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Sample records for activating intrinsic pathway

  1. The Role of Intrinsic Pathway in Apoptosis Activation and Progression in Peyronie's Disease

    PubMed Central

    Loreto, Carla; Caltabiano, Rosario; Vespasiani, Giuseppe; Castorina, Sergio; Ralph, David J.; Musumeci, Giuseppe; Djinovic, Rados; Sansalone, Salvatore

    2014-01-01

    Peyronie's disease (PD) is characterized with formation of fibrous plaques which result in penile deformity, pain, and erectile dysfunction. The aim of this study was to investigate the activation of the intrinsic apoptotic pathway in plaques from PD patients. Tunica albuginea from either PD or control patients was assessed for the expression of bax, bcl-2 and caspases 9 and 3 using immunohistochemistry and by measurement of apoptotic cells using TUNEL assay. Bax overexpression was observed in metaplastic bone tissue, in fibroblasts, and in myofibroblast of plaques from PD patients. Little or no bcl-2 immunostaining was detected in samples from either patients or controls. Caspase 3 immunostaining was very strong in fibrous tissue, in metaplasic bone osteocytes, and in primary ossification center osteoblasts. Moderate caspase 9 immunostaining was seen in fibrous cells plaques and in osteocytes and osteoblasts of primary ossification centers from PD patients. Control samples were negative for caspase 9 immunostaining. In PD patients the TUNEL immunoassay showed intense immunostaining of fibroblasts and myofibroblasts, the absence of apoptotic cells in metaplasic bone tissue and on the border between fibrous and metaplastic bone tissue. Apoptosis occurs in stabilized PD plaques and is partly induced by the intrinsic pathway. PMID:25197653

  2. Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions.

    PubMed

    Thangam, Ramar; Sathuvan, Malairaj; Poongodi, Arasu; Suresh, Veeraperumal; Pazhanichamy, Kalailingam; Sivasubramanian, Srinivasan; Kanipandian, Nagarajan; Ganesan, Nalini; Rengasamy, Ramasamy; Thirumurugan, Ramasamy; Kannan, Soundarapandian

    2014-07-17

    Essential oils of Cymbopogon citratus were already reported to have wide ranging medical and industrial applications. However, information on polysaccharides from the plant and their anticancer activities are limited. In the present study, polysaccharides from C. citratus were extracted and fractionated by anion exchange and gel filtration chromatography. Two different polysaccharide fractions such as F1 and F2 were obtained, and these fractions were found to have distinct acidic polysaccharides as characterized by their molecular weight and sugar content. NMR spectral analysis revealed the presence of (1→4) linked b-d-Xylofuranose moiety in these polysaccharides. Using these polysaccharide fractions F1 and F2, anti-inflammatory and anticancer activities were evaluated against cancer cells in vitro and the mechanism of action of the polysaccharides in inducing apoptosis in cancer cells via intrinsic pathway was also proposed. Two different reproductive cancer cells such as Siha and LNCap were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation, changes in mitochondrial membrane potential, and profiles of gene and protein expression in response to treatment of cells by the polysaccharide fractions. These polysaccharide fractions exhibited potential cytotoxic and apoptotic effects on carcinoma cells, and they induced apoptosis in these cells through the events of up-regulation of caspase 3, down-regulation of bcl-2 family genes followed by cytochrome c release. PMID:24702929

  3. Cytotoxic activity of the novel heterocyclic compound G-11 is primarily mediated through intrinsic apoptotic pathway.

    PubMed

    Saleh, Ayman M; Aziz, Mohammad A; Abdou, Ibrahim M; Taha, Mutasem O; Al-Qudah, Mahmoud A; Abadleh, Mohammed M; Aljada, Ahmad; Rizvi, Syed A

    2016-07-01

    Natural and chemically synthesized heterocyclic compounds have been explored for their potential use as anticancer agents. We had synthesized non-natural heterocyclic analogs and evaluated their anti-tumor activity by measuring effect on cell proliferation and induction of apoptosis in different cell lines. Previously, we identified a pyrazole-containing compound (G-11) showing cytotoxic effect towards leukemia and lymphoma cell lines. In this study, we further investigated the mechanistic aspects of anticancer properties of G-11 in HL-60 cell line. We demonstrated that cytotoxic effect of G-11 is mediated by caspase-dependent apoptosis. However, the involvement of mitochondrial dysfunction induced by G-11 was independent of caspases. G-11 triggered generation of ROS, caused disruption of mitochondrial transmembrane potential, increased release of cytochrome c to the cytosol, and altered the expression of Bcl-2 and Bax proteins. These results suggest significant involvement of intrinsic apoptotic pathway. This study comprehensively details the possible mechanisms of action of a novel heterocyclic compound which could find its potential use as an anticancer agent. PMID:27154302

  4. Umbelliprenin from Ferula szowitsiana Activates both Intrinsic and Extrinsic Pathways of Apoptosis in Jurkat T-CLL cell line.

    PubMed

    Gholami, Omid; Jeddi-Tehrani, Mahmood; Iranshahi, Mehrdad; Zarnani, Amir Hassan; Ziai, Seyed Ali

    2013-01-01

    Umbelliprenin is a prenylated compound, which belongs to the class of sesquiterpene coumarins. It is extracted from dried roots of Ferula szwitsiana collected from the mountains of Golestan forest (Golestan Province, north of Iran). Induction of apoptosis in Jurkat T-CLL cells has been previously shown. In this study, effect of umbelliprenin on proapoptotic caspases (caspase-8 and -9) and antiapoptotic Bcl-2 family protein was studied. Jurkat cells were incubated with umbelliprenin. Cells were then lysed and activation of proteins was studied by Western blot analysis. In this study, we showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and -9 respectively. Inhibition of Bcl-2 was also shown. In conclusion, umbelliprenin induced apoptosis in Jurkat cells through caspase-dependent apoptosis pathway. PMID:24250644

  5. Umbelliprenin from Ferula szowitsiana Activates both Intrinsic and Extrinsic Pathways of Apoptosis in Jurkat T-CLL cell line

    PubMed Central

    Gholami, Omid; Jeddi-Tehrani, Mahmood; Iranshahi, Mehrdad; Zarnani, Amir Hassan; Ziai, Seyed Ali

    2013-01-01

    Umbelliprenin is a prenylated compound, which belongs to the class of sesquiterpene coumarins. It is extracted from dried roots of Ferula szwitsiana collected from the mountains of Golestan forest (Golestan Province, north of Iran). Induction of apoptosis in Jurkat T-CLL cells has been previously shown. In this study, effect of umbelliprenin on proapoptotic caspases (caspase-8 and -9) and antiapoptotic Bcl-2 family protein was studied. Jurkat cells were incubated with umbelliprenin. Cells were then lysed and activation of proteins was studied by Western blot analysis. In this study, we showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and -9 respectively. Inhibition of Bcl-2 was also shown. In conclusion, umbelliprenin induced apoptosis in Jurkat cells through caspase-dependent apoptosis pathway. PMID:24250644

  6. Highly efficient synthetic iron-dependent nucleases activate both intrinsic and extrinsic apoptotic death pathways in leukemia cancer cells.

    PubMed

    Horn, Adolfo; Fernandes, Christiane; Parrilha, Gabrieli L; Kanashiro, Milton M; Borges, Franz V; de Melo, Edésio J T; Schenk, Gerhard; Terenzi, Hernán; Pich, Claus T

    2013-11-01

    The nuclease activity and the cytotoxicity toward human leukemia cancer cells of iron complexes, [Fe(HPClNOL)Cl2]NO3 (1), [Cl(HPClNOL)Fe(μ-O)Fe(HPClNOL)Cl]Cl2·2H2O (2), and [(SO4)(HPClNOL)Fe(μ-O)Fe(HPClNOL)(SO4)]·6H2O (3) (HPClNOL=1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol), were investigated. Each complex was able to promote plasmid DNA cleavage and change the supercoiled form of the plasmid to circular and linear ones. Kinetic data revealed that (1), (2) and (3) increase the rate of DNA hydrolysis about 278, 192 and 339 million-fold, respectively. The activity of the complexes was inhibited by distamycin, indicating that they interact with the minor groove of the DNA. The cytotoxic activity of the complexes toward U937, HL-60, Jukart and THP-1 leukemia cancer cells was studied employing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fluorescence and electronic transmission microscopies, flow cytometry and a cytochrome C release assay. Compound (2) has the highest activity toward cancer cells and is the least toxic for normal ones (i.e. peripheral blood mononuclear cells (PBMCs)). In contrast, compound (1) is the least active toward cancer cells but displays the highest toxicity toward normal cells. Transmission electronic microscopy indicates that cell death shows features typical of apoptotic cells, which was confirmed using the annexin V-FITC/PI (fluorescein isothiocyanate/propidium iodide) assay. Furthermore, our data demonstrate that at an early stage during the treatment with complex (2) mitochondria lose their transmembrane potential, resulting in cytochrome C release. A quantification of caspases 3, 9 (intrinsic apoptosis pathway) and caspase 8 (extrinsic apoptosis pathway) indicated that both the intrinsic (via mitochondria) and extrinsic (via death receptors) pathways are involved in the apoptotic stimuli. PMID:23933562

  7. BisGMA-induced cytotoxicity and genotoxicity in macrophages are attenuated by wogonin via reduction of intrinsic caspase pathway activation.

    PubMed

    Huang, Fu-Mei; Chang, Yu-Chao; Lee, Shiuan-Shinn; Yeh, Chung-Hsin; Lee, Kevin Gee; Huang, Yi-Chun; Chen, Chun-Jung; Chen, Wen-Ying; Pan, Pin-Ho; Kuan, Yu-Hsiang

    2016-02-01

    Bisphenol-A-glycidyldimethacrylate (BisGMA) is a frequently used monomer in dental restorative resins. However, BisGMA could leach from dental restorative resins after polymerization leading to inflammation in the peripheral environment. Wogonin, a natural flavone derivative, has several benefits, such as antioxidative, anti-inflammatory and neuroprotective properties. Pretreatment of macrophage RAW264.7 cells with wogonin inhibited cytotoxicity which is induced by BisGMA in a concentration-dependent manner. BisGMA induced apoptotic responses, such as redistribution of phosphatidylserine from the internal to the external membrane and DNA fragmentation, were decreased by wogonin in a concentration-dependent manner. In addition, BisGMA-induced genotoxicity, which detected by cytokinesis-blocked micronucleus and single-cell gel electrophoresis assays, were inhibited by wogonin in a concentration-dependent manner. Furthermore, wogonin suppressed BisGMA-induced activation of intrinsic caspase pathways, such as caspases-3 and -8. Parallel trends were observed in inhibition of caspase-3 and -8 activities, apoptosis, and genotoxicity. These results indicate wogonin suppressed the BisGMA-induced apoptosis and genotoxicity mainly via intrinsic caspase pathway in macrophages. PMID:26756871

  8. Distinct spatial activation of intrinsic and extrinsic apoptosis pathways in natural scrapie: association with prion-related lesions

    PubMed Central

    Serrano, Carmen; Lyahyai, Jaber; Bolea, Rosa; Varona, Luis; Monleón, Eva; Badiola, Juan J.; Zaragoza, Pilar; Martín-Burriel, Inmaculada

    2009-01-01

    Neurodegeneration and gliosis are the main neuropathological features of prion diseases. However, the molecular mechanisms involved in these processes remain unclear. Several studies have demonstrated changes in the expression of apoptotic factors and inflammatory cytokines in animals with experimental infection. Here we present the expression profiles of 15 genes implicated in the intrinsic and extrinsic apoptotic pathways in the central nervous systems of sheep naturally infected with scrapie. Expression changes obtained by real-time RT-PCR were also compared with the extent of classical scrapie lesions, such as prion deposition, neuronal vacuolisation, spongiosis, and astrogliosis as well as with the activation of caspase-3, using a stepwise regression. The results suggest that the factors assessed participate in apoptotic or inflammatory functions, depending on the affected area. The mitochondrial apoptosis pathway was associated with prion deposition in the prefrontal cortex (the less affected area), and with activation of caspase-3-mediated cell death via over-expression of BAK. In addition to its known association with astroglial activation, the extrinsic apoptosis pathway was also related to cell death and neuronal vacuolisation. PMID:19401142

  9. Photodynamic therapy activated STAT3 associated pathways: Targeting intrinsic apoptotic pathways to increase PDT efficacy in human squamous carcinoma cells.

    PubMed

    Qiao, Li; Xu, Chengshan; Li, Qiang; Mei, Zhusong; Li, Xinji; Cai, Hong; Liu, Wei

    2016-06-01

    5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) has been used for part of squamous cell carcinoma (premalignant conditions or in situ cutaneous SCC-Bowen disease). However, mechanism of ALA-PDT is not fully understood yet on the cell apoptosis pathway. The aim of this study was to further investigate the effect and mechanism of 5-ALA-PDT on human squamous carcinoma A431cells. Apoptosis and cell viability after PDT were evaluated using Annexin V-FITC apoptosis detection kit and MTT assay. The mRNA and protein levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Our data showed that 5-ALA-PDT significantly inhibited cell proliferation (p<0.05), but there was no significant difference when the photosensitizer reached to 4.8mM. The inhibition in cell proliferation after 5-ALA-PDT treatment was correlated to more cells being arrested in the G0/G1 phase of the cell cycle (p<0.01). Immunocytochemical observations using anti-active caspase-3 antibodies showed active caspase-3 was translocated from cytoplasm to nuclear during apoptosis. STAT3 and its downstream gene Bax and BCL-2 were changed after 5-ALA-PDT treatment for the mRNA and protein expression. Our studies confirmed that 5-ALA-PDT might be an effective treatment for human squamous carcinoma by inhibiting the tumor cell A431growth and for the first time demonstrated that the expression of STAT3 was significantly reduced at 24h after 5-ALA-PDT treatment. PMID:26607555

  10. Prostaglandin D2 induces apoptosis of human osteoclasts by activating the CRTH2 receptor and the intrinsic apoptosis pathway.

    PubMed

    Yue, Li; Durand, Marianne; Lebeau Jacob, M Christian; Hogan, Philippe; McManus, Stephen; Roux, Sophie; de Brum-Fernandes, Artur J

    2012-09-01

    Prostaglandin D(2) (PGD(2)) is a lipid mediator synthesized from arachidonic acid that directly activates two specific receptors, the D-type prostanoid (DP) receptor and chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). PGD(2) can affect bone metabolism by influencing both osteoblast and osteoclast (OC) functions, both cells involved in bone remodeling and in in vivo fracture repair as well. The objective of the present study was to determine the effects of PGD(2), acting through its two specific receptors, on human OC apoptosis. Human OCs were differentiated in vitro from peripheral blood mononuclear cells in the presence of receptor activator for nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and treated with PGD(2), its specific agonists and antagonists. Treatment with PGD(2) for 24hours in the presence of naproxen (10μM) to inhibit endogenous prostaglandin production increased the percentage of apoptotic OCs in a dose-dependent manner, as did the specific CRTH2 agonist compound DK-PGD(2) but not the DP agonist compound BW 245C. In the absence of naproxen, the CRTH2 antagonist compound CAY 10471 reduced OC apoptosis rate but the DP antagonist BW A868C had no effect. The induction of PGD(2)-CRTH2 dependent apoptosis was associated with the activation of caspase-9, but not caspase-8, leading to caspase-3 cleavage. These data show that PGD(2) induces human OC apoptosis through activation of CRTH2 and the apoptosis intrinsic pathway. PMID:22705147

  11. Dichloromethane fraction of Melissa officinalis induces apoptosis by activation of intrinsic and extrinsic pathways in human leukemia cell lines.

    PubMed

    Ebrahimnezhad Darzi, Salimeh; Amirghofran, Zahra

    2013-06-01

    Various components from medicinal plants are currently used in cancer therapy because of their apoptosis-inducing effects. The present study has aimed to investigate the growth inhibitory and apoptotic effects of Melissa officinalis on tumor cells. We prepared different fractions of this plant to investigate their inhibitory effects on two leukemia cell lines, Jurkat and K562. Fractions with the highest inhibitory effects were examined for induction of apoptosis by the annexin V/propidium iodide assay and cell cycle changes by flow cytometry. Real-time polymerase chain reaction evaluated the changes in expression of apoptosis-related genes. Among different fractions, dichloromethane and n-hexane dose-dependent showed the strongest inhibitory effects on both K562 and Jurkat cells. The dichloromethane fraction significantly induced apoptosis at concentration of 50 µg/ml on Jurkat (85.66 ± 4.9%) and K562 cells (65.04 ± 0.93%) at 24 h after treatment (p < 0.002). According to cell cycle analysis, more than 70% of the cells accumulated in the sub-G1 phase when cultured in the presence of the dichloromethane fraction. This fraction up-regulated Fas and Bax mRNA expression as well as the Bax/Bcl-2 ratio according to cell type, showing its effect on the activation of both extrinsic and intrinsic pathways of apoptosis. The expression of apoptosis-related genes did not significantly change following treatment with the n-hexane fraction. These data indicated that the dichloromethane fraction of M. officinalis had the ability to induce apoptosis and change apoptosis-related gene expression in leukemia cells. PMID:23432355

  12. Predicting intrinsic brain activity.

    PubMed

    Craddock, R Cameron; Milham, Michael P; LaConte, Stephen M

    2013-11-15

    Multivariate supervised learning methods exhibit a remarkable ability to decode externally driven sensory, behavioral, and cognitive states from functional neuroimaging data. Although they are typically applied to task-based analyses, supervised learning methods are equally applicable to intrinsic effective and functional connectivity analyses. The obtained models of connectivity incorporate the multivariate interactions between all brain regions simultaneously, which will result in a more accurate representation of the connectome than the ones available with standard bivariate methods. Additionally the models can be applied to decode or predict the time series of intrinsic brain activity of a region from an independent dataset. The obtained prediction accuracy provides a measure of the integration between a brain region and other regions in its network, as well as a method for evaluating acquisition and preprocessing pipelines for resting state fMRI data. This article describes a method for learning multivariate models of connectivity. The method is applied in the non-parametric prediction accuracy, influence, and reproducibility-resampling (NPAIRS) framework, to study the regional variation of prediction accuracy and reproducibility (Strother et al., 2002). The resulting spatial distribution of these metrics is consistent with the functional hierarchy proposed by Mesulam (1998). Additionally we illustrate the utility of the multivariate regression connectivity modeling method for optimizing experimental parameters and assessing the quality of functional neuroimaging data. PMID:23707580

  13. Farnesol activates the intrinsic pathway of apoptosis and the ATF4-ATF3-CHOP cascade of ER stress in human T lymphoblastic leukemia Molt4 cells.

    PubMed

    Joo, Joung Hyuck; Ueda, Eiichiro; Bortner, Carl D; Yang, Xiao-Ping; Liao, Grace; Jetten, Anton M

    2015-10-01

    In this study, we demonstrate that treatment of T lymphoblastic leukemic Molt4 cells with farnesol activates the apoptosome via the intrinsic pathway of apoptosis. This induction was associated with changes in the level of intracellular potassium and calcium, the dissipation of the mitochondrial and plasma membrane potential, release of cytochrome c, activation of several caspases, and PARP cleavage. The induction of apoptosis by farnesol was inhibited by the addition of the pan-caspase inhibitor Z-VAD-fmk and by the exogenous expression of the anti-apoptotic protein Bcl2. Analysis of the gene expression profiles by microarray analysis revealed that farnesol increased the expression of several genes related to the unfolded protein response (UPR), including CHOP and CHAC1. This induction was associated with the activation of the PERK-eIF2α-ATF3/4 cascade, but not the XBP-1 branch of the UPR. Although farnesol induced activation of the ERK1/2, p38, and JNK pathways, inhibition of these MAPKs had little effect on farnesol-induced apoptosis or the induction of UPR-related genes. Our data indicate that the induction of apoptosis in leukemic cells by farnesol is mediated through a pathway that involves activation of the apoptosome via the intrinsic pathway and induction of the PERK-eIF2α-ATF3/4 cascade in a manner that is independent of the farnesol-induced activation of MAPKs. PMID:26275811

  14. Arsenite induces apoptosis in human mesenchymal stem cells by altering Bcl-2 family proteins and by activating intrinsic pathway

    SciTech Connect

    Yadav, Santosh; Shi Yongli; Wang Feng; Wang He

    2010-05-01

    Purpose: Environmental exposure to arsenic is an important public health issue. The effects of arsenic on different tissues and organs have been intensively studied. However, the effects of arsenic on bone marrow mesenchymal stem cells (MSCs) have not been reported. This study is designed to investigate the cell death process caused by arsenite and its related underlying mechanisms on MSCs. The rationale is that absorbed arsenic in the blood circulation can reach to the bone marrow and may affect the cell survival of MSCs. Methods: MSCs of passage 1 were purchased from Tulane University, grown till 70% confluency level and plated according to the experimental requirements followed by treatment with arsenite at various concentrations and time points. Arsenite (iAs{sup III}) induced cytotoxic effects were confirmed by cell viability and cell cycle analysis. For the presence of canonic apoptosis markers; DNA damage, exposure of intramembrane phosphotidylserine, protein and m-RNA expression levels were analyzed. Results: iAs{sup III} induced growth inhibition, G2-M arrest and apoptotic cell death in MSCs, the apoptosis induced by iAs{sup III} in the cultured MSCs was, via altering Bcl-2 family proteins and by involving intrinsic pathway. Conclusion: iAs{sup III} can induce apoptosis in bone marrow-derived MSCs via Bcl-2 family proteins, regulating intrinsic apoptotic pathway. Due to the multipotency of MSC, acting as progenitor cells for a variety of connective tissues including bone, adipose, cartilage and muscle, these effects of arsenic may be important in assessing the health risk of the arsenic compounds and understanding the mechanisms of arsenic-induced harmful effects.

  15. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

    PubMed Central

    Ruiz-Magaña, María J.; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

    2016-01-01

    Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect. PMID:26942461

  16. Ferulago angulata activates intrinsic pathway of apoptosis in MCF-7 cells associated with G1 cell cycle arrest via involvement of p21/p27

    PubMed Central

    Karimian, Hamed; Moghadamtousi, Soheil Zorofchian; Fadaeinasab, Mehran; Golbabapour, Shahram; Razavi, Mahboubeh; Hajrezaie, Maryam; Arya, Aditya; Abdulla, Mahmood Ameen; Mohan, Syam; Ali, Hapipah Mohd; Noordin, Mohamad Ibrahim

    2014-01-01

    Ferulago angulata is a medicinal plant that is traditionally known for its anti-inflammatory and antiulcer properties. The present study was aimed to evaluate its anticancer activity and the possible mechanism of action using MCF-7 as an in vitro model. F. angulata leaf extracts were prepared using solvents in the order of increasing polarity. As determined by MTT assay, F. angulata leaves hexane extract (FALHE) revealed the strongest cytotoxicity against MCF-7 cells with the half maximal inhibitory concentration (IC50) value of 5.3±0.82 μg/mL. The acute toxicity study of FALHE provided evidence of the safety of the plant extract. Microscopic and flow cytometric analysis using annexin-V probe showed an induction of apoptosis in MCF-7 by FALHE. Treatment of MCF-7 cells with FALHE encouraged the intrinsic pathway of apoptosis, with cell death transducing signals that reduced the mitochondrial membrane potential with cytochrome c release from mitochondria to cytosol. The released cytochrome c triggered the activation of caspase-9. Meanwhile, the overexpression of caspase-8 suggested the involvement of an extrinsic pathway in the induced apoptosis at the late stage of treatment. Moreover, flow cytometric analysis showed that FALHE treatment significantly arrested MCF-7 cells in the G1 phase, which was associated with upregulation of p21 and p27 assessed by quantitative polymerase chain reaction. Immunofluorescence and the quantitative polymerase chain reaction analysis of MCF-7 cells after treatment with FALHE revealed an upregulation of Bax and a downregulation of Bcl-2 proteins. These findings proposed that FALHE suppressed the proliferation of MCF-7 cells via cell cycle arrest and the induction of apoptosis through intrinsic pathway. PMID:25278746

  17. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis.

    PubMed

    Tao, Weikang; South, Victoria J; Diehl, Ronald E; Davide, Joseph P; Sepp-Lorenzino, Laura; Fraley, Mark E; Arrington, Kenneth L; Lobell, Robert B

    2007-01-01

    The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors. PMID:17101792

  18. NT-3 promotes proprioceptive axon regeneration when combined with activation of the mTor intrinsic growth pathway but not with reduction of myelin extrinsic inhibitors.

    PubMed

    Liu, Yingpeng; Kelamangalath, Lakshmi; Kim, Hyukmin; Han, Seung Baek; Tang, Xiaoqing; Zhai, Jinbin; Hong, Jee W; Lin, Shen; Son, Young-Jin; Smith, George M

    2016-09-01

    Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons. PMID:27264357

  19. Zinc ferrite nanoparticles activate IL-1b, NFKB1, CCL21 and NOS2 signaling to induce mitochondrial dependent intrinsic apoptotic pathway in WISH cells

    SciTech Connect

    Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ahmad, Javed; Siddiqui, Maqsood A.; Dwivedi, Sourabh; Khan, Shams T.; Musarrat, Javed

    2013-12-01

    The present study has demonstrated the translocation of zinc ferrite nanoparticles (ZnFe{sub 2}O{sub 4}-NPs) into the cytoplasm of human amnion epithelial (WISH) cells, and the ensuing cytotoxicity and genetic damage. The results suggested that in situ NPs induced oxidative stress, alterations in cellular membrane and DNA strand breaks. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) and neutral red uptake (NRU) cytotoxicity assays indicated 64.48 ± 1.6% and 50.73 ± 2.1% reduction in cell viability with 100 μg/ml of ZnFe{sub 2}O{sub 4}-NPs exposure. The treated WISH cells exhibited 1.2-fold higher ROS level with 0.9-fold decline in membrane potential (ΔΨm) and 7.4-fold higher DNA damage after 48 h of ZnFe{sub 2}O{sub 4}-NPs treatment. Real-time PCR (qPCR) analysis of p53, CASP 3 (caspase-3), and bax genes revealed 5.3, 1.6, and 14.9-fold upregulation, and 0.18-fold down regulation of bcl 2 gene vis-à-vis untreated control. RT{sup 2} Profiler™ PCR array data elucidated differential up-regulation of mRNA transcripts of IL-1b, NFKB1, NOS2 and CCL21 genes in the range of 1.5 to 3.7-folds. The flow cytometry based cell cycle analysis suggested the transfer of 15.2 ± 2.1% (p < 0.01) population of ZnFe{sub 2}O{sub 4}-NPs (100 μg/ml) treated cells into apoptotic phase through intrinsic pathway. Over all, the data revealed the potential of ZnFe{sub 2}O{sub 4}-NPs to induce cellular and genetic toxicity in cells of placental origin. Thus, the significant ROS production, reduction in ΔΨm, DNA damage, and activation of genes linked to inflammation, oxidative stress, proliferation, DNA damage and repair could serve as the predictive toxicity and stress markers for ecotoxicological assessment of ZnFe{sub 2}O{sub 4}-NPs induced cellular and genetic damage. - Highlights: • First report on the molecular toxicity of ZnFe{sub 2}O{sub 4}-NPs in cells of placental origin • WISH cells treated with ZnFe{sub 2}O{sub 4}-NPs exhibited cytoplasmic

  20. Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways

    PubMed Central

    Chen, Chien-Wen; Wu, Ming-Shan; Huang, Yi-Jen; Lin, Pei-Wen; Shih, Chueh-Ju; Lin, Fu-Pang; Chang, Chi-Yao

    2015-01-01

    Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways. PMID:26047333

  1. Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway

    PubMed Central

    Venugopal, Jessica; Blanco, Gustavo

    2016-01-01

    Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key “executioner” caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression. PMID:27047392

  2. Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway.

    PubMed

    Venugopal, Jessica; Blanco, Gustavo

    2016-01-01

    Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key "executioner" caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression. PMID:27047392

  3. BCL-2 Antagonism to Target the Intrinsic Mitochondrial Pathway of Apoptosis.

    PubMed

    Gibson, Christopher J; Davids, Matthew S

    2015-11-15

    Despite significant improvements in treatment, cure rates for many cancers remain suboptimal. The rise of cytotoxic chemotherapy has led to curative therapy for a subset of cancers, though intrinsic treatment resistance is difficult to predict for individual patients. The recent wave of molecularly targeted therapies has focused on druggable-activating mutations, and is thus limited to specific subsets of patients. The lessons learned from these two disparate approaches suggest the need for therapies that borrow aspects of both, targeting biologic properties of cancer that are at once distinct from normal cells and yet common enough to make the drugs widely applicable across a range of cancer subtypes. The intrinsic mitochondrial pathway of apoptosis represents one such promising target for new therapies, and successfully targeting this pathway has the potential to alter the therapeutic landscape of therapy for a variety of cancers. Here, we discuss the biology of the intrinsic pathway of apoptosis, an assay known as BH3 profiling that can interrogate this pathway, early attempts to target BCL-2 clinically, and the recent promising results with the BCL-2 antagonist venetoclax (ABT-199) in clinical trials in hematologic malignancies. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy." PMID:26567361

  4. The restless brain: how intrinsic activity organizes brain function

    PubMed Central

    Raichle, Marcus E.

    2015-01-01

    Traditionally studies of brain function have focused on task-evoked responses. By their very nature such experiments tacitly encourage a reflexive view of brain function. While such an approach has been remarkably productive at all levels of neuroscience, it ignores the alternative possibility that brain functions are mainly intrinsic and ongoing, involving information processing for interpreting, responding to and predicting environmental demands. I suggest that the latter view best captures the essence of brain function, a position that accords well with the allocation of the brain's energy resources, its limited access to sensory information and a dynamic, intrinsic functional organization. The nature of this intrinsic activity, which exhibits a surprising level of organization with dimensions of both space and time, is revealed in the ongoing activity of the brain and its metabolism. As we look to the future, understanding the nature of this intrinsic activity will require integrating knowledge from cognitive and systems neuroscience with cellular and molecular neuroscience where ion channels, receptors, components of signal transduction and metabolic pathways are all in a constant state of flux. The reward for doing so will be a much better understanding of human behaviour in health and disease. PMID:25823869

  5. The restless brain: how intrinsic activity organizes brain function.

    PubMed

    Raichle, Marcus E

    2015-05-19

    Traditionally studies of brain function have focused on task-evoked responses. By their very nature such experiments tacitly encourage a reflexive view of brain function. While such an approach has been remarkably productive at all levels of neuroscience, it ignores the alternative possibility that brain functions are mainly intrinsic and ongoing, involving information processing for interpreting, responding to and predicting environmental demands. I suggest that the latter view best captures the essence of brain function, a position that accords well with the allocation of the brain's energy resources, its limited access to sensory information and a dynamic, intrinsic functional organization. The nature of this intrinsic activity, which exhibits a surprising level of organization with dimensions of both space and time, is revealed in the ongoing activity of the brain and its metabolism. As we look to the future, understanding the nature of this intrinsic activity will require integrating knowledge from cognitive and systems neuroscience with cellular and molecular neuroscience where ion channels, receptors, components of signal transduction and metabolic pathways are all in a constant state of flux. The reward for doing so will be a much better understanding of human behaviour in health and disease. PMID:25823869

  6. Multiple oxygen entry pathways in globin proteins revealed by intrinsic pathway identification method

    NASA Astrophysics Data System (ADS)

    Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

    2015-12-01

    Each subunit of human hemoglobin (HbA) stores an oxygen molecule (O2) in the binding site (BS) cavity near the heme group. The BS is buried in the interior of the subunit so that there is a debate over the O2 entry pathways from solvent to the BS; histidine gate or multiple pathways. To elucidate the O2 entry pathways, we executed ensemble molecular dynamics (MD) simulations of T-state tetramer HbA in high concentration O2 solvent to simulate spontaneous O2 entry from solvent into the BS. By analyzing 128 independent 8 ns MD trajectories by intrinsic pathway identification by clustering (IPIC) method, we found 141 and 425 O2 entry events into the BS of the α and β subunits, respectively. In both subunits, we found that multiple O2 entry pathways through inside cavities play a significant role for O2 entry process of HbA. The rate constants of O2 entry estimated from the MD trajectories correspond to the experimentally observed values. In addition, by analyzing monomer myoglobin, we verified that the high O2 concentration condition can reproduce the ratios of each multiple pathway in the one-tenth lower O2 concentration condition. These indicate the validity of the multiple pathways obtained in our MD simulations.

  7. Apoptosis Induction by 13-Acetoxyrolandrolide through the Mitochondrial Intrinsic Pathway

    PubMed Central

    Acuña, Ulyana Muñoz; Matthew, Susan; Pan, Li; Kinghorn, A. Douglas; Swanson, Steven M.; Carcache de Blanco, Esperanza J.

    2014-01-01

    The aim of this study was to evaluate the mechanisms of cytotoxicity of the sesquiterpene lactone 13-acetoxyrolandrolide, an NF-κB inhibitor that was previously isolated from Rolandra fruticosa. The effects associated with inhibition of the NFκB pathway included dose-dependent inhibition of the NF-κB subunit p65 (RelA) and inhibition of upstream mediators IKKβ and oncogenic K-Ras. The inhibitory concentration (IC50) of 13-acetoxyrolandrolide on K-Ras was 7.7 μM. The downstream effects of the inhibition of NF-κB activation were also investigated in vitro. After 24 h of treatment with 13-acetoxyrolandrolide, the mitochondrial transmembrane potential (ΔΨm) was depolarized in human colon cancer (HT-29) cells. The mitochondrial oxidative phosphorylation was also negatively affected and reduced levels of NAD(P)H were detected after 2 h of 13-acetoxyrolandrolide exposure. Furthermore, the expression of the pro-apoptotic protein caspase-3 increased in a concentration-dependent manner. Cell flow cytometry showed that 13-acetoxyrolandrolide induced cell cycle arrest at G1, suggesting that treated cells had undergone caspase-3-mediated apoptosis, suggesting negative effects on cancer cell proliferation. These results suggest that 13-acetoxyrolandrolide inhibits NF-κB and K-Ras as well as promotes cell death mediated through the mitochondrial apoptotic pathway. PMID:24338805

  8. Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway.

    PubMed

    Elumalai, P; Gunadharini, D N; Senthilkumar, K; Banudevi, S; Arunkumar, R; Benson, C S; Sharmila, G; Arunakaran, J

    2012-11-30

    We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy. PMID:23089555

  9. Longitudinal Pathways from Math Intrinsic Motivation and Achievement to Math Course Accomplishments and Educational Attainment

    ERIC Educational Resources Information Center

    Gottfried, Adele Eskeles; Marcoulides, George A.; Gottfried, Allen W.; Oliver, Pamella H.

    2013-01-01

    Across 20 years, pathways from math intrinsic motivation and achievement (ages 9-17) to high school math course accomplishments and educational attainment (age 29) were analyzed. Academic intrinsic motivation was the theoretical foundation. To determine how initial status and change in motivation and achievement related to course accomplishments…

  10. 2-Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway.

