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Sample records for cells involves activation

  1. Protease activation involved in resistance of human cells to x-ray cell killing

    International Nuclear Information System (INIS)

    Zhang, Hong-Chang; Takahashi, Shuji; Karata, Kiyonobu; Kita, Kazuko; Suzuki, Nobuo

    2003-01-01

    Little is known of proteases that play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa-R cells after X-ray irradiation. The activity was identified as fibrinolytic, using 125 I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa-R cells showed an increased level of protease activity 10 min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with the X-ray susceptibility of cells. Leupeptin, a serine-cysteine protease inhibitor, inhibited the protease activity in samples obtained from X-ray-irradiated RSa-R cells. Treatment of RSa-R cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. However, the treatment of cells with other inhibitors tested did not modulate the X-ray susceptibility. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. (author)

  2. Activation of PPARγ is not involved in butyrate-induced epithelial cell differentiation

    International Nuclear Information System (INIS)

    Ulrich, S.; Waechtershaeuser, A.; Loitsch, S.; Knethen, A. von; Bruene, B.; Stein, J.

    2005-01-01

    Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor γ (PPARγ) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPARγ in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPARγ in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPARγ ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPARγ ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPARγ mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPARγ is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation

  3. Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

    Science.gov (United States)

    Zeng, Yizhou; Tian, Xiaofang; Wang, Quan; He, Weiyang; Fan, Jing; Gou, Xin

    2018-01-01

    Aim The mammalian target of rapamycin (mTOR) pathway is a critical target for cancer treatment and the mTOR inhibitor everolimus (RAD001) has been approved for treatment of renal cell carcinoma (RCC). However, the limited efficacy of RAD001 has led to the development of drug resistance. Autophagy is closely related to cell survival and death, which may be activated under RAD001 stimulation. The aim of the present study was to identify the underlying mechanisms of RAD001 resistance in RCC cells through cytoprotective autophagy involving activation of the extracellular signal-regulated kinase (ERK) pathway. Methods and results: RAD001 strongly induced autophagy of RCC cells in a dose- and time-dependent manner, as confirmed by Western blot analysis. Importantly, suppression of autophagy by the pharmacological inhibitor chloroquine effectively enhanced RAD001-induced apoptotic cytotoxicity, as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis, indicating a cytoprotective role for RAD001-induced autophagy. In addition, as was shown by the MTT assay, flow cytometry, and Western blot analysis, RAD001 robustly activated ERK, but not c-Jun N-terminal kinase and p38. Activation of ERK was inhibited by the pharmacological inhibitor selumetinib (AZD6244), which effectively promoted RAD001-induced cell death. Moreover, employing AZD6244 markedly attenuated RAD001-induced autophagy and enhanced RAD001-induced apoptosis, which play a central role in RAD001-induced cell death. Furthermore, RAD001-induced autophagy is regulated by ERK-mediated phosphorylation of Beclin-1 and B-cell lymphoma 2, as confirmed by Western blot analysis. Conclusion These results suggest that RAD001-induced autophagy involves activation of the ERK, which may impair cytotoxicity of RAD001 in RCC cells. Thus, inhibition of the activation of ERK pathway-mediated autophagy may be useful to overcome chemoresistance to RAD001. PMID:29719377

  4. NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

    International Nuclear Information System (INIS)

    Nishina, Takashi; Yamaguchi, Noritaka; Gohda, Jin; Semba, Kentaro; Inoue, Jun-ichiro

    2009-01-01

    Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.

  5. NIK is involved in constitutive activation of the alternative NF-{kappa}B pathway and proliferation of pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Nishina, Takashi [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Yamaguchi, Noritaka [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041 (Japan); Gohda, Jin [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Semba, Kentaro [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan)

    2009-10-09

    Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-{kappa}B is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-{kappa}B activation. Here, we show that the alternative pathway is constitutively activated and NF-{kappa}B-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.

  6. Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway

    Energy Technology Data Exchange (ETDEWEB)

    Nagane, Masaki; Yasui, Hironobu; Sakai, Yuri; Yamamori, Tohru [Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Niwa, Koichi [Laboratory of Biochemistry, Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri 099-2493 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Inanami, Osamu, E-mail: inanami@vetmed.hokudai.ac.jp [Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan)

    2015-01-02

    Highlights: • eNOS activity is increased in BAECs exposed to X-rays. • ATM is involved in this increased eNOS activity. • HSP90 modulates the radiation-induced activation of ATM and eNOS. - Abstract: In this study, the involvement of ataxia telangiectasia mutated (ATM) kinase and heat shock protein 90 (HSP90) in endothelial nitric oxide synthase (eNOS) activation was investigated in X-irradiated bovine aortic endothelial cells. The activity of nitric oxide synthase (NOS) and the phosphorylation of serine 1179 of eNOS (eNOS-Ser1179) were significantly increased in irradiated cells. The radiation-induced increases in NOS activity and eNOS-Ser1179 phosphorylation levels were significantly reduced by treatment with either an ATM inhibitor (Ku-60019) or an HSP90 inhibitor (geldanamycin). Geldanamycin was furthermore found to suppress the radiation-induced phosphorylation of ATM-Ser1181. Our results indicate that the radiation-induced eNOS activation in bovine aortic endothelial cells is regulated by ATM and HSP90.

  7. Acrolein activates cell survival and apoptotic death responses involving the endoplasmic reticulum in A549 lung cells.

    Science.gov (United States)

    Tanel, André; Pallepati, Pragathi; Bettaieb, Ahmed; Morin, Patrick; Averill-Bates, Diana A

    2014-05-01

    Acrolein, a highly reactive α,β-unsaturated aldehyde, is a product of endogenous lipid peroxidation. It is a ubiquitous environmental pollutant that is generated mainly by smoke, overheated cooking oil and vehicle exhaust. Acrolein damages cellular proteins, which could lead to accumulation of aberrantly-folded proteins in the endoplasmic reticulum (ER). This study determines the mechanisms involved in acrolein-induced apoptosis mediated by the ER and possible links with the ER stress response in human A549 lung cells. The exposure of cells to acrolein (15-50μM) for shorter times of 15 to 30min activated several ER stress markers. These included the ER chaperone protein BiP and the three ER sensors: (i) the survival/rescue molecules protein kinase RNA (PKR)-like ER kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α) were phosphorylated; (ii) cleavage of activating transcription factor 6 (ATF6) occurred, and (iii) inositol-requiring protein-1 alpha (IRE1α) was phosphorylated. Acrolein (25-50μM) caused apoptotic cell death mediated by the ER after 2h, which was characterised by the induction of CHOP and activation of ER proteases calpain and caspase-4. Calpain and caspase-7 were the initiating factors for caspase-4 activation in acrolein-induced apoptosis. These results increase our knowledge about cellular responses to acrolein in lung cells, which have implications for human health. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ.

    Directory of Open Access Journals (Sweden)

    Khetam Ghannam

    Full Text Available OBJECTIVE: In idiopathic inflammatory myopathies (IIM infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. METHODS: Expression of constitutive (PSMB5, -6, -7 and corresponding immunoproteasomal subunits (PSMB8, -9, -10 was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM and healthy donors (HD. Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. RESULTS: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10 in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. CONCLUSIONS: Immunoproteasomes seem to indicate

  9. Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ

    Science.gov (United States)

    Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R.

    2014-01-01

    Objective In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions Immunoproteasomes seem to indicate IIM activity and

  10. Involvement of multiple cell lineages in atherogenesis | Ogeng'o ...

    African Journals Online (AJOL)

    Involvement of multiple cell lineages in atherogenesis. ... mast cells, dendritic cells, macrophages and immigrant cells usually found in blood, namely ... which influence inflammation, migration, proliferation and secretory activity of each other in ...

  11. Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

    International Nuclear Information System (INIS)

    Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

    2008-01-01

    CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

  12. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    International Nuclear Information System (INIS)

    Wang, Yi; Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing

    2011-01-01

    Highlights: → Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. → Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. → P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. → Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam

  13. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi, E-mail: wangyi2004a@126.com [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China); Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China)

    2011-08-05

    Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the

  14. Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity

    Science.gov (United States)

    Almaguel, Frankis G.; Liu, Jo-Wen; Pacheco, Fabio J.; De Leon, Daisy; Casiano, Carlos A.; De Leon, Marino

    2010-01-01

    Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity. PMID:20043885

  15. Endoplasmic reticulum involvement in yeast cell death

    International Nuclear Information System (INIS)

    Nicanor Austriaco, O.

    2012-01-01

    Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death.

  16. Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

    International Nuclear Information System (INIS)

    Wang, Bing; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

    2013-01-01

    Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells

  17. Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bing, E-mail: wangbin69@yahoo.com; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

    2013-07-19

    Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells.

  18. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

  19. NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells

    International Nuclear Information System (INIS)

    Pecorelli, Alessandra; Bocci, Velio; Acquaviva, Alessandra; Belmonte, Giuseppe; Gardi, Concetta; Virgili, Fabio; Ciccoli, Lucia; Valacchi, Giuseppe

    2013-01-01

    During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O 3 per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H 2 O 2 ) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis. - Highlights: ► Endothelial HO1 is upregulated by ozonated plasma ► This activation is induced by NRF2 and it is ERK independent. ► 4HNE and H 2 O 2 are the main molecules involved in this process. ► Ozonated plasma induced a hormetic effect ► Combination of orthodox medicine and ozonated plasma can be a useful treatment

  20. NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pecorelli, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Child Neuropsychiatry Unit, University Hospital, AOUS, Siena (Italy); Bocci, Velio [Department of Physiology, University of Siena (Italy); Acquaviva, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Belmonte, Giuseppe [Department of Biomedical Sciences, University of Siena (Italy); Gardi, Concetta [Department of Molecular and Developmental Medicine, University of Siena (Italy); Virgili, Fabio [INRAN, Rome (Italy); Ciccoli, Lucia [Department of Molecular and Developmental Medicine, University of Siena (Italy); Valacchi, Giuseppe, E-mail: giuseppe.valacchi@unife.it [Department of Life Sciences and Biotechnology, University of Ferrara (Italy); Department of Food and Nutrition, Kyung Hee University, Seoul (Korea, Republic of)

    2013-02-15

    During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O{sub 3} per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H{sub 2}O{sub 2}) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis. - Highlights: ► Endothelial HO1 is upregulated by ozonated plasma ► This activation is induced by NRF2 and it is ERK independent. ► 4HNE and H{sub 2}O{sub 2} are the main molecules involved in this process. ► Ozonated plasma induced a hormetic effect ► Combination of orthodox medicine and ozonated plasma can be a useful treatment.

  1. Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

    Science.gov (United States)

    Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

    2002-05-01

    Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

  2. Rosmarinic acid counteracts activation of hepatic stellate cells via inhibiting the ROS-dependent MMP-2 activity: Involvement of Nrf2 antioxidant system

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Changfang; Zou, Yu; Liu, Yuzhang; Niu, Yingcai, E-mail: nyc1968@126.com

    2017-03-01

    Recently, oxidative stress is involved in hepatofibrogenesis. Matrix metalloproteinase-2 (MMP-2) is required for activation of hepatic stellate cells (HSCs) in response to reactive oxygen species (ROS). This study was designed to explore the hypothesis that the inhibitory effect of rosmarinic acid (RA) on HSCs activation might mainly result from its antioxidant capability by increasing the synthesis of glutathione (GSH) involved in nuclear factor kappa B (NF-κB)-dependent inhibition of MMP-2 activity. Here, we demonstrate that RA reverses activated HSCs to quiescent cells. Concomitantly, RA inhibits MMP-2 activity. RNA interference-imposed knockdown of NF-κB abolished down-regulation of MMP-2 by RA. RA-mediated inactivation of NF-κB could be blocked by the diphenyleneiodonium chloride (DPI; a ROS inhibitor). Conversely, transfection of dominant-negative (DN) mutant of extracellular signal-regulated kinases 2 (ERK2), c-Jun N-terminal kinase 1 (JNK1), or p38α kinase had no such effect. Simultaneously, RA suppresses ROS generation and lipid peroxidation (LPO) whereas increases cellular GSH in HSC-T6 cells. Furthermore, RA significantly increased antioxidant response element (ARE)-mediated luciferase activity, nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and catalytic subunits from glutamate cysteine ligase (GCLc) expression, but not modulatory subunits from GCL (GCLm). RA-mediated up-regulation of GClc is inhibited by the shRNA-induced Nrf2 knockdown. The knocking down of Nrf2 or buthionine sulfoximine (a GCL inhibitor) abolished RA-mediated inhibition of ROS. Collectively, these results provide novel insights into the mechanisms of RA as an antifibrogenic candidate in the prevention and treatment of liver fibrosis. - Highlights: • RA reverses activated HSCs to quiescent cells. • RA suppresses MMP-2 activity through a NF-κB-dependent pathway. • Inhibition of oxidative stress by RA is dependent on nuclear translocation of Nrf2

  3. Rosmarinic acid counteracts activation of hepatic stellate cells via inhibiting the ROS-dependent MMP-2 activity: Involvement of Nrf2 antioxidant system

    International Nuclear Information System (INIS)

    Lu, Changfang; Zou, Yu; Liu, Yuzhang; Niu, Yingcai

    2017-01-01

    Recently, oxidative stress is involved in hepatofibrogenesis. Matrix metalloproteinase-2 (MMP-2) is required for activation of hepatic stellate cells (HSCs) in response to reactive oxygen species (ROS). This study was designed to explore the hypothesis that the inhibitory effect of rosmarinic acid (RA) on HSCs activation might mainly result from its antioxidant capability by increasing the synthesis of glutathione (GSH) involved in nuclear factor kappa B (NF-κB)-dependent inhibition of MMP-2 activity. Here, we demonstrate that RA reverses activated HSCs to quiescent cells. Concomitantly, RA inhibits MMP-2 activity. RNA interference-imposed knockdown of NF-κB abolished down-regulation of MMP-2 by RA. RA-mediated inactivation of NF-κB could be blocked by the diphenyleneiodonium chloride (DPI; a ROS inhibitor). Conversely, transfection of dominant-negative (DN) mutant of extracellular signal-regulated kinases 2 (ERK2), c-Jun N-terminal kinase 1 (JNK1), or p38α kinase had no such effect. Simultaneously, RA suppresses ROS generation and lipid peroxidation (LPO) whereas increases cellular GSH in HSC-T6 cells. Furthermore, RA significantly increased antioxidant response element (ARE)-mediated luciferase activity, nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and catalytic subunits from glutamate cysteine ligase (GCLc) expression, but not modulatory subunits from GCL (GCLm). RA-mediated up-regulation of GClc is inhibited by the shRNA-induced Nrf2 knockdown. The knocking down of Nrf2 or buthionine sulfoximine (a GCL inhibitor) abolished RA-mediated inhibition of ROS. Collectively, these results provide novel insights into the mechanisms of RA as an antifibrogenic candidate in the prevention and treatment of liver fibrosis. - Highlights: • RA reverses activated HSCs to quiescent cells. • RA suppresses MMP-2 activity through a NF-κB-dependent pathway. • Inhibition of oxidative stress by RA is dependent on nuclear translocation of Nrf2

  4. Endothelial cell permeability during hantavirus infection involves factor XII-dependent increased activation of the kallikrein-kinin system.

    Directory of Open Access Journals (Sweden)

    Shannon L Taylor

    Full Text Available Hemorrhagic fever with renal syndrome (HFRS and hantavirus pulmonary syndrome (HPS are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF. To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS. We show that incubation of factor XII (FXII, prekallikrein (PK, and high molecular weight kininogen (HK plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL and increased liberation of bradykinin (BK. Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS, we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation

  5. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

    Directory of Open Access Journals (Sweden)

    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  6. Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

    Science.gov (United States)

    Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

    2017-03-16

    Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

  7. Involvement of JAK2 upstream of the PI 3-kinase in cell-cell adhesion regulation by gastrin

    International Nuclear Information System (INIS)

    Ferrand, Audrey; Kowalski-Chauvel, Aline; Bertrand, Claudine; Pradayrol, Lucien; Fourmy, Daniel; Dufresne, Marlene; Seva, Catherine

    2004-01-01

    The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway has been implicated in cell transformation and proliferation. Besides aberrant cell proliferation, loss of cell-cell adhesion during epithelial-mesenchymal transition (EMT) is an important event which occurs during development of epithelial cancers. However, the role of JAK-dependent pathways in this process is not known. We analyzed the involvement of these pathways in the regulation of E-cadherin-dependent cell-cell adhesion by gastrin, a mitogenic factor for gastrointestinal (GI) tract. We identified JAK2/STAT3 as a new pathway in gastrin signaling. We demonstrated that JAK2 functions as an upstream mediator of the phosphatidylinositol 3 (PI 3)-kinase activity in gastrin signaling. Indeed, we observed a coprecipitation of both kinases and an inhibition of gastrin-induced PI 3-kinase activation when JAK2 activity is blocked. We also demonstrated that loss of cell-cell adhesion and the increase in cell motility induced by gastrin required the activation of JAK2 and the PI 3-kinase. Indeed, the modifications in localization of adherens junctions proteins and the migration, observed in gastrin-stimulated cells, were reversed by inhibition of both kinases. These results described the involvement of JAK2 in the modulation of cell-cell adhesion in epithelial cells. They support a possible role of JAK2 in the epithelial-mesenchymal transition which occurs during malignant development

  8. Discovery of a novel gene involved in autolysis of Clostridium cells.

    Science.gov (United States)

    Yang, Liejian; Bao, Guanhui; Zhu, Yan; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2013-06-01

    Cell autolysis plays important physiological roles in the life cycle of clostridial cells. Understanding the genetic basis of the autolysis phenomenon of pathogenic Clostridium or solvent producing Clostridium cells might provide new insights into this important species. Genes that might be involved in autolysis of Clostridium acetobutylicum, a model clostridial species, were investigated in this study. Twelve putative autolysin genes were predicted in C. acetobutylicum DSM 1731 genome through bioinformatics analysis. Of these 12 genes, gene SMB_G3117 was selected for testing the in tracellular autolysin activity, growth profile, viable cell numbers, and cellular morphology. We found that overexpression of SMB_G3117 gene led to earlier ceased growth, significantly increased number of dead cells, and clear electrolucent cavities, while disruption of SMB_G3117 gene exhibited remarkably reduced intracellular autolysin activity. These results indicate that SMB_G3117 is a novel gene involved in cellular autolysis of C. acetobutylicum.

