruce; Strickland, Sidney

    2005-12-13

    Repeated stress can impair function in the hippocampus, a brain structure essential for learning and memory. Although behavioral evidence suggests that severe stress triggers cognitive impairment, as seen in major depression or posttraumatic stress disorder, little is known about the molecular mediators of these functional deficits in the hippocampus. We report here both pre- and postsynaptic effects of chronic stress, manifested as a reduction in the number of NMDA receptors, dendritic spines, and expression of growth-associated protein-43 in the cornu ammonis 1 region. Strikingly, the stress-induced decrease in NMDA receptors coincides spatially with sites of plasminogen activation, thereby predicting a role for tissue plasminogen activator (tPA) in this form of stress-induced plasticity. Consistent with this possibility, tPA-/- and plasminogen-/- mice are protected from stress-induced decrease in NMDA receptors and reduction in dendritic spines. At the behavioral level, these synaptic and molecular signatures of stress-induced plasticity are accompanied by impaired acquisition, but not retrieval, of hippocampal-dependent spatial learning, a deficit that is not exhibited by the tPA-/- and plasminogen-/- mice. These findings establish the tPA/plasmin system as an important mediator of the debilitating effects of prolonged stress on hippocampal function at multiple levels of neural organization.

  19. Role of the N-terminal activation domain of coactivator CoCoA in mediating transcriptional activation by β-catenin*

    OpenAIRE

    Yang, Catherine K.; Kim, Jeong Hoon; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is involved in transcriptional activation of target genes by nuclear receptors and the xenobiotic aryl hydrocarbon receptor, as well as target genes of the Wnt signaling pathway, which is mediated by the lymphocyte enhancer factor (LEF)/T cell factor transcription factors and the coactivator β-catenin. The recruitment of CoCoA by nuclear receptors is accomplished by the interaction of the central coiled-coiled domain of CoCoA with p160 coactivators; the C-t...

  20. MD-2 determinants of nickel and cobalt-mediated activation of human TLR4.

    Directory of Open Access Journals (Sweden)

    Alja Oblak

    Full Text Available Recent findings unexpectedly revealed that human TLR4 can be directly activated by nickel ions. This activation is due to the coordination of nickel by a cluster of histidine residues on the ectodomain of human TLR4, which is absent in most other species. We aimed to elucidate the role of MD-2 in the molecular mechanism of TLR4/MD-2 activation by nickel, as nickel binding site on TLR4 is remote from MD-2, which directly binds the endotoxin as the main pathological activator of TLR4. We identified MD-2 and TLR4 mutants which abolished TLR4/MD-2 receptor activation by endotoxin but could nevertheless be significantly activated by nickel, which acts in synergy with LPS. Human TLR4/MD-2 was also activated by cobalt ions, while copper and cadmium were toxic in the tested concentration range. Activation of TLR4 by cobalt required MD-2 and was abolished by human TLR4 mutations of histidine residues at positions 456 and 458. We demonstrated that activation of TLR4 by nickel and cobalt ions can trigger both the MyD88-dependent and the -independent pathway. Based on our results we propose that predominantly hydrophobic interactions between MD-2 and TLR4 contribute to the stabilization of the TLR4/MD-2/metal ion complex in a conformation that enables activation.

  1. Evidence That GABA Mediates Dopaminergic and Serotonergic Pathways Associated with Locomotor Activity in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)

    Science.gov (United States)

    Clements, S.; Schreck, C.B.

    2004-01-01

    The authors examined the control of locomotor activity in juvenile salmon (Oncorhynchus tshawytscha) by manipulating 3 neurotransmitter systems-gamma-amino-n-butyric acid (GABA), dopamine, and serotonin-as well as the neuropeptide corticotropin releasing hormone (CRH). Intracerebroventricular (ICV) injections of CRH and the GABAAagonist muscimol stimulated locomotor activity. The effect of muscimol was attenuated by administration of a dopamine receptor antagonist, haloperidol. Conversely, the administration of a dopamine uptake inhibitor (4???,4??? -difluoro-3-alpha-[diphenylmethoxy] tropane hydrochloride [DUI]) potentiated the effect of muscimol. They found no evidence that CRH-induced hyperactivity is mediated by dopaminergic systems following concurrent injections of haloperidol or DUI with CRH. Administration of muscimol either had no effect or attenuated the locomotor response to concurrent injections of CRH and fluoxetine, whereas the GABAA antagonist bicuculline methiodide potentiated the effect of CRH and fluoxetine.

  2. Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

    Institute of Scientific and Technical Information of China (English)

    Beverly Z Packard; Akira Komoriya

    2008-01-01

    Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

  3. PERK Utilizes Intrinsic Lipid Kinase Activity To Generate Phosphatidic Acid, Mediate Akt Activation, and Promote Adipocyte Differentiation

    Science.gov (United States)

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J.; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A.; Witze, Eric S.

    2012-01-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought. PMID:22493067

  4. PERK utilizes intrinsic lipid kinase activity to generate phosphatidic acid, mediate Akt activation, and promote adipocyte differentiation.

    Science.gov (United States)

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A; Witze, Eric S; Diehl, J Alan

    2012-06-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.

  5. Synthesis and Characterization of Optically Active Fractal Seed Mediated Silver Nickel Bimetallic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Joseph Adeyemi Adekoya

    2014-01-01

    Full Text Available The synthesis of new seed mediated AgNi allied bimetallic nanocomposites was successfully carried out by the successive reduction of the metal ions in diethylene glycol, ethylene glycol, glycerol, and pentaerythritol solutions, with concomitant precipitation of Ag/Ni bimetal sols. The optical measurement revealed the existence of distinct band edge with surface plasmon resonance (SPR in the region of 400–425 nm and excitonic emission with maximum peak at 382 nm which were reminiscent of cluster-in-cluster surface enriched bimetallic silver-nickel sols. The morphological characterization by transmission electron microscopy, high resolution transmission electron microscopy, and X-ray diffraction analyses complimented by surface scan using X-ray photoelectron spectroscopy strongly supported the formation of intimately alloyed face-centered silver/nickel nanoclusters.

  6. 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.

  7. Cool-temperature-mediated activation of phospholipase C-γ2 in the human hereditary disease PLAID.

    Science.gov (United States)

    Schade, Anja; Walliser, Claudia; Wist, Martin; Haas, Jennifer; Vatter, Petra; Kraus, Johann M; Filingeri, Davide; Havenith, George; Kestler, Hans A; Milner, Joshua D; Gierschik, Peter

    2016-09-01

    Deletions in the gene encoding signal-transducing inositol phospholipid-specific phospholipase C-γ2 (PLCγ2) are associated with the novel human hereditary disease PLAID (PLCγ2-associated antibody deficiency and immune dysregulation). PLAID is characterized by a rather puzzling concurrence of augmented and diminished functions of the immune system, such as cold urticaria triggered by only minimal decreases in temperature, autoimmunity, and immunodeficiency. Understanding of the functional effects of the genomic alterations at the level of the affected enzyme, PLCγ2, is currently lacking. PLCγ2 is critically involved in coupling various cell surface receptors to regulation of important functions of immune cells such as mast cells, B cells, monocytes/macrophages, and neutrophils. PLCγ2 is unique by carrying three Src (SH) and one split pleckstrin homology domain (spPH) between the two catalytic subdomains (spPHn-SH2n-SH2c-SH3-spPHc). Prevailing evidence suggests that activation of PLCγ2 is primarily due to loss of SH-region-mediated autoinhibition and/or enhanced plasma membrane translocation. Here, we show that the two PLAID PLCγ2 mutants lacking portions of the SH region are strongly (>100-fold), rapidly, and reversibly activated by cooling by only a few degrees. We found that the mechanism(s) underlying PLCγ2 PLAID mutant activation by cool temperatures is distinct from a mere loss of SH-region-mediated autoinhibition and dependent on both the integrity and the pliability of the spPH domain. The results suggest a new mechanism of PLCγ activation with unique thermodynamic features and assign a novel regulatory role to its spPH domain. Involvement of this mechanism in other human disease states associated with cooling such as exertional asthma and certain acute coronary events appears an intriguing possibility. PMID:27196803

  8. 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

  9. Platelet surface-associated activation and secretion-mediated inhibition of coagulation factor XII.

    Science.gov (United States)

    Zakharova, Natalia V; Artemenko, Elena O; Podoplelova, Nadezhda A; Sveshnikova, Anastasia N; Demina, Irina A; Ataullakhanov, Fazly I; Panteleev, Mikhail A

    2015-01-01

    Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (ki/ka = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis. PMID:25688860

  10. Platelet surface-associated activation and secretion-mediated inhibition of coagulation factor XII.

    Directory of Open Access Journals (Sweden)

    Natalia V Zakharova

    Full Text Available Coagulation factor XII (fXII is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (ki/ka = 180 molecules/platelet. Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa, particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.