    PubMed

    Schweikl, Helmut; Petzel, Christine; Bolay, Carola; Hiller, Karl-Anton; Buchalla, Wolfgang; Krifka, Stephanie

    2014-03-01

    Resin monomers of dental composites like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation of dental pulp-derived cells, or induce cell death via apoptosis. The induction of apoptosis is related to the availability of the antioxidant glutathione, although a detailed understanding of the signaling pathways is still unknown. The present study provides insight into the causal relationship between oxidative stress, oxidative DNA damage, and the specific signaling pathway leading to HEMA-induced apoptosis in RAW264.7 mouse macrophages. The differential expression of the antioxidative enzymes superoxide dismutase, glutathione peroxidase, and catalase in HEMA-exposed cells indicated oxidative stress, which was associated with the cleavage of pro-caspase 3 as a critical apoptosis executioner. A 2-fold increase in the amount of mitochondrial superoxide anions after a 24 h exposure to HEMA (6-8 mM) was paralleled by a considerable decrease in the mitochondrial membrane potential (MMP). Additionally, expression of proteins critical for the signaling of apoptosis through the intrinsic mitochondrial pathway was detected. Transcription-dependent and transcription-independent mechanisms of p53-regulated apoptosis were activated, and p53 was translocated from the cytosol to mitochondria. HEMA-induced transcriptional activity of p53 was indicated by increased levels of PUMA localized to mitochondria as a potent inducer of apoptosis. The expression of Bcl-xL and Bax suggested that cells responded to stress caused by HEMA via the activation of a complicated and antagonistic machinery of pro- and anti-apoptotic Bcl-2 family members. A HEMA-induced and oxidative stress-sensitive delay of the cell cycle, indicating a DNA damage response, occurred independent of the influence of KU55399, a potent inhibitor of ATM (ataxia-telangiectasia mutated) activity. However, ATM, a protein kinase which

  11. Methyl angolensate, a natural tetranortriterpenoid induces intrinsic apoptotic pathway in leukemic cells.

    PubMed

    Chiruvella, Kishore K; Kari, Vijayalakshmi; Choudhary, Bibha; Nambiar, Mridula; Ghanta, Rama Gopal; Raghavan, Sathees C

    2008-12-10

    Methyl angolensate (MA), a natural tetranortriterpenoid, purified from Soymida febrifuga is examined for the first time for its anticancer properties. We find that MA inhibits growth of T-cell leukemia and chronic myelogenous leukemia cells in a time- and dose-dependent manner. Accumulation of cells in the subG1 peak, annexin V binding and DNA fragmentation suggested induction of apoptosis. Besides, upregulation of BAD (proapoptotic) and downregulation of BCL2 (antiapoptotic) gene products further supported induction of apoptosis. Loss of mitochondrial membrane potential, activation of caspase 9, caspase 3, cleavage of PARP, downregulation of Ku70/80 and phosphorylation of MAP kinases suggested that MA could induce intrinsic pathway of apoptosis in leukemic cells. PMID:19022252

  12. Induction of intrinsic and extrinsic apoptosis pathways in the human leukemic MOLT-4 cell line by terpinen-4-ol.

    PubMed

    Khaw-on, Patompong; Banjerdpongchai, Ratana

    2012-01-01

    Terpinen-4-ol is a terpene found in the rhizome of Plai (Zingiber montanum (Koenig) Link ex Dietr.). In this study apoptogenic activity and mechanisms of cell death induced by terpinen-4-ol were investigated in the human leukemic MOLT-4 cell line. Terpinen-4-ol exhibited cytotoxicity in MOLT-4 cells, with characteristic morphological features of apoptosis by Wright's staining. The mode of cell death was confirmed to be apoptosis by flow cytometric analysis after staining with annexin V-FITC and propidium iodide. A sub-G1 peak in DNA histograms of cell cycle assays was observed. Terpinen-4-ol induced-MOLT-4 cell apoptosis mediated through an intrinsic pathway involving the loss of mitochondrial transmembrane potential (MTP) and release of cytochrome c into the cytosol. In addition, terpinen-4-ol also induced apoptosis via an extrinsic pathway by caspase-8 activation resulting in the cleavage of cytosolic Bid. Truncated-Bid (tBid) translocated to mitochondria and activated the mitochondrial pathway in conjunction with down-regulation of Bcl-2 protein expression. Caspase-3 activity also increased. In conclusion, terpinen-4-ol can induce human leukemic MOLT-4 cell apoptosis via both intrinsic and extrinsic pathways. PMID:22994712

  13. Intrinsic free energy in active nematics

    NASA Astrophysics Data System (ADS)

    Thampi, Sumesh P.; Doostmohammadi, Amin; Golestanian, Ramin; Yeomans, Julia M.

    2015-10-01

    Basing our arguments on the theory of active liquid crystals, we demonstrate, both analytically and numerically, that the activity can induce an effective free energy which enhances ordering in extensile systems of active rods and in contractile suspensions of active discs. We argue that this occurs because any ordering fluctuation is enhanced by the flow field it produces. A phase diagram in the temperature-activity plane compares ordering due to a thermodynamic free energy to that resulting from the activity. We also demonstrate that activity can drive variations in concentration, but for a different physical reason that relies on the separation of hydrodynamic and diffusive time scales.

  14. The roles of intrinsic disorder in orchestrating the Wnt-pathway.

    PubMed

    Xue, Bin; Dunker, A Keith; Uversky, Vladimir N

    2012-01-01

    The canonical Wnt-pathway plays a number of crucial roles in the development of organism. Malfunctions of this pathway lead to various diseases including cancer. In the inactivated state, this pathway involves five proteins, Axin, CKI-α, GSK-3β, APC, and β-catenin. We analyzed these proteins by a number of computational tools, such as PONDR(r)VLXT, PONDR(r)VSL2, MoRF-II predictor and Hydrophobic Cluster Analysis (HCA) to show that each of the Wnt-pathway proteins contains several intrinsically disordered regions. Based on a comprehensive analysis of published data we conclude that these disordered regions facilitate protein-protein interactions, post-translational modifications, and signaling. The scaffold protein Axin and another large protein, APC, act as flexible concentrators in gathering together all other proteins involved in the Wnt-pathway, emphasizing the role of intrinsically disordered regions in orchestrating the complex protein-protein interactions. We further explore the intricate roles of highly disordered APC in regulation of β-catenin function. Intrinsically disordered APC helps the collection of β-catenin from cytoplasm, facilitates the b-catenin delivery to the binding sites on Axin, and controls the final detachment of β-catenin from Axin. PMID:22292947

  15. Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells.

    PubMed

    Ramirez-Tagle, Rodrigo; Escobar, Carlos A; Romero, Valentina; Montorfano, Ignacio; Armisén, Ricardo; Borgna, Vincenzo; Jeldes, Emanuel; Pizarro, Luis; Simon, Felipe; Echeverria, Cesar

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4'-trimethoxy-2'-hydroxy-chalcone (CH1) and 3'-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas. PMID:26907262

  16. Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells

    PubMed Central

    Ramirez-Tagle, Rodrigo; Escobar, Carlos A.; Romero, Valentina; Montorfano, Ignacio; Armisén, Ricardo; Borgna, Vincenzo; Jeldes, Emanuel; Pizarro, Luis; Simon, Felipe; Echeverria, Cesar

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4′-trimethoxy-2′-hydroxy-chalcone (CH1) and 3′-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas. PMID:26907262

  17. Prep1 directly regulates the intrinsic apoptotic pathway by controlling Bcl-XL levels.

    PubMed

    Micali, Nicola; Ferrai, Carmelo; Fernandez-Diaz, Luis C; Blasi, Francesco; Crippa, Massimo P

    2009-03-01

    The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression. PMID:19103748

  18. Artesunate induces apoptosis via a ROS-independent and Bax-mediated intrinsic pathway in HepG2 cells.

    PubMed

    Qin, Guiqi; Wu, Liping; Liu, Hongyu; Pang, Yilin; Zhao, Chubiao; Wu, Shengnan; Wang, Xiaoping; Chen, Tongsheng

    2015-08-15

    This study aims to explore the detail molecular mechanism by which artesunate (ARS), an artemisinin derivative, induces apoptosis in HepG2 cells. ARS induced a loss of mitochondrial transmemberane potential (ΔΨm), phosphatidylserine (PS) externalization, as well as activations of Bax/Bak and caspases indicative of apoptosis induction. Silencing Bax but not Bak significantly inhibited ARS-induced apoptosis, demonstrating the key role of the Bax-mediated intrinsic pathway. Although ARS increased intracellular reactive oxygen species (ROS), ARS-induced apoptosis was neither prevented by pretreatment with ROS scavengers nor potentiated by pretreatment with l-buthionine-sulfoximine (BSO) that enhanced the ARS-induced intracellular ROS generation, demonstrating that ROS was not involved in ARS-induced apoptosis. In addition, ARS did not induce Bid translocation to mitochondria, and the cytotoxicity of ARS was not prevented by silencing Bim, Puma or Mcl-1, but was significantly enhanced by HA14-1 pretreatment, demonstrating that Bcl-2/-xl instead of Bid and Bim as well as Puma may be the upstream factor to regulate the Bax-mediated intrinsic pathway. Collectively, our data demonstrate that ARS induces ROS-independent apoptosis via the Bax-mediated intrinsic pathway in HepG2 cells. PMID:26163896

  19. The Potential of Brittle Star Extracted Polysaccharide in Promoting Apoptosis via Intrinsic Signaling Pathway

    PubMed Central

    Baharara, Javad; Amini, Elaheh

    2015-01-01

    Background: Anti-cancer potential of marine natural products such as polysaccharides represented therapeutic potential in oncological researches. In this study, total polysaccharide from brittle star [Ophiocoma erinaceus (O. erinaceus)] was extracted and chemopreventive efficacy of Persian Gulf brittle star polysaccharide was investigated in HeLa human cervical cancer cells. Methods: To extract polysaccharide, dried brittle stars were ground and extracted mechanically. Then, detection of polysaccharide was performed by phenol sulfuric acid, Ultra Violet (UV)-sulfuric acid method and FTIR. The anti proliferative activity of isolated polysaccharide was examined by MTT assay and evaluation of cell death was done through morphological cell changes; Propodium Iodide staining, fluorescence microscopy and caspase-3, -9 enzymatic measurements. To assess its underlying mechanism, expression of Bax, Bcl-2 was evaluated. Results: The polysaccharide detection methods demonstrated isolation of crude polysaccharide from Persian Gulf brittle star. The results revealed that O. erinaceus polysaccharide suppressed the proliferation of HeLa cells in a dose and time dependent manner. Morphological observation of DAPI and Acridine Orange/Propodium Iodide staining was documented by typical characteristics of apoptotic cell death. Flow cytometry analyses exhibited the accumulation of treated cells in sub-G1 region. Additionally, polysaccharide extracted induced intrinsic apoptosis via up-regulation of caspase-3, caspase-9 and Bax along with down-regulation of Bcl-2 in HeLa cells. Conclusion: Taken together, the apoptosis inducing effect of brittle star polysaccharide via intrinsic pathway confirmed the anti tumor potential of marine polysaccharide. Therefore, these findings proposed new insight into anti cancer properties of brittle star polysaccharide as a promising agent in cervical cancer treatment. PMID:26605009

  20. Effects of 10-hydroxycamptothecin on intrinsic mitochondrial pathway in silkworm BmN-SWU1 cells.

    PubMed

    Pan, Chun; Hu, Yan-Fen; Song, Juan; Yi, Hua-Shan; Wang, La; Yang, Yi-Ying; Wang, Yong-Ping; Zhang, Man; Pan, Min-Hui; Lu, Cheng

    2016-02-01

    10-Hydroxycamptothecin (HCPT), a plant alkaloid isolated from Camptotheca acuminate, is known as a planted-derived insecticide, however, the specific mechanism in insect cells is still unclear. In this study, we treated the ovarian cell line of the silkworm, BmN-SWU1, with different HCPT doses for durations ranging from 0 to 72h. The apoptosis morphology was evident after 72h of incubation and included cell protuberance, concentrated cytoplasm and apoptotic bodies. We observed DNA fragmentation and cell apoptosis after HCPT treatment. The disruption of mitochondrial distribution, activation of the intracellular mitochondrial permeability transition pore, and release of cytochrome c during HCPT-induced apoptosis in dose and time-dependent manner indicate the involvement of mitochondria in BmN-SWU1 cells. Caspase-9 and -3 activities increased gradually with the duration of incubation time. In conclusion, HCPT has a significant effect to initiate the intrinsic mitochondrial pathway in silkworm cells, providing a theoretical basis for better application of plant-derived insecticide in pest control. PMID:26821653

  1. Apoptosis Cell Death Effect of Scrophularia Variegata on Breast Cancer Cells via Mitochondrial Intrinsic Pathway

    PubMed Central

    Azadmehr, Abbas; Hajiaghaee, Reza; Baradaran, Behzad; Haghdoost-Yazdi, Hashem

    2015-01-01

    Purpose: Scrophularia variegata M. Beib. (Scrophulariaceae) is an Iranian medicinal plant which is used for various inflammatory disorders in traditional medicine. In this study we evaluated the anti-cancer and cytotoxic effects of the Scrophularia variegata (S. variegata) ethanolic extract on the human breast cancer cell line. Methods: The cytotoxicity effect of the extract on MCF-7 cells was evaluated by MTT assay. In addition, Caspase activity, DNA ladder and Cell death were evaluated by ELISA, gel electrophoresis and Annexin V-FITC/PI staining, respectively. Results: The S. variegata extract showed significant effect cytotoxicity on MCF-7 human breast cancer cell line. Treatment with the extract induced apoptosis on the breast cancer cells by cell cycle arrest in G2/M phase. The results indicated that cytotoxicity activity was associated with an increase of apoptosis as demonstrated by DNA fragmentation as well as an increase of the amount of caspase 3 and caspase 9. In addition, the phytochemical assay showed that the extract had antioxidant capacity and also flavonoids, phenolic compounds and phenyl propanoids were presented in the extract. Conclusion: Our findings indicated that S. variegata extract induced apoptosis via mitochondrial intrinsic pathway on breast cancer by cell cycle arrest in G2/M phase and an increase of caspase 3 and caspase 9. However future studies are needed. PMID:26504768

  2. Hyperammonia induces specific liver injury through an intrinsic Ca2+-independent apoptosis pathway

    PubMed Central

    2014-01-01

    Background Numerous pathological processes that affect liver function in patients with liver failure have been identified. Among them, hyperammonia is one of the most common phenomena.The purpose of this study was to determine whether hyperammonia could induced specific liver injury. Methods Hyperammonemic cells were established using NH4Cl. The cells were assessed by MTT, ELISA, and flow cytometric analyses. The expression levels of selected genes and proteins were confirmed by quantitative RT-PCR and western blot analyses. Results The effects of 20 mM NH4Cl pretreatment on the cell proliferation and apoptosis of primary hepatocytes and other cells were performed by MTT assays and flow cytometric analyses. Significant increasing in cytotoxicity and apoptosis were only observed in hepatocytes. The cell damage was reduced after adding BAPTA-AM but unchanged after adding EGTA. The expression levels of caspase-3, cytochrome C, calmodulin, and inducible nitric oxide synthase were increased and that of bcl-2 was reduced. The Na+-K+-ATPase activities in hyperammonia liver cells was no signiaficant difference compaired with the control group, but was decreased in astrocytes. NH4Cl pretreatment of primary hepatocytes promoted the activation of mitochondrial permeability transition pores and the mitochondria swelled irregularly. Conclusions Hyperammonia induces specific liver injury through an intrinsic Ca2+-independent apoptosis pathway. PMID:25145683

  3. Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway.

    PubMed

    Rahman, Md Ataur; Bishayee, Kausik; Huh, Sung-Oh

    2016-02-01

    Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy. PMID:26674967

  4. Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway

    PubMed Central

    Rahman, Md. Ataur; Bishayee, Kausik; Huh, Sung-Oh

    2016-01-01

    Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy. PMID:26674967

  5. On the intrinsic disorder status of the major players in programmed cell death pathways

    PubMed Central

    Uversky, Vladimir N

    2013-01-01

    Earlier computational and bioinformatics analysis of several large protein datasets across 28 species showed that proteins involved in regulation and execution of programmed cell death (PCD) possess substantial amounts of intrinsic disorder. Based on the comprehensive analysis of these datasets by a wide array of modern bioinformatics tools it was concluded that disordered regions of PCD-related proteins are involved in a multitude of biological functions and interactions with various partners, possess numerous posttranslational modification sites, and have specific evolutionary patterns (Peng et al. 2013). This study extends our previous work by providing information on the intrinsic disorder status of some of the major players of the three major PCD pathways: apoptosis, autophagy, and necroptosis. We also present a detailed description of the disorder status and interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathways. PMID:24358900

  6. Hypercholesterolemic diet induces vascular smooth muscle cell apoptosis in sympathectomized rats via intrinsic pathway.

    PubMed

    Hachani, Rafik; Dab, Houcine; Feriani, Anouar; Saber, Sami; Sakly, Mohsen; Vicaut, Eric; Callebert, Jacques; Sercombe, Richard; Kacem, Kamel

    2014-07-01

    In this study, we intend to investigate the role of hypercholesterolemic diet, a high risk factor for atherosclerosis, on vascular cell apoptosis in rats that have been previously sympathectomized. Thus, newborn male Wistar rats received injections of guanethidine for sympathectomy. Sham received injections of vehicle. The two groups were fed 1% cholesterol diet for 3months. Sympathectomy alone group was also exploited. Apoptosis in abdominal aortic tissue was identified by TUNEL method and conventional agarose gel electrophoresis to detect specific DNA fragmentation. Caspases 3 and 9, Bcl-2, Bax and cytochrome c were examined by immunoblotting. Oil Red O staining was used to reveal lipid in the arterial wall. Vascular smooth muscle cells (VSMCs) and macrophages were identified by immunostaining for α-smooth muscle actin and rat macrophage marker (ED1), respectively. The efficacy of sympathectomy was evaluated by analysis of perivascular sympathetic fibers. Our study showed that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, 1) increased aortic TUNEL-positive cells compared to sham and sympathectomy alone groups, 2) illustrated a typical apoptotic DNA ladder on agarose gel electrophoresis, 3) induced Bax translocation from cytosol to mitochondria, 4) enhanced cytochrome c release from mitochondria to cytosol, 5) increased expression of active caspases 3 and 9, and 6) decreased Bcl-2 expression. VSMCs are identified as the major cell type exhibiting apoptosis in this model. Taken together, it can be concluded that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, induces vascular cell apoptosis in an intrinsic pathway. PMID:24708922

  7. Parkia javanica Extract Induces Apoptosis in S-180 Cells via the Intrinsic Pathway of Apoptosis.

    PubMed

    Patra, Kartick; Jana, Samarjit; Sarkar, Arnab; Karmakar, Subrata; Jana, Jagannath; Gupta, Mradu; Mukherjee, Gopeswar; De, Utpal Chandra; Mandal, Deba Prasad; Bhattacharjee, Shamee

    2016-01-01

    Parkia javanica is a leguminous tree, various parts of which are used as food and folklore medicine by the ethnic groups of northeastern India. The present study investigates the in vitro and in vivo anticancer effect of aqueous methanol extract of P. javanica fruit (PJE). HPLC analysis was done to establish the fingerprint chromatogram of PJE and its in vitro radical scavenging activity was measured. PJE caused significant cytotoxicity in sarcoma-180 (S-180), A549, AGS, and MDA-MB435S cancer cells in vitro. Exploration of the mechanistic details in S-180 cells suggested that the reduced cell viability was mediated by induction of apoptosis. Increased expression of proapoptotic proteins such as p53, p21, Bax/Bcl2, cytochrome c (Cyt c), caspase 9, and cleaved poly(ADP-ribose) polymerase, and decrease in proliferative and antiapoptotic markers (Ki-67, Proliferating Cell Nuclear Antigen [PCNA], Bcl-2) validated the anticancer effect of PJE. A decline in the relative fluorescence emission upon staining S-180 cells with Rhodamine 123 (Rh 123), enhanced expression of cytosolic Cyt c and mitochondrial Bax, and inhibition of apoptosis in the presence of caspase-9 inhibitor in PJE-treated cells indicated intrinsic pathway of apoptosis. Liver function test and hepatic antioxidant enzymes demonstrated non-toxicity of PJE. Finally, the detection of PJE in sera by HPLC confirmed its bioavailability. PMID:27144503

  8. APP Overexpression Causes Aβ-Independent Neuronal Death through Intrinsic Apoptosis Pathway.

    PubMed

    Cheng, Ning; Jiao, Song; Gumaste, Ankita; Bai, Li; Belluscio, Leonardo

    2016-01-01

    Accumulation of amyloid-β (Aβ) peptide in the brain is a central hallmark of Alzheimer's disease (AD) and is thought to be the cause of the observed neurodegeneration. Many animal models have been generated that overproduce Aβ yet do not exhibit clear neuronal loss, questioning this Aβ hypothesis. We previously developed an in vivo mouse model that expresses a humanized amyloid precursor protein (hAPP) in olfactory sensory neurons (OSNs) showing robust apoptosis and olfactory dysfunction by 3 weeks of age, which is consistent with early OSN loss and smell deficits, as observed in AD patients. Here we show, by deleting the β-site APP cleaving enzyme 1 (BACE1) in two distinct transgenic mouse models, that hAPP-induced apoptosis of OSNs is Aβ independent and remains cell autonomous. In addition, we reveal that the intrinsic apoptosis pathway is responsible for hAPP-induced OSN death, as marked by mitochondrial damage and caspase-9 activation. Given that hAPP expression causes OSN apoptosis despite the absence of BACE1, we propose that Aβ is not the sole cause of hAPP-induced neurodegeneration and that the early loss of olfactory function in AD may be based on a cell-autonomous mechanism, which could mark an early phase of AD, prior to Aβ accumulation. Thus, the olfactory system could serve as an important new platform to study the development of AD, providing unique insight for both early diagnosis and intervention. PMID:27517085

  9. APP Overexpression Causes Aβ-Independent Neuronal Death through Intrinsic Apoptosis Pathway

    PubMed Central

    Cheng, Ning; Jiao, Song; Gumaste, Ankita; Bai, Li

    2016-01-01

    Abstract Accumulation of amyloid-β (Aβ) peptide in the brain is a central hallmark of Alzheimer’s disease (AD) and is thought to be the cause of the observed neurodegeneration. Many animal models have been generated that overproduce Aβ yet do not exhibit clear neuronal loss, questioning this Aβ hypothesis. We previously developed an in vivo mouse model that expresses a humanized amyloid precursor protein (hAPP) in olfactory sensory neurons (OSNs) showing robust apoptosis and olfactory dysfunction by 3 weeks of age, which is consistent with early OSN loss and smell deficits, as observed in AD patients. Here we show, by deleting the β-site APP cleaving enzyme 1 (BACE1) in two distinct transgenic mouse models, that hAPP-induced apoptosis of OSNs is Aβ independent and remains cell autonomous. In addition, we reveal that the intrinsic apoptosis pathway is responsible for hAPP-induced OSN death, as marked by mitochondrial damage and caspase-9 activation. Given that hAPP expression causes OSN apoptosis despite the absence of BACE1, we propose that Aβ is not the sole cause of hAPP-induced neurodegeneration and that the early loss of olfactory function in AD may be based on a cell-autonomous mechanism, which could mark an early phase of AD, prior to Aβ accumulation. Thus, the olfactory system could serve as an important new platform to study the development of AD, providing unique insight for both early diagnosis and intervention. PMID:27517085

  10. Luteolin induces cell cycle arrest and apoptosis through extrinsic and intrinsic signaling pathways in MCF-7 breast cancer cells.

    PubMed

    Park, Su-Ho; Ham, Sunyoung; Kwon, Tae Ho; Kim, Man Sub; Lee, Dong Hun; Kang, Jeoung-Woo; Oh, Sei-Ryang; Yoon, Do-Young

    2014-01-01

    Luteolin is a common flavonoid that exists in medicinal herbs, fruits, and vegetables. Luteolin has biochemical functions including anti-allergy, anti-inflammation, and anti-cancer functions. However, its efficacy and precise mode of action against breast cancer are still under study. To elucidate whether luteolin exhibits an anticancer effect in breast cancer, MCF-7 breast cancer cells were incubated with luteolin, and apoptosis was assessed by observing nuclear morphological changes and by performing cell viability assay, cell cycle analysis, annexin V-FITC/PI double staining, western blotting, RT-PCR, and mitochondrial membrane potential measurements. Luteolin inhibited growth through perturbation of cell cycle progression at the sub-G1 and G1 phases in MCF-7 cells. Furthermore, luteolin enhanced the expression of death receptors, such as DR5, and activated caspase cascades. It enhanced the activities of caspase-8/-9/-3 in a dose-dependent manner, followed by inactivation of PARP. Activation of caspase-8 and caspase-9 induced caspase-3 activity, respectively, in apoptosis of extrinsic and intrinsic pathways. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and increased Bax expression by inhibiting expression of Bcl-2. Taken together, these results suggest that luteolin provokes cell cycle arrest and induces apoptosis by activating the extrinsic and intrinsic pathways. PMID:25272060

  11. Drug-mediated sensitization to TRAIL-induced apoptosis in caspase-8-complemented neuroblastoma cells proceeds via activation of intrinsic and extrinsic pathways and caspase-dependent cleavage of XIAP, Bcl-xL and RIP.

    PubMed

    Mühlethaler-Mottet, Annick; Bourloud, Katia Balmas; Auderset, Katya; Joseph, Jean-Marc; Gross, Nicole

    2004-07-15

    Neuroblastoma (NB) is a childhood neoplasm which heterogeneous behavior can be explained by differential regulation of apoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces rapid apoptosis in most tumor cells and thus represents a promising anticancer agent. We have reported silencing of caspase-8 expression in highly malignant NB cells as a possible mechanism of resistance to TRAIL-induced apoptosis. To explore the particular contribution of caspase-8 in such resistance, retroviral-mediated stable caspase-8 expression was induced in the IGR-N91 cells. As a result, sensitivity to TRAIL was fully restored in the caspase-8-complemented cells. TRAIL-induced cell death could be further enhanced by cotreatment of IGR-N91-C8 and SH-EP cells with cycloheximide or subtoxic concentrations of chemotherapeutic drugs in a caspase-dependent manner. Sensitization to TRAIL involved enhanced death receptor DR5 expression, activation of Bid and the complete caspases cascade. Interestingly, combined treatments also enhanced the cleavage-mediated inactivation of antiapoptotic molecules, XIAP, Bcl-x(L) and RIP. Our results show that restoration of active caspase-8 expression in a caspase-8-deficient NB cell line is necessary and sufficient to fully restore TRAIL sensitivity. Moreover, the synergistic effect of drugs and TRAIL results from activation of the caspase cascade via a mitochondrial pathway-mediated amplification loop and from the inactivation of apoptosis inhibitors. PMID:15094781

  12. Involvement of the extrinsic and intrinsic pathways in ultraviolet B-induced apoptosis of corneal epithelial cells.

    PubMed

    Ubels, John L; Glupker, Courtney D; Schotanus, Mark P; Haarsma, Loren D

    2016-04-01

    The goal of this study was to elucidate the pathway by which UVB initiates efflux of K(+) and subsequently apoptosis in human corneal limbal epithelial (HCLE) cells. The initial focus of the study was on the extrinsic pathway involving Fas. HCLE cells transfected with Fas siRNA were exposed to 80-150 mJ/cm(2) UVB and incubated in culture medium with 5.5 mM K(+). Knockdown of Fas resulted in limited reduction in UVB-induced caspase-8 and -3 activity. Patch-clamp recordings showed no difference in UVB-induced normalized K(+) currents between Fas transfected and control cells. Knockdown of caspase-8 had no effect on the activation of caspase-3 following UVB exposure, while a caspase-8 inhibitor completely eliminated UVB activation of caspase-3. This suggests that caspase-8 is a robust enzyme, able to activate caspase-3 via residual caspase-8 present after knockdown, and that caspase-8 is directly involved in the UVB activation of caspase-3. Inhibition of caspase-9 significantly decreased the activation of caspases-8 and -3 in response to UVB. Knockdown of Apaf-1, required for activation of caspase-9, resulted in a significant reduction in UVB-induced activation of caspases-9, -8, and -3. Knockdown of Apaf-1 also inhibited intrinsic and UVB-induced levels of apoptosis, as determined by DNA fragmentation measured by TUNEL assay. In UVB exposed cultures treated with caspase-3 inhibitor, the percentage of apoptotic cells was reduced to control levels, confirming the necessity of caspase-3 activation in DNA fragmentation. The lack of effect of Fas knockdown on K(+) channel activation, as well as the limited effect on activation of caspases-8 and -3, strongly suggest that Fas and the extrinsic pathway is not of primary importance in the initiation of apoptosis in response to UVB in HCLE cells. Inhibition of caspase-8 and -3 activation following inhibition of caspase-9, as well as reduction in activation of caspases-9, -8, and -3 and DNA fragmentation in response to Apaf

  13. H9 induces apoptosis via the intrinsic pathway in non-small-cell lung cancer A549 cells.

    PubMed

    Kwon, Sae-Bom; Kim, Min-Je; Ham, Sun Young; Park, Ga Wan; Choi, Kang-Duk; Jung, Seung Hyun; Yoon, Do-Young

    2015-03-01

    H9 is an ethanol extract prepared from nine traditional/medicinal herbs. This study was focused on the anticancer effect of H9 in non-small-cell lung cancer cells. The effects of H9 on cell viability, apoptosis, mitochondrial membrane potential (MMP; Δφm), and apoptosis-related protein expression were investigated in A549 human lung cancer cells. In this study, H9-induced apoptosis was confirmed by propidium iodide staining, expression levels of mRNA were determined by reverse transcriptase polymerase chain reaction, protein expression levels were checked by western blot analysis, and MMP (Δφm) was measured by JC- 1 staining. Our results indicated that H9 decreased the viability of A549 cells and induced cell morphological changes in a dose-dependent manner. H9 also altered expression levels of molecules involved in the intrinsic signaling pathway. H9 inhibited Bcl-xL expression, whereas Bax expression was enhanced and cytochrome C was released. Furthermore, H9 treatment led to the activation of caspase-3/caspase-9 and proteolytic cleavage of poly(ADPribose) polymerase; the MMP was collapsed by H9. However, the expression levels of extrinsic pathway molecules such as Fas/FasL, TRAIL/TRAIL-R, DR5, and Fas-associated death receptor were downregulated by H9. These results indicated that H9 inhibited proliferation and induced apoptosis by activating intrinsic pathways but not extrinsic pathways in human lung cancer cells. Our results suggest that H9 can be used as an alternative remedy for human non-small-cell lung cancer. PMID:25563417

  14. Expression of selected proteins of the extrinsic and intrinsic pathways of apoptosis in human leukocytes exposed to N-nitrosodimethylamine.

    PubMed

    Iwaniuk, A; Jabłońska, E; Jabłoński, J; Ratajczak-Wrona, W; Garley, M

    2015-06-01

    N-nitrosodimethylamine (NDMA) is a xenobiotic widespread in human environment capable of regulating the lifespan of immune cells. In this study, we examined the roles of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5) complex and the Fas molecule in the induction of the extrinsic apoptosis pathway in human neutrophils (polymorphonuclear neutrophils (PMNs)) and peripheral blood mononuclear cells (PBMCs) exposed to NDMA. Also we assessed these proteins ability to trigger the intrinsic apoptosis pathway in those cells. For this purpose, we examined the expression of Fas-associated protein with death domain, truncated Bid (tBid) proteins, and apoptogenic factors such as apoptosis-inducing factor, Smac/Diablo, Omi/HtrA2, and caspase-3 as an indication of accomplished apoptosis phenomenon. PMNs and PBMCs were isolated from whole blood by density gradient centrifugation using Polymorphrep. Apoptotic cells were assessed with flow cytometry using a ready-made kit. The expression of proapoptotic molecules was investigated by Western blot analysis of PMNs and PBMCs treated with NDMA and/or rhTRAIL. The obtained results confirm the proapoptotic effects of NDMA on the examined human leukocytes and indicate an active participation of the TRAIL/DR5 complex and Fas protein in the process of apoptosis. Moreover, the research revealed distinct mechanisms of intrinsic apoptosis pathway activation between PMNs and PBMCs exposed to NDMA, as confirmed by the different levels of tBid, Smac/Diablo, Omi/HtrA2, and caspase-3 expression in those cells. PMID:25304970

  15. Radiation Sensitivity in a Preclinical Mouse Model of Medulloblastoma Relies on the Function of the Intrinsic Apoptotic Pathway.

    PubMed

    Crowther, Andrew J; Ocasio, Jennifer K; Fang, Fang; Meidinger, Jessica; Wu, Jaclyn; Deal, Allison M; Chang, Sha X; Yuan, Hong; Schmid, Ralf; Davis, Ian; Gershon, Timothy R

    2016-06-01

    While treatments that induce DNA damage are commonly used as anticancer therapies, the mechanisms through which DNA damage produces a therapeutic response are incompletely understood. Here we have tested whether medulloblastomas must be competent for apoptosis to be sensitive to radiotherapy. Whether apoptosis is required for radiation sensitivity has been controversial. Medulloblastoma, the most common malignant brain tumor in children, is a biologically heterogeneous set of tumors typically sensitive to radiation and chemotherapy; 80% of medulloblastoma patients survive long-term after treatment. We used functional genetic studies to determine whether the intrinsic apoptotic pathway is required for radiation to produce a therapeutic response in mice with primary, Shh-driven medulloblastoma. We found that cranial radiation extended the survival of medulloblastoma-bearing mice and induced widespread apoptosis. Expression analysis and conditional deletion studies showed that Trp53 (p53) was the predominant transcriptional regulator activated by radiation and was strictly required for treatment response. Deletion of Bax, which blocked apoptosis downstream of p53, was sufficient to render tumors radiation resistant. In apoptosis-incompetent, Bax-deleted tumors, radiation activated p53-dependent transcription without provoking cell death and caused two discrete populations to emerge. Most radiated tumor cells underwent terminal differentiation. Perivascular cells, however, quickly resumed proliferation despite p53 activation, behaved as stem cells, and rapidly drove recurrence. These data show that radiation must induce apoptosis in tumor stem cells to be effective. Mutations that disable the intrinsic apoptotic pathways are sufficient to impart radiation resistance. We suggest that medulloblastomas are typically sensitive to DNA-damaging therapies, because they retain apoptosis competence. Cancer Res; 76(11); 3211-23. ©2016 AACR. PMID:27197166

  16. Perceived Intrinsic Barriers to Physical Activity Among Rural Mothers

    PubMed Central

    Drake, Keith M.; MacKenzie, Todd A.; Titus-Ernstoff, Linda; Longacre, Meghan R.; Hendricks, Kristy M.; Beach, Michael L.; Dalton, Madeline A.