  9. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    Directory of Open Access Journals (Sweden)

    Vivas Yurena

    2011-12-01

    Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

  10. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-05-29

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

    Science.gov (United States)

    Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

    2017-09-15

    Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

  12. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway

    Science.gov (United States)

    Pinsino, Annalisa; Russo, Roberta; Bonaventura, Rosa; Brunelli, Andrea; Marcomini, Antonio; Matranga, Valeria

    2015-01-01

    Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue. PMID:26412401

  13. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

    Science.gov (United States)

    Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-01

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression. PMID:26700461

  14. TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

    Science.gov (United States)

    Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

    2013-11-01

    The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John

  15. Clinical implications of mast cell involvement in allergic conjunctivitis.

    Science.gov (United States)

    Elieh Ali Komi, D; Rambasek, T; Bielory, L

    2018-03-01

    The conjunctiva is a common site for the allergic inflammatory response due to it being highly vascularized, having constant exposure to environmental pollutants and allergenic pollens and having a unique conjunctival associated lymphoid tissue. The primary morbidity of anterior surface conjunctival disorders that include allergic conjunctivitis and tear film disorders is associated with its high frequency of involvement rather than its severity, although the more chronic forms can involve the cornea and lead to sight-threatening conditions. Ocular allergy is associated with IgE-mediated mast cell activation in conjunctival tissue leading to the release of preformed mediators including histamine and proteases and subsequent de novo formation of lipid-derived mediators and cytokines that trigger a cascade of cellular and molecular events leading to extensive migration and infiltration of inflammatory cells to the ocular surface. The trafficking of neutrophils, eosinophils, and lymphocytes to the ocular surface is due to establishing various chemokine gradients (mainly CCL11, CCL24, CCL5, MCP-3, and MCP-4), cell surface expression of adhesion molecules (such as VCAM-1 the ligand for VLA-4), and leukocyte adhesion to vascular endothelium. The release of preformed mediators underlies the acute ocular surface response while the secondary influx of inflammatory cells leading to the recruitment and activation of eosinophils and the subsequent activation of Th2 and Th1 lymphocytes at the level of the conjunctiva reflects the late-phase reaction. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

  16. Activation of JNK and c-Jun is involved in glucose oxidase-mediated cell death of human lymphoma cells.

    Science.gov (United States)

    Son, Young-Ok; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

    2009-12-31

    Mitogen-activated protein kinases (MAPK) affect the activation of activator protein-1 (AP-1), which plays an important role in regulating a range of cellular processes. However, the roles of these signaling factors on hydrogen peroxide (H(2)O(2))-induced cell death are unclear. This study examined the effects of H(2)O(2) on the activation of MAPK and AP-1 by exposing the cells to H(2)O(2) generated by either glucose oxidase or a bolus addition. Exposing BJAB or Jurkat cells to H(2)O(2) affected the activities of MAPK differently according to the method of H(2)O(2) exposure. H(2)O(2) increased the AP-1-DNA binding activity in these cells, where continuously generated H(2)O(2) led to an increase in mainly the c-Fos, FosB and c-Jun proteins. The c-Jun-NH(2)-terminal kinase (JNK)-mediated activation of c-Jun was shown to be related to the H(2)O(2)-induced cell death. However, the suppression of H(2)O(2)-induced oxidative stress by either JNK inhibitor or c-Jun specific antisense transfection was temporary in the cells exposed to glucose oxidase but not to a bolus H(2)O(2). This was associated with the disruption of death signaling according to the severe and prolonged depletion of reduced glutathione. Overall, these results suggest that H(2)O(2) may decide differently the mode of cell death by affecting the intracellular redox state of thiol-containing antioxidants, and this depends more closely on the duration exposed to H(2)O(2) than the concentration of this agent.

  17. Activation of NF-κB is involved in 6-hydroxydopamine-but not MPP+-induced dopaminergic neuronal cell death: its potential role as a survival determinant

    International Nuclear Information System (INIS)

    Park, Seong H.; Choi, Won-Seok; Yoon, So-Young; Ahn, Young Soo; Oh, Young J.

    2004-01-01

    The nuclear factor-kappaB (NF-κB) family plays an important role in the control of the apoptotic response. Its activation has been demonstrated in both neurons and glial cells in many neurological disorders. In the present study, we specifically examined whether and to what extent NF-κB activation is involved in culture models of Parkinson's disease following exposure of MN9D dopaminergic neuronal cells to 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-4-phenylpyridinium ion (MPP + ). Both analysis by immunocytochemistry and of immunoblots revealed that NF-κB-p65 was translocated into the nuclei following 6-OHDA but not MPP + -treatment. A time-dependent activation of NF-κB induced by 6-OHDA but not MPP + was also demonstrated by an electrophoretic mobility shift assay. A competition assay indicated that not only NF-κB-p65 but also -p50 is involved in 6-OHDA-induced NF-κB activity. Co-treatment with an antioxidant, N-acetyl-L-cysteine, blocked 6-OHDA-induced activation of NF-κB signaling. In the presence of an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), 6-OHDA-induced cell death was accelerated while PDTC did not affect MPP + -induced cell death. Our data may point to a drug-specific activation of NF-κB as a survival determinant for dopaminergic neurons

  18. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    DEFF Research Database (Denmark)

    Knoch, Eva

    and tissues, their functions and synthesis are still poorly understood. The aim of the research presented in the thesis was to characterize carbohydrate active enzymes involved in AGP biosynthesis and modification to gain insights into the biosynthesis of the glycoproteins in plants. Candidate...... glycosyltransferases and glycoside hydrolases were selected based on co-expression profiles from a transcriptomics analysis. Reverse genetics approach on a novel glucuronosyltransferase involved in AGP biosynthesis has revealed that the enzyme activity is required for normal cell elongation in etiolated seedlings....... The enzymatic activity of a hydrolase from GH family 17 was investigated, without successful determination of the activity. Members of hydrolase family 43 appeared to be localized in the Golgi-apparatus, which is also the compartment for glycan biosynthesis. The localization of these glycoside hydrolases...

  19. Hindbrain medulla catecholamine cell group involvement in lactate-sensitive hypoglycemia-associated patterns of hypothalamic norepinephrine and epinephrine activity.

    Science.gov (United States)

    Shrestha, P K; Tamrakar, P; Ibrahim, B A; Briski, K P

    2014-10-10

    Cell-type compartmentation of glucose metabolism in the brain involves trafficking of the oxidizable glycolytic end product, l-lactate, by astrocytes to fuel neuronal mitochondrial aerobic respiration. Lactate availability within the hindbrain medulla is a monitored function that regulates systemic glucostasis as insulin-induced hypoglycemia (IIH) is exacerbated by lactate repletion of that brain region. A2 noradrenergic neurons are a plausible source of lactoprivic input to the neural gluco-regulatory circuit as caudal fourth ventricular (CV4) lactate infusion normalizes IIH-associated activation, e.g. phosphorylation of the high-sensitivity energy sensor, adenosine 5'-monophosphate-activated protein kinase (AMPK), in these cells. Here, we investigated the hypothesis that A2 neurons are unique among medullary catecholamine cells in directly screening lactate-derived energy. Adult male rats were injected with insulin or vehicle following initiation of continuous l-lactate infusion into the CV4. Two hours after injections, A1, C1, A2, and C2 neurons were collected by laser-microdissection for Western blot analysis of AMPKα1/2 and phosphoAMPKα1/2 proteins. Results show that AMPK is expressed in each cell group, but only a subset, e.g. A1, C1, and A2 neurons, exhibit increased sensor activity in response to IIH. Moreover, hindbrain lactate repletion reversed hypoglycemic augmentation of pAMPKα1/2 content in A2 and C1 but not A1 cells, and normalized hypothalamic norepinephrine and epinephrine content in a site-specific manner. The present evidence for discriminative reactivity of AMPK-expressing medullary catecholamine neurons to the screened energy substrate lactate implies that that lactoprivation is selectively signaled to the hypothalamus by A2 noradrenergic and C1 adrenergic cells. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Signal transduction pathways involved in mechanotransduction in bone cells

    International Nuclear Information System (INIS)

    Liedert, Astrid; Kaspar, Daniela; Blakytny, Robert; Claes, Lutz; Ignatius, Anita

    2006-01-01

    Several in vivo and in vitro studies with different loading regimens showed that mechanical stimuli have an influence on proliferation and differentiation of bone cells. Prerequisite for this influence is the transduction of mechanical signals into the cell, a phenomenon that is termed mechanotransduction, which is essential for the maintenance of skeletal homeostasis in adults. Mechanoreceptors, such as the integrins, cadherins, and stretch-activated Ca 2+ channels, together with various signal transduction pathways, are involved in the mechanotransduction process that ultimately regulates gene expression in the nucleus. Mechanotransduction itself is considered to be regulated by hormones, the extracellular matrix of the osteoblastic cells and the mode of the mechanical stimulus

  1. The Fas pathway is involved in pancreatic beta cell secretory function

    DEFF Research Database (Denmark)

    Schumann, Desiree M; Maedler, Kathrin; Franklin, Isobel

    2007-01-01

    Pancreatic beta cell mass and function increase in conditions of enhanced insulin demand such as obesity. Failure to adapt leads to diabetes. The molecular mechanisms controlling this adaptive process are unclear. Fas is a death receptor involved in beta cell apoptosis or proliferation, depending...... on the activity of the caspase-8 inhibitor FLIP. Here we show that the Fas pathway also regulates beta cell secretory function. We observed impaired glucose tolerance in Fas-deficient mice due to a delayed and decreased insulin secretory pattern. Expression of PDX-1, a beta cell-specific transcription factor...... regulating insulin gene expression and mitochondrial metabolism, was decreased in Fas-deficient beta cells. As a consequence, insulin and ATP production were severely reduced and only partly compensated for by increased beta cell mass. Up-regulation of FLIP enhanced NF-kappaB activity via NF...

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

    Directory of Open Access Journals (Sweden)

    Cristina Antonella Nadalutti

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

  3. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation

    International Nuclear Information System (INIS)

    Chou, C.-T.; He Shiping; Jan, C.-R.

    2007-01-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca 2+ ] i increases which involved the mobilization of intracellular Ca 2+ stored in the endoplasmic reticulum and Ca 2+ influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca 2+ chelator, to prevent paroxetine-induced [Ca 2+ ] i increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca 2+ -independent apoptosis via inducing p38 MAPK-associated caspase-3 activation

  4. Dengue Virus-Infected Dendritic Cells, but Not Monocytes, Activate Natural Killer Cells through a Contact-Dependent Mechanism Involving Adhesion Molecules.

    Science.gov (United States)

    Costa, Vivian Vasconcelos; Ye, Weijian; Chen, Qingfeng; Teixeira, Mauro Martins; Preiser, Peter; Ooi, Eng Eong; Chen, Jianzhu

    2017-08-01

    Natural killer (NK) cells play a protective role against dengue virus (DENV) infection, but the cellular and molecular mechanisms are not fully understood. Using an optimized humanized mouse model, we show that human NK cells, through the secretion of gamma interferon (IFN-γ), are critical in the early defense against DENV infection. Depletion of NK cells or neutralization of IFN-γ leads to increased viremia and more severe thrombocytopenia and liver damage in humanized mice. In vitro studies using autologous human NK cells show that DENV-infected monocyte-derived dendritic cells (MDDCs), but not monocytes, activate NK cells in a contact-dependent manner, resulting in upregulation of CD69 and CD25 and secretion of IFN-γ. Blocking adhesion molecules (LFA-1, DNAM-1, CD2, and 2β4) on NK cells abolishes NK cell activation, IFN-γ secretion, and the control of DENV replication. NK cells activated by infected MDDCs also inhibit DENV infection in monocytes. These findings show the essential role of human NK cells in protection against acute DENV infection in vivo , identify adhesion molecules and dendritic cells required for NK cell activation, and delineate the sequence of events for NK cell activation and protection against DENV infection. IMPORTANCE Dengue is a mosquito-transmitted viral disease with a range of symptoms, from mild fever to life-threatening dengue hemorrhagic fever. The diverse disease manifestation is thought to result from a complex interplay between viral and host factors. Using mice engrafted with a human immune system, we show that human NK cells inhibit virus infection through secretion of the cytokine gamma interferon and reduce disease pathogenesis, including depletion of platelets and liver damage. During a natural infection, DENV initially infects dendritic cells in the skin. We find that NK cells interact with infected dendritic cells through physical contact mediated by adhesion molecules and become activated before they can control

  5. Activation of ion transport systems during cell volume regulation

    International Nuclear Information System (INIS)

    Eveloff, J.L.; Warnock, D.G.

    1987-01-01

    This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na + -H + and Cl - -HCO 3 - exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K + and Cl - conductances, a K-Cl cotransport system, or parallel K + -H + and Cl - -HCO 3 - exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca 2+ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that transverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na + -H + and Cl - -HCO 3 - exchange systems

  6. Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

    Science.gov (United States)

    Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

    2010-12-15

    Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    Science.gov (United States)

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  8. The Inhibition of microRNA-128 on IGF-1-Activating mTOR Signaling Involves in Temozolomide-Induced Glioma Cell Apoptotic Death.

    Directory of Open Access Journals (Sweden)

    Peng-Hsu Chen

    Full Text Available Temozolomide (TMZ, an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug used in the clinical therapy of glioblastoma multiforme, the most common and high-grade primary glioma in adults. Micro (miRNAs, which are small noncoding RNAs, post-transcriptionally regulate gene expressions and are involved in gliomagenesis. However, no studies have reported relationships between TMZ and miRNA gene regulation. We investigated TMZ-mediated miRNA profiles and its molecular mechanisms underlying the induction of glioma cell death. By performing miRNA microarray and bioinformatics analyses, we observed that expression of 248 miRNAs was altered, including five significantly upregulated and 17 significantly downregulated miRNAs, in TMZ-treated U87MG cells. miR-128 expression levels were lower in different glioma cells and strongly associated with poor survival. TMZ treatment significantly upregulated miR-128 expression. TMZ significantly enhanced miR-128-1 promoter activity and transcriptionally regulated miR-128 levels through c-Jun N-terminal kinase 2/c-Jun pathways. The overexpression and knockdown of miR-128 expression significantly affected TMZ-mediated cell viability and apoptosis-related protein expression. Furthermore, the overexpression of miR-128 alone enhanced apoptotic death of glioma cells through caspase-3/9 activation, poly(ADP ribose polymerase degradation, reactive oxygen species generation, mitochondrial membrane potential loss, and non-protective autophagy formation. Finally, we identified that key members in mammalian target of rapamycin (mTOR signaling including mTOR, rapamycin-insensitive companion of mTOR, insulin-like growth factor 1, and PIK3R1, but not PDK1, were direct target genes of miR-128. TMZ inhibited mTOR signaling through miR-128 regulation. These results indicate that miR-128-inhibited mTOR signaling is involved in TMZ-mediated cytotoxicity. Our findings may provide a better understanding

  9. Involvement of CD244 in regulating CD4+ T cell immunity in patients with active tuberculosis.

    Directory of Open Access Journals (Sweden)

    Bingfen Yang

    Full Text Available CD244 (2B4 is a member of the signaling lymphocyte activation molecule (SLAM family of immune cell receptors and it plays an important role in modulating NK cell and CD8(+ T cell immunity. In this study, we investigated the expression and function of CD244/2B4 on CD4(+ T cells from active TB patients and latent infection individuals. Active TB patients had significantly elevated CD244/2B4 expression on M. tuberculosis antigen-specific CD4(+ T cells compared with latent infection individuals. The frequencies of CD244/2B4-expressing antigen-specific CD4(+ T cells were significantly higher in retreatment active TB patients than in new active TB patients. Compared with CD244/2B4-dull and -middle CD4(+ T cells, CD244/2B4-bright CD4(+ T cell subset had significantly reduced expression of IFN-γ, suggesting that CD244/2B4 expression may modulate IFN-γ production in M. tuberculosis antigen-responsive CD4(+ T cells. Activation of CD244/2B4 signaling by cross-linking led to significantly decreased production of IFN-γ. Blockage of CD244/2B4 signaling pathway of T cells from patients with active TB resulted in significantly increased production of IFN-γ, compared with isotype antibody control. In conclusion, CD244/2B4 signaling pathway has an inhibitory role on M. tuberculosis antigen-specific CD4(+ T cell function.