  11. Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

    OpenAIRE

    Zakharova, Natalia V.; Artemenko, Elena O.; Podoplelova, Nadezhda A.; Anastasia N Sveshnikova; Demina, Irina A.; Ataullakhanov, Fazly I.; Panteleev, Mikhail A.

    2015-01-01

    Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey p...

  12. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation

    OpenAIRE

    Cuff, Carolyn A.; Kothapalli, Devashish; Azonobi, Ijeoma; Chun, Sam; Zhang, Yuanming; Belkin, Richard; Yeh, Christine; Secreto, Anthony; Richard K Assoian; Rader, Daniel J; Puré, Ellen

    2001-01-01

    Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD4...

  13. Cholesterol-mediated activation of P-glycoprotein: distinct effects on basal and drug-induced ATPase activities.

    Science.gov (United States)

    Belli, Sara; Elsener, Priska M; Wunderli-Allenspach, Heidi; Krämer, Stefanie D

    2009-05-01

    Cholesterol promotes basal and verapamil-induced ATPase activity of P-glycoprotein (P-gp). We investigated whether these effects are related to each other and to the impact of the sterol on bilayer fluidity and verapamil membrane affinity. P-gp was reconstituted in egg-phosphatidylcholine (PhC) liposomes with or without cholesterol, 1,2-dipalmitoyl-phosphatidylcholine (DPPC), alpha-tocopherol (alpha-Toc) or 2,2,5,7,8-pentamethyl-6-chromanol (PMC). Basal and verapamil-induced ATPase activities were studied with an enzymatic assay. Membrane fluidity was characterized with diphenyl-hexatriene anisotropy measurements and membrane affinity by equilibrium dialysis. DPPC (70% mol/mol) decreased the fluidity of PhC bilayers to the same level as 20% cholesterol. PMC (20%) and alpha-Toc (20%) decreased the fluidity to lesser extents. alpha-Toc and PMC, but not DPPC increased the verapamil membrane affinity. While 20% cholesterol strikingly enhanced the basal ATPase activity, none of the other constituents had a similar effect. In contrast, verapamil stimulation of P-gp ATPase activity was not only enabled by cholesterol but also by alpha-Toc and DPPC. PMC had no effect. In conclusion, cholesterol exerts distinct effects on basal and verapamil-induced ATPase activity. The influence on basal ATPase activity is sterol-specific while its effect on verapamil-induced ATPase activity is unspecific and not related to its influence on membrane fluidity and on verapamil membrane affinity.

  14. Gene specificity of suppression of transgene-mediated insertional transcriptional activation by the chicken HS4 insulator.

    Directory of Open Access Journals (Sweden)

    Romain Desprat

    Full Text Available Insertional mutagenesis has emerged as a major obstacle for gene therapy based on vectors that integrate randomly in the genome. Reducing the genotoxicity of genomic viral integration can, in first approximation, be equated with reducing the risk of oncogene activation, at least in the case of therapeutic payloads that have no known oncogenic potential, such as the globin genes. An attractive solution to the problem of oncogene activation is the inclusion of insulators/enhancer-blockers in the viral vectors. In this study we have used Recombinase-Mediated Cassette Exchange to characterize the effect of integration of globin therapeutic cassettes in the presence or absence of the chicken HS4 and three other putative insulators inserted near Stil, Tal1 and MAP17, three well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that insertion of a Locus Control Region-driven globin therapeutic globin transgene had a dramatic activating effect on Tal1 and Map17, the two closest genes, a minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four element tested, cHS4 was the only one that was able to suppress this transgene-mediated insertional transcriptional activation. cHS4 had a strong suppressive effect on the activation expression of Map17 but has little or no effect on expression of Tal1. The suppressive activity of cHS4 is therefore promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 activation did not depend on its intercalation between the LCR and the Map 17 promoter. Rather, presence of one or two copies of cHS4 anywhere within the transgene was sufficient to almost completely block the activation of Map17. Therefore, at this complex locus, suppression of transgene-mediated insertional transcriptional activation by cHS4 could not be adequately explained by models that predict that cHS4 can only suppress expression through an enhancer-blocking activity that requires intercalation

  15. EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells.

    Science.gov (United States)

    Martínez-Palacián, A; del Castillo, G; Herrera, B; Fernández, M; Roncero, C; Fabregat, I; Sánchez, A

    2012-02-01

    Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met(flx/flx) and Met(-/-) oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-β)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met(flx/flx) cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met(flx/flx) and Met(-/-) oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in

  16. Lineage-specific STAT5 target gene activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype.

    Science.gov (United States)

    Müller, T A; Grundler, R; Istvanffy, R; Rudelius, M; Hennighausen, L; Illert, A L; Duyster, J

    2016-08-01

    Mutations that activate FMS-like tyrosine kinase 3 (FLT3) are frequent occurrences in acute myeloid leukemia. Two distinct types of mutations have been described: internal duplication of the juxtamembranous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both mutations lead to constitutive FLT3 signaling, only FLT3-ITD strongly activates signal transducer and activator of transcription 5 (STAT5). In a murine transplantation model, FLT3-ITD induces a myeloproliferative neoplasm, whereas FLT3-TKD leads to a lymphoid malignancy with significantly longer latency. Here we report that the presence of STAT5 is critical for the development of a myeloproliferative disease by FLT3-ITD in mice. Deletion of Stat5 in FLT3-ITD-induced leukemogenesis leads not only to a significantly longer survival (82 vs 27 days) of the diseased mice, but also to an immunophenotype switch with expansion of the lymphoid cell compartment. Interestingly, we were able to show differential STAT5 activation in FLT3-ITD(+) myeloid and lymphoid murine progenitors. STAT5 target genes such as Oncostatin M were highly expressed in FLT3-ITD(+) myeloid but not in FLT3-ITD(+) lymphoid progenitor cells. Strikingly, FLT3-TKD expression in combination with Oncostatin M is sufficient to reverse the phenotype to a myeloproliferative disease in FLT3-TKD mice. Thus, lineage-specific STAT5 activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype in mice. PMID:27046463

  17. 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.

  18. Extra Copper-mediated Enhancement of the DNA Cleavage Activity Supported with Wild-type Cu, Zn Superoxide Dismutase

    Institute of Scientific and Technical Information of China (English)

    ZHOU Ruo-Yu; JIANG Wei; ZHANG Li-Na; WANG Li; LIU Chang-Lin

    2008-01-01

    It is well known that the primary function of wild type Cu, Zn superoxide dismutase (holo SOD) is to catalyze the conversion of the superoxide anion to H2O2 and O2 as an antioxidant enzyme. However, the aberrant copper-mediated oxidation chemistry in the enzyme (including its mutation forms) that damages nucleic acids, proteins including itself and cell membrane has attracted extensive attention in the past decade. The present study examined the hydrogen peroxide-dependent DNA cleavage activity supported with the combinations between holo SOD and extra copper (holo SOD+nCu(Ⅱ)). The results indicate that the presence of extra copper can enhance the DNA cleavage activity and a cooperative effect between holo SOD and the extra Cu(Ⅱ) occurs in DNA cleavage. The relative activity and kinetic assay showed that the DNA cleavage activity of holo SOD+nCu(Ⅱ) was enhanced upon addition of extra Cu(Ⅱ). The favorable pH regions for the DNA cleavage were observed to be 3.6-5.6 and 9.0-10, suggesting the species responsible for the DNA cleavage are different in different pH regions. In addition,to obtain an insight into DNA cleavage pathways, the effect of free radical scavengers and inhibitors on the DNA cleavage activity was probed.

  19. Participation of the cell polarity protein PALS1 to T-cell receptor-mediated NF-κB activation.

    Directory of Open Access Journals (Sweden)

    Gabrielle Carvalho

    Full Text Available BACKGROUND: Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR-mediated activation. METHODOLOGY/PRINCIPAL FINDINGS: By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. CONCLUSIONS: The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

  20. Rice Phospholipase Dα is Involved in Salt Tolerance by the Mediation of H+-ATPase Activity and Transcription

    Institute of Scientific and Technical Information of China (English)

    Peng Shen; Rong Wang; Wen Jing; Wenhua Zhang

    2011-01-01

    Phospholipase Dα (PLDα) is involved in plant response to salt stress, but the mechanisms remain unclear.We investigated rice PLDα (OsPLDα) localization and its effect on tonoplast (TP) and plasma membrane (PM) H+-ATPase activity and transcription in response to NaCl. When rice suspension-cultured cells were treated with 100 mM NaCI, PLDα activity in cell extracts showed a transient activation with a threefold increase at 1 h. The amount of OsPLDα protein decreased slightly in the cytosolic fractions, whereas it increased significantly in the TP after NaCI treatment. OsPLDα1 knockdown cells were developed using RNA interference (RNAi) methods. The increase in TP and PM H+-ATPase activity induced by NaCl was significantly inhibited in OsPLDα1-RNAi cells. Knockdown of OsPLDα1 prevented the NaCl-induced increase in the transcript level of OsVHA-A (encodes TP H+-ATPase) and OSA2 (encodes PM H+-ATPase),as well as OsNHX1 (encodes TP Na+/H+ antiporter). The cells died more in OsPLDα1-RNAi mutant than in wild type when they were treated with NaCl. These results suggest that OsPLDα is involved in salt tolerance in rice through the mediation of H+-ATPase activity and transcription.