    2010-01-01

    Abstract Background The purpose of this study was to identify and determine the influence of perceived intrinsic barriers to physical activity among mothers living in rural areas. Methods Mothers were identified through a study of child-parent dyads in the predominantly rural states of New Hampshire and Vermont. Using a telephone interview, we asked mothers (n = 1691) about their level of physical activity and assessed eight potential barriers to physical activity. Data were analyzed using chi-square tests, t tests, and analysis of variance (ANOVA) comparisons for groups within each variable. We used multiple regression analysis to assess associations between perceived barriers to physical activity and self-reported levels of physical activity. Results Each barrier was inversely associated with physical activity. Multivariate models that included terms for all potential barriers and covariates identified three barriers associated with lower levels of physical activity: lack of self-discipline, lack of time, and lack of interest. Conclusions Rural mothers are less likely to be physically active if they identify lack of self-discipline, time, or interest as barriers, suggesting that they have difficulty prioritizing exercise for themselves. Interventions aimed at increasing physical activity for mothers should specifically consider these barriers. One possible solution may be to support infrastructure that facilitates active living as the default option, to remove the issue of having to purposefully engage in physical activity as a separate aspect of a mother's life. PMID:20973674

  17. Robust active binocular vision through intrinsically motivated learning.

    PubMed

    Lonini, Luca; Forestier, Sébastien; Teulière, Céline; Zhao, Yu; Shi, Bertram E; Triesch, Jochen

    2013-01-01

    The efficient coding hypothesis posits that sensory systems of animals strive to encode sensory signals efficiently by taking into account the redundancies in them. This principle has been very successful in explaining response properties of visual sensory neurons as adaptations to the statistics of natural images. Recently, we have begun to extend the efficient coding hypothesis to active perception through a form of intrinsically motivated learning: a sensory model learns an efficient code for the sensory signals while a reinforcement learner generates movements of the sense organs to improve the encoding of the signals. To this end, it receives an intrinsically generated reinforcement signal indicating how well the sensory model encodes the data. This approach has been tested in the context of binocular vison, leading to the autonomous development of disparity tuning and vergence control. Here we systematically investigate the robustness of the new approach in the context of a binocular vision system implemented on a robot. Robustness is an important aspect that reflects the ability of the system to deal with unmodeled disturbances or events, such as insults to the system that displace the stereo cameras. To demonstrate the robustness of our method and its ability to self-calibrate, we introduce various perturbations and test if and how the system recovers from them. We find that (1) the system can fully recover from a perturbation that can be compensated through the system's motor degrees of freedom, (2) performance degrades gracefully if the system cannot use its motor degrees of freedom to compensate for the perturbation, and (3) recovery from a perturbation is improved if both the sensory encoding and the behavior policy can adapt to the perturbation. Overall, this work demonstrates that our intrinsically motivated learning approach for efficient coding in active perception gives rise to a self-calibrating perceptual system of high robustness. PMID:24223552

  18. Robust active binocular vision through intrinsically motivated learning

    PubMed Central

    Lonini, Luca; Forestier, Sébastien; Teulière, Céline; Zhao, Yu; Shi, Bertram E.; Triesch, Jochen

    2013-01-01

    The efficient coding hypothesis posits that sensory systems of animals strive to encode sensory signals efficiently by taking into account the redundancies in them. This principle has been very successful in explaining response properties of visual sensory neurons as adaptations to the statistics of natural images. Recently, we have begun to extend the efficient coding hypothesis to active perception through a form of intrinsically motivated learning: a sensory model learns an efficient code for the sensory signals while a reinforcement learner generates movements of the sense organs to improve the encoding of the signals. To this end, it receives an intrinsically generated reinforcement signal indicating how well the sensory model encodes the data. This approach has been tested in the context of binocular vison, leading to the autonomous development of disparity tuning and vergence control. Here we systematically investigate the robustness of the new approach in the context of a binocular vision system implemented on a robot. Robustness is an important aspect that reflects the ability of the system to deal with unmodeled disturbances or events, such as insults to the system that displace the stereo cameras. To demonstrate the robustness of our method and its ability to self-calibrate, we introduce various perturbations and test if and how the system recovers from them. We find that (1) the system can fully recover from a perturbation that can be compensated through the system's motor degrees of freedom, (2) performance degrades gracefully if the system cannot use its motor degrees of freedom to compensate for the perturbation, and (3) recovery from a perturbation is improved if both the sensory encoding and the behavior policy can adapt to the perturbation. Overall, this work demonstrates that our intrinsically motivated learning approach for efficient coding in active perception gives rise to a self-calibrating perceptual system of high robustness. PMID:24223552

  19. Pulchrin A, a New Natural Coumarin Derivative of Enicosanthellum pulchrum, Induces Apoptosis in Ovarian Cancer Cells via Intrinsic Pathway

    PubMed Central

    Nordin, Noraziah; Fadaeinasab, Mehran; Mohan, Syam; Mohd Hashim, Najihah; Othman, Rozana; Karimian, Hamed; Iman, Venus; Ramli, Noorlela; Mohd Ali, Hapipah; Abdul Majid, Nazia

    2016-01-01

    Drug resistance presents a challenge in chemotherapy and has attracted research interest worldwide and particular attention has been given to natural compounds to overcome this difficulty. Pulchrin A, a new compound isolated from natural products has demonstrated novel potential for development as a drug. The identification of pulchrin A was conducted using several spectroscopic techniques such as nuclear magnetic resonance, liquid chromatography mass spectrometer, infrared and ultraviolet spectrometry. The cytotoxicity effects on CAOV-3 cells indicates that pulchrin A is more active than cisplatin, which has an IC50 of 22.3 μM. Significant changes in cell morphology were present, such as cell membrane blebbing and formation of apoptotic bodies. The involvement of phosphatidylserine (PS) in apoptosis was confirmed by Annexin V-FITC after a 24 h treatment. Apoptosis was activated through the intrinsic pathway by activation of procaspases 3 and 9 as well as cleaved caspases 3 and 9 and ended at the executioner pathway, with the occurrence of DNA laddering. Apoptosis was further confirmed via gene and protein expression levels, in which Bcl-2 protein was down-regulated and Bax protein was up-regulated. Furthermore, the CAOV-3 cell cycle was disrupted at the G0/G1 phase, leading to apoptosis. Molecular modeling of Bcl-2 proteins demonstrated a high- binding affinity, which inhibited the function of Bcl-2 proteins and led to cell death. Results of the current study can shed light on the development of new therapeutic agents, particularly, human ovarian cancer treatments. PMID:27136097

  20. Lag threads organize the brain’s intrinsic activity

    PubMed Central

    Mitra, Anish; Snyder, Abraham Z.; Blazey, Tyler; Raichle, Marcus E.

    2015-01-01

    It has been widely reported that intrinsic brain activity, in a variety of animals including humans, is spatiotemporally structured. Specifically, propagated slow activity has been repeatedly demonstrated in animals. In human resting-state fMRI, spontaneous activity has been understood predominantly in terms of zero-lag temporal synchrony within widely distributed functional systems (resting-state networks). Here, we use resting-state fMRI from 1,376 normal, young adults to demonstrate that multiple, highly reproducible, temporal sequences of propagated activity, which we term “lag threads,” are present in the brain. Moreover, this propagated activity is largely unidirectional within conventionally understood resting-state networks. Modeling experiments show that resting-state networks naturally emerge as a consequence of shared patterns of propagation. An implication of these results is that common physiologic mechanisms may underlie spontaneous activity as imaged with fMRI in humans and slowly propagated activity as studied in animals. PMID:25825720

  1. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Gao, Lizeng; Zhuang, Jie; Nie, Leng; Zhang, Jinbin; Zhang, Yu; Gu, Ning; Wang, Taihong; Feng, Jing; Yang, Dongling; Perrett, Sarah; Yan, Xiyun

    2007-09-01

    Nanoparticles containing magnetic materials, such as magnetite (Fe3O4), are particularly useful for imaging and separation techniques. As these nanoparticles are generally considered to be biologically and chemically inert, they are typically coated with metal catalysts, antibodies or enzymes to increase their functionality as separation agents. Here, we report that magnetite nanoparticles in fact possess an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, which are widely used to oxidize organic substrates in the treatment of wastewater or as detection tools. Based on this finding, we have developed a novel immunoassay in which antibody-modified magnetite nanoparticles provide three functions: capture, separation and detection. The stability, ease of production and versatility of these nanoparticles makes them a powerful tool for a wide range of potential applications in medicine, biotechnology and environmental chemistry.

  2. Brain Mechanical Property Measurement Using MRE with Intrinsic Activation

    PubMed Central

    Pattison, Adam J.; McGarry, Matthew D.; Perreard, Irina M.; Swienckowski, Jessica G.; Eskey, Clifford J.; Lollis, S. Scott; Paulsen, Keith D.

    2013-01-01

    the MRE procedures were repeated on the same day. Cardiac pulsation, termed intrinsic activation, produces sufficient motion to allow mechanical properties to be recovered. The poroelastic model is more consistent with the measured data from brain at low frequencies than the linear elastic model. Intrinsic activation allows MR elastography to be performed without a device shaking the head so the patient notices no differences between it and the other sequences in an MR examination. PMID:23079508

  3. Brain mechanical property measurement using MRE with intrinsic activation

    NASA Astrophysics Data System (ADS)

    Weaver, John B.; Pattison, Adam J.; McGarry, Matthew D.; Perreard, Irina M.; Swienckowski, Jessica G.; Eskey, Clifford J.; Lollis, S. Scott; Paulsen, Keith D.

    2012-11-01

    , termed intrinsic activation, produces sufficient motion to allow mechanical properties to be recovered. The poroelastic model is more consistent with the measured data from brain at low frequencies than the linear elastic model. Intrinsic activation allows MRE to be performed without a device shaking the head so the patient notices no differences between it and the other sequences in an MR examination.

  4. Cordycepin induces apoptosis in human liver cancer HepG2 cells through extrinsic and intrinsic signaling pathways

    PubMed Central

    Shao, Le-Wen; Huang, Li-Hua; Yan, Sheng; Jin, Jian-Di; Ren, Shao-Yan

    2016-01-01

    Cordycepin, also termed 3′-deoxyadenosine, is a nucleoside analogue from Cordyceps sinensis and has been reported to demonstrate numerous biological and pharmacological properties. Our previous study illustrated that the anti-tumor effect of cordycepin may be associated with apoptosis. In the present study, the apoptotic effect of cordycepin on HepG2 cells was investigated using 4′,6-diamidino-2-phenylindole, tetraethylbenzimidazolylcarbocyanine iodide and propidium iodide staining analysis and flow cytometry. The results showed that cordycepin exhibited the ability to inhibit HepG2 cells in a time- and dose-dependent manner when cells produced typical apoptotic morphological changes, including chromatin condensation, the accumulation of sub-G1 cells and change mitochondrial permeability. A potential mechanism for cordycepin-induced apoptosis of human liver cancer HepG2 cells may occur through the extrinsic signaling pathway mediated by the transmembrane Fas-associated with death domain protein. Apoptosis was also associated with Bcl-2 family protein regulation, leading to altered mitochondrial membrane permeability and resulting in the release of cytochrome c into the cytosol. The activation of the caspase cascade is responsible for the execution of apoptosis. In conclusion, cordycepin-induced apoptosis in HepG2 cells involved the extrinsic and intrinsic signaling pathway and was primarily regulated by the Bcl-2 family proteins. PMID:27446383

  5. INOSITOL HEXAKISPHOSPHATE MEDIATES APOPTOSIS IN HUMAN BREAST ADENOCARCINOMA MCF-7 CELL LINE VIA INTRINSIC PATHWAY

    SciTech Connect

    Agarwal, Rakhee; Ali, Nawab

    2010-04-12

    Inositol polyphosphates (InsP{sub s}) are naturally occurring compounds ubiquitously present in plants and animals. Inositol hexakisphosphate (InsP{sub 6}) is the most abundant among all InsP{sub s} and constitutes the major portion of dietary fiber in most cereals, legumes and nuts. Certain derivatives of InsP{sub s} also regulate cellular signaling mechanisms. InsP{sub s} have also been shown to reduce tumor formation and induce apoptosis in cancerous cells. Therefore, in this study, the effects of InsPs on apoptosis were studied in an attempt to investigate their potential anti-cancer therapeutic application and understand their mechanism of action. Acridine orange and ethidium bromide staining suggested that InsP{sub 6} dose dependently induced apoptosis in human breast adenocarcinoma MCF-7 cells. Among InsP{sub s} tested (InsP{sub 3}, InsP{sub 4}, InsP{sub 5}, and InsP{sub 6}), InsP{sub 6} was found to be the most effective in inducing apoptosis. Furthermore, effects of InsP{sub 6} were found most potent inducing apoptosis. Etoposide, the drug known to induce apoptosis in both in vivo and in vitro, was used as a positive control. Western blotting experiments using specific antibodies against known apoptotic markers suggested that InsP{sub 6} induced apoptotic changes were mediated via an intrinsic apoptotic pathway.

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

    NASA Astrophysics Data System (ADS)

    Agarwal, Rakhee; Ali, Nawab

    2010-04-01

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

  7. Protein denaturation in vacuo: intrinsic unfolding pathways associated with the native tertiary structure of lysozyme

    NASA Astrophysics Data System (ADS)

    Arteca, Gustavo A.; Tapia, O.

    Using computer-simulated molecular dynamics, we study the effect of sequence mutation on the unfolding mechanism of a native fold. The system considered is the native fold of hen egg-white lysozyme, exposed to centrifugal unfolding in vacuo. This unfolding bias elicits configurational transitions that imitate the behaviour of anhydrous proteins diffusing after electrospraying from neutral-pH solutions. By changing the sequences threaded onto the native fold of lysozyme, we probe the role of disulfide bridges and the effect of a global mutation. We find that the initial denaturing steps share common characteristics for the tested sequences. Recurrent features are: (i) the presence of dumbbell conformers with significant residual secondary structure, (ii) the ubiquitous formation of hairpins and two-stranded β-sheets regardless of disulfide bridges, and (iii) an unfolding pattern where the reduction in folding complexity is highly correlated with the decrease in chain compactness. These findings appear to be intrinsic to the shape of the native fold, suggesting that similar unfolding pathways may be accessible to many protein sequences.

  8. Extracts of Strawberry Fruits Induce Intrinsic Pathway of Apoptosis in Breast Cancer Cells and Inhibits Tumor Progression in Mice

    PubMed Central

    Somasagara, Ranganatha R.; Hegde, Mahesh; Chiruvella, Kishore K.; Musini, Anjaneyulu; Choudhary, Bibha; Raghavan, Sathees C.

    2012-01-01

    Background The consumption of berry fruits, including strawberries, has been suggested to have beneficial effects against oxidative stress mediated diseases. Berries contain multiple phenolic compounds and secondary metabolites that contribute to their biological properties. Methodology/Principal Findings Current study investigates the anticancer activity of the methanolic extract of strawberry (MESB) fruits in leukaemia (CEM) and breast cancer (T47D) cell lines ex vivo, and its cancer therapeutic and chemopreventive potential in mice models. Results of MTT, trypan blue and LDH assays suggested that MESB can induce cytotoxicity in cancer cells, irrespective of origin, in a concentration- and time-dependent manner. Treatment of mice bearing breast adenocarcinoma with MESB blocked the proliferation of tumor cells in a time-dependent manner and resulted in extended life span. Histological and immunohistochemical studies suggest that MESB treatment affected tumor cell proliferation by activating apoptosis and did not result in any side effects. Finally, we show that MESB can induce intrinsic pathway of apoptosis by activating p73 in breast cancer cells, when tumor suppressor gene p53 is mutated. Conclusions/Significance The present study reveals that strawberry fruits possess both cancer preventive and therapeutic values and we discuss the mechanism by which it is achieved. PMID:23071702

  9. Ultrasonication processed Panax ginseng berry extract induces apoptosis through an intrinsic apoptosis pathway in HepG2 cells.

    PubMed

    Jung, Hyunwoo; Bae, Jinhyung; Ko, Sung Kwon; Sohn, Uy Dong

    2016-06-01

    Ginseng's major active components, ginsenosides, have been known to show anti-cancer, neuroprotective, and anti-inflammatory activities. Ultrasonication processed Panax ginseng berry extract (UGB) contains various ginsenosides. The components are different from Panax ginseng berry extract (GBE). This study was aimed to investigate the cytotoxic mechanism of UGB in HepG2 cells, human hepatocellular carcinoma cell line. HepG2 cells were treated with UGB (0, 10, 20 μg/ml). Cell growth and cellular apoptosis were evaluated by MTT assay and Annexin V/Pi staining, respectively. Intracellular Reactive oxygen species (ROS) levels were also determined by 2', 7'-dichlorofluorescin diacetate (DCFDA) staining. The expressions of Bax, Bcl-2 and caspase-3, the apoptotic markers, were evaluated by Western Blot. UGB dose-dependently inhibited cell growth and induced apoptotic cell death. Intracellular ROS levels were increased. UGB increased the expression of the cleaved form of caspase-3. Furthermore, UGB induced apoptosis of HepG2 cells through Bax activation and Bcl-2 inhibition. In conclusion, UGB induced apoptosis through an intrinsic pathway in HepG2 cells suggesting that UGB might play a role as a novel substance for anti-cancer effect. PMID:27233905

  10. MOFzyme: Intrinsic protease-like activity of Cu-MOF.

    PubMed

    Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

    2014-01-01

    The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu₂(C₉H₃O₆)₄/₃ MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA. PMID:25342169

  11. MOFzyme: Intrinsic protease-like activity of Cu-MOF

    NASA Astrophysics Data System (ADS)

    Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

    2014-10-01

    The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

  12. Dietary chlorophyllin inhibits the canonical NF-κB signaling pathway and induces intrinsic apoptosis in a hamster model of oral oncogenesis.

    PubMed

    Thiyagarajan, P; Senthil Murugan, R; Kavitha, K; Anitha, P; Prathiba, D; Nagini, S

    2012-03-01

    Chlorophyllin, a water-soluble, semi-synthetic derivative of the ubiquitous green pigment chlorophyll is shown to exert potent anticarcinogenic effects. In the present study, we investigated the chemopreventive effects of chlorophyllin on 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by analyzing the expression of NF-κB family members and markers of intrinsic apoptosis. Dietary administration of chlorophyllin (4 mg/kg bw) suppressed the development of HBP carcinomas by inhibiting the canonical NF-κB signaling pathway by downregulating IKKβ, preventing the phosphorylation of IκB-α, and reducing the expression of nuclear NF-κB. Inactivation of NF-κB signaling by chlorophyllin was associated with the induction of intrinsic apoptosis as evidenced by modulation of Bcl-2 family proteins, enforced nuclear localization of survivin, upregulation of apoptogenic molecules, activation of caspases, and cleavage of PARP. The results of the present study demonstrate that chlorophyllin inhibits the development of DMBA-induced HBP carcinogenesis by targeting NF-κB and the intrinsic apoptotic pathway. Thus, dietary agents such as chlorophyllin that simultaneously target divergent pathways of cell survival and cell death are novel candidates for cancer chemoprevention. PMID:22210229

  13. Oxytocin Neurones: Intrinsic Mechanisms Governing the Regularity of Spiking Activity.

    PubMed

    Maícas Royo, J; Brown, C H; Leng, G; MacGregor, D J

    2016-04-01

    Oxytocin neurones of the rat supraoptic nucleus are osmoresponsive and, with all other things being equal, they fire at a mean rate that is proportional to the plasma sodium concentration. However, individual spike times are governed by highly stochastic events, namely the random occurrences of excitatory synaptic inputs, the probability of which is increased by increasing extracellular osmotic pressure. Accordingly, interspike intervals (ISIs) are very irregular. In the present study, we show, by statistical analyses of firing patterns in oxytocin neurones, that the mean firing rate as measured in bins of a few seconds is more regular than expected from the variability of ISIs. This is consistent with an intrinsic activity-dependent negative-feedback mechanism. To test this, we compared observed neuronal firing patterns with firing patterns generated by a leaky integrate-and-fire model neurone, modified to exhibit activity-dependent mechanisms known to be present in oxytocin neurones. The presence of a prolonged afterhyperpolarisation (AHP) was critical for the ability to mimic the observed regularisation of mean firing rate, although we also had to add a depolarising afterpotential (DAP; sometimes called an afterdepolarisation) to the model to match the observed ISI distributions. We tested this model by comparing its behaviour with the behaviour of oxytocin neurones exposed to apamin, a blocker of the medium AHP. Good fits indicate that the medium AHP actively contributes to the firing patterns of oxytocin neurones during non-bursting activity, and that oxytocin neurones generally express a DAP, even though this is usually masked by superposition of a larger AHP. PMID:26715365

  14. Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

    PubMed

    Kahl, M; Gökçen, A; Fischer, S; Bäumer, M; Wiesner, J; Lochnit, G; Wygrecka, M; Vilcinskas, A; Preissner, K T

    2015-08-01

    For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds. PMID:25948398

  15. Intrinsic mechanisms of pain inhibition: activation by stress.

    PubMed

    Terman, G W; Shavit, Y; Lewis, J W; Cannon, J T; Liebeskind, J C

    1984-12-14

    Portions of the brain stem seem normally to inhibit pain. In man and laboratory animals these brain areas and pathways from them to spinal sensory circuits can be activated by focal stimulation. Endogenous opioids appear to be implicated although separate nonopioid mechanisms are also evident. Stress seems to be a natural stimulus triggering pain suppression. Properties of electric footshock have been shown to determine the opioid or nonopioid basis of stress-induced analgesia. Two different opioid systems can be activated by different footshock paradigms. This dissection of stress analgesia has begun to integrate divergent findings concerning pain inhibition and also to account for some of the variance that has obscured the reliable measurement of the effects of stress on tumor growth and immune function. PMID:6505691

  16. Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BK(Ca) channels through IP₃ pathway.

    PubMed

    Che, Tongtong; Sun, Hui; Li, Jingxin; Yu, Xiao; Zhu, Dexiao; Xue, Bing; Liu, Kejing; Zhang, Min; Kunze, Wolfgang; Liu, Chuanyong

    2012-05-01

    Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 16-24 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca²⁺ concentration ([Ca²⁺](i) ) of isolated neurons was measured using calcium imaging. The concentration of IP(3) in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BK(Ca)) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10⁻⁷ to 10⁻⁵ M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BK(Ca) channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BK(Ca) channel opening. OTR and the BK(Ca) α subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10⁻⁵ M, a BK(Ca) channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca²⁺](i) and the OT-evoked outward current was significantly attenuated by thapsigargin (10⁻⁶ M) or CdCl₂. The effect of OT on the BK(Ca) current was also blocked by pre-treatment with the IP₃ receptor antagonist 2-APB (10⁻⁴ M

  17. A quantitative structure--activity relationship model for the intrinsic activity of uncouplers of oxidative phosphorylation.

    PubMed

    Spycher, Simon; Escher, Beate I; Gasteiger, Johann

    2005-12-01

    A quantitative structure-activity relationship (QSAR) has been derived for the prediction of the activity of phenols in uncoupling oxidative and photophosphorylation. Twenty-one compounds with experimental data for uncoupling activity as well as for the acid dissociation constant, pKa, and for partitioning constants of the neutral and the charged species into model membranes were analyzed. From these measured data, the effective concentration in the membrane was derived, which allowed the study of the intrinsic activity of uncouplers within the membrane. A linear regression model for the intrinsic activity could be established using the following three descriptors: solvation free energies of the anions, an estimate for heterodimer formation describing transport processes, and pKa values describing the speciation of the phenols. In a next step, the aqueous effect concentrations were modeled by combining the model for the intrinsic uncoupling activity with descriptors accounting for the uptake into membranes. Results obtained with experimental membrane-water partitioning data were compared with the results obtained with experimental octanol-water partition coefficients, log Kow, and with calculated log Kow values. The properties of these different measures of lipophilicity were critically discussed. PMID:16359176

  18. Plastic Change along the Intact Crossed Pathway in Acute Phase of Cerebral Ischemia Revealed by Optical Intrinsic Signal Imaging

    PubMed Central

    Guo, Xiaoli; He, Yongzhi; Lu, Hongyang; Li, Yao; Su, Xin; Jiang, Ying; Tong, Shanbao

    2016-01-01

    The intact crossed pathway via which the contralesional hemisphere responds to the ipsilesional somatosensory input has shown to be affected by unilateral stroke. The aim of this study was to investigate the plasticity of the intact crossed pathway in response to different intensities of stimulation in a rodent photothrombotic stroke model. Using optical intrinsic signal imaging, an overall increase of the contralesional cortical response was observed in the acute phase (≤48 hours) after stroke. In particular, the contralesional hyperactivation is more prominent under weak stimulations, while a strong stimulation would even elicit a depressed response. The results suggest a distinct stimulation-response pattern along the intact crossed pathway after stroke. We speculate that the contralesional hyperactivation under weak stimulations was due to the reorganization for compensatory response to the weak ipsilateral somatosensory input. PMID:27144032

  19. Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture.

    PubMed

    Kaszuba-Zwoinska, J; Chorobik, P; Juszczak, K; Zaraska, W; Thor, P J

    2012-10-01

    Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early

  20. Oxidative Dehydrogenation on Nanocarbon: Intrinsic Catalytic Activity and Structure-Function Relationships.

    PubMed

    Qi, Wei; Liu, Wei; Guo, Xiaoling; Schlögl, Robert; Su, Dangsheng

    2015-11-01

    Physical and chemical insights into the nature and quantity of the active sites and the intrinsic catalytic activity of nanocarbon materials in alkane oxidative dehydrogenation (ODH) reactions are reported using a novel in situ chemical titration process. A study on the structure-function relationship reveals that the active sites are identical both in nature and function on various nanocarbon catalysts. Additionally, the quantity of the active sites could be used as a metric to normalize the reaction rates, and thus to evaluate the intrinsic activity of nanocarbon catalysts. The morphology of the nanocarbon catalysts at the microscopic scale exhibits a minor influence on their intrinsic ODH catalytic activity. The number of active sites calculated from the titration process indicates the number of catalytic centers that are active (that is, working) under the reaction conditions. PMID:26388451

  1. Exogenous modulation of intrinsic optic nerve neuroprotective activity

    PubMed Central

    Lazic, Tatjana; Kuehn, Markus H.; Harper, Matthew M.; Kardon, Randy H.; Kwon, Young H.; Lavik, Erin B.; Sakaguchi, Donald S.

    2013-01-01

    Background To characterize the molecular and functional status of the rat retina and optic nerve after acute elevation of intraocular pressure (IOP). Methods Retinal ischemia was induced in rats by increasing the IOP (110 mmHg/60 minutes). Microarray analysis, quantitative RT-PCR (qRT-PCR) and immunohistochemistry were used to characterize retinal tissue. PLGA microspheres containing neurotrophic factors (BDNF, GDNF, or CNTF) or empty microspheres were injected into the vitreous of operated animals 1 day after elevation of IOP. Pupil light reflex (PLR) parameters and electroretinograms (ERG) were monitored at multiple time points during the 60-day postoperative recovery period. Results Molecular analysis showed a significant intrinsic up-regulation of CNTF at 10 and 25 days after induction of the acute ocular hypertension (p=0.0067). Molecular tissue analysis of GDNF and its receptors (GDNFR1, GDNFR2), and BDNF and its receptor (trkB) showed no change in expression. Animals that received CNTF microspheres had no significant functional recovery compared to animals which received blank microspheres (p>0.05). Animals that received GDNF or BDNF microspheres showed significant PLR recovery (p<0.05 and p<0.001 respectively) compared to non-treated animals. Conclusions Continuous release of neurotrophic growth factors (NGFs) significantly protects optic nerve function in the experimental model of retinal ischemia observed by PLR analysis. PMID:20229104

  2. Activation of InsP3 receptors is sufficient for inducing graded intrinsic plasticity in rat hippocampal pyramidal neurons

    PubMed Central

    Ashhad, Sufyan; Johnston, Daniel

    2014-01-01

    The synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signaling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. In this study, we complemented the recently established necessity of inositol trisphosphate (InsP3) receptors (InsP3R) in a form of intrinsic plasticity by asking if InsP3R activation was sufficient to induce intrinsic plasticity in hippocampal neurons. Specifically, incorporation of d-myo-InsP3 in the recording pipette reduced input resistance, maximal impedance amplitude, and temporal summation but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead. Strikingly, the magnitude of plasticity in all these measurements was dependent on InsP3 concentration, emphasizing the graded dependence of such plasticity on InsP3R activation. Mechanistically, we found that this InsP3-induced plasticity depended on hyperpolarization-activated cyclic nucleotide-gated channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP3Rs, the influx of calcium through N-methyl-d-aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. Our results delineate a causal role for InsP3Rs in graded adaptation of neuronal response dynamics, revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis. PMID:25552640

  3. Differential Contributions of Intrinsic and Extrinsic Pathways to Thrombin Generation in Adult, Maternal and Cord Plasma Samples

    PubMed Central

    Rice, Nicklaus T.; Szlam, Fania; Varner, Jeffrey D.; Bernstein, Peter S.; Szlam, Arthur D.; Tanaka, Kenichi A.

    2016-01-01

    Background Thrombin generation (TG) is a pivotal process in achieving hemostasis. Coagulation profiles during pregnancy and early neonatal period are different from that of normal (non-pregnant) adults. In this ex vivo study, the differences in TG in maternal and cord plasma relative to normal adult plasma were studied. Methods Twenty consented pregnant women and ten consented healthy adults were included in the study. Maternal and cord blood samples were collected at the time of delivery. Platelet-poor plasma was isolated for the measurement of TG. In some samples, anti-FIXa aptamer, RB006, or a TFPI inhibitor, BAX499 were added to elucidate the contribution of intrinsic and extrinsic pathway to TG. Additionally, procoagulant and inhibitor levels were measured in maternal and cord plasma, and these values were used to mathematically simulate TG. Results Peak TG was increased in maternal plasma (393.6±57.9 nM) compared to adult and cord samples (323.2±38.9 nM and 209.9±29.5 nM, respectively). Inhibitory effects of RB006 on TG were less robust in maternal or cord plasma (52% vs. 12% respectively) than in adult plasma (81%). Likewise the effectiveness of BAX499 as represented by the increase in peak TG was much greater in adult (21%) than in maternal (10%) or cord plasma (12%). Further, BAX499 was more effective in reversing RB006 in adult plasma than in maternal or cord plasma. Ex vivo data were reproducible with the results of the mathematical simulation of TG. Conclusion Normal parturient plasma shows a large intrinsic pathway reserve for TG compared to adult and cord plasma, while TG in cord plasma is sustained by extrinsic pathway, and low levels of TFPI and AT. PMID:27196067

  4. High and Low Activity Rats: Elevated intrinsic physical activity drives resistance to diet induced obesity in non-bred rats

    PubMed Central

    Perez-Leighton, Claudio E.; Boland, Kelsey; Billington, Charles; Kotz, Catherine M.

    2012-01-01

    Humans and rodents show large variability in their individual sensitivity to diet-induced obesity, which has been associated with differences in intrinsic spontaneous physical activity (SPA). Evidence from genetic and out-bred rat obesity models shows that higher activity of the orexin peptides results in higher intrinsic SPA and protection against diet-induced obesity. Based on this, we hypothesized that naturally occurring variation in SPA and orexin signaling activity is sufficient to drive differences in sensitivity to diet-induced obesity. We analyzed orexin activity and sensitivity to diet-induced obesity in non-manipulated male Sprague Dawley rats selected for high and low intrinsic SPA. Our results defined a new model of differential DIO sensitivity, the high-activity and low activity-rats, and suggest that naturally occurring variations in intrinsic SPA cause differences in energy expenditure that are mediated by orexin signaling and alter DIO sensitivity. PMID:23404834

  5. Influenza A Virus and Influenza B Virus Can Induce Apoptosis via Intrinsic or Extrinsic Pathways and Also via NF-κB in a Time and Dose Dependent Manner

    PubMed Central

    El-Sayed, Ibrahim; Nokaly, Aziz; Abdelghani, Ahmed S.

    2016-01-01

    Influenza viruses are able to cause annual epidemics and pandemics due to their mutation rates and reassortment capabilities leading to antigenic shifts and drifts. To identify host response to influenza A and B viruses on A549 and MDCK II cells at low and high MOIs, expressions of MxA and caspases 3, 8, and 9 and BAD, TNFα, and IκBα genes were measured in the cells supernatants. H1N1 and H3N2 prefer to initially enhance the intrinsic pathway, determined by higher caspase 9 activity in MDCK II cells compared to caspase 8 activity and vice versa in A549 cells at different MOIs, while INF B prefers extrinsic pathway in A549 cells according to significant low or undetectable caspase 9 activity and high activity of caspase 8 but also can induce intrinsic pathway in MDCK II cells as determined by significant low or undetectable activity of caspase 8 and high caspase 9 activity at different MOIs; the considerable MxA expression was found in influenza A and B viruses infected A549 and MDCK II cells at low MOIs. In conclusion, influenza A and B viruses induced extrinsic and intrinsic apoptosis in parallel, and the induction was associated with viral infection in a dose dependent manner. PMID:27042352

  6. Influenza A Virus and Influenza B Virus Can Induce Apoptosis via Intrinsic or Extrinsic Pathways and Also via NF-κB in a Time and Dose Dependent Manner.

    PubMed

    El-Sayed, Ibrahim; Bassiouny, Khalid; Nokaly, Aziz; Abdelghani, Ahmed S; Roshdy, Wael

    2016-01-01

    Influenza viruses are able to cause annual epidemics and pandemics due to their mutation rates and reassortment capabilities leading to antigenic shifts and drifts. To identify host response to influenza A and B viruses on A549 and MDCK II cells at low and high MOIs, expressions of MxA and caspases 3, 8, and 9 and BAD, TNFα, and IκBα genes were measured in the cells supernatants. H1N1 and H3N2 prefer to initially enhance the intrinsic pathway, determined by higher caspase 9 activity in MDCK II cells compared to caspase 8 activity and vice versa in A549 cells at different MOIs, while INF B prefers extrinsic pathway in A549 cells according to significant low or undetectable caspase 9 activity and high activity of caspase 8 but also can induce intrinsic pathway in MDCK II cells as determined by significant low or undetectable activity of caspase 8 and high caspase 9 activity at different MOIs; the considerable MxA expression was found in influenza A and B viruses infected A549 and MDCK II cells at low MOIs. In conclusion, influenza A and B viruses induced extrinsic and intrinsic apoptosis in parallel, and the induction was associated with viral infection in a dose dependent manner. PMID:27042352

  7. Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation

    PubMed Central

    Smorodinsky-Atias, Karina; Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Shir, Alexei; Mooshayef, Navit; Beenstock, Jonah; Karamansha, Yael; Darlyuk-Saadon, Ilona; Livnah, Oded; Ahn, Natalie G.; Admon, Arie; Engelberg, David

    2016-01-01

    The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk’s activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation. PMID:26658610

  8. The methane monooxygenase intrinsic activity of kinds of methanotrophs.

    PubMed

    Zhang, Yingxin; Xin, Jiaying; Chen, Linlin; Xia, Chungu

    2009-06-01

    Methanotrophs have promising applications in the epoxidation of some alkenes and some chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (poly-3-hydroxybutyrate; PHB). In contrast with methane monooxygenase (MMO) activity and ability of PHB synthesis of four kinds of methanotrophic bacteria Methylosinus trichosporium OB3b, M. trichosporium IMV3011, Methylococcus capsulatus HD6T, Methylomonas sp. GYJ3, and the mixture of the four kinds of strains, M. trichosporium OB3b is the highest of the four in the activity of propene epoxidation (10.72 nmol/min mg dry weight of cell [dwc]), the activity of naphthalene oxidation (22.7 mmol/mg dwc), and ability in synthesis of PHB(11% PHB content in per gram dry weight of cell in 84 h). It could be feasible to improve the MMO activity by mixing four kinds of methanotrophs. The MMO activity dramatically decreased when the cellular PHB accumulated in the second stage. The reason for this may be the dilution of the MMO system in the cells with increasing PHB contents. It has been found that the PHB contents at the level of 1-5% are beneficial to the cells for maintenance of MMO epoxidation activity when enough PHB have been accumulated. Moreover, it was also found that high particulate methane monooxygenase activity may contribute to the synthesis of PHB in the cell, which could be used to improve the yield of PHB in methanotrophs. PMID:19052919

  9. A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway.

    PubMed

    Zhang, Hailong; Liu, Xiaogang; Wu, Fengbo; Qin, Feifei; Feng, Ping; Xu, Ting; Li, Xiang; Yang, Li

    2016-01-01

    Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG's effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa. PMID:27196894

  10. A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway

    PubMed Central

    Zhang, Hailong; Liu, Xiaogang; Wu, Fengbo; Qin, Feifei; Feng, Ping; Xu, Ting; Li, Xiang; Yang, Li

    2016-01-01

    Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG’s effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa. PMID:27196894

  11. Role of DNA damage response pathways in preventing carcinogenesis caused by intrinsic replication stress.

    PubMed

    Wallace, M D; Southard, T L; Schimenti, K J; Schimenti, J C

    2014-07-10

    Defective DNA replication can result in genomic instability, cancer and developmental defects. To understand the roles of DNA damage response (DDR) genes on carcinogenesis in mutants defective for core DNA replication components, we utilized the Mcm4(Chaos3/Chaos3) ('Chaos3') mouse model that, by virtue of an amino-acid alteration in MCM4 that destabilizes the MCM2-7 DNA replicative helicase, has fewer dormant replication origins and an increased number of stalled replication forks. This leads to genomic instability and cancer in most Chaos3 mice. We found that animals doubly mutant for Chaos3 and components of the ataxia telangiectasia-mutated (ATM) double-strand break response pathway (Atm, p21/Cdkn1a and Chk2/Chek2) had decreased tumor latency and/or increased tumor susceptibility. Tumor latency and susceptibility differed between genetic backgrounds and genders, with females demonstrating an overall greater cancer susceptibility to Atm and p21 deficiency than males. Atm deficiency was semilethal in the Chaos3 background and impaired embryonic fibroblast proliferation, suggesting that ATM drug inhibitors might be useful against tumors with DNA replication defects. Hypomorphism for the 9-1-1 component Hus1 did not affect tumor latency or susceptibility in Chaos3 animals, and tumors in these mice did not exhibit impaired ATR pathway signaling. These and other data indicate that under conditions of systemic replication stress, the ATM pathway is particularly important both for cancer suppression and viability during development. PMID:23975433

  12. Intrinsic Brain Activity in Altered States of Consciousness

    PubMed Central

    Boly, M.; Phillips, C.; Tshibanda, L.; Vanhaudenhuyse, A.; Schabus, M.; Dang-Vu, T.T.; Moonen, G.; Hustinx, R.; Maquet, P.; Laureys, S.