  10. Involvement of regulatory T cells and selected cytokines in the pathogenesis of bronchial asthma

    Directory of Open Access Journals (Sweden)

    Monika Zuśka-Prot

    2016-06-01

    Full Text Available Asthma pathogenesis is complex and involves the interplay of many factors and actions. Airway inflammation in allergic asthma is characterized by an exaggerated activation of T helper type 2 cells, IgE production and infiltration and activation of eosinophils. The results of studies conducted in recent years indicate that the deficit of naturally occurring Foxp3+CD25+CD4+ and Foxp3+CD25+CD8+ regulatory T cells and type 1 regulatory T cells plays a pivotal role in the development of this disease. Moreover, numerous studies have provided convincing evidence that a decrease in IL-10 production and an increase in IL-17 production have an important place in the pathophysiology of asthma. TGF-β is another important cytokine involved in this disease. TGF-β has a paradoxical status in relation to asthma pathogenesis because it seems to play a role in both suppressing and promoting asthma development. This review discusses briefly clinical and experimental data concerning the involvement of T regulatory cells and IL-10, IL-17 and TGF-β in the pathogenesis of asthma.

  11. POLYGALACTURONASE INVOLVED IN EXPANSION1 functions in cell elongation and flower development in Arabidopsis.

    Science.gov (United States)

    Xiao, Chaowen; Somerville, Chris; Anderson, Charles T

    2014-03-01

    Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1's involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.

  12. Supporting Active User Involvment in Prototyping

    DEFF Research Database (Denmark)

    Grønbæk, Kaj

    1990-01-01

    The term prototyping has in recent years become a buzzword in both research and practice of system design due to a number of claimed advantages of prototyping techniques over traditional specification techniques. In particular it is often stated that prototyping facilitates the users' involvement...... in the development process. But prototyping does not automatically imply active user involvement! Thus a cooperative prototyping approach aiming at involving users actively and creatively in system design is proposed in this paper. The key point of the approach is to involve users in activities that closely couple...... development of prototypes to early evaluation of prototypes in envisioned use situations. Having users involved in such activities creates new requirements for tool support. Tools that support direct manipulation of prototypes and simulation of behaviour have shown promise for cooperative prototyping...

  13. Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

    Energy Technology Data Exchange (ETDEWEB)

    Shibata, Ayano; Tanabe, Eriko; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2013-04-12

    Highlights: •Hydrogen peroxide stimulates cell motility of WB-F344 cells. •LPA{sub 3} is induced by hydrogen peroxide in WB-F344 cells. •Cell motility by hydrogen peroxide is inhibited in LPA{sub 3} knockdown cells. •LPA signaling is involved in cell migration by hydrogen peroxide. -- Abstract: Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA{sub 3} on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA{sub 3} may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.

  14. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  15. Human retinal pigment epithelial cells inhibit proliferation and IL2R expression of activated T cells

    DEFF Research Database (Denmark)

    Kaestel, Charlotte G; Jørgensen, Annette; Nielsen, Mette

    2002-01-01

    -Thymidine incorporation assay, respectively. T cells and RPE cells were cultured directly together or in a transwell system for determination of the effect of cell contact. The importance of cell surface molecules was examined by application of a panel of blocking antibodies (CD2, CD18, CD40, CD40L, CD54, CD58......) in addition to use of TCR negative T cell lines. The expression of IL2R-alpha -beta and -gamma chains of activated T cells was analysed by flow cytometry after incubation of T cells alone or with RPE cells. Human RPE cells were found to inhibit the proliferation of activated T cells by a cell contact......-beta and -gamma chain expression within 24 hr after removal from the coculture. It is concluded that the cultured human adult and foetal RPE cells inhibit the proliferation of activated T cells by a process that does not involve apoptosis. It depends on cell contact but the involved surface molecules were...

  16. Involvement of Lgl and Mahjong/VprBP in cell competition.

    Directory of Open Access Journals (Sweden)

    Yoichiro Tamori

    2010-07-01

    Full Text Available During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby "loser cells" undergo apoptosis are not clearly understood. Lgl (lethal giant larvae is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj(-/- do not have obvious patterning defects during embryonic or larval development. However, mahj(-/- cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl(-/- cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK is increased in mahj(-/- or lgl(-/- mutant cells, and expression of Puckered (Puc, an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl(-/- cells strongly suppresses JNK activation and blocks apoptosis of lgl(-/- cells in the wild

  17. Genes involved in cell division in mycoplasmas

    Directory of Open Access Journals (Sweden)

    Frank Alarcón

    2007-01-01

    Full Text Available Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

  18. Langerhans Cell Histiocytosis Involving Maxilla and Mandible

    Directory of Open Access Journals (Sweden)

    M. Guna Shekhar

    2009-06-01

    Full Text Available Langerhans cell histiocytosis is a relatively rare unique disease process characterized by an abnormal proliferation of immature dendritic cells usually affecting children and young adults. Jaws are involved in less than 10% of children with the disease while mandibular involvement in young children is uncommon and bilateral affection is very rare. The purpose of this report is to describe a unique and very rare case of simultaneous and bilateral occurrence of Langerhans cell histiocytosis in both the jaws of a four-year-old boy.

  19. Obesity-induced vascular inflammation involves elevated arginase activity.

    Science.gov (United States)

    Yao, Lin; Bhatta, Anil; Xu, Zhimin; Chen, Jijun; Toque, Haroldo A; Chen, Yongjun; Xu, Yimin; Bagi, Zsolt; Lucas, Rudolf; Huo, Yuqing; Caldwell, Ruth B; Caldwell, R William

    2017-11-01

    Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-( S )-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 μM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

  20. Mechanisms involved in alternariol-induced cell cycle arrest

    Energy Technology Data Exchange (ETDEWEB)

    Solhaug, A., E-mail: Anita.Solhaug@vetinst.no [Norwegian Veterinary Institute, Oslo (Norway); Vines, L.L. [Michigan State University, Department of Food Science and Human Nutrition, East Lansing, MI (United States); Ivanova, L.; Spilsberg, B. [Norwegian Veterinary Institute, Oslo (Norway); Holme, J.A. [Norwegian Institute of Public Health, Division of Environmental Medicine, Oslo (Norway); Pestka, J. [Michigan State University, Department of Food Science and Human Nutrition, East Lansing, MI (United States); Collins, A. [University of Oslo, Department of Nutrition, Faculty of Medicine, Oslo (Norway); Eriksen, G.S. [Norwegian Veterinary Institute, Oslo (Norway)

    2012-10-15

    Alternariol (AOH), a mycotoxin produced by Alternaria sp, is often found as a contaminant in fruit and cereal products. Here we employed the murine macrophage cell line RAW 264.7 to test the hypothesis that AOH causes toxicity as a response to DNA damage. AOH at concentrations of 15-30 {mu}M almost completely blocked cell proliferation. Within 30 min treatment, AOH (30 {mu}M) significantly increased the level of reactive oxygen species (ROS). Furthermore, DNA base oxidations as well as DNA strand breaks and/or alkaline labile sites were detected by the comet assay after 2 h exposure of AOH. Cell death (mostly necrosis) was observed after prolonged exposure to the highest concentration of AOH (60 {mu}M for 24 and 48 h) in our study. The DNA damage response involved phosphorylation (activation) of histone H2AX and check point kinase-1- and 2 (Chk-1/2). Moreover, AOH activated p53 and increased the expression of p21, Cyclin B, MDM2, and Sestrin 2; likewise the level of several miRNA was affected. AOH-induced Sestrin 2 expression was regulated by p53 and could at least partly be inhibited by antioxidants, suggesting a role of ROS in the response. Interestingly, the addition of antioxidants did not inhibit cell cycle arrest. Although the formation of ROS by itself was not directly linked cell proliferation, AOH-induced DNA damage and resulting transcriptional changes in p21, MDM2, and Cyclin B likely contribute to the reduced cell proliferation; while Sestrin 2 would contribute to the oxidant defense.

  1. Mechanisms involved in alternariol-induced cell cycle arrest

    International Nuclear Information System (INIS)

    Solhaug, A.; Vines, L.L.; Ivanova, L.; Spilsberg, B.; Holme, J.A.; Pestka, J.; Collins, A.; Eriksen, G.S.

    2012-01-01

    Alternariol (AOH), a mycotoxin produced by Alternaria sp, is often found as a contaminant in fruit and cereal products. Here we employed the murine macrophage cell line RAW 264.7 to test the hypothesis that AOH causes toxicity as a response to DNA damage. AOH at concentrations of 15–30 μM almost completely blocked cell proliferation. Within 30 min treatment, AOH (30 μM) significantly increased the level of reactive oxygen species (ROS). Furthermore, DNA base oxidations as well as DNA strand breaks and/or alkaline labile sites were detected by the comet assay after 2 h exposure of AOH. Cell death (mostly necrosis) was observed after prolonged exposure to the highest concentration of AOH (60 μM for 24 and 48 h) in our study. The DNA damage response involved phosphorylation (activation) of histone H2AX and check point kinase-1- and 2 (Chk-1/2). Moreover, AOH activated p53 and increased the expression of p21, Cyclin B, MDM2, and Sestrin 2; likewise the level of several miRNA was affected. AOH-induced Sestrin 2 expression was regulated by p53 and could at least partly be inhibited by antioxidants, suggesting a role of ROS in the response. Interestingly, the addition of antioxidants did not inhibit cell cycle arrest. Although the formation of ROS by itself was not directly linked cell proliferation, AOH-induced DNA damage and resulting transcriptional changes in p21, MDM2, and Cyclin B likely contribute to the reduced cell proliferation; while Sestrin 2 would contribute to the oxidant defense.

  2. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  3. δ-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    International Nuclear Information System (INIS)

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-01-01

    δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen 2,5 ]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  4. Activated NKT cells imprint NK-cell differentiation, functionality and education.

    Science.gov (United States)

    Riese, Peggy; Trittel, Stephanie; May, Tobias; Cicin-Sain, Luka; Chambers, Benedict J; Guzmán, Carlos A

    2015-06-01

    NK cells represent a vital component of the innate immune system. The recent discoveries demonstrating that the functionality of NK cells depends on their differentiation and education status underscore their potential as targets for immune intervention. However, to exploit their full potential, a detailed understanding of the cellular interactions involved in these processes is required. In this regard, the cross-talk between NKT cells and NK cells needs to be better understood. Our results provide strong evidence for NKT cell-induced effects on key biological features of NK cells. NKT-cell activation results in the generation of highly active CD27(high) NK cells with improved functionality. In this context, degranulation activity and IFNγ production were mainly detected in the educated subset. In a mCMV infection model, we also demonstrated that NKT-cell stimulation induced the generation of highly functional educated and uneducated NK cells, crucial players in viral control. Thus, our findings reveal new fundamental aspects of the NKT-NK cell axis that provide important hints for the manipulation of NK cells in clinical settings. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Hongyu Zhang

    2015-09-01

    Full Text Available Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB, we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP activity in MSCs as well as late osteogenic markers osteopontin (OPN, osteocalcin (OCN and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.

  6. Outer membrane protein A (OmpA of Shigella flexneri 2a induces TLR2-mediated activation of B cells: involvement of protein tyrosine kinase, ERK and NF-κB.

    Directory of Open Access Journals (Sweden)

    Rajsekhar Bhowmick

    Full Text Available B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs. The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs, ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an

  7. Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation

    International Nuclear Information System (INIS)

    Moretta, A.; Poggi, A.; Olive, D.; Bottino, C.; Fortis, C.; Pantaleo, G.; Moretta, L.

    1987-01-01

    A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3 + , Ti + , T44 + , T11 + , T40 + ) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of γ-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, negative cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 - Ti - T44 + Leu1 + T11 + T40 + 4F2 + HLA class I + surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3 - Ti - T44 - Leu1 - T11 - T40 - 4F2 - HLA class I + phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate

  8. Activation-induced cell death of dendritic cells is dependent on sphingosine kinase 1

    Directory of Open Access Journals (Sweden)

    Anja eSchwiebs

    2016-04-01

    Full Text Available Sphingosine 1-phosphate (S1P is an immune modulatory lipid mediator and has been implicated in numerous pathophysiological processes. S1P is produced by sphingosine kinase 1 (Sphk1 and Sphk2. Dendritic cells (DCs are central for the direction of immune responses and crucially involved in autoimmunity and cancerogenesis. In this study we examined the function and survival of bone marrow-derived DCs under long-term inflammatory stimulation. We observed that differentiated cells undergo activation-induced cell death upon LPS stimulation with an increased metabolic activity shortly after stimulation, followed by a rapid activation of caspase 3 and subsequent augmented apoptosis. Importantly, we highlight a profound role of Sphk1 in secretion of inflammatory cytokines and survival of dendritic cells that might be mediated by a change in sphingolipid levels as well as by a change in STAT3 expression. Cell growth during differentiation of Sphk1-deficient cells treated with the functional S1P receptor antagonist FTYP was reduced. Importantly, in dendritic cells we did not observe a compensatory regulation of Sphk2 mRNA in Sphk1-deficient cells. Instead, we discovered a massive increase in Sphk1 mRNA concentration upon long-term stimulation with LPS in wild type cells that might function as an attempt to rescue from inflammation-caused cell death. Taken together, in this investigation we describe details of a crucial involvement of sphingolipids and Sphk1 in activation-induced cell death during long-term immunogenic activity of DCs that might play an important role in autoimmunity and might explain the differences in immune response observed in in vivo studies of Sphk1 modulation.

  9. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

    Science.gov (United States)

    Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV ) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

  10. Chemokines: a new dendritic cell signal for T cell activation

    Directory of Open Access Journals (Sweden)

    Christoph A Thaiss

    2011-08-01

    Full Text Available Dendritic cells (DCs are the main inducers and regulators of cytotoxic T lymphocyte (CTL responses against viruses and tumors. One checkpoint to avoid misguided CTL activation, which might damage healthy cells of the body, is the necessity for multiple activation signals, involving both antigenic as well as additional signals that reflect the presence of pathogens. DCs provide both signals when activated by ligands of pattern recognition receptors and licensed by helper lymphocytes. Recently, it has been established that such T cell licensing can be facilitated by CD4+ T helper cells (classical licensing or by NKT cells (alternative licensing. Licensing regulates the DC/CTL cross-talk at multiple layers. Direct recruitment of CTLs through chemokines released by licensed DCs has recently emerged as a common theme and has a crucial impact on the efficiency of CTL responses. Here, we discuss recent advances in our understanding of DC licensing for cross-priming and implications for the temporal and spatial regulation underlying this process. Future vaccination strategies will benefit from a deeper insight into the mechanisms that govern CTL activation.

  11. Ion Channels Involved in Cell Volume Regulation

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay

    2011-01-01

    regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation......This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume...

  12. Immunoregulatory T Cells May Be Involved in Preserving CD4 T Cell Counts in HIV-Infected Long-Term Nonprogressors and Controllers

    DEFF Research Database (Denmark)

    Gaardbo, Julie C; Ronit, Andreas; Hartling, Hans J

    2014-01-01

    BACKGROUND: HIV-infected controllers control viral replication and maintain normal CD4 T cell counts. Long-term nonprogressors (LTNPs) also maintain normal CD4 T cell counts but have ongoing viral replication. We hypothesized that immunoregulatory mechanisms are involved in preserved CD4 T cell...... of patients and controls. However, both ECs and LTNPs displayed a large proportion of activated Tregs suggesting immunoregulatory mechanisms to be involved in preserving CD4 T cell counts in HIV-infected nonprogressors....... counts in controllers and in LTNPs. METHODS: Twenty HIV-infected viremic controllers, 5 elite controllers (ECs), and 14 LTNPs were included in this cross-sectional study. For comparison, 25 progressors and 34 healthy controls were included. Regulatory T cells (Tregs), Treg subpopulations, CD161+Th17...