  1. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression.

    Science.gov (United States)

    Pinton, Laura; Solito, Samantha; Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-12

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression.

  2. Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF

    NARCIS (Netherlands)

    Schuijers, Jurian; Mokry, Michal; Hatzis, Pantelis; Cuppen, Edwin; Clevers, Hans

    2014-01-01

    Active canonical Wnt signaling results in recruitment of β-catenin to DNA by TCF/LEF family members, leading to transcriptional activation of TCF target genes. However, additional transcription factors have been suggested to recruit β-catenin and tether it to DNA. Here, we describe the genome-wide p

  3. Kindergarten Teachers' Orchestration of Mathematical Activities Afforded by Technology: Agency and Mediation

    Science.gov (United States)

    Carlsen, Martin; Erfjord, Ingvald; Hundeland, Per Sigurd; Monaghan, John

    2016-01-01

    This paper focuses on kindergarten teachers' interactions with young children during mathematical learning activities involving the use of digital tools. We aim to characterise the teachers' roles and actions in these activities and extend considerations of teachers' orchestrations current in the research literature with regard to agency and…

  4. NADPH oxidase mediates β-amyloid peptide-induced activation of ERK in hippocampal organotypic cultures

    Science.gov (United States)

    Serrano, Faridis; Chang, Angela; Hernandez, Caterina; Pautler, Robia G; Sweatt, J David; Klann, Eric

    2009-01-01

    Background Previous studies have shown that beta amyloid (Aβ) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either Aβ (1-42) or nicotine. Results We observed that Aβ and nicotine increased the levels of active ERK in distinct cellular localizations. We also examined whether phospho-ERK was regulated by redox signaling mechanisms and found that increases in active ERK induced by Aβ and nicotine were blocked by inhibitors of NADPH oxidase. Conclusion Our findings indicate that NADPH oxidase-dependent redox signaling is required for Aβ-induced activation of ERK, and suggest a similar mechanism may occur during early stages of Alzheimer's disease. PMID:19804648

  5. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.

    Science.gov (United States)

    Li, Fang; Malik, Kafait U

    2005-03-01

    Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt. PMID:15525798

  6. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.

    Science.gov (United States)

    Li, Fang; Malik, Kafait U

    2005-03-01

    Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt.

  7. Physical Activity and Health Perception in Aging: Do Body Mass and Satisfaction Matter? A Three-Path Mediated Link.

    Science.gov (United States)

    Condello, Giancarlo; Capranica, Laura; Stager, Joel; Forte, Roberta; Falbo, Simone; Di Baldassarre, Angela; Segura-Garcia, Cristina; Pesce, Caterina

    2016-01-01

    Although ageing people could benefit from healthy diet and physical activity to maintain health and quality of life, further understandings of the diet- and physical activity-related mechanisms that may cause changes in health and quality of life perception are necessary. The purpose of the study was to investigate the effect of eating attitudes, body mass and image satisfaction, and exercise dependence in the relationship between physical activity and health and quality of life perception in older individuals. Hundred and seventy-nine late middle-aged, (55-64 yrs), young-old (65-74 yrs), and old (75-84 yrs) senior athletes (n = 56), physically active (n = 58) or sedentary adults (n = 65) were submitted to anthropometric evaluations (body mass, height) and self-reported questionnaires: Body Image Dimensional Assessment, Exercise Dependence Scale, Eating Attitude Test, and Short Form Health Survey (Physical Component Summary [PCS] and Mental Component Summary [MCS] of and health and quality of life perception). Senior athletes, physically active, and sedentary participants subgroups differed (Pbody mass index (BMI) and several components of body image and exercise dependence. Senior athletes showed, compared to their sedentary counterparts, further differences (Pbody image (dis)satisfaction. Findings confirm the relevant role of physically active life habits for older individuals to perceive good physical and mental health. The novelty of the three-path mediated link between physical activity level and mental health perception suggests that the beneficial effect of a physically active lifestyle on weight control can positively impinge on the cognitive-emotional dimension of mental health by ensuring the maintenance, also at older age, of a satisfactory body image. PMID:27611689

  8. Commensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-kappaB activation.

    Directory of Open Access Journals (Sweden)

    Caitlin O'Mahony

    Full Text Available Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-kappaB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-kappaB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-kappaB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis-fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-kappaB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kappaB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.

  9. Flavonoids activate pregnane × receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells

    Directory of Open Access Journals (Sweden)

    Wu Jing

    2010-06-01

    Full Text Available Abstract Background The expression of the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4 is regulated by the pregnane × receptor (PXR, which is modulated by numerous signaling pathways, including the cyclin-dependent kinase (Cdk pathway. Flavonoids, commonly consumed by humans as dietary constituents, have been shown to modulate various signaling pathways (e.g., inhibiting Cdks. Flavonoids have also been shown to induce CYPs expression, but the underlying mechanism of action is unknown. Here, we report the mechanism responsible for flavonoid-mediated PXR activation and CYP expression. Results In a cell-based screen designed to identify compounds that activate PXR-mediated CYP3A4 gene expression in HepG2 human carcinoma cells, we identified several flavonoids, such as luteolin and apigenin, as PXR activators. The flavonoids did not directly bind to PXR, suggesting that an alternative mechanism may be responsible for flavonoid-mediated PXR activation. Consistent with the Cdk5-inhibitory effect of flavonoids, Cdk5 and p35 (a non-cyclin regulatory subunit required to activate Cdk5 were expressed in HepG2. The activation of Cdk5 attenuated PXR-mediated CYP3A4 expression whereas its downregulation enhanced it. The Cdk5-mediated downregulation of CYP3A4 promoter activity was restored by flavonoids, suggesting that flavonoids activate PXR by inactivating Cdk5. In vitro kinase assays showed that Cdk5 directly phosphorylates PXR. The Cdk kinase profiling assay showed that apigenin inhibits multiple Cdks, suggesting that several Cdks may be involved in activation of PXR by flavonoids. Conclusions Our results for the first time link the stimulatory effect of flavonoids on CYP expression to their inhibitory effect on Cdks, through a PXR-mediated mechanism. These results may have important implications on the pharmacokinetics of drugs co-administered with herbal remedy and herbal-drug interactions.

  10. The role of HSP27 in RACK1-mediated PKC activation in THP-1 cells.

    Science.gov (United States)

    Corsini, Emanuela; Galbiati, Valentina; Papale, Angela; Kummer, Elena; Pinto, Antonella; Guaita, Antonio; Racchi, Marco

    2016-08-01

    Receptor for Activated C Kinase 1 (RACK1) pseudosubstrate is a commercially available peptide that directly activates protein kinase C-β (PKCβ). We have recently shown that RACK1 pseudosubstrate, alone or in combination with classical immune activators, results in increased cytokine production and CD86 upregulation in primary leukocytes. Furthermore, we demonstrated a role of PKCβ and RACK1 in chemical allergen-induced CD86 expression and IL-8 production in both THP-1 cells and primary human dendritic cells. Aim of this study was to shed light on the mechanisms underlying RACK1 pseudosubstrate-induced immune activation and to compare it to lipopolysaccharide (LPS). The human promyelocytic cell line THP-1 was used throughout the study. RACK1 pseudosubstrate induced rapid (5 min) and dose-related PKCβ activation as assessed by its membrane translocation. Among the proteins phosphorylated, we identified Hsp27. Both RACK1 pseudosubstrate and LPS induce its phosphorylation and release in culture medium. The release of Hsp27 induced by RACK1 pseudosubstrate was also confirmed in peripheral blood mononuclear cells. To evaluate the role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, we conducted Hsp27 silencing and neutralization experiments. Both strategies confirmed the central role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, as assessed by IL-8 production and upregulation of CD86. PMID:27178349

  11. Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation

    OpenAIRE

    Liszewski, M. Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G.; Fara, Antonella F.; Subias, Marta; Pickering, Matthew C.; Drouet, Christian; Meri, Seppo; Arstila, T. Petteri; Pekkarinen, Pirkka T.; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas

    2013-01-01

    Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generate...