    2010-01-01

    Spontaneous brain activity has recently received increasing interest in the neuroimaging community. However, the value of resting-state studies to a better understanding of brain–behavior relationships has been challenged. That altered states of consciousness are a privileged way to study the relationships between spontaneous brain activity and behavior is proposed, and common resting-state brain activity features observed in various states of altered consciousness are reviewed. Early positron emission tomography studies showed that states of extremely low or high brain activity are often associated with unconsciousness. However, this relationship is not absolute, and the precise link between global brain metabolism and awareness remains yet difficult to assert. In contrast, voxel-based analyses identified a systematic impairment of associative frontoparieto–cingulate areas in altered states of consciousness, such as sleep, anesthesia, coma, vegetative state, epileptic loss of consciousness, and somnambulism. In parallel, recent functional magnetic resonance imaging studies have identified structured patterns of slow neuronal oscillations in the resting human brain. Similar coherent blood oxygen level–dependent (BOLD) systemwide patterns can also be found, in particular in the default-mode network, in several states of unconsciousness, such as coma, anesthesia, and slow-wave sleep. The latter results suggest that slow coherent spontaneous BOLD fluctuations cannot be exclusively a reflection of conscious mental activity, but may reflect default brain connectivity shaping brain areas of most likely interactions in a way that transcends levels of consciousness, and whose functional significance remains largely in the dark. PMID:18591474

  13. N-methyl-D-aspartate receptor encephalitis mediates loss of intrinsic activity measured by functional MRI.

    PubMed

    Brier, Matthew R; Day, Gregory S; Snyder, Abraham Z; Tanenbaum, Aaron B; Ances, Beau M

    2016-06-01

    Spontaneous brain activity is required for the development and maintenance of normal brain function. Many disease processes disrupt the organization of intrinsic brain activity, but few pervasively reduce the amplitude of resting state blood oxygen level dependent (BOLD) fMRI fluctuations. We report the case of a female with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, longitudinally studied during the course of her illness to determine the contribution of NMDAR signaling to spontaneous brain activity. Resting state BOLD fMRI was measured at the height of her illness and 18 weeks following discharge from hospital. Conventional resting state networks were defined using established methods. Correlation and covariance matrices were calculated by extracting the BOLD time series from regions of interest and calculating either the correlation or covariance quantity. The intrinsic activity was compared between visits, and to expected activity from 45 similarly aged healthy individuals. Near the height of the illness, the patient exhibited profound loss of consciousness, high-amplitude slowing of the electroencephalogram, and a severe reduction in the amplitude of spontaneous BOLD fMRI fluctuations. The patient's neurological status and measures of intrinsic activity improved following treatment. We conclude that NMDAR-mediated signaling plays a critical role in the mechanisms that give rise to organized spontaneous brain activity. Loss of intrinsic activity is associated with profound disruptions of consciousness and cognition. PMID:27025853

  14. Dissociation Pathways of the CH2CH2ONO Radical: NO2 + Ethene, NO + Oxirane, and a Non-Intrinsic Reaction Coordinate HNO + Vinoxy Pathway.

    PubMed

    Scrape, Preston G; Roberts, Trevor D; Lee, Shih-Huang; Butler, Laurie J

    2016-07-14

    We first characterize the dissociation pathways of BrCH2CH2ONO, a substituted alkyl nitrite, upon photoexcitation at 193 nm under collision-free conditions, in a crossed laser-molecular beam scattering apparatus using vacuum ultraviolet photoionization detection. Three primary photodissociation pathways occur: photoelimination of HNO, leading to the products HNO + BrCH2CHO; C-Br bond photofission, leading to Br + CH2CH2ONO; and O-NO bond photofission, leading to NO + BrCH2CH2O. The data show that alkyl nitrites can eliminate HNO via a unimolecular mechanism in addition to the commonly accepted bulk disproportionation mechanism. Some of the products from the primary photodissociation pathways are highly vibrationally excited, so we then probe the product branching from the unimolecular dissociation of these unstable intermediates. Notably, the vibrationally excited CH2CH2ONO radicals undergo two channels predicted by statistical transition-state theory, and an additional non-intrinsic reaction coordinate channel, HNO elimination. CH2CH2ONO is formed with high rotational energy; by employing rotational models based on conservation of angular momentum, we predict, and verify experimentally, the kinetic energies of stable CH2CH2ONO radicals and the angular distribution of dissociation products. The major dissociation pathway of CH2CH2ONO is NO2 + ethene, and some of the NO2 is formed with sufficient internal energy to undergo further photodissociation. Nascent BrCH2CHO and CH2Br are also photodissociated upon absorption of a second 193 nm photon; we derive the kinetic energy release of these dissociations based on our data, noting similarities to the analogous photodissociation of ClCH2CHO and CH2Cl. PMID:27124098

  15. Display of cardiac activation pathways with echocardiography

    NASA Astrophysics Data System (ADS)

    Olstad, Bjoern; Brodin, Lars A.; Berg, Sevald

    1997-05-01

    The study of cardiac activation dynamics is an important factor in the characterization of the cardiac function. One such example is the localization of WPW-pathways inside the myocardium. Accurate localization of these pathways can be used to determine if the patient should be treated with catheter techniques or surgical techniques. This paper analyzes the temporal information in tissue velocity imaging with both qualitative and quantitative methods. The clinical experiments indicate that echocardiography can become an alternative technique for non-invasive electrophysiology in these kinds of applications.

  16. Effect of Autaptic Activity on Intrinsic Coherence Resonance in Newman-Watts Networks of Stochastic Hodgkin-Huxley Neurons

    NASA Astrophysics Data System (ADS)

    Wang, Qi; Gong, Yubing

    2016-06-01

    In this paper, we study the effect of autaptic activity on intrinsic coherence resonance (CR) induced by channel noise in Newman-Watts (NW) networks of stochastic Hodgkin-Huxley (HH) neurons. It is found that autaptic strength and autaptic delay have a big effect on the intrinsic CR. As autaptic strength increases, there is optimal autaptic strength by which the intrinsic CR is most highly enhanced. Autaptic delay can enhance, reduce, or destroy the intrinsic CR, depending on the delay length. Moreover, there are optimal coupling strength and network randomness by which autaptic activity can most highly enhance the intrinsic CR. These results show that autaptic activity has different effects on the intrinsic CR in the neuronal networks, and it can most highly enhance the intrinsic CR at optimal coupling strength and network randomness. These findings could find potential implications of channel noise and autaptic activity for the information processing and transmission in neural systems.

  17. Flavonoids of Korean Citrus aurantium L. Induce Apoptosis via Intrinsic Pathway in Human Hepatoblastoma HepG2 Cells.

    PubMed

    Lee, Seung Hwan; Yumnam, Silvia; Hong, Gyeong Eun; Raha, Suchismita; Saralamma, Venu Venkatarame Gowda; Lee, Ho Jeong; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won-Sup; Kim, Eun-Hee; Park, Hyeon Soo; Kim, Gon Sup

    2015-12-01

    Korean Citrus aurantium L. has long been used as a medicinal herb for its anti-inflammatory, antioxidant, and anticancer properties. The present study investigates the anticancer role of flavonoids extracted from C. aurantium on human hepatoblastoma cell, HepG2. The Citrus flavonoids inhibit the proliferation of HepG2 cells in a dose-dependent manner. This result was consistent with the in vivo xenograft results. Apoptosis was detected by cell morphology, cell cycle analysis, and immunoblot. Flavonoids decreased the level of pAkt and other downstream targets of phosphoinositide-3-kinase/Akt pathway - P-4EBP1 and P-p70S6K. The expressions of cleaved caspase 3, Bax, and Bak were increased, while those of Bcl-2 and Bcl-xL were decreased with an increase in the expression of Bax/Bcl-xL ratio in treated cells. Loss of mitochondrial membrane potential was also observed in flavonoid-treated HepG2 cells. It was also observed that the P-p38 protein level was increased both dose and time dependently in flavonoid-treated cells. Collectively, these results suggest that flavonoid extracted from Citrus inhibits HepG2 cell proliferation by inducing apoptosis via an intrinsic pathway. These findings suggest that flavonoids extracted from C. aurantium L. are potential chemotherapeutic agents against liver cancer. PMID:26439681

  18. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  19. The induction of apoptosis in HepG-2 cells by ruthenium(II) complexes through an intrinsic ROS-mediated mitochondrial dysfunction pathway.

    PubMed

    Zeng, Chuan-Chuan; Lai, Shang-Hai; Yao, Jun-Hua; Zhang, Cheng; Yin, Hui; Li, Wei; Han, Bing-Jie; Liu, Yun-Jun

    2016-10-21

    Four new ruthenium(II) polypyridyl complexes [Ru(N-N)2(dhbn)](ClO4)2 (N-N = dmb: 4,4'-dimethyl-2,2'-bipyridine 1; bpy = 2,2'-bipyridine 2; phen = 1,10-phenanthroline 3; dmp = 2,9-dimethyl-1,10-phenanthroline 4) were synthesized and characterized. The cytotoxicity in vitro of the ligand and complexes toward HepG-2, HeLa, MG-63 and A549 were assayed by MTT method. The IC50 values of the complexes against the above cells range from 17.7 ± 1.1 to 45.1 ± 2.8 μM. The cytotoxic activity of the complexes against HepG-2 cells follows the order of 4 > 2 > 3 > 1. Ligand shows no cytotoxic activity against the selected cell lines. Cellular uptake, apoptosis, comet assay, reactive oxygen species, mitochondrial membrane potential, cell cycle arrest, and the expression of proteins involved in apoptosis pathway induced by the complexes were investigated. The results indicate that complexes 1-4 induce apoptosis in HepG-2 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway. PMID:27344489

  20. The importance of challenge for the enjoyment of intrinsically motivated, goal-directed activities.

    PubMed

    Abuhamdeh, Sami; Csikszentmihalyi, Mihaly

    2012-03-01

    Although early interview-based analyses of the enjoyment of intrinsically motivated, goal-directed activities (e.g., chess, rock climbing, art making) suggested the importance of relatively difficult, "optimal" challenges, subsequent findings derived from a wider range of activities have not provided consistent support for this proposition. Two studies were conducted to clarify the relation between challenge and enjoyment. Study 1 focused on a single activity-Internet chess. The importance of challenge was evident at the subjective level (perceived challenge strongly predicted enjoyment) as well as the objective level (games against superior opponents were more enjoyable than games against inferior opponents, and close games were more enjoyable than blowouts). In Study 2, the experience sampling method was used to examine the enjoyment of challenge across a wide range of everyday activities. Activity motivation (intrinsically motivated, non-intrinsically motivated) and activity type (goal directed, non-goal directed) moderated the relation. Implications for theories of intrinsic motivation are discussed. PMID:22067510

  1. Overexpression of Amyloid- β Protein Precursor Induces Mitochondrial Oxidative Stress and Activates the Intrinsic Apoptotic Cascade

    PubMed Central

    Bartley, Matthew G.; Marquardt, Kristin; Kirchhof, Danielle; Wilkins, Heather M.; Patterson, David; Linseman, Daniel A.

    2015-01-01

    Alzheimer's disease (AD) is a debilitating cognitive disorder which is characterized pathologically by amyloid-β plaques and neurofibrillary tangles. Aberrant processing of amyloid beta protein precursor (AβPP) into amyloid-β fragments underlies the formation of senile plaques. Moreover, amyloid-β fragments, particularly Aβ42, exert direct toxic effects within neurons including the induction of mitochondrial oxidative stress (MOS). Interestingly, individuals with Down Syndrome (DS) frequently develop early onset AD as a major co-morbid phenotype. One hypothesis for AD associated with DS involves the overexpression of wild type (WT) AβPP protein, due to its location on chromosome 21. However, the mechanism by which the overexpression of WT AβPP might trigger MOS and induce cell death is presently unclear. Here we show that transient overexpression of DsRed2-tagged AβPP (WT) in CHO cells induces the activation of caspase-3 and nuclear fragmentation indicative of apoptosis. AβPP localizes to the mitochondrial fraction of transfected CHO cells and its overexpression causes glutathione (GSH)-sensitive opening of the mitochondrial permeability transition pore (mPTP) and cytochrome c release. MOS and intrinsic apoptosis induced by AβPP were significantly inhibited by the co-expression of Bcl-2 or treatment with either GSH or Boc, a pan-caspase inhibitor. Furthermore, the mPTP inhibitor, cyclosporin A, also significantly protected CHO cells from apoptosis induced by AβPP overexpression. Finally, co-treatment with a β-secretase inhibitor did not significantly protect CHO cells from AβPP overexpression and Aβ42 levels were undetectable in transfected CHO cells. Therefore, the mechanism of AβPP induced MOS and apoptosis is independent of the production of Aβ42. However, a γ-secretase inhibitor showed significant protection of the CHO cells against AβPP overexpression. Thus, suggesting a possible role of the AβPP intracellular domain in this apoptotic

  2. Cell Intrinsic and Extrinsic Activators of the Unfolded Protein Response in Cancer: Mechanisms and Targets for Therapy

    PubMed Central

    Tameire, Feven; Verginadis, Ioannis I.; Koumenis, Constantinos

    2015-01-01

    A variety of cell intrinsic or extrinsic stresses evoke perturbations in the folding environment of the endoplasmic reticulum (ER), collectively known as ER stress. Adaptation to stress and reestablishment of ER homeostasis is achieved by activation of an integrated signal transduction pathway called the unfolded protein response (UPR). Both ER stress and UPR activation have been implicated in a variety of human cancers. Although at early stages, or physiological conditions of ER stress, the UPR generally promotes survival, when the stress becomes more stringent or prolonged, its role can switch to a pro-cell death one. Here, we discuss historical and recent evidence supporting an involvement of the UPR in malignancy, describe the main mechanisms by which how tumor cells overcome ER stress to promote their survival, tumor progression and metastasis and discuss the current state of efforts to develop therapeutic approaches of targeting the UPR. PMID:25920797

  3. Aqueous synthesis of porous platinum nanotubes at room temperature and their intrinsic peroxidase-like activity.

    PubMed

    Cai, Kai; Lv, Zhicheng; Chen, Kun; Huang, Liang; Wang, Jing; Shao, Feng; Wang, Yanjun; Han, Heyou

    2013-07-11

    Platinum nanotubes (PtNTs) exhibiting high porosity were constructed by sacrificing the exterior of tellurium nanowires (TeNWs) and disintegrating the inner part spontaneously in aqueous solution at room temperature, in which the Kirkendall effect may play an important role. The present PtNTs exhibited intrinsic peroxidase-like activity in the presence of H2O2. PMID:23598712

  4. Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways

    SciTech Connect

    Park, Hyunjin; Bergeron, Eric; Senta, Helena; Guillemette, Kim; Beauvais, Sabrina; Blouin, Richard; Sirois, Joel; Faucheux, Nathalie

    2010-08-27

    Research highlights: {yields} We show for the first time the effect of sanguinarine (SA) on MG63 and SaOS-2 cells. {yields} SA altered osteosarcoma cell viability in a concentration and time dependent manner. {yields} SA induced osteosarcoma cell apoptosis and increased caspase-8 and -9 activities. {yields} SA decreased dose dependently the Bcl-2 protein level only in MG63 cells. {yields} SaOS-2 which are osteoblast-derived, seemed more resistant to SA than MG63. -- Abstract: The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma, a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 {mu}mol/L sanguinarine for 4 and 24 h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 {mu}mol/L sanguinarine killed almost 100% of both cell populations within 24 h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1 h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.

  5. Crosstalk between tumor suppressors p53 and PKCδ: Execution of the intrinsic apoptotic pathways.

    PubMed

    Dashzeveg, Nurmaa; Yoshida, Kiyotsugu

    2016-07-28

    p53 and PKCδ are tumor suppressors that execute apoptotic mechanisms in response to various cellular stresses. p53 is a transcription factor that is frequently mutated in human cancers; it regulates apoptosis in transcription-dependent and -independent ways in response to genotoxic stresses. PKCδ is a serine/threonine protein kinase and mutated in human cancers. Available evidence shows that PKCδ activates p53 by direct and/or indirect mechanisms. Moreover, PKCδ is also implicated in the transcriptional regulation of p53 in response to DNA damage. Recent findings demonstrated that p53, in turn, binds onto the PKCδ promoter and induces its expression upon DNA damage to facilitate apoptosis. Both p53 and PKCδ are associated with the apoptotic mechanisms in the mitochondria by regulating Bcl-2 family proteins to provide mitochondrial outer membrane permeabilization. This review discusses the crosstalk between p53 and PKCδ in the context of apoptotic cell death and cancer therapy. PMID:27130668

  6. Momordica charantia seed extract exhibits strong anticoagulant effect by specifically interfering in intrinsic pathway of blood coagulation and dissolves fibrin clot.

    PubMed

    Manjappa, Bhagyalakshmi; Gangaraju, Sowmyashree; Girish, Kesturu S; Kemparaju, Kempaiah; Gonchigar, Sathish J; Shankar, Rohit L; Shinde, Manohar; Sannaningaiah, Devaraja

    2015-03-01

    The current study explores the anticoagulant and fibrin clot-hydrolyzing properties of Momordica charantia seed extract (MCSE). MCSE hydrolyzed casein with the specific activity of 0.780 units/mg per min. Interestingly, it enhanced the clot formation process of citrated human plasma from control 146 to 432 s. In addition, the intravenous injection of MCSE significantly prolonged the bleeding time in a dose-dependent manner from control 150 to more than 800 s, and strengthened its anticoagulant activity. Interestingly, MCSE specifically prolonged the clotting time of only activated partial thromboplastin time, but not prothrombin time, and revealed the participation of MCSE in the intrinsic pathway of the blood coagulation cascade. Furthermore, MCSE completely hydrolyzed both Aα and Bβ chains of the human fibrinogen and partially hydrolyzed the γ chain. However, it hydrolyzed all the chains (α polymer, α chain, β chain and γ-γ dimmers) of partially cross-linked human fibrin clot. The proteolytic activity followed by the anticoagulant effect of the MCSE was completely abolished by the 1,10-phenanthroline and phenyl methyl sulphonyl fluoride, but iodoacetic acid, EDTA, and ethylene glycol-N,N,N',N'-tetra acetic acid did not. Curiously, MCSE did not hydrolyze any other plasma proteins except the plasma fibrinogen. Moreover, MCSE was devoid of RBC lysis, edema and hemorrhagic properties, suggesting its nontoxic nature. Taken together, MCSE may be a valuable candidate in the treatment of blood clot/thrombotic disorders. PMID:25192240

  7. Altered Intrinsic Regional Activity and Interregional Functional Connectivity in Post-stroke Aphasia

    PubMed Central

    Yang, Mi; Li, Jiao; Li, Yibo; Li, Rong; Pang, Yajing; Yao, Dezhong; Liao, Wei; Chen, Huafu

    2016-01-01

    Several neuroimaging studies have examined cerebral function in patients who suffer from aphasia, but the mechanism underlying this disorder remains poorly understood. In this study, we examined alterations in the local regional and remote interregional network cerebral functions in aphasia combined with amplitude of low-frequency fluctuations and interregional functional connectivity (FC) using resting-state functional magnetic resonance imaging. A total of 17 post-stroke aphasic patients, all having suffered a stroke in the left hemisphere, as well as 20 age- and sex-matched healthy controls, were enrolled in this study. The aphasic patients showed significantly increased intrinsic regional activity mainly in the contralesional mesial temporal (hippocampus/parahippocampus, [HIP/ParaHIP]) and lateral temporal cortices. In addition, intrinsic regional activity in the contralesional HIP/ParaHIP was negatively correlated with construction score. Aphasic patients showed increased remote interregional FC between the contralesional HIP/ParaHIP and fusiform gyrus, but reduced FC in the ipsilesional occipital and parietal cortices. These findings suggested that the intrinsic regional brain dysfunctions in aphasia were related to interregional functional connectivity. Changes in the intrinsic regional brain activity and associated remote functional connectivity pattern would provide valuable information to enhance the understanding of the pathophysiological mechanisms of aphasia. PMID:27091494

  8. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway)

    NASA Astrophysics Data System (ADS)

    Ganesh Kumar, C.; Poornachandra, Y.; Chandrasekhar, Cheemalamarri

    2015-11-01

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications.The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2

  9. Bright light activates a trigeminal nociceptive pathway

    PubMed Central

    Okamoto, Keiichiro; Tashiro, Akimasa; Chang, Zheng; Bereiter, David A.

    2010-01-01

    Bright light can cause ocular discomfort and/or pain; however, the mechanism linking luminance to trigeminal nerve activity is not known. In this study we identify a novel reflex circuit necessary for bright light to excite nociceptive neurons in superficial laminae of trigeminal subnucleus caudalis (Vc/C1). Vc/C1 neurons encoded light intensity and displayed a long delay (>10 s) for activation. Microinjection of lidocaine into the eye or trigeminal root ganglion (TRG) inhibited light responses completely, whereas topical application onto the ocular surface had no effect. These findings indicated that light-evoked Vc/C1 activity was mediated by an intraocular mechanism and transmission through the TRG. Disrupting local vasomotor activity by intraocular microinjection of the vasoconstrictive agents, norepinephrine or phenylephrine, blocked light-evoked neural activity, whereas ocular surface or intra-TRG microinjection of norepinephrine had no effect. Pupillary muscle activity did not contribute since light-evoked responses were not altered by atropine. Microinjection of lidocaine into the superior salivatory nucleus diminished light-evoked Vc/C1 activity and lacrimation suggesting that increased parasympathetic outflow was critical for light-evoked responses. The reflex circuit also required input through accessory visual pathways since both Vc/C1 activity and lacrimation were prevented by local blockade of the olivary pretectal nucleus. These findings support the hypothesis that bright light activates trigeminal nerve activity through an intraocular mechanism driven by a luminance-responsive circuit and increased parasympathetic outflow to the eye. PMID:20206444

  10. Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

    PubMed

    Kim, Geun-Young; Park, Soon Yong; Jo, Ara; Kim, Mira; Leem, Sun-Hee; Jun, Woo-Jin; Shim, Sang In; Lee, Sang Chul; Chung, Jin Woong

    2015-09-01

    Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536]. PMID:26246284

  11. Reduced Hyperpolarization-Activated Current Contributes to Enhanced Intrinsic Excitability in Cultured Hippocampal Neurons from PrP−/− Mice

    PubMed Central

    Fan, Jing; Stemkowski, Patrick L.; Gandini, Maria A.; Black, Stefanie A.; Zhang, Zizhen; Souza, Ivana A.; Chen, Lina; Zamponi, Gerald W.

    2016-01-01

    Genetic ablation of cellular prion protein (PrPC) has been linked to increased neuronal excitability and synaptic activity in the hippocampus. We have previously shown that synaptic activity in hippocampi of PrP-null mice is increased due to enhanced N-methyl-D-aspartate receptor (NMDAR) function. Here, we focused on the effect of PRNP gene knock-out (KO) on intrinsic neuronal excitability, and in particular, the underlying ionic mechanism in hippocampal neurons cultured from P0 mouse pups. We found that the absence of PrPC profoundly affected the firing properties of cultured hippocampal neurons in the presence of synaptic blockers. The membrane impedance was greater in PrP-null neurons, and this difference was abolished by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (100 μM). HCN channel activity appeared to be functionally regulated by PrPC. The amplitude of voltage sag, a characteristic of activating HCN channel current (Ih), was decreased in null mice. Moreover, Ih peak current was reduced, along with a hyperpolarizing shift in activation gating and slower kinetics. However, neither HCN1 nor HCN2 formed a biochemical complex with PrPC. These results suggest that the absence of PrP downregulates the activity of HCN channels through activation of a cell signaling pathway rather than through direct interactions. This in turn contributes to an increase in membrane impedance to potentiate neuronal excitability. PMID:27047338

  12. Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole Quinuclidine analogues

    PubMed Central

    Franks, Lirit N.; Ford, Benjamin M.; Madadi, Nikhil R.; Penthala, Narsimha R.; Crooks, Peter A.; Prather, Paul L.

    2014-01-01

    Our laboratory recently reported that a group of novel indole quinuclidine analogues bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analogue exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogues acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. PMID:24858620

  13. Intrinsic brain activity as a diagnostic biomarker in children with benign epilepsy with centrotemporal spikes.

    PubMed

    Zhu, Yihong; Yu, Yang; Shinkareva, Svetlana V; Ji, Gong-Jun; Wang, Jue; Wang, Zhong-Jin; Zang, Yu-Feng; Liao, Wei; Tang, Ye-Lei

    2015-10-01

    Benign epilepsy with centrotemporal spikes (BECTS) is often associated with neural circuit dysfunction, particularly during the transient active state characterized by interictal epileptiform discharges (IEDs). Little is known, however, about the functional neural circuit abnormalities in BECTS without IEDs, or if such abnormalities could be used to differentiate BECTS patients without IEDs from healthy controls (HCs) for early diagnosis. To this end, we conducted resting-state functional magnetic resonance imaging (RS-fMRI) and simultaneous Electroencephalogram (EEG) in children with BECTS (n = 43) and age-matched HC (n = 28). The simultaneous EEG recordings distinguished BECTS with IEDs (n = 20) from without IEDs (n = 23). Intrinsic brain activity was measured in all three groups using the amplitude of low frequency fluctuation at rest. Compared to HC, BECTS patients with IEDs exhibited an intrinsic activity abnormality in the thalamus, suggesting that thalamic dysfunction could contribute to IED emergence while patients without IEDs exhibited intrinsic activity abnormalities in middle frontal gyrus and superior parietal gyrus. Using multivariate pattern classification analysis, we were able to differentiate BECTS without IEDs from HCs with 88.23% accuracy. BECTS without epileptic transients can be distinguished from HC and BECTS with IEDs by unique regional abnormalities in resting brain activity. Both transient abnormalities as reflected by IEDs and chronic abnormalities as reflected by RS-fMRI may contribute to BECTS development and expression. Intrinsic brain activity and multivariate pattern classification techniques are promising tools to diagnose and differentiate BECTS syndromes. Hum Brain Mapp 36:3878-3889, 2015. © 2015 Wiley Periodicals, Inc. PMID:26173095

  14. Changes in At-Risk Boys' Intrinsic Motivation toward Physical Activity: A Three-Year Longitudinal Study

    ERIC Educational Resources Information Center

    Liu, Jiling; Xiang, Ping; McBride, Ron E.; Su, Xiaoxia; Juzaily, Nasnoor

    2015-01-01

    Intrinsic motivation (IM) is an important predictor of children's physical activity participation. The present 3-year longitudinal study examined changes in IM toward physical activity among a group of at-risk boys (N = 92) at a summer sports camp. Results showed the boys were intrinsically motivated in their first camp year, but their IM levels…

  15. A chemical bonding model for photo-induced defects in hydrogenated amorphous silicon (a-Si:H): Intrinsic and extrinsic reaction pathways

    SciTech Connect

    Lucovsky, G.; Yang, H.

    1997-07-01

    In device grade a-Si:H photo- or light-induced defect generation is an intrinsic effect for impurity concentrations of oxygen and nitrogen below about 10{sup 19} to 10{sup 20} cm{sup {minus}3}; however, at higher concentrations it increases with increasing impurity content. Charged defect configurations are identified by empirical chemistry and are studied by ab initio calculations. This paper addresses: (1) the chemical stability of charged defects; (2) the reaction pathways for defect metastability; and (3) the transition between extrinsic and intrinsic behavior.

  16. Plasma Membrane Intrinsic Proteins from Maize Cluster in Two Sequence Subgroups with Differential Aquaporin Activity1

    PubMed Central

    Chaumont, François; Barrieu, François; Jung, Rudolf; Chrispeels, Maarten J.

    2000-01-01

    The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins. PMID:10759498

  17. A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

    PubMed Central

    2011-01-01

    Background Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-)C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. Methods B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Results Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. Conclusions The

  18. Identification of the Cell-Intrinsic and -Extrinsic Pathways Downstream of EGFR and IFNγ That Induce PD-L1 Expression in Head and Neck Cancer.

    PubMed

    Concha-Benavente, Fernando; Srivastava, Raghvendra M; Trivedi, Sumita; Lei, Yu; Chandran, Uma; Seethala, Raja R; Freeman, Gordon J; Ferris, Robert L

    2016-03-01

    Many cancer types, including head and neck cancers (HNC), express programmed death ligand 1 (PD-L1). Interaction between PD-L1 and its receptor, programmed death 1 (PD-1), inhibits the function of activated T cells and results in an immunosuppressive microenvironment, but the stimuli that induce PD-L1 expression are not well characterized. Interferon gamma (IFNγ) and the epidermal growth factor receptor (EGFR) utilize Janus kinase 2 (JAK2) as a common signaling node to transmit tumor cell-mediated extrinsic or intrinsic signals, respectively. In this study, we investigated the mechanism by which these factors upregulate PD-L1 expression in HNC cells in the context of JAK/STAT pathway activation, Th1 inflammation, and HPV status. We found that wild-type, overexpressed EGFR significantly correlated with JAK2 and PD-L1 expression in a large cohort of HNC specimens. Furthermore, PD-L1 expression was induced in an EGFR- and JAK2/STAT1-dependent manner, and specific JAK2 inhibition prevented PD-L1 upregulation in tumor cells and enhanced their immunogenicity. Collectively, our findings suggest a novel role for JAK2/STAT1 in EGFR-mediated immune evasion, and therapies targeting this signaling axis may be beneficial to block PD-L1 upregulation found in a large subset of HNC tumors. PMID:26676749

  19. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway).

    PubMed

    Kumar, C Ganesh; Poornachandra, Y; Chandrasekhar, Cheemalamarri

    2015-11-28

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications. PMID:26503300

  20. Rapid Eye Movement Sleep Deprivation Induces Neuronal Apoptosis by Noradrenaline Acting on Alpha1 Adrenoceptor and by Triggering Mitochondrial Intrinsic Pathway

    PubMed Central

    Somarajan, Bindu I.; Khanday, Mudasir A.; Mallick, Birendra N.

    2016-01-01

    Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate

  1. Rapid Eye Movement Sleep Deprivation Induces Neuronal Apoptosis by Noradrenaline Acting on Alpha1 Adrenoceptor and by Triggering Mitochondrial Intrinsic Pathway.

    PubMed

    Somarajan, Bindu I; Khanday, Mudasir A; Mallick, Birendra N

    2016-01-01

    Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate

  2. Phosphorylation in intrinsically disordered regions regulates the activity of Neurogenin2

    PubMed Central

    2014-01-01

    Background Neuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear. Results Here we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks). Conclusions Phosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation. PMID:25374254

  3. Propagated infra-slow intrinsic brain activity reorganizes across wake and slow wave sleep

    PubMed Central

    Mitra, Anish; Snyder, Abraham Z; Tagliazucchi, Enzo; Laufs, Helmut; Raichle, Marcus E

    2015-01-01

    Propagation of slow intrinsic brain activity has been widely observed in electrophysiogical studies of slow wave sleep (SWS). However, in human resting state fMRI (rs-fMRI), intrinsic activity has been understood predominantly in terms of zero-lag temporal synchrony (functional connectivity) within systems known as resting state networks (RSNs). Prior rs-fMRI studies have found that RSNs are generally preserved across wake and sleep. Here, we use a recently developed analysis technique to study propagation of infra-slow intrinsic blood oxygen level dependent (BOLD) signals in normal adults during wake and SWS. This analysis reveals marked changes in propagation patterns in SWS vs. wake. Broadly, ordered propagation is preserved within traditionally defined RSNs but lost between RSNs. Additionally, propagation between cerebral cortex and subcortical structures reverses directions, and intra-cortical propagation becomes reorganized, especially in visual and sensorimotor cortices. These findings show that propagated rs-fMRI activity informs theoretical accounts of the neural functions of sleep. DOI: http://dx.doi.org/10.7554/eLife.10781.001 PMID:26551562

  4. The experience matters more than you think: People value intrinsic incentives more inside than outside an activity.

    PubMed

    Woolley, Kaitlin; Fishbach, Ayelet

    2015-12-01

    We document a shift in the value assigned to intrinsic incentives: people value these incentives more inside an activity than outside the activity (i.e., during vs. before or after pursuit). For example, people care more about the level of interest of their present work task than of past or future work tasks. We document this shift across a variety of activities (exercising, visiting a museum, and lab tasks) and using various measures, including rated importance of intrinsic incentives inside and outside pursuit, actual and planned persistence on activities that offer these incentives, and regret when choosers outside pursuit forgo intrinsic incentives that pursuers later seek. This shift in valuation occurs because intrinsic incentives improve the experience during action pursuit, and therefore, this shift is unique to intrinsic incentives. Extrinsic incentives, by contrast, are valued similarly inside and outside pursuit. PMID:26371401

  5. Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases*S⃞♦

    PubMed Central

    Levin-Salomon, Vered; Kogan, Konstantin; Ahn, Natalie G.; Livnah, Oded; Engelberg, David

    2008-01-01

    MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants (each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation (producing Erk2R65S+D319N and RolledR80S+D334N) resulted in even higher activity (45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2R65S and Erk2R65S+D319N were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained. PMID:18829462

  6. Intrinsic survival advantage of social insect queens depends on reproductive activation.

    PubMed

    Rueppell, O; Königseder, F; Heinze, J; Schrempf, A

    2015-12-01

    The central trade-off between reproduction and longevity dominates most species' life history. However, no mortality cost of reproduction is apparent in eusocial species, particularly social insects in the order Hymenoptera: one or a few individuals (typically referred to as queens) in a group specialize on reproduction and are generally longer lived than all other group members (typically referred to as workers), despite having the same genome. However, it is unclear whether this survival advantage is due to social facilitation by the group or an intrinsic, individual property. Furthermore, it is unknown whether the correlation between reproduction and longevity is due to a direct mechanistic link or an indirect consequence of the social role of the reproductives. To begin addressing these questions, we performed a comparison of queen and worker longevity in the ant Cardiocondyla obscurior under social isolation conditions. Survival of single queens and workers was compared under laboratory conditions, monitoring and controlling for brood production. Our results indicate that there is no intrinsic survival advantage of queens relative to workers unless individuals are becoming reproductively active. This interactive effect of caste and reproduction on life expectancy outside of the normal social context suggests that the positive correlation between reproduction and longevity in social insect queens is due to a direct link that can activate intrinsic survival mechanisms to ensure queen longevity. PMID:26348543

  7. Network burst activity in hippocampal neuronal cultures: the role of synaptic and intrinsic currents.

    PubMed

    Suresh, Jyothsna; Radojicic, Mihailo; Pesce, Lorenzo L; Bhansali, Anita; Wang, Janice; Tryba, Andrew K; Marks, Jeremy D; van Drongelen, Wim

    2016-06-01

    The goal of this work was to define the contributions of intrinsic and synaptic mechanisms toward spontaneous network-wide bursting activity, observed in dissociated rat hippocampal cell cultures. This network behavior is typically characterized by short-duration bursts, separated by order of magnitude longer interburst intervals. We hypothesize that while short-timescale synaptic processes modulate spectro-temporal intraburst properties and network-wide burst propagation, much longer timescales of intrinsic membrane properties such as persistent sodium (Nap) currents govern burst onset during interburst intervals. To test this, we used synaptic receptor antagonists picrotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP) to selectively block GABAA, AMPA, and NMDA receptors and riluzole to selectively block Nap channels. We systematically compared intracellular activity (recorded with patch clamp) and network activity (recorded with multielectrode arrays) in eight different synaptic connectivity conditions: GABAA + NMDA + AMPA, NMDA + AMPA, GABAA + AMPA, GABAA + NMDA, AMPA, NMDA, GABAA, and all receptors blocked. Furthermore, we used mixed-effects modeling to quantify the aforementioned independent and interactive synaptic receptor contributions toward spectro-temporal burst properties including intraburst spike rate, burst activity index, burst duration, power in the local field potential, network connectivity, and transmission delays. We found that blocking intrinsic Nap currents completely abolished bursting activity, demonstrating their critical role in burst onset within the network. On the other hand, blocking different combinations of synaptic receptors revealed that spectro-temporal burst properties are uniquely associated with synaptic functionality and that excitatory connectivity is necessary for the presence of network-wide bursting. In addition to confirming the critical contribution of direct

  8. The characteristics of intrinsic complex micro-contractile activity in isolated strips of the rat bladder.

    PubMed

    Gillespie, J I; Rouget, C; Palea, S; Granato, C; Birder, L; Korstanje, C

    2015-07-01

    In the resting and un-stimulated state, the bladder wall is not quiescent and discrete contractile events, microcontractions, can be recorded in almost all species. This activity contributes to the active element of compliance and to the basal resting tension. This intrinsic activity underpins the more complex phasic activity, non-voiding activity (NVA) that can be seen to increase progressively as the bladder is filled. The NVA represents the motor component of a motor sensory system that relays information to the CNS on bladder volume. Despite the importance of this intrinsic motor activity, little is known about the mechanisms involved in its generation and modulation. The present experiments were done on isolated hemi-bladders from normal rats and measurements made of the intrinsic motor activity. Detailed analysis of the resting state reveals the presence of discrete phasic contractile events, micro-contractions that range in amplitude from 0.1-0.6 mN. These events seem to occur randomly and the basal activity has the appearance of ‘noise’. An analysis of the frequency amplitude distribution of the contractile events, reveals that the total activity appears to be the sum of a number of discrete contractile units, each generating a phasic contraction about a specific mean value and with characteristic frequency. In a hemi-bladder, there are between 20-30 units generating the activity at rest. Using the timed integral of the activity (product of amplitude and frequency), it was noted that the activity was increased by the muscarinic agonist carbachol, but it was decreased by the β-adrenergic agonist isoprenaline. Stretching the preparations also increased the activity. Using these observations, a simple model of the structural and functional organisation of the isolated rat bladder is proposed: the wall appears to be arranged into a number of discrete motor units acting independently. However, the activity can be stimulated or inhibited by pharmacological

  9. p210 Bcr-Abl confers overexpression of inosine monophosphate dehydrogenase : an intrinsic pathway to drug resistance mediated by oncogene.