  13. TSA-induced cell death in prostate cancer cell lines is caspase-2 dependent and involves the PIDDosome.

    Science.gov (United States)

    Taghiyev, Agshin F; Guseva, Natalya V; Glover, Rebecca A; Rokhlin, Oskar W; Cohen, Michael B

    2006-09-01

    The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

  14. Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Diego Sanchez-Martinez

    2016-10-01

    Full Text Available Mutational status of TP53 together with expression of wild type (wt IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL patients. Adoptive cell therapy using allogeneic HLA mismatched Natural Killer (NK cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs the most effective stimulus to activate NK cells. Here we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell activating receptors (NKG2D and NCRs and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.□

  15. Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

    Science.gov (United States)

    Dennig, Jörg

    Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

  16. CD44 is involved in mineralization of dental pulp cells.

    Science.gov (United States)

    Chen, Kuan-Liang; Huang, Yu-Yuan; Lung, Jrhau; Yeh, Ying-Yi; Yuan, Kuo

    2013-03-01

    CD44 is a transmembrane glycoprotein with various biological functions. Histologic studies have shown that CD44 is strongly expressed in odontoblasts at the appositional stage of tooth development. We investigated whether CD44 is involved in the mineralization of dental pulp cells. Ten human third molars with incomplete root formation were collected and processed for immunohistochemistry of CD44. Dental pulp cells isolated from another 5 human third molars were assayed for their viability, alkaline phosphatase activity, and alizarin red staining in vitro after silencing stably their expression of CD44 by using the short hairpin RNA technique. The CD44 knockdown cells were cultured on a collagen sponge and transplanted subcutaneously into the dorsal surfaces of immunocompromised mice. After 6 weeks, the subcutaneous tissues were processed for alizarin red staining and immunohistochemistry of human specific antigen. The dental pulp cells transduced with control short hairpin RNA were used as the control in all assays. CD44 is expressed in odontogenic cells with active mineral deposition during tooth development. Odontoblasts in the root ends of immature teeth express a stronger CD44 signal compared with those in the crown portion. When CD44 expression was stably suppressed in dental pulp cells, their mineralization activities were substantially decreased in both in vitro and in vivo assays. CD44 may play a crucial role in the initial mineralization of tooth-associated structures. However, further studies are required to clarify the underlying mechanisms. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  17. Leukotriene B4 induces EMT and vimentin expression in PANC-1 pancreatic cancer cells: Involvement of BLT2 via ERK2 activation.

    Science.gov (United States)

    Kim, You Ri; Park, Mi Kyung; Kang, Gyeong Jin; Kim, Hyun Ji; Kim, Eun Ji; Byun, Hyun Jung; Lee, Moo-Yeol; Lee, Chang Hoon

    2016-12-01

    Leukotriene B 4 (LTB 4 ) is a leukocyte chemoattractant and plays a major role controlling inflammatory responses including pancreatitis. LTB 4 is known to be correlated with cancer progression. LTB 4 induces keratin phosphorylation and reorganization by activating extracellular regulated kinase (ERK) in PANC-1 pancreatic cancer cell lines. However, the role of LTB 4 in epithelial mesenchymal transition (EMT) and vimentin expression in pancreatic cancer cells is unknown. We examined whether LTB 4 induces EMT and vimentin expression by Western blot, si-RNA, and RT-PCR. LTB 4 induced morphological change, decreased E-cadherin expression and increased N-cadherin and vimentin expression. LTB4 increased migration and invasion of PANC-1 cancer cells. LTB 4 dose-dependently upregulated expression of vimentin in PANC-1 cancer cells. LTB 4 -induced vimentin expression was suppressed by LY255283 (BLT2 antagonist). Comp A, a BLT2 agonist, further increased vimentin expression. Gene silencing of BLT2 suppressed LTB 4 -or Comp A-induced vimentin expression in PANC-1 cells. The MEK inhibitor, PD98059 suppressed Comp A-induced vimentin expression. Comp A or transfection of plasmid containing BLT2 cDNA (pC BLT2 ) activated ERK, and BLT2 gene silencing suppressed Comp A-induced ERK activation. ERK2 siRNA abrogated Comp A-induced vimentin expression and ERK2 overexpression enhanced vimentin expression. One of well-known cause of ras mutation, cigarette smoke extracts increased BLT2 expression in PANC-1 cancer cells. Taken together, these results suggest that BLT2 is involved in LTB 4 -induced vimentin expression through ERK2 in PANC-1 cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Liver involvement in Langerhans cell histiocytosis

    International Nuclear Information System (INIS)

    Wong, Adelaine; Ortiz-Neira, Clara L.; Abou Reslan, Walid; Kaura, Deepak; Sharon, Raphael; Anderson, Ronald; Pinto-Rojas, Alfredo

    2006-01-01

    Liver involvement in Langerhans cell histiocytosis (LCH) typically presents with hepatomegaly and other signs of liver dysfunction. We present an 11-month-old child having only minimally elevated liver enzymes as an indication of liver involvement. Using sonography as the initial diagnostic tool followed by MRI, LCH of the liver was revealed. A review of sonographic, CT, MRI and MR cholangiopancreatography findings in liver LCH is presented. We recommend that physicians consider sonography and MRI screening for liver involvement in patients with newly diagnosed LCH, as periportal involvement may be present with little or no liver function abnormality present, as in this patient. (orig.)

  19. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    OpenAIRE

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs ...

  20. Increased cell surface metallopeptidase activity in cells undergoing UV-induced apoptosis

    International Nuclear Information System (INIS)

    Piva, T.J.; Davern, C.M.; Ellem, K.A.O.

    1999-01-01

    Full text: We have previously shown that UVC irradiation activated a range of cell surface peptidases (CSP) in HeLa cell monolayer cultures 20 h post-irradiation (1). In cells undergoing apoptosis there is an increase in CSP activity compared to control viable cells in cultures which have been treated by a wide range of agents including UV-irradiation (2). In order to further understand the mechanism involved in this process, we induced apoptosis in HeLa cells using 500 Jm -2 UVB. The separation of viable, apoptotic and necrotic cells of irradiated HeLa cell cultures was made by FACS analysis and sorting. The three populations were distinguished by their staining with PI and Hoechst 33342 dyes. CSP activity was measured using the P9 assay developed in this laboratory (1-3). The viable fraction of the irradiated cells had a higher level of CSP activity compared to unirradiated controls. The level of CSP activity in the apoptotic fraction was higher than that of the viable fraction, however that of the necrotic fraction was significantly lower. This finding agreed with that seen in UVC-irradiated (50 Jm -2 ) cultures (2). In order to elucidate the mechanism by which CSP activity was increased in UVB-irradiated cells undergoing apoptosis, the cultures were treated with the following agents: bestatin, aminopeptidase inhibitor, DEVD, caspase 3 inhibitor, and 3-aminobenzamide (3AB), PARP activation inhibitor. Bestatin and DEVD did not affect the level of CSP activity in the different cell subpopulations following UVB-irradiation. Treatment with 3AB abolished the increased CSP activity seen in the viable and apoptotic fraction following UVB-irradiation. All treated cells had the same morphology as observed under EM. The degree of phosphatidylserine eversion on the cell membrane was similar as were the cleavage profiles of PARP and actin. Only DEVD-treated cells had reduced caspase 3 activity which confirmed that the activation of CSP activity in apoptotic cells is

  1. Immune receptors involved in Streptococcus suis recognition by dendritic cells.

    Directory of Open Access Journals (Sweden)

    Marie-Pier Lecours

    Full Text Available Streptococcus suis is an important swine pathogen and an emerging zoonotic agent of septicemia and meningitis. Knowledge on host immune responses towards S. suis, and strategies used by this pathogen for subversion of these responses is scarce. The objective of this study was to identify the immune receptors involved in S. suis recognition by dendritic cells (DCs. Production of cytokines and expression of co-stimulatory molecules by DCs were shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize S. suis and become activated mostly through Toll-like receptor (TLR signaling. Supporting this fact, TLR2(-/- DCs were severely impaired in the release of several cytokines and the surface expression of CD86 and MHC-II. The release of IL-12p70 and CXC10, and the expression of CD40 were found to depend on signaling by both TLR2 and TLR9. The release of IL-23 and CXCL1 were partially dependent on NOD2. Finally, despite the fact that MyD88 signaling was crucial for DC activation and maturation, MyD88-dependent pathways were not implicated in S. suis internalization by DCs. This first study on receptors involved in DC activation by S. suis suggests a major involvement of MyD88 signaling pathways, mainly (but not exclusively through TLR2. A multimodal recognition involving a combination of different receptors seems essential for DC effective response to S. suis.

  2. Liver involvement in Langerhans' cell histiocytosis. Case report.

    Science.gov (United States)

    Dina, Ion; Copaescu, Catalin; Herlea, Vlad; Wrba, Fritz; Iacobescu, Claudia

    2006-03-01

    Langerhans'cell histiocytosis (Histiocytosis X) is a rare disease of unknown cause characterized by oligoclonal proliferation of Langerhans cells. It occurs mostly in children and young adults and involves one or more body systems such as bone, hypothalamus, posterior pituitary gland, lymph nodes, liver or various soft tissues. The diagnosis is always made by a histological approach. We report a case of Langerhans'cell histiocytosis in a young patient with clinical signs of diabetes insipidus and hepatic involvement in whom the immunohistochemical analysis of the liver tissue led to the definitive diagnosis.

  3. IGF-II-mediated downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α in myoblast cells involves PI3K/Akt/FoxO1 signaling pathway.

    Science.gov (United States)

    Mu, Xiaoyu; Qi, Weihong; Liu, Yunzhang; Zhou, Jianfeng; Li, Yun; Rong, Xiaozhi; Lu, Ling

    2017-08-01

    Insulin-like growth factor II (IGF-II) can stimulate myogenesis and is critically involved in skeletal muscle differentiation. The presence of negative regulators of this process, however, is not well explored. Here, we showed that in myoblast cells, IGF-II negatively regulated peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA expression, while constitutive expression of PGC-1α induced myoblast differentiation. These results suggest that the negative regulation of PGC-1α by IGF-II may act as a negative feedback mechanism in IGF-II-induced myogenic differentiation. Reporter assays demonstrated that IGF-II suppresses the basal PGC-1α promoter activity. Blocking the IGF-II signaling pathway increased the endogenous PGC-1α levels. In addition, pharmacological inhibition of PI3 kinase activity prevented the downregulation of PGC-1α but the activation of mTOR was not required for this process. Importantly, further analysis showed that forkhead transcription factor FoxO1 contributes to mediating the effects of IGF-II on PGC-1 promoter activity. These findings indicate that IGF-II reduces PGC-1α expression in skeletal muscle cells through a mechanism involving PI3K-Akt-FoxO1 but not p38 MAPK or Erk1/2 MAPK pathways.

  4. Activation of acetyl-coenzyme A carboxylase is involved in Taxol-induced ovarian cancer cell death.

    Science.gov (United States)

    Wu, Jiang; Ji, Fang; DI, Wen; Chen, Hongduo; Wan, Yinsheng

    2011-05-01

    Acetyl-coenzyme A carboxylase (ACC) is an attractive target for research into the treatment of a variety of human diseases, including diabetes, obesity and cancer. Mounting evidence suggests that the inhibition of ACC induced of cancer cell apoptosis. However, whether the inhibition of ACC regulates apoptosis in CaOV3 cancer cells has yet to be addressed. This study investigated the cytotoxic mechanism of action of ACC inhibition. Results showed that 5-(tetradecyloxy)-2-furoic acid (TOFA), an ACC inhibitor, enhanced Taxol-induced CaOV3 human ovarian cancer cell apoptosis. Notably, when TOFA was administered as a monotherapy, it induced CaOV3 cell apoptosis. Pre-treatment with the EGFR inhibitor PD153035 was found to markedly enhance ACC phosphorylation, whereas AMP-activated protein kinase (AMPK) activator AICAR was found to marginally enhance ACC phosphorylation. Taken together, the data showed ACC is a potential novel molecular target of Taxol. Additionally, ACC inhibition partially contributed to the cytotoxic effect of Taxol in ovarian cancer cells.

  5. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    International Nuclear Information System (INIS)

    Li, Yan; Ohms, Stephen J.; Shannon, Frances M.; Sun, Chao; Fan, Jun Y.

    2012-01-01

    Highlights: ► DNA methylation is dynamic and flexible and changes rapidly upon cell activation. ► DNA methylation controls the inducible gene expression in a given cell type. ► Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  6. cAMP-dependent cell differentiation triggered by activated CRHR1 in hippocampal neuronal cells.

    Science.gov (United States)

    Inda, Carolina; Bonfiglio, Juan José; Dos Santos Claro, Paula A; Senin, Sergio A; Armando, Natalia G; Deussing, Jan M; Silberstein, Susana

    2017-05-16

    Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.

  7. Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjaer; Bergdahl, Andreas; Christophersen, Palle

    2007-01-01

    Recent evidence implicates the volume-regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl- currents in Ehrlich Lettre Ascites (ELA) cells were monitored...... during cell cycle progression, under three conditions: (i) after osmotic swelling (i.e., VRAC), (ii) after an increase in the free intracellular Ca2+ concentration (i.e., the Ca2+-activated Cl- current, CaCC), and (iii) under steady-state isotonic conditions. The maximal swelling-activated VRAC current......+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high-affinity anion channel inhibitor, the acidic di-aryl-urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle...

  8. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR and NKG2D

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2006-01-01

    Full Text Available Human natural killer (NK lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis. Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

  9. AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks.

    Science.gov (United States)

    Chun, Min Jeong; Kim, Sunshin; Hwang, Soo Kyung; Kim, Bong Sub; Kim, Hyoun Geun; Choi, Hae In; Kim, Jong Heon; Goh, Sung Ho; Lee, Chang-Hun

    2016-08-16

    Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.

  10. Neuron–Glia Crosstalk and Neuropathic Pain: Involvement in the Modulation of Motor Activity in the Orofacial Region

    Science.gov (United States)

    Unno, Shumpei; Ando, Hiroshi; Masuda, Yuji; Kitagawa, Junichi

    2017-01-01

    Neuropathic orofacial pain (NOP) is a debilitating condition. Although the pathophysiology remains unclear, accumulating evidence suggests the involvement of multiple mechanisms in the development of neuropathic pain. Recently, glial cells have been shown to play a key pathogenetic role. Nerve injury leads to an immune response near the site of injury. Satellite glial cells are activated in the peripheral ganglia. Various neural and immune mediators, released at the central terminals of primary afferents, lead to the sensitization of postsynaptic neurons and the activation of glia. The activated glia, in turn, release pro-inflammatory factors, further sensitizing the neurons, and resulting in central sensitization. Recently, we observed the involvement of glia in the alteration of orofacial motor activity in NOP. Microglia and astroglia were activated in the trigeminal sensory and motor nuclei, in parallel with altered motor functions and a decreased pain threshold. A microglial blocker attenuated the reduction in pain threshold, reduced the number of activated microglia, and restored motor activity. We also found an involvement of the astroglial glutamate–glutamine shuttle in the trigeminal motor nucleus in the alteration of the jaw reflex. Neuron–glia crosstalk thus plays an important role in the development of pain and altered motor activity in NOP. PMID:28954391

  11. Neuron-Glia Crosstalk and Neuropathic Pain: Involvement in the Modulation of Motor Activity in the Orofacial Region.

    Science.gov (United States)

    Hossain, Mohammad Zakir; Unno, Shumpei; Ando, Hiroshi; Masuda, Yuji; Kitagawa, Junichi

    2017-09-26

    Neuropathic orofacial pain (NOP) is a debilitating condition. Although the pathophysiology remains unclear, accumulating evidence suggests the involvement of multiple mechanisms in the development of neuropathic pain. Recently, glial cells have been shown to play a key pathogenetic role. Nerve injury leads to an immune response near the site of injury. Satellite glial cells are activated in the peripheral ganglia. Various neural and immune mediators, released at the central terminals of primary afferents, lead to the sensitization of postsynaptic neurons and the activation of glia. The activated glia, in turn, release pro-inflammatory factors, further sensitizing the neurons, and resulting in central sensitization. Recently, we observed the involvement of glia in the alteration of orofacial motor activity in NOP. Microglia and astroglia were activated in the trigeminal sensory and motor nuclei, in parallel with altered motor functions and a decreased pain threshold. A microglial blocker attenuated the reduction in pain threshold, reduced the number of activated microglia, and restored motor activity. We also found an involvement of the astroglial glutamate-glutamine shuttle in the trigeminal motor nucleus in the alteration of the jaw reflex. Neuron-glia crosstalk thus plays an important role in the development of pain and altered motor activity in NOP.

  12. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi 712100 (China); Department of Genome Biology, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia); Ohms, Stephen J. [ACRF Biomolecular Resource Facility, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia); Shannon, Frances M. [Department of Genome Biology, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia); The University of Canberra, ACT 2602 (Australia); Sun, Chao, E-mail: sunchao2775@163.com [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi 712100 (China); Fan, Jun Y., E-mail: jun.fan@anu.edu.au [Department of Genome Biology, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer DNA methylation is dynamic and flexible and changes rapidly upon cell activation. Black-Right-Pointing-Pointer DNA methylation controls the inducible gene expression in a given cell type. Black-Right-Pointing-Pointer Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  13. Effect of platelet lysate on human cells involved in different phases of wound healing.

    Science.gov (United States)

    Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

    2013-01-01

    Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (pplatelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  14. Fbxl10 overexpression in murine hematopoietic stem cells induces leukemia involving metabolic activation and upregulation of Nsg2

    DEFF Research Database (Denmark)

    Ueda, Takeshi; Nagamachi, Akiko; Takubo, Keiyo

    2015-01-01

    We previously reported deficiency for Samd9L, which was cloned as a candidate gene for -7/7q- syndrome, accelerated leukemia cooperatively with enhanced expression of a histone demethylase F-box and leucine-rich repeat protein 10 (Fbxl10, also known as Jhdm1b, Kdm2b, and Ndy1). To further...... with a normal G0 to G1 entry, resulting in pleiotropic progenitor cell expansion. Fbxl10 Tg HSCs displayed enhanced expression of neuron specific gene family member 2 (Nsg2) and forced expression of Nsg2 in primary bone marrow cells resulted in expansion of immature cells. In addition, the genes involved....... These findings define Fbxl10 as a bona fide oncogene, whose deregulated expression contributes to the development of leukemia involving metabolic proliferative advantage and Nsg2-mediated impaired differentiation....