  12. Protein Kinase Cδ mediates the activation of Protein Kinase D2 in Platelets

    OpenAIRE

    Bhavanasi, Dheeraj; Kim, Soochong; Goldfinger, Lawrence E.; Kunapuli, Satya P.

    2011-01-01

    Protein Kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCν in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well ...

  13. Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates

    Directory of Open Access Journals (Sweden)

    Yinliang Yang

    2013-10-01

    Full Text Available Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions.

  14. Effect of bile on nisin-mediated antibacterial activity and the expression of nisin genes of Lactococcus lactis W8.

    Science.gov (United States)

    Mitra, Suranjita; Mukhopadhyay, Bidhan Chandra; Chakrabartty, Pran Krishna; Biswas, Swadesh Ranjan

    2013-12-01

    The capability of Lactococcus lactis to produce nisin in the presence of bile in the intestinal environment remains an intriguing question. The aim of this study was to determine the effects of bile on production of nisin and the mRNA expression of nisin genes of L. lactis W8. The strain L. lactis W8 was grown on glucose in the absence and presence of bile (0.005-0.08 %) and the antibacterial activities of culture supernatants were determined. In culture with 0.035 % bile, the nisin activity was significantly reduced (400 AU/mL) within 5 h compared to that in the control without bile (2000 AU/mL), while growth of the cells was only slightly affected. In the presence of 0.07 % bile no nisin activity of the strain was manifested. Consistent with these results, mRNA expression of nisin-biosynthetic genes nisZ, nisRK, nisI, and nisF was down-regulated by 7.5-, 2.5-, 1.7-, and 6.0-fold, respectively in cells grown in the presence of bile (0.07 %) as compared to control culture without bile. The present study suggested that bile inhibited transcription of nisin genes. Nisin-production in intestine by orally administered L. lactis, thus, does not occur since complete inhibition of nisin-production by bile is observed at a concentration much lower than the physiological concentration (0.3 %) of bile present in the human intestine. The molecular mechanism underlying the bile-mediated inhibition of nisin genes remains to be elucidated. This is the first report on bile-mediated inhibition of nisin genes.

  15. CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling

    Science.gov (United States)

    Rodríguez-Nieves, José A.; Patalano, Susan C.; Almanza, Diego; Gharaee-Kermani, Mehrnaz; Macoska, Jill A.

    2016-01-01

    Benign prostate hyperplasia (BPH), an enlargement of the prostate common in aging in men, is associated with urinary voiding dysfunction manifest as Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal smooth muscle contractions are known to play key roles in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the unregulated differentiation of fibroblasts or other precursor cell types into myofibroblasts, which is usually accomplished by activation of the TGFβ/TGFβR axis. Previously we reported that the CXC-type chemokines, CXCL5, CXCL8 and CXCL12, which are up-regulated in the aging in the prostate, can drive this differentiation process as well in the absence of TGFβ. Based on this data we sought to elucidate the molecular mechanisms employed by CXCL12, and its receptor CXCR4, during prostate myofibroblast phenoconversion. The results of these studies suggest that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGFβ/TGFβR axis activation or Smad signaling. Here we report that CXCL12/CXCR4 axis activation promotes signaling through the EGFR and downstream MEK/ERK and PI3K/Akt pathways during myofibroblast phenoconversion, but not through TGFβ/TGFβR and downstream Smad signaling, in prostate fibroblasts undergoing myofibroblast phenoconversion. We document that EGFR transactivation is required for CXCL12-mediated signaling and expression of genes associate with myofibroblast phenoconversion (α-SMA, COL1a1). Our study successfully identified TGFβ/TGFβR-independent molecular mechanisms that promote CXCL12/CXCR4-induced myofibroblast phenoconversion. This information may be crucial for the development of novel therapies and potential biomarkers for prostatic fibrosis. PMID:27434301

  16. Use of serum C-reactive protein as an early marker of inflammatory activity in canine type II immune-mediated polyarthritis: case report

    Directory of Open Access Journals (Sweden)

    Kristensen Annemarie T

    2006-06-01

    Full Text Available Abstract Background Monitoring systemic inflammatory activity during steroid therapy of canine immune-mediated polyarthritis (IMPA is difficult and mainly relies on clinical signs. Case presentation Canine serum C-reactive protein (CRP was measured serially and blinded during a 27-week follow-up period of a case of Anaplasma phagocytophilia induced type II immune-mediated polyarthritis. Conclusion WBC was, as expected, observed not to reflect the inflammatory activity during steroid treatment in a clinical useful manner, whereas, CRP is suggested a valuable unbiased marker of inflammatory activity during steroid treatment in this case.

  17. Use of serum C-reactive protein as an early marker of inflammatory activity in canine type II immune-mediated polyarthritis: case report

    Science.gov (United States)

    Kjelgaard-Hansen, Mads; Jensen, Asger Lundorff; Houser, Geoffrey A; Jessen, Lisbeth Rem; Kristensen, Annemarie T

    2006-01-01

    Background Monitoring systemic inflammatory activity during steroid therapy of canine immune-mediated polyarthritis (IMPA) is difficult and mainly relies on clinical signs. Case presentation Canine serum C-reactive protein (CRP) was measured serially and blinded during a 27-week follow-up period of a case of Anaplasma phagocytophilia induced type II immune-mediated polyarthritis. Conclusion WBC was, as expected, observed not to reflect the inflammatory activity during steroid treatment in a clinical useful manner, whereas, CRP is suggested a valuable unbiased marker of inflammatory activity during steroid treatment in this case. PMID:16987405

  18. CARD9 mediates Dectin-1–induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity

    OpenAIRE

    Jia, Xin-ming; Tang, Bing; Zhu, Le-Le; Liu, Yan-Hui; Zhao, Xue-Qiang; Gorjestani, Sara; Hsu, Yen-Michael S.; Yang, Long; Guan, Jian-Hong; Xu, Guo-Tong; Lin, Xin

    2014-01-01

    Dectin-1 functions as a pattern recognition receptor for sensing fungal infection. It has been well-established that Dectin-1 induces innate immune responses through caspase recruitment domain-containing protein 9 (CARD9)–mediated NF-κB activation. In this study, we find that CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands, such as curdlan or Candida albicans yeast. In contrast, we find that CARD9 regulates H-Ras activation by linking Ras-GRF1 to H-Ras, which mediates De...

  19. Aspirin Augments IgE-Mediated Histamine Release from Human Peripheral Basophils via Syk Kinase Activation

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsuo

    2013-01-01

    Conclusions: Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.

  20. Substrate-induced activation of a trapped IMC-mediated protein folding intermediate.

    Science.gov (United States)

    Inouye, M; Fu, X; Shinde, U

    2001-04-01

    While several unfolded proteins acquire native structures through distinct folding intermediates, the physiological relevance and importance of such states in the folding kinetics remain controversial. The intramolecular chaperone (IMC) of subtilisin was used to trap a partially folded, stable crosslinked intermediate conformer (CLIC) through a disulfide bond between mutated IMC and subtilisin. The trapped CLIC contains non-native interactions. Here we show that CLIC can be induced into a catalytically active form by incubating it with small peptide substrates. The structure and catalytic properties of the activated crosslinked intermediate conformer (A-CLIC) differ from those of the fully folded enzyme in that A-CLIC lacks any endopeptidase activity toward a large protein substrate. Our results show that a disulfide-linked partially folded protein can be induced to acquire catalytic activity with a substrate specificity that is different from completely folded subtilisin. These results also suggest that protein folding intermediates may also participate in catalytic reactions.

  1. PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY FROM NERIUM OLEANDER AND EVALUVATE THEIR PLANT MEDIATED NANOPARTICLE SYNTHESIS

    Directory of Open Access Journals (Sweden)

    R.S. Suganya

    2012-05-01

    Full Text Available Phytochemical and antibacterial activity of essential oils obtained from Nerium oleander leaf with the help of three different extracts like ethanol, Methanol, and acetone. In this aqueous leaf extracts confirms the presence of various phytochemicals. To evaluate the antibacterial activities of these aqueous extracts were determined by disc diffusion method. From these three extracts methanol shows strong antibacterial effect on Klebsiella, Pseudomonas, Alkaligenes except Acinetobacter. None of the leaf extracts show no more activity in Acinetobacter. Biologically synthesized nanoparticles have been widely used in the field of medicine. Shade dried leaves of Nerium oleander was used for the synthesis of silver nanoparticles. UV- Visible spectroscopy studies were carried out to assess the formation of Ag nanoparticles. Scanning electron microscope (SEM was used to characterize the Ag nanoparticle. To compare the antimicrobial activity of silver nanoparticles with the different leaf extracts.

  2. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator.