    SciTech Connect

    Gharehbaghi, K.; Burgess, G. S.; Collart, F. R.; Litz-Jackson, S.; Huberman, E.; Jayaram, H. N.; Boswell, H. S.; Center for Mechanistic Biology and Biotechnology; Lab. for Experimental Oncology; Indiana Univ. School of Medicine

    1994-01-01

    The p210 bcr-abl fusion protein tyrosine kinase oncogene has been implicated in the pathogenesis of chronic granulocytic leukemia (CGL). Specific intracellular functions performed by p210 bcr-abl have recently been delineated. We considered the possibility that p210 bcr-abl may also regulate the abundance of inosine 5'-monophosphate dehydrogenase (IMPDH) which is a rate-limiting enzyme for de novo guanylate synthesis. We performed studies of the inhibition of IMPDH by tiazofurin, which acts as a competitive inhibitor through its active species that mimics nicotinamide adenine dinucleotide (NAD), i.e. thiazole-4-carboxamide adenine dinucleotide (TAD). The mean inhibitory concentration (IC50) of tiazofurin for cellular proliferation inhibition was 2.3-2.8-fold greater in cells expressing p210 bcr-abl than in their corresponding parent cells proliferating under the influence of growth factors or in growth factor-independent derivative cells not expressing detectable p210 bcr-abl. IMPDH activity was 1.5-2.3-fold greater within cells expressing p210 bcr-abl than in their parent cells. This increase in enzyme activity was a result of 2-fold increased IMPDH protein as determined by immunoblotting. In addition, an increase in the Km value for NAD utilization by IMPDH was observed in p210 bcr-abl transformed cells, but this increase was within the range of resident NAD concentrations observed in the cells. Increased IMPDH protein in p210 bcr-abl transformed cells was traced to an increased level of IMP dehydrogenase II messenger RNA. Thus, regulation of IMPDH gene expression is mediated at least in part by the bcr-abl gene product and may therefore be indicative of a specific mechanism of intrinsic resistance to tiazofurin.

  10. p210 bcr-abl confers overexpression of inosine monophosphate dehydrogenase: an intrinsic pathway to drug resistance mediated by oncogene.

    PubMed

    Gharehbaghi, K; Burgess, G S; Collart, F R; Litz-Jackson, S; Huberman, E; Jayaram, H N; Boswell, H S

    1994-08-01

    The p210 bcr-abl fusion protein tyrosine kinase oncogene has been implicated in the pathogenesis of chronic granulocytic leukemia (CGL). Specific intracellular functions performed by p210 bcr-abl have recently been delineated. We considered the possibility that p210 bcr-abl may also regulate the abundance of inosine 5'-monophosphate dehydrogenase (IMPDH) which is a rate-limiting enzyme for de novo guanylate synthesis. We performed studies of the inhibition of IMPDH by tiazofurin, which acts as a competitive inhibitor through its active species that mimics nicotinamide adenine dinucleotide (NAD), i.e. thiazole-4-carboxamide adenine dinucleotide (TAD). The mean inhibitory concentration (IC50) of tiazofurin for cellular proliferation inhibition was 2.3-2.8-fold greater in cells expressing p210 bcr-abl than in their corresponding parent cells proliferating under the influence of growth factors or in growth factor-independent derivative cells not expressing detectable p210 bcr-abl. IMPDH activity was 1.5-2.3-fold greater within cells expressing p210 bcr-abl than in their parent cells. This increase in enzyme activity was a result of 2-fold increased IMPDH protein as determined by immunoblotting. In addition, an increase in the Km value for NAD utilization by IMPDH was observed in p210 bcr-abl transformed cells, but this increase was within the range of resident NAD concentrations observed in the cells. Increased IMPDH protein in p210 bcr-abl transformed cells was traced to an increased level of IMP dehydrogenase II messenger RNA. Thus, regulation of IMPDH gene expression is mediated at least in part by the bcr-abl gene product and may therefore be indicative of a specific mechanism of intrinsic resistance to tiazofurin. PMID:7520100

  11. Minireview: Targeting GPCR Activated ERK Pathways for Drug Discovery

    PubMed Central

    Eishingdrelo, Haifeng; Kongsamut, Sathapana

    2013-01-01

    It has become clear in recent years that multiple signal transduction pathways are employed upon GPCR activation. One of the major cellular effectors activated by GPCRs is extracellular signal-regulated kinase (ERK). Both G-protein and β-arrestin mediated signaling pathways can lead to ERK activation. However, depending on activation pathway, the subcellular destination of activated ERK1/2 may be different. G-protein -dependent ERK activation results in the translocation of active ERK to the nucleus, whereas ERK activated via an arrestin-dependent mechanism remains largely in the cytoplasm. The subcellular location of activated ERK1/2 determines the downstream signaling cascade. Many substrates of ERK1/2 are found in the nucleus: nuclear transcription factors that participate in gene transcription, cell proliferation and differentiation. ERK1/2 substrates are also found in cytosol and other cellular organelles: they may play roles in translation, mitosis, apoptosis and cross-talk with other signaling pathways. Therefore, determining specific subcellular locations of activated ERK1/2 mediated by GPCR ligands would be important in correlating signaling pathways with cellular physiological functions. While GPCR-stimulated selective ERK pathway activation has been studied in several receptor systems, exploitation of these different signaling cascades for therapeutics has not yet been seriously pursued. Many old drug candidates were identified from screens based on G-protein signaling assays, and their activity on β-arrestin signaling pathways being mostly unknown, especially regarding their subcellular ERK pathways. With today’s knowledge of complicated GPCR signaling pathways, drug discovery can no longer rely on single-pathway approaches. Since ERK activation is an important signaling pathway and associated with many physiological functions, targeting the ERK pathway, especially specific subcellular activation pathways should provide new avenues for GPCR drug

  12. Glucocorticoid receptor (GR) {beta} has intrinsic, GR{alpha}-independent transcriptional activity

    SciTech Connect

    Kino, Tomoshige; Manoli, Irini; Kelkar, Sujata; Wang, Yonghong; Su, Yan A.; Chrousos, George P.

    2009-04-17

    The human glucocorticoid receptor (GR) gene produces C-terminal GR{beta} and GR{alpha} isoforms through alternative use of specific exons 9{beta} and {alpha}, respectively. We explored the transcriptional activity of GR{beta} on endogenous genes by developing HeLa cells stably expressing EGFP-GR{beta} or EGFP. Microarray analyses revealed that GR{beta} had intrinsic gene-specific transcriptional activity, regulating mRNA expression of a large number of genes negatively or positively. Majority of GR{beta}-responsive genes was distinct from those modulated by GR{alpha}, while GR{beta} and GR{alpha} mutually modulated each other's transcriptional activity in a subpopulation of genes. We did not observe in HCT116 cells nuclear translocation of GR{beta} and activation of this receptor by RU 486, a synthetic steroid previously reported to bind GR{beta} and to induce nuclear translocation. Our results indicate that GR{beta} has intrinsic, GR{alpha}-independent, gene-specific transcriptional activity, in addition to its previously reported dominant negative effect on GR{alpha}-induced transactivation of GRE-driven promoters.

  13. Spatial distribution of intrinsic and scattering seismic attenuation in active volcanic islands - II: Deception Island images

    NASA Astrophysics Data System (ADS)

    Prudencio, Janire; Ibáñez, Jesús M.; García-Yeguas, Araceli; Del Pezzo, Edoardo; Posadas, Antonio M.

    2013-12-01

    In this work, we present regional maps of the inverse intrinsic quality factor (Qi-1), the inverse scattering quality factor (Qs-1) and total inverse quality factor (Qt-1) for the volcanic environment of Deception Island (Antarctica). Our attenuation study is based on diffusion approximation, which permits us to obtain the attenuation coefficients for every single couple source-receiver separately. The data set used in this research is derived from an active seismic experiment using more than 5200 offshore shots (air guns) recorded at 32 onshore seismic stations and four ocean bottom seismometers. To arrive at a regional distribution of these values, we used a new mapping technique based on a Gaussian space probability function. This approach led us to create `2-D probabilistic maps' of values of intrinsic and scattering seismic attenuation. The 2-D tomographic images confirm the existence of a high attenuation body below an inner bay of Deception Island. This structure, previously observed in 2-D and 3-D velocity tomography of the region, is associated with a massive magma reservoir. Magnetotelluric studies reach a similar interpretation of this strong anomaly. Additionally, we observed areas with lower attenuation effects that bear correlation with consolidated structures described in other studies and associated with the crystalline basement of the area. Our calculations of the transport mean-free path and absorption length for intrinsic attenuation gave respective values of ≈ 950 m and 5 km, which are lower than the values obtained in tectonic regions or volcanic areas such as Tenerife Island. However, as observed in other volcanic regions, our results indicate that scattering effects dominate strongly over the intrinsic attenuation.

  14. How Intrinsic Molecular Dynamics Control Intramolecular Communication in Signal Transducers and Activators of Transcription Factor STAT5

    PubMed Central

    Langenfeld, Florent; Guarracino, Yann; Arock, Michel; Trouvé, Alain; Tchertanov, Luba

    2015-01-01

    Signal Transducer and Activator of Transcription STAT5 is a key mediator of cell proliferation, differentiation and survival. While STAT5 activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated with a broad range of hematological and solid tumor cancers. Therefore the development of compounds able to modulate pathogenic activation of this protein is a very challenging endeavor. A crucial step of drug design is the understanding of the protein conformational features and the definition of putative binding site(s) for such modulators. Currently, there is no structural data available for human STAT5 and our study is the first footprint towards the description of structure and dynamics of this protein. We investigated structural and dynamical features of the two STAT5 isoforms, STAT5a and STAT5b, taken into account their phosphorylation status. The study was based on the exploration of molecular dynamics simulations by different analytical methods. Despite the overall folding similarity of STAT5 proteins, the MD conformations display specific structural and dynamical features for each protein, indicating first, sequence-encoded structural properties and second, phosphorylation-induced effects which contribute to local and long-distance structural rearrangements interpreted as allosteric event. Further examination of the dynamical coupling between distant sites provides evidence for alternative profiles of the communication pathways inside and between the STAT5 domains. These results add a new insight to the understanding of the crucial role of intrinsic molecular dynamics in mediating intramolecular signaling in STAT5. Two pockets, localized in close proximity to the phosphotyrosine-binding site and adjacent to the channel for communication pathways across STAT5, may constitute valid targets to develop inhibitors able to modulate the function-related communication properties of this signaling

  15. Spaceflight Activates Lipotoxic Pathways in Mouse Liver.

    PubMed

    Jonscher, Karen R; Alfonso-Garcia, Alba; Suhalim, Jeffrey L; Orlicky, David J; Potma, Eric O; Ferguson, Virginia L; Bouxsein, Mary L; Bateman, Ted A; Stodieck, Louis S; Levi, Moshe; Friedman, Jacob E; Gridley, Daila S; Pecaut, Michael J

    2016-01-01

    Spaceflight affects numerous organ systems in the body, leading to metabolic dysfunction that may have long-term consequences. Microgravity-induced alterations in liver metabolism, particularly with respect to lipids, remain largely unexplored. Here we utilize a novel systems biology approach, combining metabolomics and transcriptomics with advanced Raman microscopy, to investigate altered hepatic lipid metabolism in mice following short duration spaceflight. Mice flown aboard Space Transportation System -135, the last Shuttle mission, lose weight but redistribute lipids, particularly to the liver. Intriguingly, spaceflight mice lose retinol from lipid droplets. Both mRNA and metabolite changes suggest the retinol loss is linked to activation of PPARα-mediated pathways and potentially to hepatic stellate cell activation, both of which may be coincident with increased bile acids and early signs of liver injury. Although the 13-day flight duration is too short for frank fibrosis to develop, the retinol loss plus changes in markers of extracellular matrix remodeling raise the concern that longer duration exposure to the space environment may result in progressive liver damage, increasing the risk for nonalcoholic fatty liver disease. PMID:27097220

  16. Spaceflight Activates Lipotoxic Pathways in Mouse Liver

    PubMed Central

    Jonscher, Karen R.; Alfonso-Garcia, Alba; Suhalim, Jeffrey L.; Orlicky, David J.; Potma, Eric O.; Ferguson, Virginia L.; Bouxsein, Mary L.; Bateman, Ted A.; Stodieck, Louis S.; Levi, Moshe; Friedman, Jacob E.; Gridley, Daila S.; Pecaut, Michael J.

    2016-01-01

    Spaceflight affects numerous organ systems in the body, leading to metabolic dysfunction that may have long-term consequences. Microgravity-induced alterations in liver metabolism, particularly with respect to lipids, remain largely unexplored. Here we utilize a novel systems biology approach, combining metabolomics and transcriptomics with advanced Raman microscopy, to investigate altered hepatic lipid metabolism in mice following short duration spaceflight. Mice flown aboard Space Transportation System -135, the last Shuttle mission, lose weight but redistribute lipids, particularly to the liver. Intriguingly, spaceflight mice lose retinol from lipid droplets. Both mRNA and metabolite changes suggest the retinol loss is linked to activation of PPARα-mediated pathways and potentially to hepatic stellate cell activation, both of which may be coincident with increased bile acids and early signs of liver injury. Although the 13-day flight duration is too short for frank fibrosis to develop, the retinol loss plus changes in markers of extracellular matrix remodeling raise the concern that longer duration exposure to the space environment may result in progressive liver damage, increasing the risk for nonalcoholic fatty liver disease. PMID:27097220

  17. Monobenzyltin Complex C1 Induces Apoptosis in MCF-7 Breast Cancer Cells through the Intrinsic Signaling Pathway and through the Targeting of MCF-7-Derived Breast Cancer Stem Cells via the Wnt/β-Catenin Signaling Pathway.

    PubMed

    Fani, Somayeh; Dehghan, Firouzeh; Karimian, Hamed; Mun Lo, Kong; Ebrahimi Nigjeh, Siyamak; Swee Keong, Yeap; Soori, Rahman; May Chow, Kit; Kamalidehghan, Behnam; Mohd Ali, Hapipah; Mohd Hashim, Najihah

    2016-01-01

    Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence. PMID:27529753

  18. Monobenzyltin Complex C1 Induces Apoptosis in MCF-7 Breast Cancer Cells through the Intrinsic Signaling Pathway and through the Targeting of MCF-7-Derived Breast Cancer Stem Cells via the Wnt/β-Catenin Signaling Pathway

    PubMed Central

    Fani, Somayeh; Dehghan, Firouzeh; Karimian, Hamed; Mun Lo, Kong; Ebrahimi Nigjeh, Siyamak; Swee Keong, Yeap; Soori, Rahman; May Chow, Kit; Kamalidehghan, Behnam; Mohd Ali, Hapipah; Mohd Hashim, Najihah

    2016-01-01

    Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence. PMID:27529753

  19. Role of CFTR's intrinsic adenylate kinase activity in gating of the Cl(-) channel.

    PubMed

    Randak, Christoph O; Welsh, Michael J

    2007-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H(2)O --> ADP + Pi). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP left arrow over right arrow ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating. PMID:17965924

  20. Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

    PubMed

    Zhang, Hai-Feng; Wang, Huan-Huan; Gao, Na; Wei, Jun-Ying; Tian, Xin; Zhao, Yan; Fang, Yan; Zhou, Jun; Wen, Qiang; Gao, Jie; Zhang, Yang-Jun; Qian, Xiao-Hong; Qiao, Hai-Ling

    2016-07-01

    Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content. PMID:27189963

  1. Intrinsic and Task-Dependent Coupling of Neuronal Population Activity in Human Parietal Cortex

    PubMed Central

    Foster, Brett L.; Rangarajan, Vinitha; Shirer, William R.; Parvizi, Josef

    2015-01-01

    Summary Human neuroimaging studies have suggested that subregions of the medial and lateral parietal cortex form key nodes of a larger brain network supporting episodic memory retrieval. To explore the electrophysiological correlates of functional connectivity between these subregions, we recorded simultaneously from medial and lateral parietal cortex using intracranial electrodes in three human subjects. We observed electrophysiological co-activation of retrosplenial/posterior cingulate cortex (RSC/PCC) and angular gyrus (AG) in the high frequency broadband (HFB, or high-gamma) range, for conditions that required episodic retrieval. During resting and sleeping states, slow fluctuations (< 1 Hz) of HFB activity were highly correlated between these task-co-activated neuronal populations. Furthermore, intrinsic electrophysiological connectivity patterns matched those obtained with resting state functional magnetic resonance imaging (fMRI) from the same subjects. Our findings quantify the spatiotemporal dynamics of parietal cortex during episodic memory retrieval and provide clear neurophysiological correlates of intrinsic and task-dependent functional connectivity in the human brain. PMID:25863718

  2. Perceptions of the activity, the social climate, and the self during group exercise classes regulate intrinsic satisfaction

    PubMed Central

    Maher, Jaclyn P.; Gottschall, Jinger S.; Conroy, David E.

    2015-01-01

    Engaging in regular physical activity is a challenging task for many adults. Intrinsic satisfaction with exercise classes is thought to promote adherence to physical activity. This study examined the characteristics of exercise classes that impact within-person changes in intrinsic satisfaction over the course of an extended group exercise program. A 30-week physical activity trial was conducted with assessments at the end of each class. Community-living adults (n = 29) were instructed to complete at least six group exercise classes each week and, following each exercise class, complete a questionnaire asking about the characteristics of the class and the participant’s evaluation of the class. Intrinsic satisfaction was high, on average, but varied as much within-person from class-to-class as it did between exercisers. Participants reported the greatest intrinsic satisfaction when classes placed greater emphasis on exercisers’ involvement with the group task, feelings of competence, and encouragement from the instructor. For the most part, exercise classes that were more intense than usual were perceived by exercisers as less intrinsically satisfying. Some overall characteristics of the exercise classes were also associated with intrinsic satisfaction. The social and motivational characteristics of group exercise classes contribute to exercisers’ intrinsic satisfaction with classes and attention to those dynamics, as well as the intensity of the exercise, may improve adherence for exercise regimens. PMID:26347696

  3. Ion channel modifying agents influence the electrical activity generated by canine intrinsic cardiac neurons in situ.

    PubMed

    Thompson, G W; Horackova, M; Armour, J A

    2000-04-01

    This study was designed to establish whether agents known to modify neuronal ion channels influence the behavior of mammalian intrinsic cardiac neurons in situ and, if so, in a manner consistent with that found previously in vitro. The activity generated by right atrial neurons was recorded extracellularly in varying numbers of anesthetized dogs before and during continuous local arterial infusion of several neuronal ion channel modifying agents. Veratridine (7.5 microM), the specific modifier of Na+-selective channels, increased neuronal activity (95% above control) in 80% of dogs tested (n = 25). The membrane depolarizing agent potassium chloride (40 mM) reduced neuronal activity (43% below control) in 84% of dogs tested (n = 19). The inhibitor of voltage-sensitive K+ channels, tetraethylammonium (10 mM), decreased neuronal activity (42% below control) in 73% of dogs tested (n = 11). The nonspecific potassium channel inhibitor barium chloride (5 mM) excited neurons (47% above control) in 13 of 19 animals tested. Cadmium chloride (200 microM), which inhibits Ca2+-selective channels and Ca2+-dependent K+ channels, increased neuronal activity (65% above control) in 79% of dogs tested (n = 14). The specific L-type Ca2+ channel blocking agent nifedipine (5 microM) reduced neuronal activity (52% blow control in 72% of 11 dogs tested), as did the nonspecific inhibitor of L-type Ca2+ channels, nickel chloride (5 mM) (36% below control in 69% of 13 dogs tested). Each agent induced either excitatory or inhibitory responses, depending on the agent tested. It is concluded that specific ion channels (I(Na), I(CaL), I(Kv), and I(KCa)) that have been associated with intrinsic cardiac neurons in vitro are involved in their capacity to generate action potentials in situ. PMID:10772056

  4. Regional Homogeneity of Intrinsic Brain Activity in Happy and Unhappy Individuals

    PubMed Central

    Luo, Yangmei; Huang, Xiting; Yang, Zhen; Li, Baolin; Liu, Jie; Wei, Dongtao

    2014-01-01

    Background Why are some people happier than others? This question has intrigued many researchers. However, limited work has addressed this question within a neuroscientific framework. Methods The present study investigated the neural correlates of trait happiness using the resting-state functional magnetic resonance imaging (rs-fMRI) approach. Specifically, regional homogeneity (ReHo) was examined on two groups of young adults: happy and unhappy individuals (N = 25 per group). Results Decreased ReHo in unhappy relative to happy individuals was observed within prefrontal cortex, medial temporal lobe, superior temporal lobe, and retrosplenial cortex. In contrast, increased ReHo in unhappy relative to happy individuals was observed within the dorsolateral prefrontal cortex, middle cingulate gyrus, putamen, and thalamus. In addition, the ReHo within the left thalamus was negatively correlated with Chinese Happiness Inventory (CHI) score within the happy group. Limitations As an exploratory study, we examined how general trait happiness is reflected in the regional homogeneity of intrinsic brain activity in a relatively small sample. Examining other types of happiness in a larger sample using a multitude of intrinsic brain activity indices are warranted for future work. Conclusions The local synchronization of BOLD signal is altered in unhappy individuals. The regions implicated in this alteration partly overlapped with previously identified default mode network, emotional circuitry, and rewarding system, suggesting that these systems may be involved in happiness. PMID:24454814

  5. A novel cationic lipid with intrinsic antitumor activity to facilitate gene therapy of TRAIL DNA.

    PubMed

    Luo, Cong; Miao, Lei; Zhao, Yi; Musetti, Sara; Wang, Yuhua; Shi, Kai; Huang, Leaf

    2016-09-01

    Metformin (dimethylbiguanide) has been found to be effective for the treatment of a wide range of cancer. Herein, a novel lipid (1,2-di-(9Z-octadecenoyl)-3-biguanide-propane (DOBP)) was elaborately designed by utilizing biguanide as the cationic head group. This novel cationic lipid was intended to act as a gene carrier with intrinsic antitumor activity. When compared with 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP), a commercially available cationic lipid with a similar structure, the blank liposomes consisting of DOBP showed much more potent antitumor effects than DOTAP in human lung tumor xenografts, following an antitumor mechanism similar to metformin. Given its cationic head group, biguanide, DOBP could encapsulate TNF-related apoptosis-inducing ligand (TRAIL) plasmids into Lipid-Protamine-DNA (LPD) nanoparticles (NPs) for systemic gene delivery. DOBP-LPD-TRAIL NPs demonstrated distinct superiority in delaying tumor progression over DOTAP-LPD-TRAIL NPs, due to the intrinsic antitumor activity combined with TRAIL-induced apoptosis in the tumor. These results indicate that DOBP could be used as a versatile and promising cationic lipid for improving the therapeutic index of gene therapy in cancer treatment. PMID:27344367

  6. Intrinsic and extrinsic cues regulate the daily profile of mouse lateral habenula neuronal activity

    PubMed Central

    Sakhi, Kanwal; Wegner, Sven; Belle, Mino D C; Howarth, Michael; Delagrange, Philippe; Brown, Timothy M; Piggins, Hugh D

    2014-01-01

    The epithalamic lateral habenula (LHb) is implicated as part of the mammalian brain's circadian system. Anatomical evidence suggests that the LHb receives extrinsic circadian timing cues from retinal ganglion cells and the master clock in the suprachiasmatic nuclei (SCN). Intriguingly, some LHb neurones contain the molecular circadian clock, but it is unclear if and how intrinsic and extrinsic circadian processes influence neuronal activity in the mouse LHb. Here, using an in vitro brain slice preparation isolating the LHb from the SCN, we show through whole-cell patch-clamp recordings that LHb neurones exhibit heterogeneity in their resting state, but the majority spontaneously fire action potentials (APs). Discharge rate of APs varied from low firing in the early day to higher firing later in the day and was absent in LHb brain slices prepared from Cry1−/−Cry2−/− mice that lack a functional molecular clock. Low amplitude circadian oscillations in the molecular circadian clock were also monitored in LHb brain slices, but were absent in Cry1−/−Cry2−/− LHb brain tissue. A putative neurochemical output signal of the SCN, prokineticin 2 (PK2), inhibited some LHb neurones by elevating the frequency of GABA release in the LHb. Using multi-electrode recordings in vivo, we found that LHb neurones sluggishly respond to retinal illumination, suggesting that they receive such information through polysynaptic processes. In summary, our results show for the first time that intrinsic circadian signals are important for regulating LHb neuronal state, while the SCN-derived signal PK2 is less influential. Moreover, we demonstrate that mouse LHb neurones have access to and can respond to visual input, but such signals are unlikely to be directly communicated to the LHb. Broadly, these findings raise the possibility that intrinsic circadian signals are likely to be influential in shaping LHb contributions to cognition and emotionality. PMID:25194046

  7. Functional imaging of glucose-evoked rat islet activities using transient intrinsic optical signals

    NASA Astrophysics Data System (ADS)

    Yao, Xin-Cheng; Cui, Wan-Xing; Li, Yi-Chao; Zhang, Wei; Lu, Rong-Wen; Thompson, Anthony; Amthor, Franklin; Wang, Xu-Jing

    2012-05-01

    We demonstrate intrinsic optical signal (IOS) imaging of intact rat islet, which consists of many endocrine cells working together. A near-infrared digital microscope was employed for optical monitoring of islet activities evoked by glucose stimulation. Dynamic NIR images revealed transient IOS responses in the islet activated by low-dose (2.75 mM) and high-dose (5.5 mM) glucose stimuli. Comparative experiments and quantitative analysis indicated that both glucose metabolism and calcium/insulin dynamics might contribute to the observed IOS responses. Further investigation of the IOS imaging technology may provide a high resolution method for ex vivo functional examination of the islet, which is important for advanced study of diabetes associated islet dysfunctions and for improved quality control of donor islets for transplantation.

  8. Mutations that bypass tRNA binding activate the intrinsically defective kinase domain in GCN2

    PubMed Central

    Qiu, Hongfang; Hu, Cuihua; Dong, Jinsheng; Hinnebusch, Alan G.

    2002-01-01

    The protein kinase GCN2 is activated in amino acid-starved cells on binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-related domain. We isolated two point mutations in the protein kinase (PK) domain, R794G and F842L, that permit strong kinase activity in the absence of tRNA binding. These mutations also bypass the requirement for ribosome binding, dimerization, and association with the GCN1/GCN20 regulatory complex, suggesting that all of these functions facilitate tRNA binding to wild-type GCN2. While the isolated wild-type PK domain was completely inert, the mutant PK was highly active in vivo and in vitro. These results identify an inhibitory structure intrinsic to the PK domain that must be overcome on tRNA binding by interactions with a regulatory region, most likely the N terminus of the HisRS segment. As Arg 794 and Phe 842 are predicted to lie close to one another and to the active site, they may participate directly in misaligning active site residues. Autophosphorylation of the activation loop was stimulated by R794G and F842L, and the autophosphorylation sites remained critical for GCN2 function in the presence of these mutations. Our results imply a two-step activation mechanism involving distinct conformational changes in the PK domain. PMID:12023305

  9. The Intrinsic Eddington Ratio Distribution of Active Galactic Nuclei in Young Galaxies from SDSS

    NASA Astrophysics Data System (ADS)

    Jones, Mackenzie L.; Hickox, Ryan C.; Black, Christine; Hainline, Kevin Nicholas; DiPompeo, Michael A.

    2016-04-01

    An important question in extragalactic astronomy concerns the distribution of black hole accretion rates, i.e. the Eddington ratio distribution, of active galactic nuclei (AGN). Specifically, it is matter of debate whether AGN follow a broad distribution in accretion rates, or if the distribution is more strongly peaked at characteristic Eddington ratios. Using a sample of galaxies from SDSS DR7, we test whether an intrinsic Eddington ratio distribution that takes the form of a broad Schechter function is in fact consistent with previous work that suggests instead that young galaxies in optical surveys have a more strongly peaked lognormal Eddington ratio distribution. Furthermore, we present an improved method for extracting the AGN distribution using BPT diagnostics that allows us to probe over one order of magnitude lower in Eddington ratio, counteracting the effects of dilution by star formation. We conclude that the intrinsic Eddington ratio distribution of optically selected AGN is consistent with a power law with an exponential cutoff, as is observed in the X-rays. This work was supported in part by a NASA Jenkins Fellowship.

  10. Increased Efferent Cardiac Sympathetic Nerve Activity and Defective Intrinsic Heart Rate Regulation in Type 2 Diabetes.

    PubMed

    Thaung, H P Aye; Baldi, J Chris; Wang, Heng-Yu; Hughes, Gillian; Cook, Rosalind F; Bussey, Carol T; Sheard, Phil W; Bahn, Andrew; Jones, Peter P; Schwenke, Daryl O; Lamberts, Regis R

    2015-08-01

    Elevated sympathetic nerve activity (SNA) coupled with dysregulated β-adrenoceptor (β-AR) signaling is postulated as a major driving force for cardiac dysfunction in patients with type 2 diabetes; however, cardiac SNA has never been assessed directly in diabetes. Our aim was to measure the sympathetic input to and the β-AR responsiveness of the heart in the type 2 diabetic heart. In vivo recording of SNA of the left efferent cardiac sympathetic branch of the stellate ganglion in Zucker diabetic fatty rats revealed an elevated resting cardiac SNA and doubled firing rate compared with nondiabetic rats. Ex vivo, in isolated denervated hearts, the intrinsic heart rate was markedly reduced. Contractile and relaxation responses to β-AR stimulation with dobutamine were compromised in externally paced diabetic hearts, but not in diabetic hearts allowed to regulate their own heart rate. Protein levels of left ventricular β1-AR and Gs (guanine nucleotide binding protein stimulatory) were reduced, whereas left ventricular and right atrial β2-AR and Gi (guanine nucleotide binding protein inhibitory regulatory) levels were increased. The elevated resting cardiac SNA in type 2 diabetes, combined with the reduced cardiac β-AR responsiveness, suggests that the maintenance of normal cardiovascular function requires elevated cardiac sympathetic input to compensate for changes in the intrinsic properties of the diabetic heart. PMID:25784543

  11. Amelioration of nandrolone decanoate-induced testicular and sperm toxicity in rats by taurine: Effects on steroidogenesis, redox and inflammatory cascades, and intrinsic apoptotic pathway

    SciTech Connect

    Ahmed, Maha A.E.

    2015-02-01

    The wide abuse of the anabolic steroid nandrolone decanoate by athletes and adolescents for enhancement of sporting performance and physical appearance may be associated with testicular toxicity and infertility. On the other hand, taurine; a free β-amino acid with remarkable antioxidant activity, is used in taurine-enriched beverages to boost the muscular power of athletes. Therefore, the purpose of this study was to investigate the mechanisms of the possible protective effects of taurine on nandrolone decanoate-induced testicular and sperm toxicity in rats. To achieve this aim, male Wistar rats were randomly distributed into four groups and administered either vehicle, nandrolone decanoate (10 mg/kg/week, I.M.), taurine (100 mg/kg/day, p.o.) or combination of taurine and nandrolone decanoate, for 8 successive weeks. Results of the present study showed that taurine reversed nandrolone decanoate-induced perturbations in sperm characteristics, normalized serum testosterone level, and restored the activities of the key steroidogenic enzymes; 3β-HSD, and 17β-HSD. Moreover, taurine prevented nandrolone decanoate-induced testicular toxicity and DNA damage by virtue of its antioxidant, anti-inflammatory, and anti-apoptotic effects. This was evidenced by taurine-induced modulation of testicular LDH-x activity, redox markers (MDA, NO, GSH contents, and SOD activity), inflammatory indices (TNF-α, ICAM-1 levels, and MMP-9 gene expression), intrinsic apoptotic pathway (cytochrome c gene expression and caspase-3 content), and oxidative DNA damage markers (8-OHdG level and comet assay). In conclusion, at the biochemical and histological levels, taurine attenuated nandrolone decanoate-induced poor sperm quality and testicular toxicity in rats. - Highlights: • Nandrolone decanoate (ND) disrupts sperm profile and steroidogenesis in rats. • ND upregulates gene expression of inflammatory and apoptotic markers. • Taurine normalizes sperm profile and serum testosterone level

  12. Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation

    PubMed Central

    Sun, Yujia; Nien, Chung-Yi; Chen, Kai; Liu, Hsiao-Yun; Johnston, Jeff; Zeitlinger, Julia; Rushlow, Christine

    2015-01-01

    The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility, but the mechanisms by which this occurs are poorly understood. Here, we analyze the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). Our results show that early enhancers are characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. We envision that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner. PMID:26335633

  13. Theoretical analysis of non-radiative multiphonon recombination activity of intrinsic defects in CdTe

    NASA Astrophysics Data System (ADS)

    Krasikov, D. N.; Scherbinin, A. V.; Knizhnik, A. A.; Vasiliev, A. N.; Potapkin, B. V.; Sommerer, T. J.