  15. Langerhans' cell histiocytosis: pathology, imaging and treatment of skeletal involvement

    Energy Technology Data Exchange (ETDEWEB)

    Azouz, E. Michel [University of Miami, Pediatric Radiology Section, Department of Radiology, Miami, FL (United States); Saigal, Gaurav [McGill University, Department of Medical Imaging, Quebec (Canada); Rodriguez, Maria M. [University of Miami, Department of Pathology, Miami, FL (United States); Podda, Antonello [University of Miami, Division of Pediatric Hematology/Oncology, Miami, FL (United States)

    2005-02-01

    Langerhans' cell histiocytosis (LCH) is manifested in a variety of ways, the most common being the eosinophilic granuloma, a localized, often solitary bone lesion that occurs predominantly in the pediatric age group. The hallmark of LCH is the proliferation and accumulation of a specific histiocyte: the Langerhans' cell. In bone this may cause pain and adjacent soft-tissue swelling, but some lesions are asymptomatic. LCH can involve any bone, but most lesions occur in the skull (especially the calvarium and temporal bones), the pelvis, spine, mandible, ribs, and tubular bones. Imaging diagnosis of the disease in bone is first based on the plain radiographic appearance, which is usually a central destructive, aggressive-looking lesion. In the skull, the lesions develop in the diploic space, are lytic, and their edges may be beveled, scalloped or confluent (geographic), or show a ''button sequestrum.'' Vertebral body involvement usually causes collapse, resulting in vertebra plana. With significant recent improvements in the quality of gamma cameras, imaging techniques, and in studying children, bone scintigraphy at diagnosis and on follow-up usually reveals the sites of active disease, especially when the involvement is polyostotic. CT and MR imaging are very useful in providing detailed cross-sectional anatomic detail of the involved bone, including the bone marrow and the adjacent soft tissues. CT is better suited for demonstrating bone detail and MR imaging for bone marrow and soft-tissue involvement. (orig.)

  16. Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

    Directory of Open Access Journals (Sweden)

    Shiming Ye

    2017-01-01

    Full Text Available Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

  17. A typology of place attachment and activity involvement

    Science.gov (United States)

    Andrew J. Mowen; Alan R. Graefe; Randy J. Virden

    1998-01-01

    While previous research suggests that place attachment and activity involvement impact visitor perceptions, it has not examined the simultaneous effects of these affective constructs. This study develops a typology of both place attachment and activity involvement. It examines variations between attachment-involvement levels and visitor evaluations of quality. Results...

  18. Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

    Science.gov (United States)

    Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

    2012-01-01

    Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-01

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

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

    International Nuclear Information System (INIS)

    Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-01-01

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

  1. Autophagy protein p62/SQSTM1 is involved in HAMLET-induced cell death by modulating apotosis in U87MG cells.

    Science.gov (United States)

    Zhang, Y-B; Gong, J-L; Xing, T-Y; Zheng, S-P; Ding, W

    2013-03-21

    HAMLET is a complex of oleic acids and decalcified α-lactalbumin that was discovered to selectively kill tumor cells both in vitro and in vivo. Autophagy is an important cellular process involved in drug-induced cell death of glioma cells. We treated U87MG human glioma cells with HAMLET and found that the cell viability was significantly decreased and accompanied with the activation of autophagy. Interestingly, we observed an increase in p62/SQSTM1, an important substrate of autophagosome enzymes, at the protein level upon HAMLET treatment for short periods. To better understand the functionality of autophagy and p62/SQSTM1 in HAMLET-induced cell death, we modulated the level of autophagy or p62/SQSTM1 with biochemical or genetic methods. The results showed that inhibition of autophagy aggravated HAMLET-induced cell death, whereas activation of authophagy attenuated this process. Meanwhile, we found that overexpression of wild-type p62/SQSTM1 was able to activate caspase-8, and then promote HAMLET-induced apoptosis, whereas knockdown of p62/SQSTM1 manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 following HAMLET treatment required its C-terminus UBA domain. Our results indicated that in addition to being a marker of autophagy activation in HAMLET-treated glioma cells, p62/SQSTM1 could also function as an important mediator for the activation of caspase-8-dependent cell death.

  2. Fucoidan extract induces apoptosis in MCF-7 cells via a mechanism involving the ROS-dependent JNK activation and mitochondria-mediated pathways.

    Directory of Open Access Journals (Sweden)

    Zhongyuan Zhang

    Full Text Available BACKGROUND: Fucoidan extract (FE, an enzymatically digested compound with a low molecular weight, is extracted from brown seaweed. As a natural compound with various actions, FE is attractive, especially in Asian countries, for improving the therapeutic efficacy and safety of cancer treatment. The present study was carried out to investigate the anti-tumor properties of FE in human carcinoma cells and further examine the underlying mechanisms of its activities. METHODOLOGY/PRINCIPAL FINDING: FE inhibits the growth of MCF-7, MDA-MB-231, HeLa, and HT1080 cells. FE-mediated apoptosis in MCF-7 cancer cells is accompanied by DNA fragmentation, nuclear condensation, and phosphatidylserine exposure. FE induces mitochondrial membrane permeabilization (MMP through loss of mitochondrial membrane potential (ΔΨm and regulation of the expression of Bcl-2 family members. Release of apoptosis-inducing factor (AIF and cytochrome c precedes MMP. AIF release causes DNA fragmentation, the final stage of apoptosis, via a caspase-independent mitochondrial pathway. Additionally, FE was found to induce phosphorylation of c-Jun N-terminal kinase (JNK, p38, and extracellular signal-regulated kinase (ERK 1/2, and apoptosis was found to be attenuated by inhibition of JNK. Furthermore, FE-mediated apoptosis was found to involve the generation of reactive oxygen species (ROS, which are responsible for the decrease of ΔΨm and phosphorylation of JNK, p38, and ERK1/2 kinases. CONCLUSIONS/SIGNIFICANCE: These data suggest that FE activates a caspase-independent apoptotic pathway in MCF-7 cancer cells through activation of ROS-mediated MAP kinases and regulation of the Bcl-2 family protein-mediated mitochondrial pathway. They also provide evidence that FE deserves further investigation as a natural anticancer and cancer preventive agent.

  3. Intact long-type DupA protein in Helicobacter pylori is an ATPase involved in multifunctional biological activities.

    Science.gov (United States)

    Wang, Ming-yi; Chen, Cheng; Shao, Chen; Wang, Shao-bo; Wang, Ai-chu; Yang, Ya-chao; Yuan, Xiao-yan; Shao, Shi-he

    2015-04-01

    The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Extraosseous Gaucher cell deposition without adjacent bone involvement.

    Science.gov (United States)

    Meyer, Brendan J; Mills, Anne M; Gaskin, Cree M

    2014-10-01

    Extraosseous Gaucher cell deposits are a rare complication of Gaucher disease that can mimic malignancy. We describe a case of Gaucher cell deposition in the subcutaneous soft tissues overlying the lower thoracic spine in an 18-year-old woman with known type III Gaucher disease. This case is unique in the literature because this subcutaneous Gaucher mass was not associated with extension from underlying bone involvement or clear lymph node origin. It demonstrated no discernible continuity with the adjacent thoracic spinous processes, the cortices of which appeared intact. Although patients with Gaucher disease are at increased risk of malignancy, Gaucher cell deposition should remain a differential consideration for soft tissue masses with or without adjacent bone involvement in patients with known Gaucher disease.

  5. Distal Regeneration Involves the Age Dependent Activity of Branchial Sac Stem Cells in the Ascidian Ciona intestinalis.

    Science.gov (United States)

    Jeffery, William R

    2015-02-01

    Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis . Analysis of regenerative potential along the proximal-distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age-related stem cells located in the branchial sac that are a source of precursors for distal body regeneration.

  6. The extrinsic and intrinsic apoptotic pathways are involved in manganese toxicity in rat astrocytoma C6 cells.

    Science.gov (United States)

    Alaimo, Agustina; Gorojod, Roxana M; Kotler, Mónica L

    2011-08-01

    Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, chronic exposure to high levels of Mn in occupational or environmental settings can lead to its accumulation in the brain resulting in a degenerative brain disorder referred to as Manganism. Astrocytes are the main Mn store in the central nervous system and several lines of evidence implicate these cells as major players in the role of Manganism development. In the present study, we employed rat astrocytoma C6 cells as a sensitive experimental model for investigating molecular mechanisms involved in Mn neurotoxicity. Our results show that C6 cells undergo reactive oxygen species-mediated apoptotic cell death involving caspase-8 and mitochondrial-mediated pathways in response to Mn. Exposed cells exhibit typical apoptotic features, such as chromatin condensation, cell shrinkage, membrane blebbing, caspase-3 activation and caspase-specific cleavage of the endogenous substrate poly (ADP-ribose) polymerase. Participation of the caspase-8 dependent pathway was assessed by increased levels of FasL, caspase-8 activation and Bid cleavage. The involvement of the mitochondrial pathway was demonstrated by the disruption of the mitochondrial membrane potential, the opening of the mitochondrial permeability transition pore, cytochrome c release, caspase-9 activation and the increased mitochondrial levels of the pro-apoptotic Bcl-2 family proteins. In addition, our data also shows for the first time that mitochondrial fragmentation plays a relevant role in Mn-induced apoptosis. Taking together, these findings contribute to a deeper elucidation of the molecular signaling mechanisms underlying Mn-induced apoptosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ9-tetrahydrocannabinol in human CD4+ T cells

    International Nuclear Information System (INIS)

    Ngaotepprutaram, Thitirat; Kaplan, Barbara L.F.; Kaminski, Norbert E.

    2013-01-01

    We have previously reported that Δ 9 -tetrahydrocannabinol (Δ 9 -THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4 + T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ 9 -THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ 9 -THC attenuated CD40L expression in human CD4 + T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ 9 -THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ 9 -THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ 9 -THC suppresses human T cell function. - Highlights: • Δ 9 -THC attenuated CD40L expression in activated human CD4+ T cells. • Δ 9 -THC suppressed DNA-binding activity of NFAT and NFκB. • Δ 9 -THC impaired elevation of intracellular Ca2+. • Δ 9 -THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β

  8. Activation of CHK1 in Supporting Cells Indirectly Promotes Hair Cell Survival

    Directory of Open Access Journals (Sweden)

    Azadeh Jadali

    2017-05-01

    Full Text Available The sensory hair cells of the inner ear are exquisitely sensitive to ototoxic insults. Loss of hair cells after exposure to ototoxic agents causes hearing loss. Chemotherapeutic agents such as cisplatin causes hair cell loss. Cisplatin forms DNA mono-adducts as well as intra- and inter-strand DNA crosslinks. DNA cisplatin adducts are repaired through the DNA damage response. The decision between cell survival and cell death following DNA damage rests on factors that are involved in determining damage tolerance, cell survival and apoptosis. Cisplatin damage on hair cells has been the main focus of many ototoxic studies, yet the effect of cisplatin on supporting cells has been largely ignored. In this study, the effects of DNA damage response in cochlear supporting cells were interrogated. Supporting cells play a major role in the development, maintenance and oto-protection of hair cells. Loss of supporting cells may indirectly affect hair cell survival or maintenance. Activation of the Phosphoinositide 3-Kinase (PI3K signaling was previously shown to promote hair cell survival. To test whether activating PI3K signaling promotes supporting cell survival after cisplatin damage, cochlear explants from the neural subset (NS Cre Pten conditional knockout mice were employed. Deletion of Phosphatase and Tensin Homolog (PTEN activates PI3K signaling in multiple cell types within the cochlea. Supporting cells lacking PTEN showed increased cell survival after cisplatin damage. Supporting cells lacking PTEN also showed increased phosphorylation of Checkpoint Kinase 1 (CHK1 levels after cisplatin damage. Nearest neighbor analysis showed increased numbers of supporting cells with activated PI3K signaling in close proximity to surviving hair cells in cisplatin damaged cochleae. We propose that increased PI3K signaling promotes supporting cell survival through phosphorylation of CHK1 and increased survival of supporting cells indirectly increases hair cell

  9. Morus alba and active compound oxyresveratrol exert anti-inflammatory activity via inhibition of leukocyte migration involving MEK/ERK signaling.

    Science.gov (United States)

    Chen, Yi-Ching; Tien, Yin-Jing; Chen, Chun-Houh; Beltran, Francesca N; Amor, Evangeline C; Wang, Ran-Juh; Wu, Den-Jen; Mettling, Clément; Lin, Yea-Lih; Yang, Wen-Chin

    2013-02-23

    Morus alba has long been used in traditional Chinese medicine to treat inflammatory diseases; however, the scientific basis for such usage and the mechanism of action are not well understood. This study investigated the action of M. alba on leukocyte migration, one key step in inflammation. Gas chromatography-mass spectrometry (GC-MS) and cluster analyses of supercritical CO2 extracts of three Morus species were performed for chemotaxonomy-aided plant authentication. Phytochemistry and CXCR4-mediated chemotaxis assays were used to characterize the chemical and biological properties of M. alba and its active compound, oxyresveratrol. fluorescence-activated cell sorting (FACS) and Western blot analyses were conducted to determine the mode of action of oxyresveratrol. Chemotaxonomy was used to help authenticate M. alba. Chemotaxis-based isolation identified oxyresveratrol as an active component in M. alba. Phytochemical and chemotaxis assays showed that the crude extract, ethyl acetate fraction and oxyresveratrol from M. alba suppressed cell migration of Jurkat T cells in response to SDF-1. Mechanistic study indicated that oxyresveratrol diminished CXCR4-mediated T-cell migration via inhibition of the MEK/ERK signaling cascade. A combination of GC-MS and cluster analysis techniques are applicable for authentication of the Morus species. Anti-inflammatory benefits of M. alba and its active compound, oxyresveratrol, may involve the inhibition of CXCR-4-mediated chemotaxis and MEK/ERK pathway in T and other immune cells.

  10. Involvement of endoplasmic reticulum stress in albuminuria induced inflammasome activation in renal proximal tubular cells.

    Directory of Open Access Journals (Sweden)

    Li Fang

    Full Text Available Albuminuria contributes to the progression of tubulointerstitial fibrosis. Although it has been demonstrated that ongoing albuminuria leads to tubular injury manifested by the overexpression of numerous proinflammatory cytokines, the mechanism remains largely unknown. In this study, we found that the inflammasome activation which has been recognized as one of the cornerstones of intracellular surveillance system was associated with the severity of albuminuria in the renal biopsies specimens. In vitro, bovine serum albumin (BSA could also induce the activation of NLRP3 inflammasome in the cultured kidney epithelial cells (NRK-52E. Since there was a significant overlap of NLRP3 with the ER marker calreticulin, the ER stress provoked by BSA seemed to play a crucial role in the activation of inflammasome. Here, we demonstrated that the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA which was proved to be an enhancer for the adaptive capacity of ER could attenuate the inflammasome activation induced by albuminuria not only in vitro but also in diabetic nephropathy. Taken together, these data suggested that ER stress seemed to play an important role in albuminuria-induced inflammasome activation, elimination of ER stress via TUDCA might hold promise as a novel avenue for preventing inflammasome activation ameliorating kidney epithelial cells injury induced by albuminuria.

  11. Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Cheng Tien [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Weng, Te I. [Department of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Chen, Li Ping [Department of Dentistry, Chang Gang Memorial Hospital, Chang Gang University, Taoyuan, Taiwan (China); Chiang, Chih Kang [Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Liu, Shing Hwa, E-mail: shinghwaliu@ntu.edu.tw [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Urology, National Taiwan University Hospital, Taipei, Taiwan (China)

    2013-01-01

    Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy. -- Highlights: ► Ionic contrast medium-urografin induces renal tubular cell apoptosis. ► Urografin induces the ER stress-regulated survival and apoptosis

  12. Cell surface GRP78 facilitates hepatoma cells proliferation and migration by activating IGF-IR.

    Science.gov (United States)

    Yin, Yancun; Chen, Chen; Chen, Jinliang; Zhan, Renhui; Zhang, Qiang; Xu, Xiaoyan; Li, Defang; Li, Minjing

    2017-07-01

    The 78kDa glucose regulated protein (GRP78) is a multifunctional chaperone that is involved in a variety of cellular processes. Insulin like growth factor I receptor (IGF-IR) often aberrant expresses in many types of tumor cells. The IGF-IR signaling plays key roles in carcinogenesis and maintenance of the malignant phenotype. The crosstalk between GRP78 and IGF-IR molecules has not well been illuminated. Here, we demonstrated a reciprocal regulation of GRP78 expression and IGF-IR pathway activation. IGF-I induced GRP78 expression in hepatoma cells. IGF-IR knockdown or IGF-IR inhibitor repressed GRP78 expression. Both phosphatidylinositol 3-kianase (PI3K) and mitogen-activated protein kinase (MAPK) pathways involved in IGF-I induction of GRP78 expression. Interestingly, treatment of hepatoma cells with IGF-I re-distributes GRP78 from endoplasmic reticulum (ER) to cell surface and promotes its physical interaction with IGF-IR. Also, GRP78 promotes IGF-IR phosphorylation and activation. Blocked of GRP78 by small interfering RNA or inhibition of GRP78 function by (-)-epigallocatechin gallate (EGCG) blocks IGF-I induced IGF-IR phosphorylation and its downstream signaling. Further, blocked cell surface GRP78 with antibody inhibits IGF-I stimulated cellular proliferation and migration. These data reveal an essential role for the molecular chaperone GRP78 in IGF-IR signaling and implicate the use of GRP78 inhibitors in blocking IGF-IR signaling in hepatoma cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

    Science.gov (United States)

    Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

    1995-08-01

    Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

  14. Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

    Science.gov (United States)

    Alrashdan, Yazan A.; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J.; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E.; Burgess, Janette K.; Armour, Carol L.; Ammit, Alaina J.