    Science.gov (United States)

    Tsirka, S E; Gualandris, A; Amaral, D G; Strickland, S

    1995-09-28

    Neuronal degeneration in the hippocampus, a region of the brain important for acquisition of memory in humans, occurs in various pathological conditions, including Alzheimer's disease, brain ischaemia and epilepsy. When neuronal activity is stimulated in the adult rat and mouse hippocampus, tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to the active protease plasmin, is transcriptionally induced. The activity of tPA in neural tissue is correlated with neurite outgrowth, regeneration and migration, suggesting that it might be involved in neuronal plasticity. Here we show that tPA is produced primarily by microglia in the hippocampus. Using excitotoxins to induce neuronal cell loss, we demonstrate that tPA-deficient mice are resistant to neuronal degeneration. These mice are also less susceptible to pharmacologically induced seizures than wild-type mice. These findings identify a role for tPA in neuronal degeneration and seizure.

  3. Microwave mediated synthesis of non-carboxylic analogues of ibuprofen with improved pharmacological activity

    Institute of Scientific and Technical Information of China (English)

    K.V. Sujith; Balakrishna Kalluraya; Adithya Adhikari; J. Ravikumar

    2011-01-01

    A series of l,2,4-triazolo [3,4-b]-thiadiazoles were synthesized following microwave irradiation method and also by conventional method. Newly synthesized compounds were evaluated for their anti-inflammatory and analgesic activities.

  4. Mediating Effects of Self-Efficacy, Benefits and Barriers on the Association between Peer and Parental Factors and Physical Activity among Adolescent Girls with a Lower Educational Level.

    Directory of Open Access Journals (Sweden)

    Maite Verloigne

    Full Text Available The prevalence of physical activity among lower educated adolescent girls is low, suggesting it is important to have insights into the complex processes that may underlie their physical activity levels. Therefore, this study aimed to examine the mediating effects of self-efficacy, perceived benefits and barriers on the associations between peer and parental variables and physical activity among lower educated adolescent girls.In total, 226 girls (mean age 16.0±1.0 years; 53% technical education; 47% vocational education from a convenience sample of 6 secondary schools in Flanders, Belgium, completed a questionnaire on their total physical activity level and related peer and parental variables (i.e. modeling of physical activity, co-participation in physical activities and encouragement to be active and personal variables (i.e. self-efficacy to be active, and specific perceived benefits of physical activity and specific barriers to be active. Mediating effects were tested using MacKinnon's product-of-coefficients test based on multilevel linear regression analyses.Higher peer and parental modeling, co-participation and encouragement were significantly related to a higher physical activity level among adolescent girls (p<0.05. Self-efficacy, the perceived benefits of having fun, being around friends or meeting new people, and not being bored and the perceived barrier of not liking physical activity mediated several associations between peer and parental variables and girls' physical activity, with some of the mediated proportions exceeding 60%.This study contributed to a better understanding of the complexity of how parental and peer factors work together with personal factors to influence the physical activity levels of adolescent girls with a lower educational level. Interventions should involve both peers and parents, as they may influence girls' physical activity both directly and indirectly through the internalisation of several personal

  5. A Highly Active Low Voltage Redox Mediator for Enhanced Rechargeability of Lithium-Oxygen Batteries.

    Science.gov (United States)

    Kundu, Dipan; Black, Robert; Adams, Brian; Nazar, Linda F

    2015-12-23

    Owing to its high theoretical specific energy, the Li-oxygen battery is one of the fundamentally most promising energy storage systems, but also one of the most challenging. Poor rechargeability, involving the oxidation of insoluble and insulating lithium peroxide (Li2O2), has remained the "Achilles' heel" of this electrochemical energy storage system. We report here on a new redox mediator tris[4-(diethylamino)phenyl]amine (TDPA), that-at 3.1 V-exhibits the lowest and closest potential redox couple compared to the equilibrium voltage of the Li-oxygen cell of those reported to date, with a second couple also at a low potential of 3.5 V. We show it is a soluble "catalyst" capable of lowering the Li2O2 charging potential by >0.8 V without requiring direct electrical contact of the peroxide and that it also facilitates high discharge capacities. Its chemical and electrochemical stability, fast diffusion kinetics, and two dynamic redox potentials represent a significant advance in oxygen-evolution catalysis. It enables Li-O2 cells that can be recharged more than 100 cycles with average round-trip efficiencies >80%, opening a new avenue for practical Li-oxygen batteries.

  6. Strain Mediated Adaptation Is Key for Myosin Mechanochemistry: Discovering General Rules for Motor Activity.

    Science.gov (United States)

    Jana, Biman; Onuchic, José N

    2016-08-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities.

  7. Activated Human T Cells Secrete Exosomes That Participate in IL-2 Mediated Immune Response Signaling

    OpenAIRE

    Wahlgren, Jessica; Tanya De L Karlson; Glader, Pernilla; Telemo, Esbjörn; Valadi, Hadi

    2012-01-01

    It has previously been shown that nano-meter sized vesicles (30–100 nm), exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3+ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3+ T cells have on resting CD3+ T cells by studying T cell proliferation, c...

  8. Physical activity and obesity mediate the association between childhood motor function and adolescents’ academic achievement

    OpenAIRE

    Kantomaa, Marko T.; Stamatakis, Emmanuel; Kankaanpää, Anna; Kaakinen, Marika; Rodriguez, Alina; Taanila, Anja; Ahonen, Timo; Järvelin, Marjo-Riitta; Tammelin, Tuija,

    2012-01-01

    The global epidemic of obesity and physical inactivity may have detrimental implications for young people’s cognitive function and academic achievement. This prospective study investigated whether childhood motor function predicts later academic achievement via physical activity, fitness, and obesity. The study sample included 8,061 children from the Northern Finland Birth Cohort 1986, which contains data about parent-reported motor function at age 8 y and self-reported physical activity, pre...

  9. The influence of Business Strategy on New Product Activity: The mediating role of Market Orientation

    OpenAIRE

    Frambach, R T; Prabhu, Jaideep; Verhallen, T.M.M.

    2000-01-01

    In this paper we examine how business strategy influences the nature and extent of firms’ market orientation and how this in turn influences the extent of firms’ new product activity. Specifically, we develop a framework linking firms’ relative emphasis on cost leadership, product differentiation and focus strategies on firms’ customer, competitor and supplier orientation as well as their new product development and introduction activity. We use this framework to develop relevant hypotheses w...

  10. The Participation Framework as a Mediating Tool in Kindergarten Journal Writing Activity

    OpenAIRE

    Larson, Joanne

    1996-01-01

    Drawing on data collected in an ethnographic study of kindergarten journal writing activity, this article demonstrates how students who are not directly participating in instruction are nevertheless key contributors to the social construction of literacy knowledge. More specifically, this study examines how the participation framework of writing activity constitutes and is constituted by the context for learning to write. Five interconnected roles in the participation framework are identified...

  11. Evaluation on Potential Contributions of Protease Activated Receptors Related Mediators in Allergic Inflammation

    OpenAIRE

    Huiyun Zhang; Xiaoning Zeng; Shaoheng He

    2014-01-01

    Protease activated receptors (PARs) have been recognized as a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) that can be cleaved by certain serine proteases. In recent years, there has been considerable interest in the role of PARs in allergic inflammation, the fundamental pathologic changes of allergy, but the potential roles of PARs in allergy remain obscure. Since many of these proteases are produced and actively involved in the pathologic process...

  12. Complement receptor 2–mediated targeting of complement inhibitors to sites of complement activation

    OpenAIRE

    Song, Hongbin; He, Chun; Knaak, Christian; GUTHRIDGE, JOEL M.; Holers, V. Michael; Tomlinson, Stephen

    2003-01-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the mem...

  13. PARP1 orchestrates variant histone exchange in signal-mediated transcriptional activation.

    Science.gov (United States)

    O'Donnell, Amanda; Yang, Shen-Hsi; Sharrocks, Andrew D

    2013-12-01

    Transcriptional activation is accompanied by multiple molecular events that remodel the local chromatin environment in promoter regions. These molecular events are often orchestrated in response to the activation of signalling pathways, as exemplified by the response of immediate early genes such as FOS to ERK MAP kinase signalling. Here, we demonstrate that inducible NFI recruitment permits PARP1 binding to the FOS promoter by a mutually reinforcing loop. PARP1 and its poly(ADP-ribosyl)ation activity are required for maintaining FOS activation kinetics. We also show that the histone variant H2A.Z associates with the FOS promoter and acts in a transcription-suppressive manner. However, in response to ERK pathway signalling, H2A.Z is replaced by H2A; PARP1 activity is required to promote this exchange. Thus, our work has revealed an additional facet of PARP1 function in promoting dynamic remodelling of promoter-associated nucleosomes to allow transcriptional activation in response to cellular signalling.