    2016-02-01

    We present an analysis of recombination activity of intrinsic defects (VCd, TeCd, VTe, and Tei) in CdTe based on the multiphonon single-mode carrier-capture model, with vibronic parameters obtained using hybrid density functional theory. This analysis allows us to determine the defects and the corresponding electronic processes that have high trapping rates for electrons, for holes, or for both. The latter, being potentially the most active recombination centers, decreases the carrier lifetime in the absorber layer of a CdTe solar cell. Taking into account the relatively high calculated capture cross-sections of the TeCd antisite defect (σ = 8.7× 10-15 cm2 for electron capture on TeCd+2 defect, σ = 6.8 × 10-14 cm2 for hole capture on TeCd+1 defect at room temperature) and its deep trapping level (0.41 eV for +2/+1 level), we conclude that this defect is the most active recombination center among the intrinsic defects in p-type CdTe. Other processes that do not lead to effective recombination are: (i) fast hole capture on Tei+1 defect (σ = 1.1 × 10-13 cm-2), (ii) electron capture on TeCd+1 defect (σ = 2.9 × 10-15 cm-2), (iii) somewhat slower hole capture on TeCd0 defect (σ = 9.4 × 10-20 cm-2), (iv) hole capture on VCd-1 defect (σ = 7 × 10-19 cm2), and (v) electron capture on Tei+1 defect (σ = 4.4 × 10-19 cm-2). The cross-sections are found to be negligibly small for the remaining capture processes.

  14. Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB).

    PubMed

    Méndez-Lorenzo, Luz; Porras-Domínguez, Jaime R; Raga-Carbajal, Enrique; Olvera, Clarita; Rodríguez-Alegría, Maria Elena; Carrillo-Nava, Ernesto; Costas, Miguel; López Munguía, Agustín

    2015-01-01

    Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final

  15. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-11-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  16. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed Central

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-01-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  17. Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB)

    PubMed Central

    Raga-Carbajal, Enrique; Olvera, Clarita; Rodríguez-Alegría, Maria Elena; Carrillo-Nava, Ernesto; Costas, Miguel; López Munguía, Agustín

    2015-01-01

    Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final

  18. Subglottal pressure, tracheal airflow, and intrinsic laryngeal muscle activity during rat ultrasound vocalization

    PubMed Central

    2011-01-01

    Vocal production requires complex planning and coordination of respiratory, laryngeal, and vocal tract movements, which are incompletely understood in most mammals. Rats produce a variety of whistles in the ultrasonic range that are of communicative relevance and of importance as a model system, but the sources of acoustic variability were mostly unknown. The goal was to identify sources of fundamental frequency variability. Subglottal pressure, tracheal airflow, and electromyographic (EMG) data from two intrinsic laryngeal muscles were measured during 22-kHz and 50-kHz call production in awake, spontaneously behaving adult male rats. During ultrasound vocalization, subglottal pressure ranged between 0.8 and 1.9 kPa. Pressure differences between call types were not significant. The relation between fundamental frequency and subglottal pressure within call types was inconsistent. Experimental manipulations of subglottal pressure had only small effects on fundamental frequency. Tracheal airflow patterns were also inconsistently associated with frequency. Pressure and flow seem to play a small role in regulation of fundamental frequency. Muscle activity, however, is precisely regulated and very sensitive to alterations, presumably because of effects on resonance properties in the vocal tract. EMG activity of cricothyroid and thyroarytenoid muscle was tonic in calls with slow or no fundamental frequency modulations, like 22-kHz and flat 50-kHz calls. Both muscles showed brief high-amplitude, alternating bursts at rates up to 150 Hz during production of frequency-modulated 50-kHz calls. A differentiated and fine regulation of intrinsic laryngeal muscles is critical for normal ultrasound vocalization. Many features of the laryngeal muscle activation pattern during ultrasound vocalization in rats are shared with other mammals. PMID:21832032

  19. Intrinsic activity and poisoning rate for HCOOH oxidation on platinum stepped surfaces.

    PubMed

    Grozovski, Vitali; Climent, Víctor; Herrero, Enrique; Feliu, Juan M

    2010-08-21

    Pulsed voltammetry has been used to study formic acid oxidation on platinum stepped surfaces to determine the kinetics of the reaction and the role of the surface structure in the reactivity. From the current transients at different potentials, the intrinsic activity of the electrode through the active intermediate reaction path (j(theta = 0)), as well as the rate constant for the CO formation (k(ads)) have been calculated. The kinetics for formic acid oxidation through the active intermediate reaction path is strongly dependent on the surface structure of the electrode, with the highest activity found for the Pt(100) surface. The presence of steps, both on (100) and (111) terraces, does not increase the activity of these surfaces. CO formation only takes place in a narrow potential window very close to the local potential of zero total charge. The extrapolation of the results obtained with stepped surfaces with (111) terraces to zero step density indicates that CO formation should not occur on an ideal Pt(111) electrode. Additionally, the analysis of the Tafel slopes obtained for the different electrodes suggests that the oxidation of formic acid is strongly affected by the presence of adsorbed anions, hydrogen and water. PMID:20539876

  20. Sequential activation of metabolic pathways: a dynamic optimization approach.

    PubMed

    Oyarzún, Diego A; Ingalls, Brian P; Middleton, Richard H; Kalamatianos, Dimitrios

    2009-11-01

    The regulation of cellular metabolism facilitates robust cellular operation in the face of changing external conditions. The cellular response to this varying environment may include the activation or inactivation of appropriate metabolic pathways. Experimental and numerical observations of sequential timing in pathway activation have been reported in the literature. It has been argued that such patterns can be rationalized by means of an underlying optimal metabolic design. In this paper we pose a dynamic optimization problem that accounts for time-resource minimization in pathway activation under constrained total enzyme abundance. The optimized variables are time-dependent enzyme concentrations that drive the pathway to a steady state characterized by a prescribed metabolic flux. The problem formulation addresses unbranched pathways with irreversible kinetics. Neither specific reaction kinetics nor fixed pathway length are assumed.In the optimal solution, each enzyme follows a switching profile between zero and maximum concentration, following a temporal sequence that matches the pathway topology. This result provides an analytic justification of the sequential activation previously described in the literature. In contrast with the existent numerical approaches, the activation sequence is proven to be optimal for a generic class of monomolecular kinetics. This class includes, but is not limited to, Mass Action, Michaelis-Menten, Hill, and some Power-law models. This suggests that sequential enzyme expression may be a common feature of metabolic regulation, as it is a robust property of optimal pathway activation. PMID:19412635

  1. Intrinsic Bayesian Active Contours for Extraction of Object Boundaries in Images

    PubMed Central

    Srivastava, Anuj

    2010-01-01

    We present a framework for incorporating prior information about high-probability shapes in the process of contour extraction and object recognition in images. Here one studies shapes as elements of an infinite-dimensional, non-linear quotient space, and statistics of shapes are defined and computed intrinsically using differential geometry of this shape space. Prior models on shapes are constructed using probability distributions on tangent bundles of shape spaces. Similar to the past work on active contours, where curves are driven by vector fields based on image gradients and roughness penalties, we incorporate the prior shape knowledge in the form of vector fields on curves. Through experimental results, we demonstrate the use of prior shape models in the estimation of object boundaries, and their success in handling partial obscuration and missing data. Furthermore, we describe the use of this framework in shape-based object recognition or classification. PMID:21076692

  2. Reduction method for intrinsic random coincidence events from (176)Lu in low activity PET imaging.

    PubMed

    Yoshida, Eiji; Tashima, Hideaki; Nishikido, Fumihiko; Murayama, Hideo; Yamaya, Taiga

    2014-07-01

    For clinical studies, the effects of the intrinsic radioactivity of lutetium-based scintillators such as LSO used in PET imaging can be ignored within a narrow energy window. However, the intrinsic radioactivity becomes problematic when used in low-count-rate situations such as gene expression imaging or in-beam PET imaging. Time-of-flight (TOF) measurement capability promises not only to improve PET image quality, but also to reduce intrinsic random coincidences. On the other hand, we have developed a new reduction method for intrinsic random coincidence events based on multiple-coincidence information. Without the energy window, an intrinsic random coincidence is detected simultaneously with an intrinsic true coincidence as a multiple coincidence. The multiple-coincidence events can serve as a guide to identification of the intrinsic coincidences. After rejection of multiple-coincidence events detected with a wide energy window, data obtained included a few intrinsic random and many intrinsic true coincidence events. We analyzed the effect of intrinsic radioactivity and used Monte Carlo simulation to test both the TOF-based method and the developed multiple-coincidence-based (MC-based) method for a whole-body LSO-PET scanner. Using the TOF- and MC-based reduction methods separately, we could reduce the intrinsic random coincidence rates by 77 and 30 %, respectively. Also, the intrinsic random coincidence rate could be reduced by 84 % when the TOF+MC reduction methods were applied. The developed MC-based method showed reduced number of the intrinsic random coincidence events, but the reduction performance was limited compared to that of the TOF-based reduction method. PMID:24496884

  3. The additional loss of Bak and not the lack of the protein tyrosine kinase p56/Lck in one JCaM1.6 subclone caused pronounced apoptosis resistance in response to stimuli of the intrinsic pathway.

    PubMed

    Rudner, J; Mueller, A-C; Matzner, N; Huber, S M; Handrick, R; Belka, C; Jendrossek, V

    2009-05-01

    Ionising radiation, hypoxia, and the cyclooxygenase-2 inhibitor Celecoxib are known agonists of the intrinsic apoptosis pathway that involves mitochondrial damage upstream of caspase activation. Mitochondrial integrity is regulated by the pro-apoptotic Bcl-2 protein family members Bak and Bax. Upstream of the mitochondria, many kinases and phosphatases control the apoptotic response. However, the role of the non-receptor tyrosine kinase p56/Lck during apoptosis is controversial. The present investigation demonstrate the existence of two JCaM1.6 subclones, one expressing and one deficient for Bak. The lack of p56/Lck expression in JCaM1.6 cells per se did hardly affect apoptosis induced by ionising radiation, hypoxia, or Celecoxib. Only the additional loss of Bak expression, as observed in one JCaM1.6 subclone, rendered the cells resistant. siRNA-mediated downregulation of Bak and p56/Lck mimicked the observed effects in the subclones. Earlier experiments performed with the Bak-negative clone might have lead to the wrong assumption that lack of p56/Lck alone, and not the additonal loss of Bak, was responsible for reduced sensitivity towards stimuli of the intrinsic apoptosis pathway. PMID:19343496

  4. HdeB chaperone activity is coupled to its intrinsic dynamic properties

    PubMed Central

    Ding, Jienv; Yang, Chengfeng; Niu, Xiaogang; Hu, Yunfei; Jin, Changwen

    2015-01-01

    Enteric bacteria encounter extreme acidity when passing through hosts’ stomach. Since the bacterial periplasmic space quickly equilibrates with outer environment, an efficient acid resistance mechanism is essential in preventing irreversible protein denaturation/aggregation and maintaining bacteria viability. HdeB, along with its homolog HdeA, was identified as a periplasmic acid-resistant chaperone. Both proteins exist as homodimers and share similar monomeric structures under neutral pH, while showing different dimeric packing interfaces. Previous investigations show that HdeA functions through an acid-induced dimer-to-monomer transition and partial unfolding at low pH (pH 2–3), resulting in exposure of hydrophobic surfaces that bind substrate proteins. In contrast, HdeB appears to have a much higher optimal activation pH (pH 4–5), under which condition the protein maintains a well-folded dimer and the mechanism for its chaperone activity remains elusive. Herein, we present an NMR study of HdeB to investigate its dynamic properties. Our results reveal that HdeB undergoes significant micro- to milli-second timescale conformational exchanges at neutral to near-neutral pH, under the later condition it exhibits optimal activity. The current study indicates that HdeB activation is coupled to its intrinsic dynamics instead of structural changes, and therefore its functional mechanism is apparently different from HdeA. PMID:26593705

  5. The unpredictability of prolonged activation of stress response pathways

    PubMed Central

    Lamech, Lilian T.

    2015-01-01

    In response to stress, cellular compartments activate signaling pathways that mediate transcriptional programs to promote survival and reestablish homeostasis. Manipulation of the magnitude and duration of the activation of stress responses has been proposed as a strategy to prevent or repair the damage associated with aging or degenerative diseases. However, as these pathways likely evolved to respond specifically to transient perturbations, the unpredictability of prolonged activation should be considered. PMID:26101215

  6. Case study of an approved corrective action integrating active remediation with intrinsic remediation

    SciTech Connect

    Teets, D.B.; Guest, P.R.; Blicker, B.R.

    1996-12-01

    Parsons Engineering Science, Inc., performed UST removals and/or site assessments at UST system locations at a former US Air Force Base (AFB) in Denver, Colorado. Four UST systems, incorporating 17 USTs, were located within the petroleum, oils, and lubricants bulk storage yard (POL Yard) of the former AFB. During the tank removals and subsequent site investigations, petroleum hydrocarbon contamination was found in soils at each site. Significant releases from two of the UST systems resulted in a dissolved benzene, toluene, ethylbenzene, and xylenes (BTEX) plume in the groundwater, and smear-zone contamination of soils beneath the majority of the POL Yard. Because of the close proximity of the UST systems, and the presence of the groundwater plume beneath the POL Yard, a corrective action plan (CAP) was prepared that encompassed all four UST systems. An innovative, risk-based CAP integrated active remediation of petroleum-contaminated soils with intrinsic remediation of groundwater. A natural attenuation evaluation for the dissolved BTEX was performed to demonstrate that natural attenuation processes are providing adequate remediation of groundwater and to predict the fate of the groundwater plume. BTEX concentrations versus distance were regressed to obtain attenuation rates, which were then used to calculate BTEX degradation rates using a one-dimensional, steady-state analytical solution. Additionally, electron acceptor concentrations in groundwater were compared to BTEX concentrations to provide evidence that natural attenuation of BTEX compounds was occurring. The natural attenuation evaluation was used in the CAP to support the intrinsic remediation with long-term monitoring alternative for groundwater, thereby avoiding the installation of an expensive groundwater remediation system.

  7. Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo.

    PubMed

    Girola, Natalia; Figueiredo, Carlos R; Farias, Camyla F; Azevedo, Ricardo A; Ferreira, Adilson K; Teixeira, Sarah F; Capello, Tabata M; Martins, Euder G A; Matsuo, Alisson L; Travassos, Luiz R; Lago, João H G

    2015-11-27

    Natural monoterpenes were isolated from the essential oil of Piper cernuum Vell. (Piperaceae) leaves. The crude oil and the individual monoterpenes were tested for cytotoxicity in human tumor cell lineages and B16F10-Nex2 murine melanoma cells. In the present work we demonstrate the activity of camphene against different cancer cells, with its mechanism of action being investigated in vitro and in vivo in murine melanoma. Camphene induced apoptosis by the intrinsic pathway in melanoma cells mainly by causing endoplasmic reticulum (ER) stress, with release of Ca(2+) together with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity. Importantly, camphene exerted antitumor activity in vivo by inhibiting subcutaneous tumor growth of highly aggressive melanoma cells in a syngeneic model, suggesting a promising role of this compound in cancer therapy. PMID:26471302

  8. Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

    PubMed Central

    Dupuis-Maurin, Valéryane; Brinza, Lilia; Baguet, Joël; Plantamura, Emilie; Schicklin, Stéphane; Chambion, Solène; Macari, Claire; Tomkowiak, Martine; Deniaud, Emmanuelle; Leverrier, Yann

    2015-01-01

    Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen. PMID:25738304

  9. Essential role of the m2R-RGS6-IKACh pathway in controlling intrinsic heart rate variability.

    PubMed

    Posokhova, Ekaterina; Ng, David; Opel, Aaisha; Masuho, Ikuo; Tinker, Andrew; Biesecker, Leslie G; Wickman, Kevin; Martemyanov, Kirill A

    2013-01-01

    Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-I(KACh) intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-I(KACh) signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-I(KACh) signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis. PMID:24204714

  10. Essential Role of the m2R-RGS6-IKACh Pathway in Controlling Intrinsic Heart Rate Variability

    PubMed Central

    Posokhova, Ekaterina; Ng, David; Opel, Aaisha; Masuho, Ikuo; Tinker, Andrew; Biesecker, Leslie G.; Wickman, Kevin; Martemyanov, Kirill A.

    2013-01-01

    Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-IKACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-IKACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-IKACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis. PMID:24204714

  11. Enhanced Intrinsic Catalytic Activity of λ-MnO2 by Electrochemical Tuning and Oxygen Vacancy Generation.

    PubMed

    Lee, Sanghan; Nam, Gyutae; Sun, Jie; Lee, Jang-Soo; Lee, Hyun-Wook; Chen, Wei; Cho, Jaephil; Cui, Yi

    2016-07-18

    Chemically prepared λ-MnO2 has not been intensively studied as a material for metal-air batteries, fuel cells, or supercapacitors because of their relatively poor electrochemical properties compared to α- and δ-MnO2 . Herein, through the electrochemical removal of lithium from LiMn2 O4 , highly crystalline λ-MnO2 was prepared as an efficient electrocatalyst for the oxygen reduction reaction (ORR). The ORR activity of the material was further improved by introducing oxygen vacancies (OVs) that could be achieved by increasing the calcination temperature during LiMn2 O4 synthesis; a concentration of oxygen vacancies in LiMn2 O4 could be characterized by its voltage profile as the cathode in a lithiun-metal half-cell. λ-MnO2-z prepared with the highest OV exhibited the highest diffusion-limited ORR current (5.5 mA cm(-2) ) among a series of λ-MnO2-z electrocatalysts. Furthermore, the number of transferred electrons (n) involved in the ORR was >3.8, indicating a dominant quasi-4-electron pathway. Interestingly, the catalytic performances of the samples were not a function of their surface areas, and instead depended on the concentration of OVs, indicating enhancement in the intrinsic catalytic activity of λ-MnO2 by the generation of OVs. This study demonstrates that differences in the electrochemical behavior of λ-MnO2 depend on the preparation method and provides a mechanism for a unique catalytic behavior of cubic λ-MnO2 . PMID:27254822

  12. Activation of the MAPK/ERK Cell-Signaling Pathway in Uterine Smooth Muscle Cells of Women With Adenomyosis.

    PubMed

    Streuli, Isabelle; Santulli, Pietro; Chouzenoux, Sandrine; Chapron, Charles; Batteux, Frédéric

    2015-12-01

    We investigated whether the myometrium might be intrinsically different in women with adenomyosis. We studied whether the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs) and phosphoinositide 3-kinase/mammalian target of rapamycin/AKT (PI3K/mTOR/AKT) cell-signaling pathways, implicated in the pathogenesis of endometriosis, might also be activated in uterine smooth muscle cells (uSMCs) of women with adenomyosis and measured the production of reactive oxygen species (ROS), proinflammatory mediators that modulate cell proliferation and have been shown to activate the MAPK/ERK pathway in endometriosis. The uSMC cultures were derived from myometrium biopsies obtained during hysterectomy or myomectomy in women with adenomyosis and controls with leiomyoma. Proliferation of uSMCs and in vitro activation of the MAPK/ERK cell-signaling pathway were increased in women with adenomyosis compared to controls. The activation of the PI3K/mTOR/AKT pathway was not significant. The ROS production and ROS detoxification pathways were not different between uSMCs of women with adenomyosis and controls suggesting an ROS-independent activation of the MAPK/ERK pathway. Our results also provide evidence that protein kinase inhibitors and the rapanalogue temsirolimus can control proliferation of uSMCs in vitro suggesting an implication of the MAPK/ERK and the PI3K/mTOR/AKT pathways in proliferation of uSMCs in women with adenomyosis and leiomyomas. PMID:26071388

  13. Intrinsic activity of human immunodeficiency virus type 1 protease heterologous fusion proteins in mammalian cells.

    PubMed

    Arrigo, S J; Haines, J K; Huffman, K M

    1995-01-01

    We have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein. PMID:7832989

  14. Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

    PubMed Central

    Wen, Junlin; Zhou, Shungui; Chen, Junhua

    2014-01-01

    Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms. PMID:24898751

  15. Cross-talk between receptors with intrinsic tyrosine kinase activity and alpha1b-adrenoceptors.

    PubMed Central

    del Carmen Medina, L; Vázquez-Prado, J; García-Sáinz, J A

    2000-01-01

    The effect of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on the phosphorylation and function of alpha(1b)-adrenoceptors transfected into Rat-1 fibroblasts was studied. EGF and PDGF increased the phosphorylation of these adrenoceptors. The effect of EGF was blocked by tyrphostin AG1478 and that of PDGF was blocked by tyrphostin AG1296, inhibitors of the intrinsic tyrosine kinase activities of the receptors for these growth factors. Wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the alpha(1b)-adrenoceptor phosphorylation induced by EGF but not that induced by PDGF. Inhibition of protein kinase C blocked the adrenoceptor phosphorylation induced by EGF and PDGF. The ability of noradrenaline to increase [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding in membrane preparations was used as an index of the functional coupling of the alpha(1b)-adrenoceptors and G-proteins. Noradrenaline-stimulated [(35)S]GTP[S] binding was markedly decreased in membranes from cells pretreated with EGF or PDGF. Our data indicate that: (i) activation of EGF and PDGF receptors induces phosphorylation of alpha(1b)-adrenoceptors, (ii) phosphatidylinositol 3-kinase is involved in the EGF response, but does not seem to play a major role in the action of PDGF, (iii) protein kinase C mediates this action of both growth factors and (iv) the phosphorylation of alpha(1b)-adrenoceptors induced by EGF and PDGF is associated with adrenoceptor desensitization. PMID:10947955

  16. Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

    NASA Astrophysics Data System (ADS)

    Wen, Junlin; Zhou, Shungui; Chen, Junhua

    2014-06-01

    Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3', 5, 5'-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms.

  17. RAF inhibitors that evade paradoxical MAPK pathway activation.

    PubMed

    Zhang, Chao; Spevak, Wayne; Zhang, Ying; Burton, Elizabeth A; Ma, Yan; Habets, Gaston; Zhang, Jiazhong; Lin, Jack; Ewing, Todd; Matusow, Bernice; Tsang, Garson; Marimuthu, Adhirai; Cho, Hanna; Wu, Guoxian; Wang, Weiru; Fong, Daniel; Nguyen, Hoa; Shi, Songyuan; Womack, Patrick; Nespi, Marika; Shellooe, Rafe; Carias, Heidi; Powell, Ben; Light, Emily; Sanftner, Laura; Walters, Jason; Tsai, James; West, Brian L; Visor, Gary; Rezaei, Hamid; Lin, Paul S; Nolop, Keith; Ibrahim, Prabha N; Hirth, Peter; Bollag, Gideon

    2015-10-22

    Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394. PMID:26466569

  18. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  19. Intrinsic HER4/4ICD transcriptional activation domains are required for STAT5A activated gene expression.

    PubMed

    Han, Wen; Sfondouris, Mary E; Semmes, Eleanor C; Meyer, Alicia M; Jones, Frank E

    2016-10-30

    The epidermal growth factor receptor family member HER4 undergoes proteolytic processing at the cell surface to release the HER4 intracellular domain (4ICD) nuclear protein. Interestingly, 4ICD directly interacts with STAT5 and functions as an obligate STAT5 nuclear chaperone. Once in the nucleus 4ICD binds with STAT5 at STAT5 target genes, dramatically potentiating STAT5 transcriptional activation. These observations raise the possibility that 4ICD directly coactivates STAT5 gene expression. Using both yeast and mammalian transactivation reporter assays, we performed truncations of 4ICD fused to a GAL4 DNA binding domain and identified two independent 4ICD transactivation domains located between residues 1022 and 1090 (TAD1) and 1192 and 1225 (TAD2). The ability of the 4ICD DNA binding domain fusions to transactivate reporter gene expression required deletion of the intrinsic tyrosine kinase domain. In addition, we identified the 4ICD carboxyl terminal TVV residues, a PDZ domain binding motif (PDZ-DBM), as a potent transcriptional repressor. The transactivation activity of the HER4 carboxyl terminal domain lacking the tyrosine kinase (CTD) was significantly lower than similar EGFR or HER2 CTD. However, deletion of the HER4 CTD PDZ-DBM enhanced HER4 CTD transactivation to levels equivalent to the EGFR and HER2 CTDs. To determine if 4ICD TAD1 and TAD2 have a physiologically relevant role in STAT5 transactivation, we coexpressed 4ICD or 4ICD lacking TAD2 or both TAD1 and TAD2 with STAT5 in a luciferase reporter assay. Our results demonstrate that each 4ICD TAD contributes additively to STAT5A transactivation and the ability of STAT5A to transactivate the β-casein promoter requires the 4ICD TADs. Taken together, published data and our current results demonstrate that both 4ICD nuclear chaperone and intrinsic coactivation activities are essential for STAT5 regulated gene expression. PMID:27502417

  20. Relation between the Intrinsic and Observed Central Engine Activity Time: Implications for Ultra-long GRBs

    NASA Astrophysics Data System (ADS)

    Gao, He; Mészáros, Peter

    2015-04-01

    The gamma-ray burst (GRB) central engine intrinsic activity time {{T}ce} is usually described through either the γ-ray duration T90 or through a generalized burst duration {{t}burst} that includes both the γ-ray emission and (when present) an extended flaring X-ray plateau. Here, we define a more specific operational description of {{T}ce}, and within the framework of the internal-external shock model, we develop a numerical code to study the relationship between T90 and {{T}ce}, as well as between {{t}burst} and {{T}ce}, for different initial conditions. We find that when {{T}ce}≲ {{10}4} s, late internal collisions or refreshed external collisions result in values of {{T}90} and {{t}burst} larger than {{T}ce}, usually by factors of 2-3. For {{T}ce}≳ {{10}4} s, the {{t}burst} is always a good estimator for {{T}ce}, while T90 can underpredict {{T}ce} when the late central engine activity is moderate. We find a clear bimodal distribution for {{T}ce}, based on our simulations as well as on the observational data for T90 and {{t}burst}. We suggest that {{t}burst} is a reliable measure for defining “ultra-long” GRBs. Bursts with T90 of order 103 s need not belong to a special population, while bursts with {{t}burst}\\gt {{10}4} s, where the late central engine activity is more moderate and shows up in X-rays, may represent a new population. These conclusions are insensitive to the initial conditions assumed in the models.

  1. Beta-adrenoceptor blockade and atrio-ventricular conduction in dogs. Role of intrinsic sympathomimetic activity.

    PubMed

    Giudicelli, J F; Lhoste, F

    1982-01-01

    1 Atrio-ventricular conduction and its modifications induced by six beta-adrenoceptor blocking agents and isoprenaline have been investigated in the anaesthetized dog using the extrastimulus technique and measuring atrial (AERP), nodal (NERP), global (GERP) effective refractory periods as well as global functional refractory period (GFRP). 2 When beta-adrenoceptor blockade was produced by (+/-)-propranolol (beta 1 + beta 2-adrenoceptor blockade) which is devoid of intrinsic sympathomimetic activity (ISA) but has membrane stabilizing effects (MSE), sotalol (beta 1 + beta 2-adrenoceptor blockade, no ISA, no MSE) and atenolol (beta 1-adrenoceptor blockade, no ISA, no MSE), all parameters were significantly increased. When beta-adrenoceptor blockade was achieved with pindolol (beta 1 + beta 2-adrenoceptor blockade) and practolol (beta 1-adrenoceptor blockade) which have ISA but no MSE, all parameters remained unchanged, as was also the case with (+)-propranolol, which has MSE but neither ISA nor beta-adrenolytic properties. 3 Isoprenaline at high doses significantly reduced the refractory periods but when infusion was stopped, marked but reversible conduction depression was observed. 4 It thus appears that beta-adrenoceptor blockade but not MSE is responsible for the onset of atrial and AV-conduction impairment and that ISA affords protection against this impairment. PMID:6125166

  2. Intrinsic predictive factors for ankle sprain in active university students: a prospective study.

    PubMed

    de Noronha, M; França, L C; Haupenthal, A; Nunes, G S

    2013-10-01

    The ankle is the joint most affected among the sports-related injuries. The current study investigated whether certain intrinsic factors could predict ankle sprains in active students. The 125 participants were submitted to a baseline assessment in a single session were then followed-up for 52 weeks regarding the occurrence of sprain. The baseline assessment were performed in both ankles and included the questionnaire Cumberland ankle instability tool - Portuguese, the foot lift test, dorsiflexion range of motion, Star Excursion Balance Test (SEBT), the side recognition task, body mass index, and history of previous sprain. Two groups were used for analysis: one with those who suffered an ankle sprain and the other with those who did not suffer an ankle sprain. After Cox regression analysis, participants with history of previous sprain were twice as likely to suffer subsequent sprains [hazard ratio (HR) 2.21 and 95% confidence interval (CI) 1.07-4.57] and people with better performance on the SEBT in the postero-lateral (PL) direction were less likely to suffer a sprain (HR 0.96 and 95% CI 0.92-0.99). History of previous sprain was the strongest predictive factor and a weak performance on SEBT PL was also considered a predictive factor for ankle sprains. PMID:22260485

  3. Enhanced efficacy (intrinsic activity) of cyclic opioid peptide analogs at the. mu. -receptor

    SciTech Connect

    Schiller, P.W.; Lemieux, C.; Nguyen, T.M.D.; Maziak, L.A.

    1986-05-01

    Side-chain to end group cyclized enkephalin analogs (e.g. H-Tyr-cyclo(-D-Lys-Gly-Phe-Leu-) and cyclic opioid peptide analogs obtained through covalent linkage of two side-chains (e.g. H-Tyr-D-Cys-Gly-Phe-Cys-NH/sub 2/ or H-Tyr-D-Lys-Gly-Phe-Glu-NH/sub 3/) were tested in the ..mu..-receptor-representative guinea pig ileum (GPI) bioassay and in a binding assay based on displacement of the ..mu..-ligand (/sup 3/H)DAGO from rat brain membranes. The cyclic analogs were 5 to 70 times more potent in the GPI assay than in the binding assay, whereas linear analogs showed equal potency in the two assays. These results suggest that the efficacy (intrinsic activity) of cyclic opioid peptide analogs at the ..mu..-receptor is increased as a consequence of the conformation constraint imposed through ring closure. This effect was most pronounced in analogs containing a long hydrophobic sidechain as part of the ring structure in the 2-position of the peptide sequence. Further experimental evidence ruled out the possibilities that these potency discrepancies may be due to differences in enzymatic degradation, dissimilar exposure of the receptors in their lipid environment or interaction with different receptor types in the two assay systems. It can be hypothesized that the semi-rigid cyclic analogs may induce a more productive conformational change in the receptor protein than the linear peptides.

  4. The transcriptional repressor Bcl6 controls the stability of regulatory T cells by intrinsic and extrinsic pathways.

    PubMed

    Sawant, Deepali V; Wu, Hao; Yao, Weiguo; Sehra, Sarita; Kaplan, Mark H; Dent, Alexander L

    2015-05-01

    Foxp3(+) regulatory T (Treg) cells are essential to maintain immune homeostasis, yet controversy exists about the stability of this cell population. Bcl6-deficient (Bcl6(-/-) ) mice develop severe and spontaneous T helper type 2 (Th2) inflammation and Bcl6-deficient Treg cells are ineffective at controlling Th2 responses. We used a lineage tracing approach to analyse the fate of Treg cells in these mice. In the periphery of Bcl6(-/-) mice, increased numbers of Foxp3-negative 'exTreg' cells were found, particularly in the CD25(+) population. ExTreg cells from Bcl6(-/-) mice expressed increased interleukin-17 (IL-17) and extremely elevated levels of Th2 cytokines compared with wild-type exTreg cells. Although Treg cells normally express only low levels of cytokines, Treg cells from Bcl6(-/-) mice secreted higher levels of IL-4, IL-5, IL-13 and IL-17 than wild-type conventional T cells. Next, Treg-specific conditional Bcl6-deficient (Bcl6(Foxp3-/-) ) mice were analysed. Bcl6(Foxp3-/-) mice do not develop inflammatory disease, indicating a requirement for non-Treg cells for inflammation in Bcl6(-/-) mice, and have normal numbers of exTreg cells. We induced Th2-type allergic airway inflammation in Bcl6(Foxp3-/-) mice, and found that while exTreg cytokine expression was normal, Bcl6-deficient Treg cells expressed higher levels of the Th2-specific regulator Gata3 than Bcl6(+) Treg cells. Bcl6(Foxp3-/-) mice had increased numbers of Th2 cells after induction of airway inflammation and increased T cells in the bronchoalveolar lavage fluid. These data show both Treg-intrinsic and Treg-extrinsic roles for Bcl6 in controlling Treg cell stability and Th2 inflammation, and support the idea that Bcl6 expression in Treg cells is critical for controlling Th2 responses. PMID:25262912

  5. Using ILP to Identify Pathway Activation Patterns in Systems Biology

    PubMed Central

    Neaves, Samuel R; Millard, Louise A C; Tsoka, Sophia

    2016-01-01

    We show a logical aggregation method that, combined with propositionalization methods, can construct novel structured biological features from gene expression data. We do this to gain understanding of pathway mechanisms, for instance, those associated with a particular disease. We illustrate this method on the task of distinguishing between two types of lung cancer; Squamous Cell Carcinoma (SCC) and Adenocarcinoma (AC). We identify pathway activation patterns in pathways previously implicated in the development of cancers. Our method identified a model with comparable predictive performance to the winning algorithm of a recent challenge, while providing biologically relevant explanations that may be useful to a biologist. PMID:27478883

  6. Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection.

    PubMed

    Oudhoff, Menno J; Antignano, Frann; Chenery, Alistair L; Burrows, Kyle; Redpath, Stephen A; Braam, Mitchell J; Perona-Wright, Georgia; Zaph, Colby

    2016-09-01

    The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7-/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection. PMID:27598373

  7. The light-activated signaling pathway in SCN-projecting rat retinal ganglion cells

    PubMed Central

    Warren, Erin J.; Allen, Charles N.; Brown, R. Lane; Robinson, David W.