    2012-01-01

    CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma. PMID:22387292

  15. Distal regeneration involves the age dependent activity of branchial sac stem cells in the ascidian Ciona intestinalis

    Science.gov (United States)

    2014-01-01

    Abstract Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis. Analysis of regenerative potential along the proximal−distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age‐related stem cells located in the branchial sac that are a source of precursors for distal body regeneration. PMID:25893097

  16. Cell-type-specific activation of mitogen-activated protein kinases in PAN-induced progressive renal disease in rats

    International Nuclear Information System (INIS)

    Park, Sang-Joon; Jeong, Kyu-Shik

    2004-01-01

    We examined the time-course activation and the cell-type specific role of MAP kinases in puromycin aminonucleoside (PAN)-induced renal disease. The maximal activation of c-Jun-NH 2 -terminal kinase (JNK), extracellular signal regulated kinase (ERK), and p38 MAP kinase was detected on Days 52, 38, and 38 after PAN-treatment, respectively. p-JNK was localized in mesangial and proximal tubular cells at the early renal injury. It was expressed, therefore, in the inflammatory cells of tubulointerstitial lesions. While, p-ERK was markedly increased in the glomerular regions and macrophages p-p38 was observed in glomerular endothelial cells, tubular cells, and some inflammatory cells. The results show that the activation of MAP kinases in the early renal injury by PAN-treatment involves cellular changes such as cell proliferation or apoptosis in renal native cells. The activation of MAP kinases in infiltrated inflammatory cells and fibrotic cells plays an important role in destructive events such as glomerulosclerosis and tubulointerstitial fibrosis

  17. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    International Nuclear Information System (INIS)

    Nakatsu, Yusuke; Kotake, Yaichiro; Hino, Atsuko; Ohta, Shigeru

    2008-01-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release

  18. Establishing a chemical genetic link between Bruton tyrosine kinase activity in malignant B cells and cell functions involved in the micro-environmental dialogue.

    Science.gov (United States)

    Göckeritz, Elisa; Vondey, Verena; Guastafierro, Anna; Pizevska, Maja; Hassenrück, Floyd; Neumann, Lars; Hallek, Michael; Krause, Günter

    2017-09-01

    To elucidate their mechanism of action, inhibitors of Bruton tyrosine kinase (BTK) and resistant BTK mutants were employed to dissect target-dependent cellular functions. BTK-C481S and -T474I, expressed in Ramos and NALM-6 cells, maintained BTK auto-phosphorylation under treatment with ibrutinib or dasatinib, respectively, which showed only modest cytotoxicity. Retained activity of BTK-T474 partially rescued cell migration from inhibition by dasatinib. Importantly, resistant BTK mutants reconstituted B cell receptor-triggered chemokine secretion in the presence of corresponding inhibitors, demonstrating that BTK activity is connected with cell-intrinsic functions of malignant B cells with importance for their dialogue with the micro-environment. © 2017 John Wiley & Sons Ltd.

  19. Extracellular Molecules Involved in Cancer Cell Invasion

    International Nuclear Information System (INIS)

    Stivarou, Theodora; Patsavoudi, Evangelia

    2015-01-01

    Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion

  20. Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

    Science.gov (United States)

    Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

    2012-04-15

    Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more

  1. Effect of platelet lysate on human cells involved in different phases of wound healing.

    Directory of Open Access Journals (Sweden)

    Maria Chiara Barsotti

    Full Text Available BACKGROUND: Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization. METHODOLOGY/PRINCIPAL FINDINGS: Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2 and inflammatory response evaluation (NFκB. Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v. Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control, comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. CONCLUSION/SIGNIFICANCE: These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  2. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment

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    Pek Siew Lim

    2016-03-01

    Full Text Available T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA and calcium ionomycin (I as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278. Keywords: EL4 T cell, Microarray, T cell activation, Inducible genes

  3. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    Science.gov (United States)

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

    Science.gov (United States)

    Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

    2016-11-01

    Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

  5. Dexamethasone-induced and estradiol-induced CREB activation and annexin 1 expression in CCRF-CEM lymphoblastic cells: evidence for the involvement of cAMP and p38 MAPK

    Directory of Open Access Journals (Sweden)

    M. Castro-caldas

    2003-01-01

    Full Text Available Aims: Annexin 1 (ANXA1, a member of the annexin family of calcium-binding and phospholipid-binding proteins, is a key mediator of the anti-inflammatory actions of steroid hormones. We have previously demonstrated that, in the human lymphoblastic CCRF-CEM cell line, both the synthetic glucocorticoid hormone, dexamethasone (Dex, and the estrogen hormone, 17β-estradiol (E2β, induce the synthesis of ANXA1, by a mechanism independent of the activation of their nuclear receptors. Recently, it was reported that the gene coding for ANXA1 contains a cAMP-responsive element (CRE. In this work, we investigated whether Dex and E2β were able to induce the activation of CRE binding proteins (CREB in the CCRF-CEM cells. Moreover, we studied the intracellular signalling pathways involved in CREB activation and ANXA1 synthesis in response to Dex and E2β; namely, the role of cAMP and the p38 mitogen-activated protein kinase (MAPK.

  6. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

    Science.gov (United States)

    Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

    2016-03-01

    T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278.

  7. Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Hanley, Cory; Layne, Janet; Feris, Kevin; Wingett, Denise [Department of Biological Sciences, Boise State University, Boise, ID 83725 (United States); Punnoose, Alex; Reddy, K M; Coombs, Isaac; Coombs, Andrew [Department of Physics, Boise State University, Boise, ID 83725 (United States)], E-mail: denisewingett@boisestate.edu

    2008-07-23

    Nanoparticles are increasingly being recognized for their potential utility in biological applications including nanomedicine. Here we examine the response of normal human cells to ZnO nanoparticles under different signaling environments and compare it to the response of cancerous cells. ZnO nanoparticles exhibit a strong preferential ability to kill cancerous T cells ({approx}28-35 x) compared to normal cells. Interestingly, the activation state of the cell contributes toward nanoparticle toxicity, as resting T cells display a relative resistance while cells stimulated through the T cell receptor and CD28 costimulatory pathway show greater toxicity in direct relation to the level of activation. Mechanisms of toxicity appear to involve the generation of reactive oxygen species, with cancerous T cells producing higher inducible levels than normal T cells. In addition, nanoparticles were found to induce apoptosis and the inhibition of reactive oxygen species was found to be protective against nanoparticle induced cell death. The novel findings of cell selective toxicity, towards potential disease causing cells, indicate a potential utility of ZnO nanoparticles in the treatment of cancer and/or autoimmunity.

  8. Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles

    International Nuclear Information System (INIS)

    Hanley, Cory; Layne, Janet; Feris, Kevin; Wingett, Denise; Punnoose, Alex; Reddy, K M; Coombs, Isaac; Coombs, Andrew

    2008-01-01

    Nanoparticles are increasingly being recognized for their potential utility in biological applications including nanomedicine. Here we examine the response of normal human cells to ZnO nanoparticles under different signaling environments and compare it to the response of cancerous cells. ZnO nanoparticles exhibit a strong preferential ability to kill cancerous T cells (∼28-35 x) compared to normal cells. Interestingly, the activation state of the cell contributes toward nanoparticle toxicity, as resting T cells display a relative resistance while cells stimulated through the T cell receptor and CD28 costimulatory pathway show greater toxicity in direct relation to the level of activation. Mechanisms of toxicity appear to involve the generation of reactive oxygen species, with cancerous T cells producing higher inducible levels than normal T cells. In addition, nanoparticles were found to induce apoptosis and the inhibition of reactive oxygen species was found to be protective against nanoparticle induced cell death. The novel findings of cell selective toxicity, towards potential disease causing cells, indicate a potential utility of ZnO nanoparticles in the treatment of cancer and/or autoimmunity

  9. Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development.

    Science.gov (United States)

    Pagnussat, Gabriela Carolina; Lanteri, María Luciana; Lombardo, María Cristina; Lamattina, Lorenzo

    2004-05-01

    Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process

  10. Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

    Science.gov (United States)

    Nguyen, Van Thi; Cancedda, Ranieri; Descalzi, Fiorella

    2018-03-01

    The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage. Copyright © 2017 John Wiley & Sons, Ltd.

  11. Radiation activation of transcription factors in mammalian cells

    International Nuclear Information System (INIS)

    Kraemer, M.; Stein, B.; Mai, S.; Kunz, E.; Koenig, H.; Ponta, H.; Herrlich, P.; Rahmsdorf, H.J.; Loferer, H.; Grunicke, H.H.

    1990-01-01

    In mammalian cells radiation induces the enhanced transcription of several genes. The cis acting elements in the control region of inducible genes have been delimited by site directed mutagenesis. Several different elements have been found in different genes. They do not only activate gene transcription in response to radiation but also in response to growth factors and to tumor promoter phorbol esters. The transcription factors binding to these elements are present also in non-irradiated cells, but their DNA binding activity and their transactivating capability is increased upon irradiation. The signal chain linking the primary radiation induced signal (damaged DNA) to the activation of transcription factors involves the action of (a) protein kinase(s). (orig.)

  12. Relevance of P-glycoprotein on CXCR4+ B cells to organ manifestation in highly active rheumatoid arthritis.

    Science.gov (United States)

    Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Kawabe, Akio; Tanaka, Yoshiya

    2018-03-01

    In rheumatoid arthritis (RA), P-glycoprotein (P-gp) expression on activated B cells is associated with active efflux of intracellular drugs, resulting in drug resistance. CXCR4 is associated with migration of B cells. This study was designed to elucidate the relevance of P-gp expression on CXCR4 + B cells to clinical manifestations in refractory RA. CD19 + B cells were analyzed using flow cytometry and immunohistochemistry. P-gp was highly expressed especially on CXCR4 + CD19 + B cells in RA. The proportion of P-gp-expressing CXCR4 + B cells correlated with disease activity, estimated by Simplified Disease Activity Index (SDAI), and showed marked expansion in RA patients with high SDAI and extra-articular involvement. In highly active RA, massive infiltration of P-gp + CXCR4 + CD19 + B cells was noted in CXCL12-expressing inflammatory lesions of RA synovitis and RA-associated interstitial pneumonitis. In RA patient with active extra-articular involvement, intracellular dexamethasone level (IDL) in lymphocytes diminished with expansion of P-gp + CXCR4 + CD19 + B cells. Adalimumab reduced P-gp + CXCR4 + CD19 + B cells, increased IDL in lymphocytes, and improved the clinical manifestation and allowed tapering of concomitant medications. Expansion of P-gp + CXCR4 + B cells seems to be associated with drug resistance, disease activity and progressive destructive arthritis with extra-articular involvement in RA.

  13. Giardia lamblia: identification of molecules that contribute to direct mast cell activation.

    Science.gov (United States)

    Muñoz-Cruz, Samira; Gomez-García, Argelia; Matadamas-Martínez, Félix; Alvarado-Torres, Juan A; Meza-Cervantez, Patricia; Arriaga-Pizano, Lourdes; Yépez-Mulia, Lilián

    2018-06-02

    Mast cells play a central role in the early clearance of the intestinal parasite Giardia lamblia. In a previous study, we reported that G. lamblia live trophozoites or trophozoite-derived total soluble extract induced direct activation (IgE-independent) of mast cells and release of IL-6 and TNF-α. To identify the Giardia molecules and the mast cell receptors involved in this activation, trophozoite-derived total soluble proteins separated into three fractions (F1-F3) were evaluated for its ability to activate mast cells in vitro. F2 activated mast cells in a greater extent than F1 and F3. Furthermore, F2 induced the release of IL-6 and TNF-α by mast cells. TLR2 and TLR4 expression increased slightly after mast cell stimulation with either F2 or total soluble extract; however, these receptors were not involved in F2 or total soluble extract-induced proinflammatory cytokine production. Proteins present in F2 as unique and high-intensity bands identified by liquid chromatography coupled with tandem mass spectrometry, include molecules with important biological activities such as enolase and arginine deiminase (ADI). Recombinant ADI and enolase were tested for their ability to activate mast cells, but only ADI induced a significant release of IL-6 and TNF-α. ADI product, citrulline but not ammonium, also induced mast cell release of TNF-α. Interestingly, recombinant ADI still stimulated the secretion of TNF-α by mast cells in a arginine-free medium, although in a lower extend that in the presence of arginine, indicating that either ADI itself can stimulate mast cells or through its metabolic product, citrulline.

  14. New protein involved in the replacement of cell molecules

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave

    2011-01-01

    In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed” compartme......In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed...

  15. Penta-acetyl geniposide-induced apoptosis involving transcription of NGF/p75 via MAPK-mediated AP-1 activation in C6 glioma cells

    International Nuclear Information System (INIS)

    Peng, C.-H.; Huang, C.-N.; Hsu, S.-P.; Wang, C.-J.

    2007-01-01

    We have demonstrated the herbal derivative penta-acetyl geniposide ((Ac) 5 GP) induces C6 glioma cell apoptosis through the critical sphingomyelinase (SMase)/nerve growth factor (NGF)/p75 and its downstream signals. It has been reported mitogen-activated protein kinase (MAPK) mediates NGF synthesis induced by SMase activation. In this study, ERK, p38 and JNK are shown to mediate (Ac) 5 GP-induced glioma cell apoptosis and elevation of NGF and p75. Treatment of PD98059 (ERK-specific inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) decreases the elevation of NGF and p75 mRNA induced by (Ac) 5 GP, indicating possible transcription regulation via MAPKs. The results of nuclear extract blotting and EMSA further confirm (Ac) 5 GP maximally increases AP-1 and NF-κB DNA binding at 6 h. Inhibition of ERK, p38 and JNK block the activation of AP-1 and NF-κB, suggesting these MAPKs are involved in (Ac) 5 GP-induced transcription regulation. We thereby used RT-PCR to analyze cells treated with (Ac) 5 GP, with or without AP-1 or NF-κB inhibitors. AP-1 inhibitor NDGA decreases NGF/p75 and expression of FasL and caspase 3 induced by (Ac) 5 GP, suggesting the importance of AP-1 in mediating NGF/p75 and their downstream apoptotic signals. However, FasL and caspase 3 do not change with the NF-κB inhibitor PDTC; NF-κB might be linked to other cellular events. Overall, we demonstrate that MAPK mediates (Ac) 5 GP-induced activation of AP-1, promoting the transcription of NGF/p75 and downstream apoptotic signals. These results further highlight the potential therapeutic effects of (Ac) 5 GP in chemoprevention or as an anti-tumor agent

  16. Hypomethylation mediated by decreased DNMTs involves in the activation of proto-oncogene MPL in TK6 cells treated with hydroquinone.