  14. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    Directory of Open Access Journals (Sweden)

    Seung Sik Lee

    2015-11-01

    Full Text Available AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  15. Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis

    Institute of Scientific and Technical Information of China (English)

    Xinhong Zhu; Meihua Gao; Shurong Ren; Qiubo Wang; Cunzhi Lin

    2008-01-01

    CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex (MAC). However, CD59 is widely overexpressed on tumor cells,which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site (Mutant 1, M1) or to change C39W40K41 to W39W40W41 (Mutant 2, M2). Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CHO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly.Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors.

  16. Calpain activation induced by glucose deprivation is mediated by oxidative stress and contributes to neuronal damage.

    Science.gov (United States)

    Páramo, Blanca; Montiel, Teresa; Hernández-Espinosa, Diego R; Rivera-Martínez, Marlene; Morán, Julio; Massieu, Lourdes

    2013-11-01

    The mechanisms leading to neuronal death during glucose deprivation have not been fully elucidated, but a role of oxidative stress has been suggested. In the present study we have investigated whether the production of reactive oxygen species during glucose deprivation, contributes to the activation of calpain, a calcium-dependent protease involved in neuronal injury associated with brain ischemia and cerebral trauma. We have observed a rapid activation of calpain, as monitored by the cleavage of the cytoskeletal protein α-spectrin, after glucose withdrawal, which is reduced by inhibitors of xanthine oxidase, phospholipase A2 and NADPH oxidase. Results suggest that phospholipase A2 and NADPH oxidase contribute to the early activation of calpain after glucose deprivation. In particular NOX2, a member of the NADPH oxidase family is involved, since reduced stimulation of calpain activity is observed after glucose deprivation in hippocampal slices from transgenic mice lacking a functional NOX2. We observed an additive effect of the inhibitors of xanthine oxidase and phospholipase A2 on both ROS production and calpain activity, suggesting a synergistic action of these two enzymes. The present results provide new evidence showing that reactive oxygen species stimulate calpain activation during glucose deprivation and that this mechanism is involved in neuronal death.

  17. The sport and the physical activity as mediators of corporal models: Genre and sexuality in learning of the masculinities

    Directory of Open Access Journals (Sweden)

    Judit Vidiella Pagés

    2007-11-01

    Full Text Available This article begins with an analysis of some theoretical contributions about the conceptualization of body in contemporary societies. These allow us to set a dialogue with the reflective experiences of a group of teenagers about how they learn masculinitiesi in their lives. Phenomenological and political emphasis on the body carried out by Feminists -with the notion of embodiment- have been essential in giving complexity to embodied issues such as gender, race, age, social class (disabilities, etc. which not only operate in a relation of power in our selves, but also as a locus of resistance and agency. Queer theory and its deconstruction of normative sexuality -with the concept of performativity- will be basic to understand the fundamental role that sexuality has in the construction of subjectivities. The accounts of teens will allow us to explore the paper that physical activities and sport have in their lives as mediators in the construction of their "masculine" subjectivities, many times as an oppressor space of their sexual and gender identity, and open an analysis of the hegemonic body representations that mediate not only physical education in schools, but also sport of competition and elite, and their representations in mass media.

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

    Science.gov (United States)

    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.

  19. Copper-mediated C-H activation/C-S cross-coupling of heterocycles with thiols

    KAUST Repository

    Ranjit, Sadananda

    2011-11-04

    We report the synthesis of a series of aryl- or alkyl-substituted 2-mercaptobenzothiazoles by direct thiolation of benzothiazoles with aryl or alkyl thiols via copper-mediated aerobic C-H bond activation in the presence of stoichiometric CuI, 2,2′-bipyridine and Na 2CO 3. We also show that the approach can be extended to thiazole, benzimidazole, and indole substrates. In addition, we present detailed mechanistic investigations on the Cu(I)-mediated direct thiolation reactions. Both computational studies and experimental results reveal that the copper-thiolate complex [(L)Cu(SR)] (L: nitrogen-based bidentate ligand such as 2,2′-bipyridine; R: aryl or alkyl group) is the first reactive intermediate responsible for the observed organic transformation. Furthermore, our computational studies suggest a stepwise reaction mechanism based on a hydrogen atom abstraction pathway, which is more energetically feasible than many other possible pathways including β-hydride elimination, single electron transfer, hydrogen atom transfer, oxidative addition/reductive elimination, and σ-bond metathesis. © 2011 American Chemical Society.

  20. Inhibition of PAR-4 and P2Y12 receptor-mediated platelet activation produces distinct hepatic pathologies in experimental xenobiotic-induced cholestatic liver disease.

    Science.gov (United States)

    Joshi, Nikita; Kopec, Anna K; Ray, Jessica L; Luyendyk, James P

    2016-07-15

    Emerging evidence supports a protective effect of platelets in experimental cholestatic liver injury and cholangiofibrosis. Coagulation-mediated platelet activation has been shown to inhibit experimental chronic cholestatic liver necrosis and biliary fibrosis. This occurs through thrombin-mediated activation of protease activated receptor-4 (PAR-4) in mice. However, it is not known whether other pathways of platelet activation, such as adenosine diphosphate (ADP)-mediated receptor P2Y12 activation is also protective. We tested the hypothesis that inhibition of P2Y12-mediated platelet activation exacerbates hepatic injury and cholangiofibrosis, and examined the impact of P2Y12 inhibition in both the presence and absence of PAR-4. Treatment of wild-type mice with the P2Y12 receptor antagonist clopidogrel increased biliary hyperplasia and cholangiofibrosis in wild-type mice exposed to the xenobiotic alpha-naphthylisothiocyanate (ANIT) for 4 weeks compared to vehicle-treated mice exposed to ANIT. Interestingly, this effect of clopidogrel occurred without a corresponding increase in hepatocellular necrosis. Whereas biliary hyperplasia and cholangiofibrosis were increased in PAR-4(-/-) mice, clopidogrel treatment failed to further increase these pathologies in PAR-4(-/-) mice. The results indicate that inhibition of receptor P2Y12-mediated platelet activation exacerbates bile duct fibrosis in ANIT-exposed mice, independent of hepatocellular necrosis. Moreover, the lack of an added effect of clopidogrel administration on the exaggerated pathology in ANIT-exposed PAR-4(-/-) mice reinforces the prevailing importance of coagulation-mediated platelet activation in limiting this unique liver pathology. PMID:27475285

  1. Titanocene(III) chloride mediated radical induced addition-elimination route to the synthesis of racemic and optically active trisubstituted tetrahydrofurans: Formal synthesis of magnofargesin and 7'-epimagnofargesin

    Indian Academy of Sciences (India)

    P CHAKRABORTY; S K MANDAL; S C ROY

    2016-07-01

    Titanocene(III) Chloride mediated radical induced synthesis of 4-benzylidene substituted tetrahydrofuran, a typical lignan skeleton, has been accomplished in good yield through addition-elimination route in racemic as well as in optically active forms. The method has been applied to the synthesis of furano lignans, magnofargesin (1) and 7'-epimagnofargesin (2) in optically active forms.

  2. HIV-1 Nef enhances dendritic cell-mediated viral transmission to CD4+ T cells and promotes T-cell activation.

    Directory of Open Access Journals (Sweden)

    Corine St Gelais

    Full Text Available HIV-1 Nef enhances dendritic cell (DC-mediated viral transmission to CD4(+ T cells, but the underlying mechanism is not fully understood. It is also unknown whether HIV-1 infected DCs play a role in activating CD4(+ T cells and enhancing DC-mediated viral transmission. Here we investigated the role of HIV-1 Nef in DC-mediated viral transmission and HIV-1 infection of primary CD4(+ T cells using wild-type HIV-1 and Nef-mutated viruses. We show that HIV-1 Nef facilitated DC-mediated viral transmission to activated CD4(+ T cells. HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+ T cells. However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+ T cells. Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+ T cells and in the activation and proliferation of resting CD4(+ T cells, which likely contribute to viral pathogenesis.