    2008-01-01

    In mammals, the master circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus. The period and phase of the circadian pacemaker are calibrated by direct photic input from retinal ganglion cells (RGCs). SCN-projecting RGCs respond to light in the absence of rod- and cone-driven synaptic input, a property for which they are termed intrinsically photosensitive. In SCN-projecting RGCs, light activates a nonselective cationic current that displays inward and outward rectification. The goal of the present study was to investigate the identity of the light-activated ion channel and the intracellular signaling pathway leading to its activation. We considered two candidate channels, cyclic nucleotide-gated (CNG) channels and transient receptor potential (TRP) channels, which mediate vertebrate and invertebrate phototransduction, respectively. We report that the intrinsic light response relies upon a G-protein-dependent process. Although our data indicate that cyclic nucleotides modulate the signaling pathway, CNG channels do not appear to conduct the light-activated current because (i) cyclic nucleotides in the pipette solution do not activate a conductance or completely block the light response, (ii) CNG channel blockers fail to inhibit the light response, (iii) the effects of internal and external divalent cations are inconsistent with their effects on CNG channels, and (iv) immunohistochemistry reveals no CNG channels in SCN-projecting RGCs. Finally, we show that the pharmacology of the light-activated channel resembles that of some TRPC channel family members; the response is blocked by lanthanides and ruthenium red and SK&F 96365, and is enhanced by flufenamic acid and 1-oleoyl-2-acetyl-sn-glycerol. Furthermore, immunohistochemical experiments reveal that TRPC6 is expressed in many RGCs, including those that express melanopsin. PMID:16706854

  8. Different pathways of macrophage activation and polarization.

    PubMed

    Juhas, Ulana; Ryba-Stanisławowska, Monika; Szargiej, Patryk; Myśliwska, Jolanta

    2015-01-01

    Monocytes are short-lived cells and undergo spontaneous apoptosis every day. Inflammatory responses may induce dramatic up-regulation of monocyte survival and differentiation. When monocytes are recruited to an area of infection they may differentiate into macrophages. In different microenvironments macrophages polarize into two types. The M1 or classically activated macrophages are characterized by the high ability to produce pro-inflammatory cytokines and the production of NO through the induced synthesis of iNOS. The M2 or alternatively activated macrophages are divided into 3 subtypes, M2 a, b and c, and they have anti-inflammatory properties. Mediators of M1 macrophage TLR-dependent polarization include transcription factors such as NF-κB, AP-1, PU.1, CCAAT/enhancer-binding protein α (C/EBP-α), STAT1 as well as interferon regulatory factor 5 (IRF5), while the transcription factors which promote M2 activation include IRF4, C/EBP-β, Krüppel-like factor 4 (KLF4), STAT6 and PPARγ receptor. PMID:25983288

  9. Contributions of contact activation pathways of coagulation factor XII in plasma.

    PubMed

    Chatterjee, Kaushik; Guo, Zhe; Vogler, Erwin A; Siedlecki, Christopher A

    2009-07-01

    Activation of human blood plasma coagulation by contact with hydrophilic or hydrophobic surfaces (procoagulants) is dominated by kallikrein (Kal)-mediated activation of the blood zymogen FXII (Hageman Factor). Mathematical modeling of prekallikrein (PK)-deficient platelet-poor plasma (d(PK)PPP) and PK-reconstituted d(PK)PPP (Rd(PK)PPP) coagulation shows that autoactivation of FXII (FXII-->[surface]FXII) produces no more than about 25% of the total FXIIa produced by the intrinsic pathway. Autoactivation and reciprocal-activation increase in the same proportion with procoagulant surface energy (water-wettability), whereas total amount of FXIIa produced per-unit-area procoagulant remains roughly constant for any particular procoagulant. These results suggest that procoagulant surfaces initiate the intrinsic cascade by producing a bolus of FXIIa in proportion to surface energy or surface area but play no additional role in subsequent molecular events in the cascade. Results further suggest that reciprocal-activation occurs in proportion to the amount of FXIIa produced by the initiating autoactivation step. PMID:18481791

  10. Wnt pathway activation by ADP-ribosylation

    PubMed Central

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P.; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S.; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)—known to target Axin for proteolysis—regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  11. Wnt pathway activation by ADP-ribosylation.

    PubMed

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  12. Abnormal intrinsic brain activity patterns in leukoaraiosis with and without cognitive impairment.

    PubMed

    Li, Chuanming; Yang, Jun; Yin, Xuntao; Liu, Chen; Zhang, Lin; Zhang, Xiaochun; Gui, Li; Wang, Jian

    2015-10-01

    The amplitude of low frequency fluctuations (ALFF) from resting-state functional MRI (rs-fMRI) signals can be used to detect intrinsic spontaneous brain activity and provide valuable insights into the pathomechanism of neural disease. In this study, we recruited 56 patients who had been diagnosed as having mild to severe leukoaraiosis. According to the neuropsychological tests, they were subdivided into a leukoaraiosis with cognitive impairment group (n = 28) and a leukoaraiosis without cognitive impairment group (n = 28). 28 volunteers were included as normal controls. We found that the three groups showed significant differences in ALFF in the brain regions of the right inferior occipital gyrus (IOG_R), left middle temporal gyrus (MTG_L), left precuneus (Pcu_L), right superior frontal gyrus (SFG_R) and right superior occipital gyrus (SOG_R). Compared with normal controls, the leukoaraiosis without cognitive impairment group exhibited significantly increased ALFF in the IOG_R, Pcu_L, SFG_R and SOG_R. While compared with leukoaraiosis without cognitive impairment group, the leukoaraiosis with cognitive impairment group showed significantly decreased ALFF in IOG_R, MTG_L, Pcu_L and SOG_R. A close negative correlation was found between the ALFF values of the MTG_L and the Montreal Cognitive Assessment (MoCA) scores. Our data demonstrate that white matter integrity and cognitive impairment are associated with different amplitude fluctuations of rs-fMRI signals. Leukoaraiosis is related to ALFF increases in IOG_R, Pcu_L, SFG_Orb_R and SOG_R. Decreased ALFF in MTG_L is characteristic of cognitive impairment and may aid in its early detection. PMID:26116811

  13. Tcf1 and Lef1 transcription factors establish CD8(+) T cell identity through intrinsic HDAC activity.

    PubMed

    Xing, Shaojun; Li, Fengyin; Zeng, Zhouhao; Zhao, Yunjie; Yu, Shuyang; Shan, Qiang; Li, Yalan; Phillips, Farrah C; Maina, Peterson K; Qi, Hank H; Liu, Chengyu; Zhu, Jun; Pope, R Marshall; Musselman, Catherine A; Zeng, Chen; Peng, Weiqun; Xue, Hai-Hui

    2016-06-01

    The CD4(+) and CD8(+) T cell dichotomy is essential for effective cellular immunity. How individual T cell identity is established remains poorly understood. Here we show that the high-mobility group (HMG) transcription factors Tcf1 and Lef1 are essential for repressing CD4(+) lineage-associated genes including Cd4, Foxp3 and Rorc in CD8(+) T cells. Tcf1- and Lef1-deficient CD8(+) T cells exhibit histone hyperacetylation, which can be ascribed to intrinsic histone deacetylase (HDAC) activity in Tcf1 and Lef1. Mutation of five conserved amino acids in the Tcf1 HDAC domain diminishes HDAC activity and the ability to suppress CD4(+) lineage genes in CD8(+) T cells. These findings reveal that sequence-specific transcription factors can utilize intrinsic HDAC activity to guard cell identity by repressing lineage-inappropriate genes. PMID:27111144

  14. Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma.

    PubMed

    Syed, Deeba N; Lall, Rahul K; Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

    2014-12-01

    The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytotoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up-regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMP-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity. PMID:25016296

  15. Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma

    PubMed Central

    Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

    2014-01-01

    The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up- regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16 days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMPK-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity. PMID:25016296

  16. Intrinsic resistance to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer with activating EGFR mutations

    PubMed Central

    Wang, Jun; Wang, Baocheng; Chu, Huili; Yao, Yunfeng

    2016-01-01

    Identifying activating EGFR mutations is a useful predictive strategy that helps select a population of advanced non-small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors (TKIs). Patients with sensitizing EGFR mutations (predominantly an in-frame deletion in exon 19 and an L858R substitution) are highly responsive to first-generation EGFR TKIs, such as gefitinib and erlotinib, and show improved progression-free survival without serious side effects. However, all patients with activating EGFR mutations who are initially responsive to EGFR TKIs eventually develop acquired resistance after a median progression-free survival of 10–16 months, followed by disease progression. Moreover, ~20%–30% of NSCLC patients have no objective tumor regression on initial EGFR TKI treatment, although they harbor an activating EGFR mutation. These patients represent an NSCLC subgroup that is defined as having intrinsic or primary resistance to EGFR TKIs. Different mechanisms of acquired EGFR TKI resistance have been identified, and several novel compounds have been developed to reverse acquired resistance, but little is known about EGFR TKI intrinsic resistance. In this review, we summarize the latest findings involving mechanisms of intrinsic resistance to EGFR TKIs in advanced NSCLC with activating EGFR mutations and present possible therapeutic strategies to overcome this resistance. PMID:27382309

  17. Pathogen-Secreted Proteases Activate a Novel Plant Immune Pathway

    PubMed Central

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z.; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J.; Sheen, Jen; Ausubel, Frederick M.

    2015-01-01

    Mitogen-Activated Protein Kinase (MAPK) cascades play central roles in innate immune signaling networks in plants and animals1,2. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive1. We report that pathogen-secreted proteases activate a previously unknown signaling pathway in Arabidopsis thaliana involving the Gα, Gβ and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of a MAPK cascade. In this pathway, Receptor for Activated C Kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G protein signaling to downstream activation of a MAPK cascade. The protease-G protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signaling pathways such as the one elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to a MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the novel protease-mediated immune signaling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel types of immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems. PMID:25731164

  18. Involvement of PI3K/Akt, ERK and p38 signaling pathways in emodin-mediated extrinsic and intrinsic human hepatoblastoma cell apoptosis.

    PubMed

    Cui, Yuting; Lu, Peiran; Song, Ge; Liu, Qian; Zhu, Di; Liu, Xuebo

    2016-06-01

    As a natural anthraquinone derivative, 1,3,8-trihydroxy-6-methylanthraquinone, known as emodin, has recently been reported to possess potential chemopreventive capacity, but the underlying molecular mechanism of its hepatocyte toxicity remains poorly clarified. The present research indicated that emodin targeted HepG2 cells without being cytotoxic to primary human hepatocyte cells in comparison with chrysophanol and rhein. The anti-proliferative effect of emodin was ascribed to occurrence of apoptosis, which characterized by higher ethidium bromide signal, brighter DAPI fluorescence, cleavages of procaspase-3 and poly (ADP-ribose) polymerase as well as quantitative result from Annexin V-FITC/PI double staining. Furthermore, emodin improved Bax/Bcl-2 ratio, elicited disruption of mitochondrial membrane potential and promoted efflux of cytochrome c to cytosol, indicative of features of mitochondria-dependent apoptotic signals. Emodin concurrently led to activations of Fas, Fas-L, caspase-8 and tBid, which provoked death receptor apoptotic signals. Notably, activated tBid relayed the Fas apoptotic signal to the mitochondrial pathway. Besides, emodin effectively attenuated phosphorylations of Akt and ERK and promoted phosphorylation of p38. Inhibitions of PI3K/Akt and ERK and activation of p38 mediated emodin-induced apoptosis through modulating the mitochondrial pathway and/or death receptor pathway. Additionally, there was a cross-talk between PI3K/Akt and MAPKs pathways in emodin-induced apoptosis. PMID:27032576

  19. Licochalcone B induces apoptosis of human oral squamous cell carcinoma through the extrinsic- and intrinsic-signaling pathways.

    PubMed

    Oh, Hana; Yoon, Goo; Shin, Jae-Cheon; Park, Seon-Min; Cho, Seung-Sik; Cho, Jin Hyoung; Lee, Mee-Hyun; Liu, Kangdong; Cho, Young Sik; Chae, Jung-Il; Shim, Jung-Hyun

    2016-04-01

    Licochalcone B (Lico B), which belongs to the retrochalcone family, is isolated from the roots of Chinese licorice. Lico B has been reported to have several other useful pharmacological properties, such as anti-inflammatory, antibacterial, antioxidant, antiulcer, anticancer, and anti-metastasis activities. We elucidated the underlying mechanism by which Lico B can induce apoptosis in oral squamous cell carcinoma (OSCC). Our results showed that exposure of OSCC cells (HN22 and HSC4) to Lico B significantly inhibited cell proliferation in a time- and concentration-dependent manner. Lico B caused cell cycle arrest at G1 phase along with downregulation of cyclin D1 and upregulation of p21 and p27 proteins. Lico B also facilitated the diffusion of phospholipid phosphatidylserine (PS) from inner to outer leaflets of the plasma membrane with chromatin condensation, DNA fragmentation, accumulated sub-G1 population in a concentration-dependent manner. Moreover, Lico B promoted the generation of reactive oxygen species (ROS), which, in turn, can induce CHOP, death receptor (DR) 4 and DR5. Lico B treatment induced downregulation of anti-apoptotic proteins (Bid and Bcl-xl and Mcl-1), and up-regulation of pro-apoptotic protein (Bax). Lico B also led to the loss of mitochondrial membrane potential (MMP), resulting in cytochrome c release. As can be expected from the above results, the apoptotic protease activating factor-1 (Apaf-1) and survivin were oppositely expressed in favor of apoptotic cell death. This notion was supported by the fact that Lico B activated multi-caspases with cleavage of poly (ADP-ribose) polymerase (PARP) protein. Therefore, it is suggested that Lico B is a promising drug for the treatment of human oral cancer via the induction of apoptotic cell death. PMID:26847145

  20. Intrinsic disc emission and the soft X-ray excess in active galactic nuclei

    NASA Astrophysics Data System (ADS)

    Done, Chris; Davis, S. W.; Jin, C.; Blaes, O.; Ward, M.

    2012-03-01

    Narrow-line Seyfert 1 (NLS1) galaxies have low-mass black holes and mass accretion rates close to (or exceeding) Eddington, so a standard blackbody accretion disc should peak in the extreme ultraviolet. However, the lack of true absorption opacity in the disc means that the emission is better approximated by a colour temperature corrected blackbody, and this colour temperature correction is large enough (˜2.4) that the bare disc emission from a zero spin black hole can extend into the soft X-ray bandpass. Part of the soft X-ray excess seen in these objects must be intrinsic emission from the disc unless the vertical structure is very different to that predicted. None the less, this is not the whole story even for the extreme NLS1 as the shape of the soft excess is much broader than predicted by a bare disc spectrum, indicating some Compton upscattering by warm, optically thick material. We associate this with the disc itself, so it must ultimately be powered by mass accretion. We build an energetically self-consistent model assuming that the emission thermalizes to a (colour temperature corrected) blackbody only at large radii. At smaller radii the gravitational energy is split between powering optically thick Comptonized disc emission (forming the soft X-ray excess) and an optically thin corona above the disc (forming the tail to higher energies). We show examples of this model fit to the extreme NLS1 RE J1034+396, and to the much lower Eddington fraction broad-line Seyfert 1 PG 1048+231. We use these to guide our fits and interpretations of three template spectra made from co-adding multiple sources to track out a sequence of active galactic nucleus (AGN) spectra as a function of L/LEdd. Both the individual objects and template spectra show the surprising result that the Compton upscattered soft X-ray excess decreases in importance with increasing L/LEdd. The strongest soft excesses are associated with low mass accretion rate AGN rather than being tied to some

  1. Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

    PubMed Central

    Mintz, C S; Schultz, D R; Arnold, P I; Johnson, W

    1992-01-01

    Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes. PMID:1612744

  2. Machine Learning Classification of Cirrhotic Patients with and without Minimal Hepatic Encephalopathy Based on Regional Homogeneity of Intrinsic Brain Activity

    PubMed Central

    Liu, Jun; Sun, Tao; Shen, Qun-Tai

    2016-01-01

    Machine learning-based approaches play an important role in examining functional magnetic resonance imaging (fMRI) data in a multivariate manner and extracting features predictive of group membership. This study was performed to assess the potential for measuring brain intrinsic activity to identify minimal hepatic encephalopathy (MHE) in cirrhotic patients, using the support vector machine (SVM) method. Resting-state fMRI data were acquired in 16 cirrhotic patients with MHE and 19 cirrhotic patients without MHE. The regional homogeneity (ReHo) method was used to investigate the local synchrony of intrinsic brain activity. Psychometric Hepatic Encephalopathy Score (PHES) was used to define MHE condition. SVM-classifier was then applied using leave-one-out cross-validation, to determine the discriminative ReHo-map for MHE. The discrimination map highlights a set of regions, including the prefrontal cortex, anterior cingulate cortex, anterior insular cortex, inferior parietal lobule, precentral and postcentral gyri, superior and medial temporal cortices, and middle and inferior occipital gyri. The optimized discriminative model showed total accuracy of 82.9% and sensitivity of 81.3%. Our results suggested that a combination of the SVM approach and brain intrinsic activity measurement could be helpful for detection of MHE in cirrhotic patients. PMID:26978777

  3. Endosulfan activates the extrinsic coagulation pathway by inducing endothelial cell injury in rats.

    PubMed

    Zhang, Lianshuang; Wei, Jialiu; Guo, Fangzi; Duan, Junchao; Li, Yanbo; Shi, Zhixiong; Yang, Yumei; Zhou, Xianqing; Sun, Zhiwei

    2015-10-01

    Endosulfan, a persistent organic pollutant, is widely used in agriculture as a pesticide. The aim of the present study was to evaluate the blood toxicity of different doses of endosulfan in Wistar rats. The experimental sample was composed of four groups, a control group that did not receive endosulfan and three endosulfan-exposed groups that respectively received 1, 5, or 10 mg/kg/day (doses below LD50), of endosulfan for 21 days. The results showed that endosulfan significantly decreased the prothrombin time (PT) and upregulated the activated coagulation factors VIIa, Xa, and XIIIa; thrombin-antithrombin complex (TAT); and P-selectin. Plasma levels of tissue factor (TF) and malondialdehyde (MDA) were increased in the endosulfan groups. The activated partial thromboplastin time (APTT) and the level of activated coagulation factor IXa showed no obvious changes. Immunohistochemical results showed increased expression of von Willebrand factor (vWF) and the inflammatory cytokine interleukin (IL)-1β in the groups exposed to endosulfan. The pathology and electron microscopy results showed impaired vascular tissue accompanied by the exfoliation of endothelial cells and mitochondrial damage in the endosulfan-exposed groups. In summary, our results suggest that endosulfan damages endothelial cells via oxidative stress and the inflammatory response, leading to the release of TF and vWF into the blood. The TF and vWF in the blood may activate extrinsic coagulation factors and platelets, thus triggering the extrinsic coagulation pathway. There were no obvious effects on the intrinsic coagulation pathway. PMID:26028348

  4. How intrinsic sympathomimetic activity modulates the haemodynamic responses to β-adrenoceptor antagonists

    PubMed Central

    Veld, A. J. Man in 'T; Schalekamp, M. A. D. H.

    1982-01-01

    1 A survey has been made of the literature on acute and long-term haemodynamic effects of ten different β-adrenoceptor antagonists. The β-adrenoceptor blockers are: pindolol, practolol, alprenolol, oxprenolol, acebutolol, penbutolol, metoprolol, atenolol, propranolol and timolol. The total numbers of patients included in this review are 396 patients in 41 acute studies and 410 patients in 36 long-term studies. 2 The effects of β-adrenoceptor blockers on the concentrations of plasma noradrenaline have also been reviewed. Ten studies including 110 patients on non-ISA-β-adrenoceptor blockers and eight studies including 116 patients on pindolol are presented. 3 In the acute studies (i.e. 15-90 min) arterial pressure was lowered by 1-7% and in the long-term studies (i.e. 3 days-5 years) by 6-17%. 4 The degree of cardio-depression induced by the various β-adrenoceptor blockers was inversely correlated with their pharmacologically defined quantity of intrinsic sympathomimetic activity (ISA) both in acute and in long-term studies. 5 In the acute studies the increments in peripheral vascular resistance were directly correlated with the degree of cardio-depression. This suggests that a fall in arterial pressure immediately after administration of a β-adrenoceptor blocker is prevented by increased vasoconstrictor nerve activity mediated through the arterial baroreflex. 6 The compensatory response of vascular resistance to cardio-depression was similar for β1-selective and non-selective blockers, thereby indicating that extra-junctional vascular β-receptors are relatively unimportant for maintaining basal vascular tone. 7 In the long-term studies the correlation between changes in cardiac output and changes in vascular resistance was shifted to a lower level of vascular resistance. This means that the onset of blood pressure reduction during β-adrenoceptor blockade was associated with a fall in vascular resistance at any level of cardiac output. Thus vascular

  5. Ligand-induced evolution of intrinsic fluorescence and catalytic activity from cobalt ferrite nanoparticles.

    PubMed

    Pal, Monalisa; Kundu, Anirban; Rakshit, Rupali; Mandal, Kalyan

    2015-06-01

    To develop CoFe(2)O(4) as magneto-fluorescent nanoparticles (NPs) for biomedical applications, it would be advantageous to identify any intrinsic fluorescence of this important magnetic material by simply adjusting the surface chemistry of the NPs themselves. Herein, we demonstrate that intrinsic multicolor fluorescence, covering the whole visible region, can be induced by facile functionalization of CoFe(2)O(4) NPs with Na-tartrate. Moreover, the functionalized CoFe(2)O(4) NPs also show unprecedented catalytic efficiency in the degradation of both biologically and environmentally harmful dyes, pioneering the potential application of these NPs in therapeutics and wastewater treatment. Detailed investigation through various spectroscopic tools unveils the story behind the emergence of this unique optical property of CoFe(2)O(4) NPs upon functionalization with tartrate ligands. We believe our developed multifunctional CoFe(2)O(4) NPs hold great promise for advanced biomedical and technological applications. PMID:25867626

  6. Long non-coding RNA ANRIL is up-regulated in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic pathway.

    PubMed

    Zhu, Hongxue; Li, Xuechao; Song, Yarong; Zhang, Peng; Xiao, Yajun; Xing, Yifei

    2015-11-13

    Antisense non-coding RNA in the INK4 locus (ANRIL) is a member of long non-coding RNAs and has been reported to be dysregulated in several human cancers. However, the role of ANRIL in bladder cancer remains unclear. This present study aimed to investigate whether and how ANRIL involved in bladder cancer. Our results showed up-regulation of ANRIL in bladder cancer tissues versus the corresponding adjacent non-tumor tissues. To explore the specific mechanisms, ANRIL was silenced by small interfering RNA or short hairpin RNA transfection in human bladder cancer T24 and EJ cells. Knockdown of ANRIL repressed cell proliferation and increased cell apoptosis, along with decreased expression of Bcl-2 and increased expressions of Bax, cytoplasmic cytochrome c and Smac and cleaved caspase-9, caspase-3 and PARP. However, no change of cleaved caspase-8 level was observed. Furthermore, in vivo experiment confirmed that knockdown of ANRIL inhibited tumorigenic ability of EJ cells in nude mice. Meanwhile, in accordance with in vitro study, knockdown of ANRIL inhibited expression of Bcl-2 and up-regulated expressions of Bax and cleaved caspase-9, but did not affect cleaved caspase-8 level. In conclusion, we first report that ANRIL possibly serves as an oncogene in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic apoptosis pathway. PMID:26449463

  7. An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway

    PubMed Central

    Robertson, Kevin A.; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J.; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J.; Enright, Anton J.; Yamamoto, Mami; Pradeepa, Madapura M.; Lennox, Kimberly A.; Behlke, Mark A.; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J.; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

    2016-01-01

    In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway. PMID:26938778

  8. Assessment of Effective Ankle Joint Positioning in Strength Training for Intrinsic Foot Flexor Muscles: A Comparison of Intrinsic Foot Flexor Muscle Activity in a Position Intermediate to Plantar and Dorsiflexion with that in Maximum Plantar Flexion Using Needle Electromyography

    PubMed Central

    Hashimoto, Takayuki; Sakuraba, Keishoku

    2014-01-01

    [Purpose] The effectiveness of intrinsic foot flexor strength training performed in the plantar flexion position was examined using needle electromyography. [Subjects] The subjects of this study were 18 healthy men. [Methods] We used needle electromyography to measure the muscle activities of the flexor hallucis brevis (FHB), and the flexor digitorum brevis (FDB) in maximum plantar and an intermediate position. [Results] Significant increases in muscle activities were observed for both FHB and FDB, and the rates of increase from the intermediate position to the plantar flexion position were 43% for FHB and 46% for FDB. [Conclusion] This study demonstrated that it is possible to evaluate intrinsic foot flexors, in addition to the numerous reports on treatment methods focusing on extrinsic foot flexors. Furthermore, the results suggest that toe flexion exercises performed during plantar flexion of the ankle joint are an effective method for intrinsic foot flexor strength training. PMID:24707106

  9. Molecular pathways: mitogen-activated protein kinase pathway mutations and drug resistance.

    PubMed

    Pritchard, Antonia L; Hayward, Nicholas K

    2013-05-01

    Receptor tyrosine kinases are a diverse family of transmembrane proteins that can activate multiple pathways upon ligation of the receptor, one of which is the series of mitogen-activated protein kinase (MAPK) signaling cascades. The MAPK pathways play critical roles in a wide variety of cancer types, from hematologic malignancies to solid tumors. Aberrations include altered expression levels and activation states of pathway components, which can sometimes be attributable to mutations in individual members. The V600E mutation of BRAF was initially described in 2002 and has been found at particularly high frequency in melanoma and certain subtypes of colorectal cancer. In the relatively short time since this discovery, a family of drugs has been developed that specifically target this mutated BRAF isoform, which, after results from phase I/II and III clinical trials, was granted U.S. Food and Drug Administration approval in August 2011. Although these drugs produce clinically meaningful increases in progression-free and overall survival, due to acquired resistance they have not improved mortality rates. New drugs targeting other members of the MAPK pathways are in clinical trials or advanced stages of development. It is hoped that combination therapies of these new drugs in conjunction with BRAF inhibitors will counteract the mechanisms of resistance and provide cures. The clinical implementation of next-generation sequencing is leading to a greater understanding of the genetic architecture of tumors, along with acquired mechanisms of drug resistance, which will guide the development of tumor-specific inhibitors and combination therapies in the future. PMID:23406774

  10. Key gravity-sensitive signaling pathways drive T cell activation.

    PubMed

    Boonyaratanakornkit, J B; Cogoli, A; Li, C-F; Schopper, T; Pippia, P; Galleri, G; Meloni, M A; Hughes-Fulford, M

    2005-12-01

    Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity. PMID:16210397

  11. Novel tungsten carbide nanorods: an intrinsic peroxidase mimetic with high activity and stability in aqueous and organic solvents.

    PubMed

    Li, Nan; Yan, Ya; Xia, Bao-Yu; Wang, Jing-Yuan; Wang, Xin

    2014-04-15

    Tungsten carbide nanorods (WC NRs) are demonstrated for the first time to possess intrinsic peroxidase-like activity towards typical peroxidase substrates, such as 3, 3', 5, 5'-tetramethylbenzidine (TMB) and ο-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2). The reactions catalyzed by these nanorods follow the Michaelis-Menten kinetics. The excellent catalytic performance of WC NRs could be attributed to their intrinsic catalytic activity to efficiently accelerate the electron-transfer process and facilitate the decomposition of H2O2 to generate more numbers of reactive oxygen species (ROS). Based upon the strong peroxidase-like activity of these WC NRs, a colorimetric sensor for H2O2 is designed, which provides good response towards H2O2 concentration over a range of 2×10(-7)-8×10(-5) M with a detection limit of 60 nM. Moreover, the peroxidase-like activities of WC NRs with TMB as the substrate are investigated in both protic and aprotic organic media, showing different colorimetric reactions from that performed in aqueous solutions. In comparison with the natural horse radish peroxidase, WC NR exhibits excellent robustness of catalytic activity and considerable reusability, thus making it a promising mimic of peroxidase catalysts. PMID:24325981

  12. The Cryptococcus neoformans Alkaline Response Pathway: Identification of a Novel Rim Pathway Activator

    PubMed Central

    Ost, Kyla S.; O’Meara, Teresa R.; Huda, Naureen; Esher, Shannon K.; Alspaugh, J. Andrew

    2015-01-01

    The Rim101/PacC transcription factor acts in a fungal-specific signaling pathway responsible for sensing extracellular pH signals. First characterized in ascomycete fungi such as Aspergillus nidulans and Saccharomyces cerevisiae, the Rim/Pal pathway maintains conserved features among very distantly related fungi, where it coordinates cellular adaptation to alkaline pH signals and micronutrient deprivation. However, it also directs species-specific functions in fungal pathogens such as Cryptococcus neoformans, where it controls surface capsule expression. Moreover, disruption of the Rim pathway central transcription factor, Rim101, results in a strain that causes a hyper-inflammatory response in animal infection models. Using targeted gene deletions, we demonstrate that several genes encoding components of the classical Rim/Pal pathway are present in the C. neoformans genome. Many of these genes are in fact required for Rim101 activation, including members of the ESCRT complex (Vps23 and Snf7), ESCRT-interacting proteins (Rim20 and Rim23), and the predicted Rim13 protease. We demonstrate that in neutral/alkaline pH, Rim23 is recruited to punctate regions on the plasma membrane. This change in Rim23 localization requires upstream ESCRT complex components but does not require other Rim101 proteolysis components, such as Rim20 or Rim13. Using a forward genetics screen, we identified the RRA1 gene encoding a novel membrane protein that is also required for Rim101 protein activation and, like the ESCRT complex, is functionally upstream of Rim23-membrane localization. Homologs of RRA1 are present in other Cryptococcus species as well as other basidiomycetes, but closely related genes are not present in ascomycetes. These findings suggest that major branches of the fungal Kingdom developed different mechanisms to sense and respond to very elemental extracellular signals such as changing pH levels. PMID:25859664

  13. Altered intrinsic properties and bursting activities of neurons in layer IV of somatosensory cortex from Fmr-1 knockout mice.

    PubMed

    Zhang, Linming; Liang, Zhanrong; Zhu, Pingping; Li, Meng; Yi, Yong-Hong; Liao, Wei-Ping; Su, Tao

    2016-06-01

    Neuroadaptations and alterations in neuronal excitability are critical in brain maturation and many neurological diseases. Fragile X syndrome (FXS) is a pervasive neurodevelopmental disorder characterized by extensive synaptic and circuit dysfunction. It is still unclear about the alterations in intrinsic excitability of individual neurons and their link to hyperexcitable circuitry. In this study, whole cell patch-clamp recordings were employed to characterize the membrane and firing properties of layer IV cells in slices of the somatosensory cortex of Fmr-1 knockout (KO) mice. These cells generally exhibited a regular spiking (RS) pattern, while there were significant increases in the number of cells that adopted intrinsic bursting (IB) compared with age-matched wild type (WT) cells. The cells subgrouped according to their firing patterns and maturation differed significantly in membrane and discharge properties between KO and WT. The changes in the intrinsic properties were consistent with highly facilitated discharges in KO cells induced by current injection. Spontaneous activities of RS neurons driven by local network were also increased in the KO cells, especially in neonate groups. Under an epileptiform condition mimicked by omission of Mg(2+) in extracellular solution, these RS neurons from KO mice were more likely to switch to burst discharges. Analysis on bursts revealed that the KO cells tended to form burst discharges and even severe events manifested as seizure-like ictal discharges. These results suggest that alterations in intrinsic properties in individual neurons are involved in the abnormal excitability of cortical circuitry and possibly account for the pathogenesis of epilepsy in FXS. PMID:27048919

  14. Sucrose activates human taste pathways differently from artificial sweetener.

    PubMed

    Frank, Guido K W; Oberndorfer, Tyson A; Simmons, Alan N; Paulus, Martin P; Fudge, Julie L; Yang, Tony T; Kaye, Walter H

    2008-02-15

    Animal models suggest that sucrose activates taste afferents differently than non-caloric sweeteners. Little information exists how artificial sweeteners engage central taste pathways in the human brain. We assessed sucrose and sucralose taste pleasantness across a concentration gradient in 12 healthy control women and applied 10% sucrose and matched sucralose during functional magnet resonance imaging. The results indicate that (1) both sucrose and sucralose activate functionally connected primary taste pathways; (2) taste pleasantness predicts left insula response; (3) sucrose elicits a stronger brain response in the anterior insula, frontal operculum, striatum and anterior cingulate, compared to sucralose; (4) only sucrose, but not sucralose, stimulation engages dopaminergic midbrain areas in relation to the behavioral pleasantness response. Thus, brain response distinguishes the caloric from the non-caloric sweetener, although the conscious mind could not. This could have important implications on how effective artificial sweeteners are in their ability to substitute sugar intake. PMID:18096409

  15. Intrinsic activity and poisoning rate for HCOOH oxidation at Pt(100) and vicinal surfaces containing monoatomic (111) steps.

    PubMed

    Grozovski, Vitali; Climent, Víctor; Herrero, Enrique; Feliu, Juan M

    2009-08-01

    Pulsed voltammetry is used to study formic acid oxidation on Pt(2n-1,1,1) surfaces and determine the effects of the size of the (100) terrace and the (111) step density on the reaction mechanism. The intrinsic activity of the electrode through the active intermediate reaction path (j(theta=) (0)), as well as the rate constant for the CO formation (k(ads)), are calculated from the current transients obtained at different potentials. For surfaces with wide terraces, j(theta=) (0) and k(ads) are almost insensitive to the step density, which suggests that step and terrace sites have a similar activity for this reaction. For narrow terraces (n<6), the intrinsic activity diminishes. The dependence of the reaction rates on the electrode potential is also elucidated. The CO formation only takes place in a narrow potential window, very close to the potential of zero total charge, while the direct oxidation takes place even when the surface is covered by anions. The different behavior for both reactions suggests that the adsorption mode of formic acid is different for each path. PMID:19569091

  16. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

    PubMed Central

    Tucker, Erik I.; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D.; Gailani, Dave; Gruber, András; McCarty, Owen J. T.

    2015-01-01

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  17. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor.

    PubMed

    Puy, Cristina; Tucker, Erik I; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D; Gailani, Dave; Gruber, András; McCarty, Owen J T

    2015-02-26

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  18. Increased Intrinsic Activity of Medial-Temporal Lobe Subregions is Associated with Decreased Cortical Thickness of Medial-Parietal Areas in Patients with Alzheimer's Disease Dementia.

    PubMed

    Pasquini, Lorenzo; Scherr, Martin; Tahmasian, Masoud; Myers, Nicholas E; Ortner, Marion; Kurz, Alexander; Förstl, Hans; Zimmer, Claus; Grimmer, Timo; Akhrif, Atae; Wohlschläger, Afra M; Riedl, Valentin; Sorg, Christian

    2016-01-21

    In Alzheimer's disease (AD), disrupted connectivity between medial-parietal cortices and medial-temporal lobes (MTL) is linked with increased MTL local functional connectivity, and parietal atrophy is associated with increased MTL memory activation. We hypothesized that intrinsic activity in MTL subregions is increased and associated with medial-parietal degeneration and impaired memory in AD. To test this hypothesis, resting-state-functional and structural-MRI was assessed in 22 healthy controls, 22 mild cognitive impairment patients, and 21 AD-dementia patients. Intrinsic activity was measured by power-spectrum density of blood-oxygenation-level-dependent signal, medial-parietal degeneration by cortical thinning. In AD-dementia patients, intrinsic activity was increased for several right MTL subregions. Raised intrinsic activity in dentate gyrus and cornu ammonis 1 was associated with cortical thinning in posterior cingulate cortices, and at-trend with impaired delayed recall. Critically, increased intrinsic activity in the right entorhinal cortex was associated with ipsilateral posterior cingulate degeneration. Our results provide evidence that in AD, intrinsic activity in MTL subregions is increased and associated with medial-parietal atrophy. Results fit a model in which medial-parietal degeneration contributes to MTL dysconnectivity from medial-parietal cortices, potentially underpinning disinhibition-like changes in MTL activity. PMID:26836175

  19. Structural studies of human Naked2: A biologically active intrinsically unstructured protein

    SciTech Connect

    Hu Tianhui; Krezel, Andrzej M.; Li Cunxi; Coffey, Robert J. . E-mail: robert.coffey@vanderbilt.edu

    2006-12-01

    Naked1 and 2 are two mammalian orthologs of Naked Cuticle, a canonical Wnt signaling antagonist in Drosophila. Naked2, but not Naked1, interacts with transforming growth factor-{alpha} (TGF{alpha}) and escorts TGF{alpha}-containing vesicles to the basolateral membrane of polarized epithelial cells. Full-length Naked2 is poorly soluble. Since most functional domains, including the Dishevelled binding region, EF-hand, vesicle recognition, and membrane targeting motifs, reside in the N-terminal half of the protein, we expressed and purified the first 217 residues of human Naked2 and performed a functional analysis of this fragment. Its circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra showed no evidence of secondary and/or tertiary structure. The fragment did not bind calcium or zinc. These results indicate that the N-terminal half of Naked2 behaves as an intrinsically unstructured protein.