    Science.gov (United States)

    Liu, Linhua; Ling, Xiaoxuan; Liang, Hairong; Gao, Yuting; Yang, Hui; Shao, Junli; Tang, Huanwen

    2012-03-25

    Hydroquinone (HQ), one of the most important metabolites derived from benzene, is known to be associated with acute myelogenous leukemia (AML) risk, however, its carcinogenic mechanism remains unclear. In this study, the epigenetic mechanism of HQ exposure was investigated. We characterized the epigenomic response of TK6 cells to HQ exposure, and examined the mRNA expression of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a and DNMT3b, methyl-CpG-binding domain protein 2 (MBD2) and six proto-oncogenes (MPL, RAF1, MYB, MYC, ERBB2 and BRAF). Compared to the control cells, HQ exposure (2.5, 5.0, 10.0 and 20.0 μM for 48 h) resulted in the decrease of DNMTs and MBD2 expression, the global hypomethylation and increase of MPL at mRNA level. Meanwhile, most of these changes were in dose-dependent manner. Moreover, inhibition of DNMTs induced by 5-aza-2'-deoxycytidine (5-AZA), an identified DNMT inhibitor, caused more induction of MPL expression at mRNA level compared to the HQ (10.0 μM) pre-treated group. Furthermore, treatment of HQ potentially led to MPL itself hypomethylation (10.0 and 20.0 μM reduced by 47% and 44%, respectively), further revealing that the activation of proto-oncogene MPL was related to hypomethylation in its DNA sequences. In conclusion, hypomethylation, including global and specific hypomethylation, might be involved in the activation of MPL, and the hypomethylation could be induced by decreased DNMTs in TK6 cells exposed to HQ. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    Directory of Open Access Journals (Sweden)

    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  18. Extracellular Molecules Involved in Cancer Cell Invasion

    Directory of Open Access Journals (Sweden)

    Theodora Stivarou

    2015-01-01

    Full Text Available Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

  19. Attenuated expression of the tight junction proteins is involved in clopidogrel-induced gastric injury through p38 MAPK activation

    International Nuclear Information System (INIS)

    Wu, Hai-Lu; Gao, Xin; Jiang, Zong-Dan; Duan, Zhao-Tao; Wang, Shu-Kui; He, Bang-Shun; Zhang, Zhen-Yu; Xie, Hong-Guang

    2013-01-01

    Highlights: ► Clopidogrel suppressed GES-1 cell viability in a concentration- and time-dependent manner. ► Clopidogrel significantly increased dextran permeability, reduced occludin and ZO-1 expression, and induced cell apoptosis. ► Clopidogrel activated p38 MAPK signaling pathway. ► Activation of p38 activity was involved in clopidogrel-induced increase in gastric epithelial cells permeability and disruption of TJ. -- Abstract: Bleeding complications and delayed healing of gastric ulcer associated with use of clopidogrel is a common clinical concern; however, the underlying mechanisms remain to be determined. This study aimed to clarify whether clopidogrel could cause the damage of the human gastric epithelial cells and to further elucidate the mechanisms involved. After human gastric epithelial cell line GES-1 had been treated with clopidogrel (0.5–2.5 mM), the cell proliferation was examined by MTT assay, apoptosis was measured with DAPI staining and flow cytometry analysis, and the barrier function of the tight junctions (TJ) was evaluated by permeability measurement and transmission electron microscopy. Moreover, expression of the TJ proteins occludin and ZO-1 and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38, ERK, and JNK were examined by western blot. In addition, three MAPK inhibitors specific to p38, ERK and JNK were used, respectively, to verify the signaling pathways responsible for regulating the expression of the TJ proteins being tested. Results showed that clopidogrel significantly increased dextran permeability, induced apoptosis, suppressed GES-1 cell viability, and reduced the expression of the TJ proteins (occludin and ZO-1), acting through p38 MAPK phosphorylation. Furthermore, these observed effects were partially abolished by SB-203580 (a p38 MAPK inhibitor), rather than by either U-0126 (an ERK inhibitor) or SP-600125 (a JNK inhibitor), suggesting that clopidogrel-induced disruption in the gastric

  20. Catalase Induced by All-Trans Retinoic Acid Is Involved in Antiproliferation of 36B10 Cells

    International Nuclear Information System (INIS)

    Park, Woo Yoon; Yu, Jae Ran

    2010-01-01

    All-trans retinoic acid (ATRA) has antiproliferative effects against brain tumor cells. Recently, ATRA has been reported to induce catalase. We investigated whether catalase induction by ATRA is associated with its antiproliferative effects. 36B10 cells were exposed to 0-50μM ATRA for 24 or 48 hours and mRNA, protein, and activity of catalase were measured. Reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescin diacetate. A clonogenic assay was used to confirm the cytotoxic effect. The mRNA, protein, and activity of catalase were found to increase in a concentration- and incubation- time-dependent manner. The increase in catalase activity induced by ATRA was decreased by the addition of 3-amino-1,2,4-triazole (ATZ). ROS was also increased with ATRA and decreased by the addition of ATZ. The decrease in cell survival induced by ATRA was partly rescued by ATZ. Catalase induction by ATRA is involved in ROS overproduction and thus inhibits the proliferation of 36B10 cells.

  1. Differential PKA activation and AKAP association determines cell fate in cancer cells

    Science.gov (United States)

    2013-01-01

    Background The dependence of malignant properties of colorectal cancer (CRC) cells on IGF1R signaling has been demonstrated and several IGF1R antagonists are currently in clinical trials. Recently, we identified a novel pathway in which cAMP independent PKA activation by TGFβ signaling resulted in the destabilization of survivin/XIAP complex leading to increased cell death. In this study, we evaluated the effect of IGF1R inhibition or activation on PKA activation and its downstream cell survival signaling mechanisms. Methods Small molecule IGF1R kinase inhibitor OSI-906 was used to test the effect of IGF1R inhibition on PKA activation, AKAP association and its downstream cell survival signaling. In a complementary approach, ligand mediated activation of IGF1R was performed and AKAP/PKA signaling was analyzed for their downstream survival effects. Results We demonstrate that the inhibition of IGF1R in the IGF1R-dependent CRC subset generates cell death through a novel mechanism involving TGFβ stimulated cAMP independent PKA activity that leads to disruption of cell survival by survivin/XIAP mediated inhibition of caspase activity. Importantly, ligand mediated activation of the IGF1R in CRC cells results in the generation of cAMP dependent PKA activity that functions in cell survival by inhibiting caspase activity. Therefore, this subset of CRC demonstrates 2 opposing pathways organized by 2 different AKAPs in the cytoplasm that both utilize activation of PKA in a manner that leads to different outcomes with respect to life and death. The cAMP independent PKA activation pathway is dependent upon mitochondrial AKAP149 for its apoptotic functions. In contrast, Praja2 (Pja2), an AKAP-like E3 ligase protein was identified as a key element in controlling cAMP dependent PKA activity and pro-survival signaling. Genetic manipulation of AKAP149 and Praja2 using siRNA KD had opposing effects on PKA activity and survivin/XIAP regulation. Conclusions We had identified 2

  2. Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation.

    Science.gov (United States)

    Alkozi, Hanan Awad; Perez de Lara, María J; Pintor, Jesús

    2017-09-01

    Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. NF-kappa B modulation is involved in celastrol induced human multiple myeloma cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Haiwen Ni

    Full Text Available Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. To investigate the effect of celastrol on human multiple myeloma cell cycle arrest and apoptosis and explore its molecular mechanism of action. The activity of celastrol on LP-1 cell proliferation was detected by WST-8 assay. The celastrol-induced cell cycle arrest was analyzed by flow cytometry after propidium iodide staining. Nuclear translocation of the nuclear factor kappa B (NF-κB was observed by fluorescence microscope. Celastrol inhibited cell proliferation of LP-1 myeloma cell in a dose-dependent manner with IC50 values of 0.8817 µM, which was mediated through G1 cell cycle arrest and p27 induction. Celastrol induced apoptosis in LP-1 and RPMI 8226 myeloma cells in a time and dose dependent manner, and it involved Caspase-3 activation and NF-κB pathway. Celastrol down-modulated antiapoptotic proteins including Bcl-2 and survivin expression. The expression of NF-κB and IKKa were decreased after celastrol treatment. Celastrol effectively blocked the nuclear translocation of the p65 subunit and induced human multiple myeloma cell cycle arrest and apoptosis by p27 upregulation and NF-kB modulation. It has been demonstrated that the effect of celastrol on NF-kB was HO-1-independent by using zinc protoporphyrin-9 (ZnPPIX, a selective heme oxygenase inhibitor. From the results, it could be inferred that celastrol may be used as a NF-kB inhibitor to inhibit myeloma cell proliferation.

  4. Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

    OpenAIRE

    Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

    2009-01-01

    High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERR...

  5. Gastrodin stimulates anticancer immune response and represses transplanted H22 hepatic ascitic tumor cell growth: Involvement of NF-κB signaling activation in CD4 + T cells

    Energy Technology Data Exchange (ETDEWEB)

    Shu, Guangwen; Yang, Tianming [College of Pharmacy, South-Central University for Nationalities, Wuhan (China); Wang, Chaoyuan [College of Life Science, South-Central University for Nationalities, Wuhan (China); Su, Hanwen, E-mail: suhanwen-1@163.com [Renmin Hospital of Wuhan University, Wuhan (China); Xiang, Meixian, E-mail: xiangmeixian99@163.com [College of Pharmacy, South-Central University for Nationalities, Wuhan (China)

    2013-06-15

    Gastrodia elata Blume (G. elata) is a famous restorative food in East Asia. It can be used as an auxiliary reagent in hepatocellular carcinoma (HCC) treatment. Previous studies unveiled that G. elata exhibited immunomodulatory activities. To explore the active ingredients contributing to its immunomodulatory activities, gastrodin, vanillin, and parishin B were purified from G. elata and their anti-HCC effects were assessed in vivo. Among these compounds, only gastrodin was capable of repressing transplanted H22 ascitic hepatic tumor cell growth in vivo with low toxicity. Further investigations were designed to explore the effects of gastrodin on the immune system of tumor-bearing mice and potential molecular mechanisms underlying these effects. Our data showed that gastrodin ameliorated tumor cell transplantation-induced activation of endogenous pro-apoptotic pathway in CD4 + T cells and abnormalities in serum cytokine profiles in host animals. These events enhanced cytotoxic activities of natural killer and CD8 + T cells against H22 hepatic cancer cells. Gastrodin administration specifically upregulated mRNA levels of several nuclear factor κB (NF-κB) responsive genes in CD4 + T cells but not in CD8 + T cells. Chromatin immunoprecipitation assay showed that gastrodin increased the association of NF-κB p65 subunit to the promoter regions of IL-2 and Bcl-2 encoding genes in CD4 + T cells. Our investigations demonstrated that gastrodin is the main active ingredient contributing to the anticancer immunomodulatory properties of G. elata. Promoting NF-κB-mediated gene transcription in CD4 + T cells is implicated in its immunomodulatory activity. - Highlights: • Gastrodin stimulates anticancer immune response. • Gastrodin represses tumor transplantation-induced CD4 + T cell apoptosis. • Gastrodin activates NF-κB activity in CD4 + T cells.

  6. Gastrodin stimulates anticancer immune response and represses transplanted H22 hepatic ascitic tumor cell growth: Involvement of NF-κB signaling activation in CD4 + T cells

    International Nuclear Information System (INIS)

    Shu, Guangwen; Yang, Tianming; Wang, Chaoyuan; Su, Hanwen; Xiang, Meixian

    2013-01-01

    Gastrodia elata Blume (G. elata) is a famous restorative food in East Asia. It can be used as an auxiliary reagent in hepatocellular carcinoma (HCC) treatment. Previous studies unveiled that G. elata exhibited immunomodulatory activities. To explore the active ingredients contributing to its immunomodulatory activities, gastrodin, vanillin, and parishin B were purified from G. elata and their anti-HCC effects were assessed in vivo. Among these compounds, only gastrodin was capable of repressing transplanted H22 ascitic hepatic tumor cell growth in vivo with low toxicity. Further investigations were designed to explore the effects of gastrodin on the immune system of tumor-bearing mice and potential molecular mechanisms underlying these effects. Our data showed that gastrodin ameliorated tumor cell transplantation-induced activation of endogenous pro-apoptotic pathway in CD4 + T cells and abnormalities in serum cytokine profiles in host animals. These events enhanced cytotoxic activities of natural killer and CD8 + T cells against H22 hepatic cancer cells. Gastrodin administration specifically upregulated mRNA levels of several nuclear factor κB (NF-κB) responsive genes in CD4 + T cells but not in CD8 + T cells. Chromatin immunoprecipitation assay showed that gastrodin increased the association of NF-κB p65 subunit to the promoter regions of IL-2 and Bcl-2 encoding genes in CD4 + T cells. Our investigations demonstrated that gastrodin is the main active ingredient contributing to the anticancer immunomodulatory properties of G. elata. Promoting NF-κB-mediated gene transcription in CD4 + T cells is implicated in its immunomodulatory activity. - Highlights: • Gastrodin stimulates anticancer immune response. • Gastrodin represses tumor transplantation-induced CD4 + T cell apoptosis. • Gastrodin activates NF-κB activity in CD4 + T cells

  7. Fasting inhibits hepatic stellate cells activation and potentiates anti-cancer activity of Sorafenib in hepatocellular cancer cells.

    Science.gov (United States)

    Lo Re, Oriana; Panebianco, Concetta; Porto, Stefania; Cervi, Carlo; Rappa, Francesca; Di Biase, Stefano; Caraglia, Michele; Pazienza, Valerio; Vinciguerra, Manlio

    2018-02-01

    Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. © 2017 Wiley Periodicals, Inc.

  8. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment

    OpenAIRE

    Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M.

    2015-01-01

    T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin...

  9. DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in human liver cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Obara, Akio; Fujita, Yoshihito; Abudukadier, Abulizi; Fukushima, Toru; Oguri, Yasuo; Ogura, Masahito; Harashima, Shin-ichi; Hosokawa, Masaya; Inagaki, Nobuya, E-mail: inagaki@metab.kuhp.kyoto-u.ac.jp

    2015-05-15

    Metformin, one of the most commonly used drugs for patients with type 2 diabetes, recently has received much attention regarding its anti-cancer action. It is thought that the suppression of mTOR signaling is involved in metformin's anti-cancer action. Although liver cancer is one of the most responsive types of cancer for reduction of incidence by metformin, the molecular mechanism of the suppression of mTOR in liver remains unknown. In this study, we investigated the mechanism of the suppressing effect of metformin on mTOR signaling and cell proliferation using human liver cancer cells. Metformin suppressed phosphorylation of p70-S6 kinase, and ribosome protein S6, downstream targets of mTOR, and suppressed cell proliferation. We found that DEPTOR, an endogenous substrate of mTOR suppression, is involved in the suppressing effect of metformin on mTOR signaling and cell proliferation in human liver cancer cells. Metformin increases the protein levels of DEPTOR, intensifies binding to mTOR, and exerts a suppressing effect on mTOR signaling. This increasing effect of DEPTOR by metformin is regulated by the proteasome degradation system; the suppressing effect of metformin on mTOR signaling and cell proliferation is in a DEPTOR-dependent manner. Furthermore, metformin exerts a suppressing effect on proteasome activity, DEPTOR-related mTOR signaling, and cell proliferation in an AMPK-dependent manner. We conclude that DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in liver, and could be a novel target for anti-cancer therapy. - Highlights: • We elucidated a novel pathway of metformin's anti-cancer action in HCC cells. • DEPTOR is involved in the suppressing effect of metformin on mTOR signaling. • Metformin increases DEPTOR protein levels via suppression of proteasome activity. • DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action.

  10. DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in human liver cancer cells

    International Nuclear Information System (INIS)

    Obara, Akio; Fujita, Yoshihito; Abudukadier, Abulizi; Fukushima, Toru; Oguri, Yasuo; Ogura, Masahito; Harashima, Shin-ichi; Hosokawa, Masaya; Inagaki, Nobuya

    2015-01-01

    Metformin, one of the most commonly used drugs for patients with type 2 diabetes, recently has received much attention regarding its anti-cancer action. It is thought that the suppression of mTOR signaling is involved in metformin's anti-cancer action. Although liver cancer is one of the most responsive types of cancer for reduction of incidence by metformin, the molecular mechanism of the suppression of mTOR in liver remains unknown. In this study, we investigated the mechanism of the suppressing effect of metformin on mTOR signaling and cell proliferation using human liver cancer cells. Metformin suppressed phosphorylation of p70-S6 kinase, and ribosome protein S6, downstream targets of mTOR, and suppressed cell proliferation. We found that DEPTOR, an endogenous substrate of mTOR suppression, is involved in the suppressing effect of metformin on mTOR signaling and cell proliferation in human liver cancer cells. Metformin increases the protein levels of DEPTOR, intensifies binding to mTOR, and exerts a suppressing effect on mTOR signaling. This increasing effect of DEPTOR by metformin is regulated by the proteasome degradation system; the suppressing effect of metformin on mTOR signaling and cell proliferation is in a DEPTOR-dependent manner. Furthermore, metformin exerts a suppressing effect on proteasome activity, DEPTOR-related mTOR signaling, and cell proliferation in an AMPK-dependent manner. We conclude that DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action in liver, and could be a novel target for anti-cancer therapy. - Highlights: • We elucidated a novel pathway of metformin's anti-cancer action in HCC cells. • DEPTOR is involved in the suppressing effect of metformin on mTOR signaling. • Metformin increases DEPTOR protein levels via suppression of proteasome activity. • DEPTOR-related mTOR suppression is involved in metformin's anti-cancer action

  11. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

    International Nuclear Information System (INIS)

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

    2012-01-01

    Highlights: ► We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. ► Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. ► Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. ► DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. ► DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X L expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

  12. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Cheah, Yew-Hoong [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur (Malaysia); Meenakshii, Nallappan [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

  13. Operationalising active involvement in the EU water framework directive

    DEFF Research Database (Denmark)

    Wright, Stuart Anthony Lewis; Fritsch, Oliver

    2011-01-01

    We identify two key stages in the river basin planning process under the Water Framework Directive: the selection of instruments for a programme of measures to achieve the environmental targets, and disproportionate cost analysis to determine whether selected measures involve high costs. Some EU...... of actively involving non-state actors, which can be summarised as increasing the effectiveness of policy and improving its implementation. Criticising the emerging economic decision-making approach, we argue that economic analyses could result in a missed opportunity to capitalise on the potential benefits...... of involvement. The article discusses the appropriateness of actively involving the public during the two aforementioned decision-making stages and suggests concrete ways in which active involvement may be operationalised. We conclude that member states should not implement a minimum form of participation...