  3. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    Science.gov (United States)

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. PMID:26980729

  4. Mediation of the Physical Activity and Healthy Nutrition Behaviors of Preschool Children by Maternal Cognition in China

    Science.gov (United States)

    Xu, Xianglong; Sharma, Manoj; Liu, Lingli; Hu, Ping; Zhao, Yong

    2016-01-01

    (1) Objective: We aimed to explore the role of social cognitive theory (SCT) of mothers in the physical activity and healthy nutrition behaviors of preschool children; (2) Methods: We used a self-administered five-point Likert common physical activity and nutrition behaviors scale in Chinese based on a social cognitive theory scale in English with established validity and reliability in the USA. The current study adopted the proportional sampling method to survey mothers of preschool children in four areas—namely, Chongqing, Chengdu, Taiyuan, and Shijiazhuang—of China; (3) Results: We included 1208 mothers (80.0% mothers of normal weight children, age 31.87 ± 4.19 years). Positive correlations were found between maternal social cognition and preschool children’s physical activity (PA) behavior (p < 0.0001). However, an insignificant correlation is observed between preschool children’s fruits and vegetables (FV) behavior, screen time (ST) behavior, and maternal social cognition; (4) Conclusions: This study provides some implications for increasing fruit and vegetable consumption, increasing physical activity time, and reducing screen time in preschool children using SCT in China. Maternal social cognition is associated with preschool children’s PA behavior, and the results suggest that maternal social cognition may not affect children FV and ST behaviors. Further research is necessary to test the mediation of maternal social cognition on preschool children’s ST behavior and the correlations between maternal social cognition and children’s ST behavior. PMID:27649215

  5. Mediation of the Physical Activity and Healthy Nutrition Behaviors of Preschool Children by Maternal Cognition in China

    Directory of Open Access Journals (Sweden)

    Xianglong Xu

    2016-09-01

    Full Text Available (1 Objective: We aimed to explore the role of social cognitive theory (SCT of mothers in the physical activity and healthy nutrition behaviors of preschool children; (2 Methods: We used a self-administered five-point Likert common physical activity and nutrition behaviors scale in Chinese based on a social cognitive theory scale in English with established validity and reliability in the USA. The current study adopted the proportional sampling method to survey mothers of preschool children in four areas—namely, Chongqing, Chengdu, Taiyuan, and Shijiazhuang—of China; (3 Results: We included 1208 mothers (80.0% mothers of normal weight children, age 31.87 ± 4.19 years. Positive correlations were found between maternal social cognition and preschool children’s physical activity (PA behavior (p < 0.0001. However, an insignificant correlation is observed between preschool children’s fruits and vegetables (FV behavior, screen time (ST behavior, and maternal social cognition; (4 Conclusions: This study provides some implications for increasing fruit and vegetable consumption, increasing physical activity time, and reducing screen time in preschool children using SCT in China. Maternal social cognition is associated with preschool children’s PA behavior, and the results suggest that maternal social cognition may not affect children FV and ST behaviors. Further research is necessary to test the mediation of maternal social cognition on preschool children’s ST behavior and the correlations between maternal social cognition and children’s ST behavior.

  6. Mediation of the Physical Activity and Healthy Nutrition Behaviors of Preschool Children by Maternal Cognition in China.

    Science.gov (United States)

    Xu, Xianglong; Sharma, Manoj; Liu, Lingli; Hu, Ping; Zhao, Yong

    2016-09-13

    (1) OBJECTIVE: We aimed to explore the role of social cognitive theory (SCT) of mothers in the physical activity and healthy nutrition behaviors of preschool children; (2) METHODS: We used a self-administered five-point Likert common physical activity and nutrition behaviors scale in Chinese based on a social cognitive theory scale in English with established validity and reliability in the USA. The current study adopted the proportional sampling method to survey mothers of preschool children in four areas-namely, Chongqing, Chengdu, Taiyuan, and Shijiazhuang-of China; (3) RESULTS: We included 1208 mothers (80.0% mothers of normal weight children, age 31.87 ± 4.19 years). Positive correlations were found between maternal social cognition and preschool children's physical activity (PA) behavior (p social cognition; (4) CONCLUSIONS: This study provides some implications for increasing fruit and vegetable consumption, increasing physical activity time, and reducing screen time in preschool children using SCT in China. Maternal social cognition is associated with preschool children's PA behavior, and the results suggest that maternal social cognition may not affect children FV and ST behaviors. Further research is necessary to test the mediation of maternal social cognition on preschool children's ST behavior and the correlations between maternal social cognition and children's ST behavior.

  7. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    Science.gov (United States)

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

  8. Induction of autophagy by valproic acid enhanced lymphoma cell chemosensitivity through HDAC-independent and IP3-mediated PRKAA activation.

    Science.gov (United States)

    Ji, Meng-Meng; Wang, Li; Zhan, Qin; Xue, Wen; Zhao, Yan; Zhao, Xia; Xu, Peng-Peng; Shen, Yang; Liu, Han; Janin, Anne; Cheng, Shu; Zhao, Wei-Li

    2015-01-01

    Autophagy is closely related to tumor cell sensitivity to anticancer drugs. The HDAC (histone deacetylase) inhibitor valproic acid (VPA) interacted synergistically with chemotherapeutic agents to trigger lymphoma cell autophagy, which resulted from activation of AMPK (AMP-activated protein kinase) and inhibition of downstream MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling. In an HDAC-independent manner, VPA potentiated the effect of doxorubicin on lymphoma cell autophagy via reduction of cellular inositol 1,4,5 trisphosphate (IP3), blockade of calcium into mitochondria and modulation of PRKAA1/2-MTOR cascade. In murine xenograft models established with subcutaneous injection of lymphoma cells, dual treatment of VPA and doxorubicin initiated IP3-mediated calcium depletion and PRKAA1/2 activation, induced in situ autophagy and efficiently retarded tumor growth. Aberrant genes involving mitochondrial calcium transfer were frequently observed in primary tumors of lymphoma patients. Collectively, these findings suggested an HDAC-independent chemosensitizing activity of VPA and provided an insight into the clinical application of targeting autophagy in the treatment of lymphoma.

  9. IL-15 Mediates Mitochondrial Activity through a PPARδ-Dependent-PPARα-Independent Mechanism in Skeletal Muscle Cells

    Science.gov (United States)

    2016-01-01

    Molecular mediators of metabolic processes, to increase energy expenditure, have become a focus for therapies of obesity. The discovery of cytokines secreted from the skeletal muscle (SKM), termed “myokines,” has garnered attention due to their positive effects on metabolic processes. Interleukin-15 (IL-15) is a myokine that has numerous positive metabolic effects and is linked to the PPAR family of mitochondrial regulators. Here, we aimed to determine the importance of PPARα and/or PPARδ as targets of IL-15 signaling. C2C12 SKM cells were differentiated for 6 days and treated every other day with IL-15 (100 ng/mL), a PPARα inhibitor (GW-6471), a PPARδ inhibitor (GSK-3787), or both IL-15 and the inhibitors. IL-15 increased mitochondrial activity and induced PPARα, PPARδ, PGC1α, PGC1β, UCP2, and Nrf1 expression. There was no effect of inhibiting PPARα, in combination with IL-15, on the aforementioned mRNA levels except for PGC1β and Nrf1. However, with PPARδ inhibition, IL-15 failed to induce the expression levels of PGC1α, PGC1β, UCP2, and Nrf1. Further, inhibition of PPARδ abolished IL-15 induced increases in citrate synthase activity, ATP production, and overall mitochondrial activity. IL-15 had no effects on mitochondrial biogenesis. Our data indicates that PPARδ activity is required for the beneficial metabolic effects of IL-15 signaling in SKM.

  10. Glycogen synthase kinase 3 β activity is required for hBora/Aurora A-mediated mitotic entry.

    Science.gov (United States)

    Lee, Yu-Cheng; Liao, Po-Chi; Liou, Yih-Cherng; Hsiao, Michael; Huang, Chi-Ying; Lu, Pei-Jung

    2013-03-15

    The synthesis and degradation of hBora is important for the regulation of mitotic entry and exist. In G 2 phase, hBora can complex with Aurora A to activate Plk1 and control mitotic entry. However, whether the post-translational modification of hBora is relevant to the mitotic entry still unclear. Here, we used the LC-MS/MS phosphopeptide mapping assay to identify 13 in vivo hBora phosphorylation sites and characterized that GSK3β can interact with hBora and phosphorylate hBora at Ser274 and Ser278. Pharmacological inhibitors of GSK3β reduced the retarded migrating band of hBora in cells and diminished the phosphorylation of hBora by in vitro kinase assay. Moreover, as well as in GSK3β activity-inhibited cells, specific knockdown of GSK3β by shRNA and S274A/S278 hBora mutant-expressing cells also exhibited the reduced Plk1 activation and a delay in mitotic entry. It suggests that GSK3β activity is required for hBora-mediated mitotic entry through Ser274 and Ser278 phosphorylation.

  11. Alternative activation of macrophages and induction of arginase are not components of pathogenesis mediated by Francisella species.