  20. Signaling pathways activated by a protease allergen in basophils

    PubMed Central

    Rosenstein, Rachel K.; Bezbradica, Jelena S.; Yu, Shuang; Medzhitov, Ruslan

    2014-01-01

    Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain’s immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils. PMID:25369937

  1. Intrinsic and chemo-sensitizing activity of SMAC-mimetics on high-risk childhood acute lymphoblastic leukemia

    PubMed Central

    Schirmer, M; Trentin, L; Queudeville, M; Seyfried, F; Demir, S; Tausch, E; Stilgenbauer, S; Eckhoff, S M; Meyer, L H; Debatin, K-M

    2016-01-01

    SMAC-mimetics represent a targeted therapy approach to overcome apoptosis resistance in many tumors. Here, we investigated the efficacy of the SMAC-mimetic BV6 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In ALL cell lines, intrinsic apoptosis sensitivity was associated with rapid cIAP degradation, NF-κB activation, TNF-α secretion and induction of an autocrine TNF-α-dependent cell death loop. This pattern of responsiveness was also observed upon ex vivo analysis of 40 primograft BCP-ALL samples. Treatment with BV6 induced cell death in the majority of ALL primografts including leukemias with high-risk and poor-prognosis features. Inhibition of cell death by the TNF receptor fusion protein etanercept demonstrated that BV6 activity is dependent on TNF-α. In a preclinical NOD/SCID/huALL model of high-risk ALL, marked anti-leukemia effectivity and significantly prolonged survival were observed upon BV6 treatment. Interestingly, also in vivo, intrinsic SMAC-mimetic activity was mediated by TNF-α. Importantly, BV6 increased the effectivity of conventional induction therapy including vincristine, dexamethasone and asparaginase leading to prolonged remission induction. These data suggest SMAC-mimetics as an important addendum to efficient therapy of pediatric BCP-ALL. PMID:26775704

  2. Intrinsic Brain Activity of Cognitively Normal Older Persons Resembles More That of Patients Both with and at Risk for Alzheimer's Disease Than That of Healthy Younger Persons

    PubMed Central

    Pasquini, Lorenzo; Tonch, Annika; Plant, Claudia; Zherdin, Andrew; Ortner, Marion; Kurz, Alexander; Förstl, Hans; Zimmer, Claus; Grimmer, Timo; Wohlschäger, Afra; Riedl, Valentin

    2014-01-01

    Abstract In Alzheimer's disease (AD), recent findings suggest that amyloid-β (Aβ)-pathology might start 20–30 years before first cognitive symptoms arise. To account for age as most relevant risk factor for sporadic AD, it has been hypothesized that lifespan intrinsic (i.e., ongoing) activity of hetero-modal brain areas with highest levels of functional connectivity triggers Aβ-pathology. This model induces the simple question whether in older persons without any cognitive symptoms intrinsic activity of hetero-modal areas is more similar to that of symptomatic patients with AD or to that of younger healthy persons. We hypothesize that due to advanced age and therefore potential impact of pre-clinical AD, intrinsic activity of older persons resembles more that of patients than that of younger controls. We tested this hypothesis in younger (ca. 25 years) and older healthy persons (ca. 70 years) and patients with mild cognitive impairment and AD-dementia (ca. 70 years) by the use of resting-state functional magnetic resonance imaging, distinct measures of intrinsic brain activity, and different hierarchical clustering approaches. Independently of applied methods and involved areas, healthy older persons' intrinsic brain activity was consistently more alike that of patients than that of younger controls. Our result provides evidence for larger similarity in intrinsic brain activity between healthy older persons and patients with or at-risk for AD than between older and younger ones, suggesting a significant proportion of pre-clinical AD cases in the group of cognitively normal older people. The observed link of aging and AD with intrinsic brain activity supports the view that lifespan intrinsic activity may contribute critically to the pathogenesis of AD. PMID:24689864

  3. Pathways to URM Retention: IBP's Professional Development and Mentoring Activities

    NASA Astrophysics Data System (ADS)

    Johnson, A.; Williamson Whitney, V.; Ricciardi, L.; Detrick, L.; Siegfried, D.; Fauver, A.; Ithier-Guzman, W.; Thomas, S. H.; Valaitis, S.

    2013-05-01

    As a not for profit organization, the Institute for Broadening Participation (IBP) hosts a variety of initiatives designed to increase the retention of underrepresented minority (URM) students pursuing pathways in STEM. IBP also assists with formative program evaluation design and implementation to help strengthen URM recruitment and retention elements. Successful initiatives include virtual and face-to-face components that bring together URM students with established URM and other scientists in academia, government and industry. These connections provide URMs with mentoring, networking opportunities, and professional skill development contributing to an improved retention rate of URM students. IBP's initiatives include the NASA One Stop Shopping Initiative (NASA OSSI), Pathways to Ocean Science and Engineering, and the Minorities Striving and Pursuing Higher Degrees of Success (MS PHD'S) in Earth System Science (ESS) Professional Development Program. The NASA OSSI recruits and facilitates student engagement in NASA education and employment opportunities. Pathways to Ocean Science connects and supports URM students with Ocean Science REU programs and serves as a resource for REU program directors. Pathways to Engineering has synthesized mentoring resources into an online mentoring manual for URM students that has been extensively vetted by mentoring experts throughout the country. The mentoring manual, which is organized by roles, provides undergraduates, graduates, postdocs, faculty and project directors with valuable resources. MS PHD'S, one of IBP's longest running and most successful initiatives, focuses on increasing the retention rate of URM students receiving advanced degrees in ESS. The program addresses barriers to retention in ESS including isolation, lack of preparation and professional development, and lack of mentoring. Program activities center on peer-to-peer community building, professional development exercises, networking experiences, one

  4. Contribution of the GABAergic pathway(s) to the correlated activities of chicken retinal ganglion cells.

    PubMed

    Liu, Xue; Zhou, Yi; Gong, Hai-Qing; Liang, Pei-Ji

    2007-10-26

    In the present study, the spatiotemporal pattern of chicken retinal ganglion cells' firing activity in response to full-field white light stimulation was investigated. Cross-correlation analysis showed that ganglion cells of sustained subtype fired in precise synchrony with their adjacent neurons of the same subtype (delay lag within 2 ms, narrow correlation). On the other hand, the activities of neighboring ganglion cells of transient subtype were correlated with distributed time lags (10-30 ms, medium correlation). Pharmacological studies demonstrated that the intensity of the medium correlations could be strengthened when exogenous GABA was applied and attenuated when GABA receptors were blocked by picrotoxin. Meanwhile, the GABAergic modulation on the narrow correlations was not consistent. These results suggest that, in the chicken retina, GABAergic pathway(s) are likely involved in the formation of medium correlations between ganglion cells. Neurons might fire at a lower rate but with higher level of synchronization to improve the efficiency of information transmission, with the mechanism involving the GABAergic inhibitory input. PMID:17919471

  5. Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration.

    PubMed

    Han, Pingping; Ivanovski, Saso; Crawford, Ross; Xiao, Yin

    2015-07-01

    Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost because of disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (1) local injection of lithium chloride; (2) local injection of sclerostin antibody; and (3) local injection of a lentiviral construct overexpressing β-catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well-organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs. PMID:25556853

  6. Phosphatidylinositol 3-kinase pathway activation in breast cancer brain metastases

    PubMed Central

    2011-01-01

    Introduction Activation status of the phosphatidylinositol 3-kinase (PI3K) pathway in breast cancer brain metastases (BCBMs) is largely unknown. We examined expression of phospho(p)-AKT, p-S6, and phosphatase and tensin homologue (PTEN) in BCBMs and their implications for overall survival (OS) and survival after BCBMs. Secondary analyses included PI3K pathway activation status and associations with time to distant recurrence (TTDR) and time to BCBMs. Similar analyses were also conducted among the subset of patients with triple-negative BCBMs. Methods p-AKT, p-S6, and PTEN expression was assessed with immunohistochemistry in 52 BCBMs and 12 matched primary BCs. Subtypes were defined as hormone receptor (HR)+/HER2-, HER2+, and triple-negative (TNBC). Survival analyses were performed by using a Cox model, and survival curves were estimated with the Kaplan-Meier method. Results Expression of p-AKT and p-S6 and lack of PTEN (PTEN-) was observed in 75%, 69%, and 25% of BCBMs. Concordance between primary BCs and matched BCBMs was 67% for p-AKT, 58% for p-S6, and 83% for PTEN. PTEN- was more common in TNBC compared with HR+/HER2- and HER2+. Expression of p-AKT, p-S6, and PTEN- was not associated with OS or survival after BCBMs (all, P > 0.06). Interestingly, among all patients, PTEN- correlated with shorter time to distant and brain recurrence. Among patients with TNBC, PTEN- in BCBMs was associated with poorer overall survival. Conclusions The PI3K pathway is active in most BCBMs regardless of subtype. Inhibition of this pathway represents a promising therapeutic strategy for patients with BCBMs, a group of patients with poor prognosis and limited systemic therapeutic options. Although expression of the PI3K pathway did not correlate with OS and survival after BCBM, PTEN- association with time to recurrence and OS (among patients with TNBC) is worthy of further study. PMID:22132754

  7. Ongoing intrinsic synchronous activity is required for the functional maturation of CA3-CA1 glutamatergic synapses.

    PubMed

    Huupponen, Johanna; Molchanova, Svetlana M; Lauri, Sari E; Taira, Tomi

    2013-11-01

    Fine-tuning of synaptic connectivity during development is guided by intrinsic activity of the immature networks characteristically consisting of intermittent bursts of synchronous activity. However, the role of synchronous versus asynchronous activity in synapse maturation in the brain is unclear. Here, we have pharmacologically prevented generation of synchronous activity in the immature rat CA3-CA1 circuitry in a manner that preserves unitary activity. Long-term desynchronization of the network resulted in weakening of AMPA-receptor-mediated glutamatergic transmission in CA1 pyramidal cells. This weakening was dependent on protein phosphatases and mGluR activity, associated with an increase in the proportion of silent synapses and a decrease in the protein levels of GluA4 suggesting postsynaptic mechanisms of expression. The findings demonstrate that synchronous activity in the immature CA3-CA1 circuitry is critical for the induction and maintenance of glutamatergic synapses and underscores the importance of temporal activity patterns in shaping the synaptic circuitry during development. PMID:22941723

  8. Synaptic activity regulates AMPA receptor trafficking through different recycling pathways.

    PubMed

    Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

    2015-01-01

    Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. PMID:25970033

  9. Activation of the TGFβ pathway impairs endothelial to haematopoietic transition

    PubMed Central

    Vargel, Özge; Zhang, Yang; Kosim, Kinga; Ganter, Kerstin; Foehr, Sophia; Mardenborough, Yannicka; Shvartsman, Maya; Enright, Anton J.; Krijgsveld, Jeroen; Lancrin, Christophe

    2016-01-01

    The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFβ signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFβ activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFβ pathway. Quantitative proteomics analysis showed that TGFβ treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFβ blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFβ signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFβ signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFβ pathway does not enhance EHT as we hypothesised but instead impairs it. PMID:26891705

  10. Metabolic activation of efferent pathways from the rat area postrema.

    PubMed

    Gross, P M; Wainman, D S; Shaver, S W; Wall, K M; Ferguson, A V

    1990-03-01

    We used the quantitative [14C]deoxyglucose method and autoradiography to evaluate metabolic activity in 47 individual cerebral structures or subregions that are part of neural pathways emanating from the brain stem circumventricular organ, area postrema. Electrical stimulation of the dorsocentral area postrema in halothane-ventilated rats produced hypotension and increased glucose metabolism by several structures within the ascending trajectories of efferent neural projections from the nucleus. Structures in the caudal medulla oblongata, including three subnuclei of the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and nucleus ambiguus-A1 noradrenergic region, had increases of metabolism during stimulation of 32-62%. Pontine activation occurred specifically in the locus coeruleus and lateral parabrachial nuclei (increases of 24-36%). Magnocellular and parvocellular subdivisions of the hypothalamic paraventricular nucleus, supraoptic and suprachiasmatic nuclei, and median eminence showed increases in metabolism of 22-34%. An 89% elevation of glucose metabolism by the pituitary neural lobe resulted. The findings are evidence for functional activation of specific structures within ascending neural pathways from area postrema to forebrain mechanisms regulating blood pressure and fluid balance. PMID:2316724

  11. Activation of the TGFβ pathway impairs endothelial to haematopoietic transition.

    PubMed

    Vargel, Özge; Zhang, Yang; Kosim, Kinga; Ganter, Kerstin; Foehr, Sophia; Mardenborough, Yannicka; Shvartsman, Maya; Enright, Anton J; Krijgsveld, Jeroen; Lancrin, Christophe

    2016-01-01

    The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFβ signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFβ activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFβ pathway. Quantitative proteomics analysis showed that TGFβ treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFβ blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFβ signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFβ signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFβ pathway does not enhance EHT as we hypothesised but instead impairs it. PMID:26891705

  12. In-vivo imaging of stimulus-evoked intrinsic optical signals correlated with retinal activation in anesthetized frog

    NASA Astrophysics Data System (ADS)

    Yao, Xin-Cheng; Zhang, Qiu-Xiang; Li, Yang-Guo

    2011-09-01

    Intrinsic optical signal imaging (IOS) promises a noninvasive method for high resolution examination of retinal function. Using freshly isolated animal retinas, we have conducted a series of experiments to test fast IOSs which have time courses comparable to electrophysiological kinetics. In this article, we demonstrate the feasibility of in vivo imaging of fast IOSs correlated with retinal activation in anesthetized frog (Rana Pipiens). A rapid (68,000 lines/s) line-scan confocal ophthalmoscope was constructed to achieve high-speed (200 frames/s) near infared (NIR) recording of fast IOSs. By rejecting out-of-focus background light, the line-scan confocal imager provided enough resolution to differentiate individual photoreceptors in vivo. With visible light stimulation, NIR confocal images disclosed transient IOSs with time courses comparable to retinal ERG kinetics. High-resolution IOS images revealed both positive (increasing) and negative (decreasing) light responses, with sub-cellular complexity, in the activated retina.

  13. Mast cell degranulation activates a pain pathway underlying migraine headache

    PubMed Central

    Levy, Dan; Burstein, Rami; Kainz, Vanessa; Jakubowski, Moshe; Strassman, Andrew M.

    2007-01-01

    Intracranial headaches such as that of migraine are generally accepted to be mediated by prolonged activation of meningeal nociceptors but the mechanisms responsible for such nociceptor activation are poorly understood. In this study, we examined the hypothesis that meningeal nociceptors can be activated locally through a neuroimmune interaction with resident mast cells, granulated immune cells that densely populate the dura mater. Using in vivo electrophysiological single unit recording of meningeal nociceptors in the rat we observed that degranulation of dural mast cells using intraperitoneal administration of the basic secretagogue agent compound 48/80 (2 mg/kg) induced a prolonged state of excitation in meningeal nociceptors. Such activation was accompanied by increased expression of the phosphorylated form of the extracellular signal-regulated kinase (pERK), an anatomical marker for nociceptor activation. Mast cell - induced nociceptor interaction was also associated with downstream activation of the spinal trigeminal nucleus as indicated by an increase in c-fos expression. Our findings provide evidence linking dural mast cell degranulation to prolonged activation of the trigeminal pain pathway believed to underlie intracranial headaches such as that of migraine. PMID:17459586

  14. The Intrinsic Eddington Ratio Distribution of Active Galactic Nuclei in Star-forming Galaxies from the Sloan Digital Sky Survey

    NASA Astrophysics Data System (ADS)

    Jones, Mackenzie L.; Hickox, Ryan C.; Black, Christine S.; Hainline, Kevin N.; DiPompeo, Michael A.; Goulding, Andy D.

    2016-07-01

    An important question in extragalactic astronomy concerns the distribution of black hole accretion rates of active galactic nuclei (AGNs). Based on observations at X-ray wavelengths, the observed Eddington ratio distribution appears as a power law, while optical studies have often yielded a lognormal distribution. There is increasing evidence that these observed discrepancies may be due to contamination by star formation and other selection effects. Using a sample of galaxies from the Sloan Digital Sky Survey Data Release 7, we test whether or not an intrinsic Eddington ratio distribution that takes the form of a Schechter function is consistent with previous work suggesting that young galaxies in optical surveys have an observed lognormal Eddington ratio distribution. We simulate the optical emission line properties of a population of galaxies and AGNs using a broad, instantaneous luminosity distribution described by a Schechter function near the Eddington limit. This simulated AGN population is then compared to observed galaxies via their positions on an emission line excitation diagram and Eddington ratio distributions. We present an improved method for extracting the AGN distribution using BPT diagnostics that allows us to probe over one order of magnitude lower in Eddington ratio, counteracting the effects of dilution by star formation. We conclude that for optically selected AGNs in young galaxies, the intrinsic Eddington ratio distribution is consistent with a possibly universal, broad power law with an exponential cutoff, as this distribution is observed in old, optically selected galaxies and X-rays.

  15. Hemispheric asymmetry of visual scene processing in the human brain: evidence from repetition priming and intrinsic activity.

    PubMed

    Stevens, W Dale; Kahn, Itamar; Wig, Gagan S; Schacter, Daniel L

    2012-08-01

    Asymmetrical specialization of cognitive processes across the cerebral hemispheres is a hallmark of healthy brain development and an important evolutionary trait underlying higher cognition in humans. While previous research, including studies of priming, divided visual field presentation, and split-brain patients, demonstrates a general pattern of right/left asymmetry of form-specific versus form-abstract visual processing, little is known about brain organization underlying this dissociation. Here, using repetition priming of complex visual scenes and high-resolution functional magnetic resonance imaging (MRI), we demonstrate asymmetrical form specificity of visual processing between the right and left hemispheres within a region known to be critical for processing of visual spatial scenes (parahippocampal place area [PPA]). Next, we use resting-state functional connectivity MRI analyses to demonstrate that this functional asymmetry is associated with differential intrinsic activity correlations of the right versus left PPA with regions critically involved in perceptual versus conceptual processing, respectively. Our results demonstrate that the PPA comprises lateralized subregions across the cerebral hemispheres that are engaged in functionally dissociable yet complementary components of visual scene analysis. Furthermore, this functional asymmetry is associated with differential intrinsic functional connectivity of the PPA with distinct brain areas known to mediate dissociable cognitive processes. PMID:21968568

  16. Complement Alternative Pathway Activation in Human Nonalcoholic Steatohepatitis

    PubMed Central

    Segers, Filip M.; Verdam, Froukje J.; de Jonge, Charlotte; Boonen, Bas; Driessen, Ann; Shiri-Sverdlov, Ronit; Bouvy, Nicole D.; Greve, Jan Willem M.; Buurman, Wim A.; Rensen, Sander S.

    2014-01-01

    The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10) or with NASH (n = 12) using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01) in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01). Hepatic properdin levels positively correlated with levels of C3c (rs = 0.69; p<0.05) and C3c/C3 activation ratio (rs = 0.59; p<0.05). C3c, C3 activation status (C3c/C3 ratio) and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05). Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;). Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28). In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05) and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08). Collectively, these data suggest a role for alternative pathway

  17. Sorafenib synergizes with metformin in NSCLC through AMPK pathway activation

    PubMed Central

    Groenendijk, Floris H; Mellema, Wouter W; van der Burg, Eline; Schut, Eva; Hauptmann, Michael; Horlings, Hugo M; Willems, Stefan M; van den Heuvel, Michel M; Jonkers, Jos; Smit, Egbert F; Bernards, René

    2015-01-01

    The multikinase inhibitor sorafenib is under clinical investigation for the treatment of many solid tumors, but in most cases, the molecular target responsible for the clinical effect is unknown. Furthermore, enhancing the effectiveness of sorafenib using combination strategies is a major clinical challenge. Here, we identify sorafenib as an activator of AMP-activated protein kinase (AMPK), in a manner that involves either upstream LKB1 or CAMKK2. We further show in a phase II clinical trial in KRAS mutant advanced non-small cell lung cancer (NSCLC) with single agent sorafenib an improved disease control rate in patients using the antidiabetic drug metformin. Consistent with this, sorafenib and metformin act synergistically in inhibiting cellular proliferation in NSCLC in vitro and in vivo. A synergistic effect of both drugs is also seen on phosphorylation of the AMPKα activation site. Our results provide a rationale for the synergistic antiproliferative effects, given that AMPK inhibits downstream mTOR signaling. These data suggest that the combination of sorafenib with AMPK activators could have beneficial effects on tumor regression by AMPK pathway activation. The combination of metformin or other AMPK activators and sorafenib could be tested in prospective clinical trials. PMID:25080865

  18. The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity

    PubMed Central

    Masson, Glenn R.; Perisic, Olga; Burke, John E.; Williams, Roger L.

    2015-01-01

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase, and both activities are necessary for its role as a tumour suppressor. PTEN activity is controlled by phosphorylation of its intrinsically disordered C-terminal tail. A recently discovered variant of PTEN, PTEN-long (PTEN-L), has a 173-residue N-terminal extension that causes PTEN-L to exhibit unique behaviour, such as movement from one cell to another. Using hydrogen/deuterium exchange mass spectrometry (HDX–MS) and biophysical assays, we show that both the N-terminal extension of PTEN-L and C-terminal tail of PTEN affect the phosphatase activity using unique mechanisms. Phosphorylation of six residues in the C-terminal tail of PTEN results in auto-inhibitory interactions with the phosphatase and C2 domains, effectively blocking both the active site and the membrane-binding interface of PTEN. Partially dephosphorylating PTEN on pThr366/pSer370 results in sufficient exposure of the active site to allow a selective activation for soluble substrates. Using HDX–MS, we identified a membrane-binding element in the N-terminal extension of PTEN-L, termed the membrane-binding helix (MBH). The MBH radically alters the membrane binding mechanism of PTEN-L compared with PTEN, switching PTEN-L to a ‘scooting’ mode of catalysis from the ‘hopping’ mode that is characteristic of PTEN. PMID:26527737

  19. The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity.

    PubMed

    Masson, Glenn R; Perisic, Olga; Burke, John E; Williams, Roger L

    2016-01-15

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase, and both activities are necessary for its role as a tumour suppressor. PTEN activity is controlled by phosphorylation of its intrinsically disordered C-terminal tail. A recently discovered variant of PTEN, PTEN-long (PTEN-L), has a 173-residue N-terminal extension that causes PTEN-L to exhibit unique behaviour, such as movement from one cell to another. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and biophysical assays, we show that both the N-terminal extension of PTEN-L and C-terminal tail of PTEN affect the phosphatase activity using unique mechanisms. Phosphorylation of six residues in the C-terminal tail of PTEN results in auto-inhibitory interactions with the phosphatase and C2 domains, effectively blocking both the active site and the membrane-binding interface of PTEN. Partially dephosphorylating PTEN on pThr(366)/pSer(370) results in sufficient exposure of the active site to allow a selective activation for soluble substrates. Using HDX-MS, we identified a membrane-binding element in the N-terminal extension of PTEN-L, termed the membrane-binding helix (MBH). The MBH radically alters the membrane binding mechanism of PTEN-L compared with PTEN, switching PTEN-L to a 'scooting' mode of catalysis from the 'hopping' mode that is characteristic of PTEN. PMID:26527737

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

    PubMed

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

    2015-07-01

    Platinum-based anticancer drugs, including cisplatin and carboplatin, have been cornerstones in the treatment of solid tumors. We report here that these DNA-damaging agents, particularly cisplatin, induce apoptosis through plasma membrane disruption, triggering FAS death receptor via mitochondrial (intrinsic) pathways. Our objectives were to: quantify the composition of membrane metabolites; and determine the potential involvement of acid sphingomyelinase (ASMase) in the FAS-mediated apoptosis in ovarian cancer after cisplatin treatment. The resulting analysis revealed enhanced apoptosis as measured by: increased phosphocholine, and glycerophosphocholine; elevated cellular energetics; and phosphocreatine and nucleoside triphosphate concentrations. The plasma membrane alterations were accompanied by increased ASMase activity, leading to the upregulation of FAS, FASL and related pro-apoptotic BAX and PUMA genes. Moreover FAS, FASL, BAX, PUMA, CASPASE-3 and -9 proteins were upregulated. Our findings implicate ASMase activity and the intrinsic pathways in cisplatin-mediated membrane demise, and contribute to our understanding of the mechanisms by which ovarian tumors may become resistant to cisplatin. PMID:25846011

  1. Hesperetin Induces Apoptosis in Breast Carcinoma by Triggering Accumulation of ROS and Activation of ASK1/JNK Pathway.

    PubMed

    Palit, Shreyasi; Kar, Susanta; Sharma, Gunjan; Das, Pijush K

    2015-08-01

    Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF-7 cells in a concentration- and time-dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF-10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl-serine externalization, DNA fragmentation, caspase-7 activation, and PARP cleavage. Apoptosis was associated with caspase-9 activation, mitochondrial membrane potential loss, release of cytochrome c, and increase in Bax:Bcl-2 ratio. Pre-treatment with caspase-9 specific inhibitor (Z-LEHD-fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow-cytometric analysis revealed triggering of ROS in a time-dependent manner. Pre-treatment with ROS scavenger N-acetylcysteine (NAC) and glutathione markedly abrogated hesperetin-mediated apoptosis whereas carbonyl cyanide m-chlorophenylhydrazone (CCCP) pretreatment along with DHR123-based flow-cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pre-treatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetin-mediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin-mediated apoptosis suggesting that hesperetin-mediated apoptosis of MCF-7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA-MB-231 cells via intrinsic pathway via activation of caspase -9 and -3 and increase in Bax:Bcl-2 ratio. PMID:25204891

  2. The Crystal Structure of Thrombin-activable Fibrinolysis Inhibitor (TAFI) Provides the Structural Basis for Its Intrinsic Activity and the Short Half-life of TAFIa*♦

    PubMed Central

    Anand, Kanchan; Pallares, Irantzu; Valnickova, Zuzana; Christensen, Trine; Vendrell, Josep; Wendt, K. Ulrich; Schreuder, Herman A.; Enghild, Jan J.; Avilés, Francesc X.

    2008-01-01

    Mature thrombin-activable fibrinolysis inhibitor (TAFIa) is a highly unstable metallocarboxypeptidase that stabilizes blood clots by clipping C-terminal lysine residues from partially degraded fibrin. In accordance with its in vitro antifibrinolytic activity, animal studies have reported that inhibition of mature TAFI aids in the prevention of thrombosis. The level of TAFI activity is stringently regulated through (i) controlled proteolytic truncation of the zymogen (TAFI), generating the mature enzyme, TAFIa, and (ii) the short half-life of TAFIa. TAFI itself exhibits an intrinsic enzymatic activity, which is likely required to provide a baseline level of antifibrinolytic activity. The novel crystal structure presented here reveals that the active site of TAFI is accessible, providing the structural explanation for the its intrinsic activity. It also supports the notion that an “instability region” exists, in agreement with site-directed mutagenesis studies. Sulfate ions, bound to this region, point toward a potential heparin-binding site and could explain how heparin stabilizes TAFIa. PMID:18669641

  3. Proteomic analysis reveals tanshinone IIA enhances apoptosis of advanced cervix carcinoma CaSki cells through mitochondria intrinsic and endoplasmic reticulum stress pathways.

    PubMed

    Pan, Tai-Long; Wang, Pei-Wen; Hung, Yu-Chiang; Huang, Chun-Hsun; Rau, Kun-Ming

    2013-12-01

    Cervix cancer is the second most common cancer among women worldwide, whereas paclitaxel, the first line chemotherapeutic drug used to treat cervical cancer, shows low chemosensitivity on the advanced cervical cancer cell line. Tanshinone IIA (Tan IIA) exhibited strong growth inhibitory effect on CaSki cells (IC50 = 5.51 μM) through promoting caspase cascades with concomitant upregulating the phosphorylation of p38 and JNK signaling. Comprehensive proteomics revealed the global protein changes and the network analysis implied that Tan IIA treatment would activate ER stress pathways that finally lead to apoptotic cell death. Moreover, ER stress inhibitor could alleviate Tan IIA caused cell growth inhibition and ameliorate C/EBP-homologous protein as well as apoptosis signal-regulating kinase 1 mediated cell death. The therapeutic interventions targeting the mitochondrial-related apoptosis and ER stress responses might be promising strategies to conquer paclitaxel resistance. PMID:24167031

  4. An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR.

    PubMed

    Randak, Christoph; Welsh, Michael J

    2003-12-26

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption. PMID:14697202

  5. A structural pathway for activation of the kinesin motor ATPase

    PubMed Central

    Yun, Mikyung; Zhang, Xiaohua; Park, Cheon-Gil; Park, Hee-Won; Endow, Sharyn A.

    2001-01-01

    Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPase. The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules. PMID:11387196

  6. Intrinsic differences in atomic ordering of calcium (alumino)silicate hydrates in conventional and alkali-activated cements

    SciTech Connect

    White, Claire E.; Daemen, Luke L.; Hartl, Monika; Page, Katharine

    2015-01-15

    The atomic structures of calcium silicate hydrate (C–S–H) and calcium (–sodium) aluminosilicate hydrate (C–(N)–A–S–H) gels, and their presence in conventional and blended cement systems, have been the topic of significant debate over recent decades. Previous investigations have revealed that synthetic C–S–H gel is nanocrystalline and due to the chemical similarities between ordinary Portland cement (OPC)-based systems and low-CO{sub 2} alkali-activated slags, researchers have inferred that the atomic ordering in alkali-activated slag is the same as in OPC–slag cements. Here, X-ray total scattering is used to determine the local bonding environment and nanostructure of C(–A)–S–H gels present in hydrated tricalcium silicate (C{sub 3}S), blended C{sub 3}S–slag and alkali-activated slag, revealing the large intrinsic differences in the extent of nanoscale ordering between C–S–H derived from C{sub 3}S and alkali-activated slag systems, which may have a significant influence on thermodynamic stability, and material properties at higher length scales, including long term durability of alkali-activated cements.

  7. Keeping women active: an examination of the impacts of self-efficacy, intrinsic motivation, and leadership on women's persistence in physical activity.

    PubMed

    Lloyd, Kathleen M; Little, Donna E

    2010-10-01

    Physical inactivity in women is a worldwide problem that has not only been well-documented but has provoked much government concern and policy activity. However, an even more important issue is encouraging women's persistence in physical activity. The purpose of this study was to examine the links between women's experiences of participation in a government-funded physical activity festival, their intentions to continue participation, and their participation behavior six months after the festival. Results from semi-structured, in-depth interviews with 20 women revealed that enhanced self-efficacy, intrinsic motivation, and supportive leadership had motivated the women's future intentions to participate. Follow-up surveys showed their levels of interest and participation in physical activity had been maintained. These results enhance our understanding of the relationship between key outcomes of women's physical activity participation and their persistence in physical activity. PMID:21104568

  8. Radiation exposure induces inflammasome pathway activation in immune cells.

    PubMed

    Stoecklein, Veit M; Osuka, Akinori; Ishikawa, Shizu; Lederer, Madeline R; Wanke-Jellinek, Lorenz; Lederer, James A

    2015-02-01

    Radiation exposure induces cell and tissue damage, causing local and systemic inflammatory responses. Because the inflammasome pathway is triggered by cell death and danger-associated molecular patterns, we hypothesized that the inflammasome may signal acute and chronic immune responses to radiation. Using a mouse radiation model, we show that radiation induces a dose-dependent increase in inflammasome activation in macrophages, dendritic cells, NK cells, T cells, and B cells as judged by cleaved caspase-1 detection in cells. Time course analysis showed the appearance of cleaved caspase-1 in cells by day 1 and sustained expression until day 7 after radiation. Also, cells showing inflammasome activation coexpressed the cell surface apoptosis marker annexin V. The role of caspase-1 as a trigger for hematopoietic cell losses after radiation was studied in caspase-1(-/-) mice. We found less radiation-induced cell apoptosis and immune cell loss in caspase-1(-/-) mice than in control mice. Next, we tested whether uric acid might mediate inflammasome activation in cells by treating mice with allopurinol and discovered that allopurinol treatment completely blocked caspase-1 activation in cells. Finally, we demonstrate that radiation-induced caspase-1 activation occurs by a Nod-like receptor family protein 3-independent mechanism because radiation-exposed Nlrp3(-/-) mice showed caspase-1 activation profiles that were indistinguishable from those of wild-type mice. In summary, our data demonstrate that inflammasome activation occurs in many immune cell types following radiation exposure and that allopurinol prevented radiation-induced inflammasome activation. These results suggest that targeting the inflammasome may help control radiation-induced inflammation. PMID:25539818

  9. Spatial distribution of intrinsic and scattering seismic attenuation in active volcanic islands - I: model and the case of Tenerife Island

    NASA Astrophysics Data System (ADS)

    Prudencio, Janire; Del Pezzo, Edoardo; García-Yeguas, Araceli; Ibáñez, Jesús M.

    2013-12-01

    The complex volcanic system of Tenerife Island is known to have a highly heterogeneous character, as recently confirmed by velocity tomography. We present new information derived from intrinsic quality factor inverse maps (Qi-1), scattering quality factor inverse maps (Qs-1) and total quality factor inverse maps (Qt-1) obtained for the same region. The data set used in this work is the result of the analysis of an active seismic experiment carried out, using offshore shots (air guns) recorded at over 85 onshore seismic stations. The estimates of the attenuation parameters are based on the assumption that the seismogram energy envelopes are determined by seismic energy diffusion processes occurring inside the island. Diffusion model parameters, proportional to Qi-1 and to Qs-1, are estimated from the inversion of the energy envelopes for any source-receiver couple. They are then weighted with a new graphical approach based on a Gaussian space probability function, which allowed us to create `2-D probabilistic maps' representing the space distribution of the attenuation parameters. The 2-D images obtained reveal the existence of a zone in the centre of the island characterized by the lowest attenuation effects. This effect is interpreted as highly rigid and cooled rocks. This low-attenuation region is bordered by zones of high attenuation, associated with the recent historical volcanic activity. We calculate the transport mean free path obtaining a value of around 4 km for the frequency range 6-12 Hz. This result is two orders of magnitude smaller than values calculated for the crust of the Earth. An absorption length between 10 and 14 km is associated with the average intrinsic attenuation parameter. These values, while small in the context of tectonic regions, are greater than those obtained in volcanic regions such as Vesuvius or Merapi. Such differences may be explained by the magnitude of the region of study, over three times larger than the aforementioned study

  10. Increased intrinsic excitability of muscle vasoconstrictor preganglionic neurons may contribute to the elevated sympathetic activity in hypertensive rats.

    PubMed

    Briant, Linford J B; Stalbovskiy, Alexey O; Nolan, Matthew F; Champneys, Alan R; Pickering, Anthony E

    2014-12-01

    Hypertension is associated with pathologically increased sympathetic drive to the vasculature. This has been attributed to increased excitatory drive to sympathetic preganglionic neurons (SPN) from brainstem cardiovascular control centers. However, there is also evidence supporting increased intrinsic excitability of SPN. To test this hypothesis, we made whole cell recordings of muscle vasoconstrictor-like (MVClike) SPN in the working-heart brainstem preparation of spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats. The MVClike SPN have a higher spontaneous firing frequency in the SH rat (3.85 ± 0.4 vs. 2.44 ± 0.4 Hz in WKY; P = 0.011) with greater respiratory modulation of their activity. The action potentials of SH SPN had smaller, shorter afterhyperpolarizations (AHPs) and showed diminished transient rectification indicating suppression of an A-type potassium conductance (IA). We developed mathematical models of the SPN to establish if changes in their intrinsic properties in SH rats could account for their altered firing. Reduction of the maximal conductance density of IA by 15-30% changed the excitability and output of the model from the WKY to a SH profile, with increased firing frequency, amplified respiratory modulation, and smaller AHPs. This change in output is predominantly a consequence of altered synaptic integration. Consistent with these in silico predictions, we found that intrathecal 4-aminopyridine (4-AP) increased sympathetic nerve activity, elevated perfusion pressure, and augmented Traube-Hering waves. Our findings indicate that IA acts as a powerful filter on incoming synaptic drive to SPN and that its diminution in the SH rat is potentially sufficient to account for the increased sympathetic output underlying hypertension. PMID:25122704