    Directory of Open Access Journals (Sweden)

    Amanda J Griffin

    Full Text Available Virulent Francisella tularensis ssp tularensis is an intracellular, Gram negative bacterium that causes acute lethal disease following inhalation of fewer than 15 organisms. Pathogenicity of Francisella infections is tied to its unique ability to evade and suppress inflammatory responses in host cells. It has been proposed that induction of alternative activation of infected macrophages is a mechanism by which attenuated Francisella species modulate host responses. In this report we reveal that neither attenuated F. tularensis Live Vaccine Strain (LVS nor virulent F. tularensis strain SchuS4 induce alternative activation of macrophages in vitro or in vivo. LVS, but not SchuS4, provoked production of arginase1 independent of alternative activation in vitro and in vivo. However, absence of arginase1 did not significantly impact intracellular replication of LVS or SchuS4. Together our data establish that neither induction of alternative activation nor expression of arginase1 are critical features of disease mediated by attenuated or virulent Francisella species.

  12. Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase 3 activation in HL-60 cells.

    Science.gov (United States)

    Huang, R F; Huang, S M; Lin, B S; Wei, J S; Liu, T Z

    2001-05-11

    The cytotoxicity of homocysteine derivatives on chromosomal damage in somatic cells is not well established. The present study used reactive homocysteine derivative of homocysteine thiolactone (Hcy) to investigate its causal effect on apoptotic DNA injury in human promyeloid HL-60 cells. Our results demonstrated that Hcy induced cell death and features of apoptosis including increased phosphotidylserine exposure on the membrane surface, increased apoptotic cells with hypoploid DNA contents, and internucleosomal DNA fragmentation, all of which occurred in a time- and concentration-dependent manner. Hcy treatment also significantly increased intracellular reactive oxygen species H2O2, which coincided with the elimination of caspase 3 proenzyme levels and increased caspase 3 activity at the time of the appearance of apoptotic DNA fragmentation. Preincubation of Hcy-treated HL-60 cells with catalase completely scavenged intracellular H2O2, thus inhibiting caspase 3 activity and protecting cells from apoptotic DNA damage. In contrast, superoxide dismutase failed to inhibit Hcy-induced DNA damage. Taken together, these results demonstrate that Hcy exerted its genotoxic effects on HL-60 cells through an apoptotic pathway, which is mediated by the activation of caspase 3 activity induced by an increase in intracellular hydrogen peroxide. PMID:11432446

  13. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

    Science.gov (United States)

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  14. Mediation of the Physical Activity and Healthy Nutrition Behaviors of Preschool Children by Maternal Cognition in China.

    Science.gov (United States)

    Xu, Xianglong; Sharma, Manoj; Liu, Lingli; Hu, Ping; Zhao, Yong

    2016-01-01

    (1) OBJECTIVE: We aimed to explore the role of social cognitive theory (SCT) of mothers in the physical activity and healthy nutrition behaviors of preschool children; (2) METHODS: We used a self-administered five-point Likert common physical activity and nutrition behaviors scale in Chinese based on a social cognitive theory scale in English with established validity and reliability in the USA. The current study adopted the proportional sampling method to survey mothers of preschool children in four areas-namely, Chongqing, Chengdu, Taiyuan, and Shijiazhuang-of China; (3) RESULTS: We included 1208 mothers (80.0% mothers of normal weight children, age 31.87 ± 4.19 years). Positive correlations were found between maternal social cognition and preschool children's physical activity (PA) behavior (p social cognition; (4) CONCLUSIONS: This study provides some implications for increasing fruit and vegetable consumption, increasing physical activity time, and reducing screen time in preschool children using SCT in China. Maternal social cognition is associated with preschool children's PA behavior, and the results suggest that maternal social cognition may not affect children FV and ST behaviors. Further research is necessary to test the mediation of maternal social cognition on preschool children's ST behavior and the correlations between maternal social cognition and children's ST behavior. PMID:27649215

  15. Silybin-mediated inhibition of Notch signaling exerts antitumor activity in human hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Song Zhang

    Full Text Available Hepatocellular carcinoma (HCC is a global health burden that is associated with limited treatment options and poor patient prognoses. Silybin (SIL, an antioxidant derived from the milk thistle plant (Silybum marianum, has been reported to exert hepatoprotective and antitumorigenic effects both in vitro and in vivo. While SIL has been shown to have potent antitumor activity against various types of cancer, including HCC, the molecular mechanisms underlying the effects of SIL remain largely unknown. The Notch signaling pathway plays crucial roles in tumorigenesis and immune development. In the present study, we assessed the antitumor activity of SIL in human HCC HepG2 cells in vitro and in vivo and explored the roles of the Notch pathway and of the apoptosis-related signaling pathway on the activity of SIL. SIL treatment resulted in a dose- and time-dependent inhibition of HCC cell viability. Additionally, SIL exhibited strong antitumor activity, as evidenced not only by reductions in tumor cell adhesion, migration, intracellular glutathione (GSH levels and total antioxidant capability (T-AOC but also by increases in the apoptotic index, caspase3 activity, and reactive oxygen species (ROS. Furthermore, SIL treatment decreased the expression of the Notch1 intracellular domain (NICD, RBP-Jκ, and Hes1 proteins, upregulated the apoptosis pathway-related protein Bax, and downregulated Bcl2, survivin, and cyclin D1. Notch1 siRNA (in vitro or DAPT (a known Notch1 inhibitor, in vivo further enhanced the antitumor activity of SIL, and recombinant Jagged1 protein (a known Notch ligand in vitro attenuated the antitumor activity of SIL. Taken together, these data indicate that SIL is a potent inhibitor of HCC cell growth that targets the Notch signaling pathway and suggest that the inhibition of Notch signaling may be a novel therapeutic intervention for HCC.

  16. Biological activity of all-trans retinol requires metabolic conversion to all-trans retinoic acid and is mediated through activation of nuclear retinoid receptors in human keratinocytes.

    Science.gov (United States)

    Kurlandsky, S B; Xiao, J H; Duell, E A; Voorhees, J J; Fisher, G J

    1994-12-30

    The biological activity of all-trans retinol, in human keratinocytes, was investigated through metabolic and functional analyses that assessed the capacity for retinol uptake and metabolism and the mechanism of retinol-induced activation of gene transcription. Human keratinocytes converted all-trans retinol predominantly to retinyl esters, which accounted for 60 and 90% of cell-associated radiolabel after a 90-min pulse and a 48-h chase, respectively. Human keratinocytes also metabolized all-trans retinol to low levels of all-trans retinoic acid (11.47-131.3 ng/mg of protein) in a dose-dependent manner, between 0.3 and 10 microM added retinol. Small amounts of 13-cis retinoic acid (5.47-8.62 ng/mg of protein) were detected, but 9-cis retinoic acid was detected only when keratinocytes were incubated with radiolabeled retinol. There was no accumulation of the oxidized catabolic metabolites 4-hydroxy- or 4-oxoretinoic acid; however, 5,6-epoxy retinoic acid was detected at pharmacological levels (10 and 30 microM) of added retinol. Biological activity of retinol was assessed through analysis of two known retinoic acid-mediated responses: 1) reduction of type I epidermal transglutaminase and 2) activation of a retinoic acid receptor-dependent reporter gene, beta RARE3-tk-CAT. Both all-trans retinol and all-trans retinoic acid reduced type I epidermal transglutaminase in a dose-dependent manner; however, the ED50 for all-trans retinol (10 nM) was 10 times greater than for all-trans retinoic acid (1 nM). All-trans retinol also stimulated beta RARE3-tk-CAT reporter gene activity in a dose-dependent manner. Half-maximal induction was observed at 30 nM retinol, which was again 10-fold greater than observed with all-trans retinoic acid. Cotransfection of human keratinocytes with expression vectors for dominant negative mutant retinoic acid and retinoid X receptors reduced retinol-induced beta RARE3-tk-CAT reporter gene activation by 80%. Inhibition of conversion of all

  17. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

    Science.gov (United States)

    Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2014-03-01

    Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

  18. The cellular bromodomain protein Brd4 has multiple functions in E2-mediated papillomavirus transcription activation.

    Science.gov (United States)

    Helfer, Christine M; Yan, Junpeng; You, Jianxin

    2014-08-01

    The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription activation, is important for E2's transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2's interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+), a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV) life cycle. PMID:25140737

  19. The Cellular Bromodomain Protein Brd4 has Multiple Functions in E2-Mediated Papillomavirus Transcription Activation

    Directory of Open Access Journals (Sweden)

    Christine M. Helfer

    2014-08-01

    Full Text Available The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb, a functional interaction partner of Brd4 in transcription activation, is important for E2’s transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2’s interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+, a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV life cycle.

  20. DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

    Science.gov (United States)

    Habibovic, Aida; Hristova, Milena; Heppner, David E.; Danyal, Karamatullah; Ather, Jennifer L.; Janssen-Heininger, Yvonne M.W.; Irvin, Charles G.; Poynter, Matthew E.; Lundblad, Lennart K.; Dixon, Anne E.; Geiszt, Miklos

    2016-01-01

    Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite–induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management.