  14. RhoA and RhoC are involved in stromal cell-derived factor-1-induced cell migration by regulating F-actin redistribution and assembly.

    Science.gov (United States)

    Luo, Jixian; Li, Dingyun; Wei, Dan; Wang, Xiaoguang; Wang, Lan; Zeng, Xianlu

    2017-12-01

    Stromal cell-derived factor-1 (SDF-1) signaling is important to the maintenance and progression of T-cell acute lymphoblastic leukemia by inducing chemotaxis migration. To identify the mechanism of SDF-1 signaling in the migration of T-ALL, Jurkat acute lymphoblastic leukemia cells were used. Results showed that SDF-1 induces Jurkat cell migration by F-actin redistribution and assembly, which is dependent on Rho activity. SDF-1 induced RhoA and RhoC activation, as well as reactive oxygen species (ROS) production, which was inhibited by Rho inhibitor. The Rho-dependent ROS production led to subsequent cytoskeleton redistribution and assembly in the process of migration. Additionally, RhoA and RhoC were involved in SDF-1-induced Jurkat cell migration. Taken together, we found a SDF-1/CXCR4-RhoA and RhoC-ROS-cytoskeleton pathway that regulates Jurkat cell migration in response to SDF-1. This work will contribute to a clearer insight into the migration mechanism of acute lymphoblastic leukemia.

  15. Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ{sup 9}-tetrahydrocannabinol in human CD4{sup +} T cells

    Energy Technology Data Exchange (ETDEWEB)

    Ngaotepprutaram, Thitirat [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States); Kaplan, Barbara L.F. [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States); Neuroscience Program, Michigan State University (United States); Kaminski, Norbert E., E-mail: kamins11@msu.edu [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States)

    2013-11-15

    We have previously reported that Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4{sup +} T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ{sup 9}-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ{sup 9}-THC attenuated CD40L expression in human CD4{sup +} T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ{sup 9}-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ{sup 9}-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ{sup 9}-THC suppresses human T cell function. - Highlights: • Δ{sup 9}-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ{sup 9}-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ{sup 9}-THC impaired elevation of intracellular Ca2+. • Δ{sup 9}-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β.

  16. CD 4 + CD 25 + T cells maintain homeostasis by promoting TER - 119 cell development and inhibiting T cell activation

    Directory of Open Access Journals (Sweden)

    Muhaimin Rifa’i

    2014-05-01

    Full Text Available CD4+ CD25+ regulatory T cells involved in the regulation of self- tolerance and normality of homeostasis. CD122 deficient mice are model animals that have an abnormal immune system characteristically have a high number of activated T cells and TER-119 cell decreased. Here we showed evidence that the transfer of CD4+ CD25+ regulatory T cells derived from normal mice to CD122- defficient neonates prevent the development of activated memory T cells and elicit TER-119 differentiation. Bone marrow reconstitution derived from CD122-/- mice to normal mice resulting tolerance to individual that genetically different. Importantly, CD4+ CD25+ regulatory T cells derived from normal mice can replace CD4+ CD25+ cells derived from CD122-/- mice. The results of this experiment suggest that regulatory T cells from normal mice exert a critical role in maintaining peripheral tolerance and controlling hematopoietic disorder.

  17. Activation of PLA2 isoforms by cell swelling and ischaemia/hypoxia

    DEFF Research Database (Denmark)

    Lambert, I. H.; Pedersen, S. F.; Poulsen, K. A.

    2006-01-01

    Phospholipase A2 (PLA2) activity is increased in mammalian cells in response to numerous stimuli such as osmotic challenge, oxidative stress and exposure to allergens. The increased PLA2 activity is seen as an increased release of free, polyunsaturated fatty acids, e.g. arachidonic acid....... Here, we review data that illustrates that stress-induced PLA2 activity involves various PLA2 subtypes and that the PLA2 in question is determined by the cell type and the physiological stress condition....

  18. Involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells.

    Science.gov (United States)

    Takahashi, Kaede; Fukushima, Kaori; Onishi, Yuka; Minami, Kanako; Otagaki, Shiho; Ishimoto, Kaichi; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

    2018-08-01

    Free fatty acid receptor 1 (FFA1) and FFA4 mediate a variety of biological responses through binding of medium- and long-chain free fatty acids. The aim of this study was to investigate an involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells. The long-term fluorouracil (5-FU) and cisplatin (CDDP) treated cells were generated from DLD1 cells (DLD-5FU and DLD-CDDP cells, respectively). FFAR1 expressions were lower in DLD-5FU and DLD-CDDP cells than in DLD1 cells. In contrast, DLD-5FU and DLD-CDDP cells showed the high FFAR4 expressions, compared with DLD1 cells. The cell motile activities of DLD-5FU and DLD-CDDP cells were reduced by GW9508 which is an agonist of FFA1 and FFA4. Moreover, GW1100, an antagonist of FFA1, inhibited the cell motile activities of DLD-5FU and DLD-CDDP cells. To evaluate whether FFA1 and FFA4 regulate the enhancement of cell motility, invasion and colony formation, highly migratory (hmDLD1) cells were established from DLD1 cells. FFAR1 expression was significantly higher in hmDLD1 cells than in DLD1 cells, but no change of FFAR4 expression was observed. The elevated cell motile and invasive activities and colony formation of hmDLD1 cells were suppressed by FFA1 inhibition. These results suggest that FFA1 and FFA4 are involved in the regulation of cellular functions during tumor progression in colon cancer DLD1 cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Langerhans cell histiocytosis with involvement of the pons: case report

    International Nuclear Information System (INIS)

    Vourtsi, A.; Papadopoulos, A.; Moulopoulos, L.A.; Vlahos, L.; Xenellis, J.

    1998-01-01

    Central nervous system involvement is uncommon in Langerhans cell histiocytosis. The suprasellar region is more frequently affected. There have been few reports of involvement of the brain parenchyma shown on CT or MRI. We present a case of involvement of the pons, showing marked contrast enhancement on MRI. (orig.)

  20. Glucose metabolism regulates T cell activation, differentiation and functions

    Directory of Open Access Journals (Sweden)

    Clovis Steve Palmer

    2015-01-01

    Full Text Available The adaptive immune system is equipped to eliminate both tumors and pathogenic microorganisms. It requires a series of complex and coordinated signals to drive the activation, proliferation and differentiation of appropriate T cell subsets. It is now established that changes in cellular activation are coupled to profound changes in cellular metabolism. In addition, emerging evidence now suggest that specific metabolic alterations associated with distinct T cell subsets may be ancillary to their differentiation and influential in their immune functions. The Warburg effect originally used to describe a phenomenon in which most cancer cells relied on aerobic glycolysis for their growth is a key process that sustain T cell activation and differentiation. Here we review how different aspects of metabolism in T cells influence their functions, focusing on the emerging role of key regulators of glucose metabolism such as HIF-1α. A thorough understanding of the role of metabolism in T cell function could provide insights into mechanisms involved in inflammatory-mediated conditions, with the potential for developing novel therapeutic approaches to treat these diseases.

  1. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer-stem-cell

  2. Mast cells in atherosclerotic cardiovascular disease - Activators and actions.

    Science.gov (United States)

    Kovanen, Petri T; Bot, Ilze

    2017-12-05

    Mast cells are potent actors involved in inflammatory reactions in various tissues, including both in the intimal and the adventitial layers of atherosclerotic arteries. In the arterial intima, the site of atherogenesis, mast cells are activated to degranulate, and thereby triggered to release an abundance of preformed inflammatory mediators, notably histamine, heparin, neutral proteases and cytokines stored in their cytoplasmic secretory granules. Depending on the stimulus, mast cell activation may also launch prolonged synthesis and secretion of single bioactive molecules, such as cytokines and derivatives of arachidonic acid. The mast cell-derived mediators may impede the functions of different types of cells present in atherosclerotic lesions, and also compromise the structural and functional integrity of the intimal extracellular matrix. In the adventitial layer of atherosclerotic coronary arteries, mast cells locate next to peptidergic sensory nerve fibers, which, by releasing neuropeptides may activate mast cells to release vasoactive compounds capable of triggering local vasoconstriction. The concerted actions of arterial mast cells have the potential to contribute to the initiation and progression of atherosclerosis, and ultimately to destabilization and rupture of an advanced atherosclerotic plaque with ensuing atherothrombotic complications. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Involvement of Endoplasmic Reticulum Stress in Capsaicin-Induced Apoptosis of Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shengzhang Lin

    2013-01-01

    Full Text Available Capsaicin, main pungent ingredient of hot chilli peppers, has been shown to have anticarcinogenic effect on various cancer cells through multiple mechanisms. In this study, we investigated the apoptotic effect of capsaicin on human pancreatic cancer cells in both in vitro and in vivo systems, as well as the possible mechanisms involved. In vitro, treatment of both the pancreatic cancer cells (PANC-1 and SW1990 with capsaicin resulted in cells growth inhibition, G0/G1 phase arrest, and apoptosis in a dose-dependent manner. Knockdown of growth arrest- and DNA damage-inducible gene 153 (GADD153, a marker of the endoplasmic-reticulum-stress- (ERS- mediated apoptosis pathway, by specific siRNA attenuated capsaicin-induced apoptosis both in PANC-1 and SW1990 cells. Moreover, in vivo studies capsaicin effectively inhibited the growth and metabolism of pancreatic cancer and prolonged the survival time of pancreatic cancer xenograft tumor-induced mice. Furthermore, capsaicin increased the expression of some key ERS markers, including glucose-regulated protein 78 (GRP78, phosphoprotein kinase-like endoplasmic reticulum kinase (phosphoPERK, and phosphoeukaryotic initiation factor-2α (phospho-eIF2α, activating transcription factor 4 (ATF4 and GADD153 in tumor tissues. In conclusion, we for the first time provide important evidence to support the involvement of ERS in the induction of apoptosis in pancreatic cancer cells by capsaicin.

  4. Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro

    NARCIS (Netherlands)

    Nieuw Amerongen, G.P. van; Koolwijk, P.; Versteilen, A.; Hinsbergh, V.W.M. van

    2003-01-01

    Objective - Growth factor-induced angiogenesis involves migration of endothelial cells (ECs) into perivascular areas and requires active remodeling of the endothelial F-actin cytoskeleton. The small GTPase RhoA previously has been implicated in vascular endothelial growth factor (VEGF)-induced

  5. Cortactin is involved in the entry of Coxiella burnetii into non-phagocytic cells.

    Directory of Open Access Journals (Sweden)

    Eliana M Rosales

    Full Text Available BACKGROUND: Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. Coxiella burnetii, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood. METHODOLOGY/PRINCIPAL FINDING: Here we analyzed the role of different cortactin motifs in the internalization of C. burnetii by non-phagocytic cells. C. burnetii internalization into HeLa cells was significantly reduced when the cells expressed GFP-cortactin W525K, which carries a mutation in the SH3 domain that renders the protein unable to bind targets such as N-WASP. However, internalization was unaffected when the cells expressed the W22A mutant, which has a mutation in the N-terminal acidic region that destroys the protein's ability to bind and activate Arp2/3. We also determined whether the phosphorylation status of cortactin is important for internalization. Expression of GFP-cortactin 3F, which lacks phosphorylatable tyrosines, significantly increased internalization of C. burnetii, while expression of GFP-cortactin 3D, a phosphotyrosine mimic, did not affect it. In contrast, expression of GFP-cortactin 2A, which lacks phosphorylatable serines, inhibited C. burnetii internalization, while expression of GFP-cortactin SD, a phosphoserine mimic, did not affect it. Interestingly, inhibitors of Src kinase and the MEK-ERK kinase pathway blocked internalization. In fact, both kinases reached maximal activity at 15 min of C. burnetii infection, after which activity decreased to basal levels. Despite the decrease in kinase activity, cortactin phosphorylation at Tyr421 reached a peak at 1 h of infection. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the SH3 domain of cortactin is implicated in C. burnetii entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation

  6. TCDD promoted EMT of hFPECs via AhR, which involved the activation of EGFR/ERK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhan [School of Public Health, Xinxiang Medical University, 453003 (China); The Fifth Affiliated Hospital, Zhengzhou University, 450052 (China); Bu, Yongjun [School of Public Health, Xinxiang Medical University, 453003 (China); Liu, Xiaozhuan [Medical College, Henan University of Science & Technology, 471023 (China); Wang, Xugang; Zhang, Guofu; Wang, Erhui; Ding, Shibin; Liu, Yongfeng; Shi, Ruling [School of Public Health, Xinxiang Medical University, 453003 (China); Li, Qiaoyun; Fu, Jianhong [The Fifth Affiliated Hospital, Zhengzhou University, 450052 (China); Yu, Zengli, E-mail: zly@zzu.edu.cn [School of Public Health, Xinxiang Medical University, 453003 (China); School of Public Health, Zhengzhou University, 450001 (China)

    2016-05-01

    One critical step of second palatal fusion is the newly formed medial epithelia seam (MES) disintegration, which involves apoptosis, epithelial to mesenchymal transition (EMT), and cell migration. Although the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate at high rates, little is known about the effects of TCDD exposure on the fate of palatal epithelial cells. By using primary epithelial cells isolated from human fetal palatal shelves (hFPECs), we show that TCDD increased cell proliferation and EMT, as demonstrated by increased the epithelial markers (E-cadherin and cytokeratin14) and enhanced the mesenchymal markers (vimentin and fibronectin), but had no effect on cell migration and apoptosis. TCDD exposure led to a dose-dependent increase in Slug protein expression. Coimmunoprecipitation revealed that TCDD promoted AhR to form a protein complex with Slug. ChIP assay confirmed that TCDD exposure recruited AhR to the xenobiotic responsive element of Slug promoter. Knockdown of AhR by siRNA remarkably weakened TCDD-induced binding of AhR to the XRE promoter of slug, thereby suppressed TCDD-induced vimentin. Further experiment showed that TCDD stimulated EGFR phosphorylation did not influence the TGFβ3/Smad signaling; whereas TCDD increased phosphorylation of ERK1/2 and p38 with no effect on activation of JNK. By using varieties of inhibitors, we confirmed that TCDD promoted proliferation and EMT of hFPECs via activation of EGFR/ERK pathway. These data make a novel contribution to the molecular mechanism of cleft palate by TCDD. - Highlights: • TCDD exposure promoted cell proliferation and EMT of hFPECs; • AhR signaling was activated and required for TCDD-induced EMT; • TCDD-mediated EMT of hFPECs involved the activation of EGFR/ERK signaling; • TCDD exposure had no effect on TGFβ3/Smad pathway.

  7. TCDD promoted EMT of hFPECs via AhR, which involved the activation of EGFR/ERK signaling

    International Nuclear Information System (INIS)

    Gao, Zhan; Bu, Yongjun; Liu, Xiaozhuan; Wang, Xugang; Zhang, Guofu; Wang, Erhui; Ding, Shibin; Liu, Yongfeng; Shi, Ruling; Li, Qiaoyun; Fu, Jianhong; Yu, Zengli

    2016-01-01

    One critical step of second palatal fusion is the newly formed medial epithelia seam (MES) disintegration, which involves apoptosis, epithelial to mesenchymal transition (EMT), and cell migration. Although the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate at high rates, little is known about the effects of TCDD exposure on the fate of palatal epithelial cells. By using primary epithelial cells isolated from human fetal palatal shelves (hFPECs), we show that TCDD increased cell proliferation and EMT, as demonstrated by increased the epithelial markers (E-cadherin and cytokeratin14) and enhanced the mesenchymal markers (vimentin and fibronectin), but had no effect on cell migration and apoptosis. TCDD exposure led to a dose-dependent increase in Slug protein expression. Coimmunoprecipitation revealed that TCDD promoted AhR to form a protein complex with Slug. ChIP assay confirmed that TCDD exposure recruited AhR to the xenobiotic responsive element of Slug promoter. Knockdown of AhR by siRNA remarkably weakened TCDD-induced binding of AhR to the XRE promoter of slug, thereby suppressed TCDD-induced vimentin. Further experiment showed that TCDD stimulated EGFR phosphorylation did not influence the TGFβ3/Smad signaling; whereas TCDD increased phosphorylation of ERK1/2 and p38 with no effect on activation of JNK. By using varieties of inhibitors, we confirmed that TCDD promoted proliferation and EMT of hFPECs via activation of EGFR/ERK pathway. These data make a novel contribution to the molecular mechanism of cleft palate by TCDD. - Highlights: • TCDD exposure promoted cell proliferation and EMT of hFPECs; • AhR signaling was activated and required for TCDD-induced EMT; • TCDD-mediated EMT of hFPECs involved the activation of EGFR/ERK signaling; • TCDD exposure had no effect on TGFβ3/Smad pathway.

  8. Molecular Mechanisms Involved in the Antitumor Activity of Cannabinoids on Gliomas: Role for Oxidative Stress

    International Nuclear Information System (INIS)

    Massi, Paola; Valenti, Marta; Solinas, Marta; Parolaro, Daniela

    2010-01-01

    Cannabinoids, the active components of Cannabis sativa, have been shown to exert antiproliferative and proapoptotic effects on a wide spectrum of tumor cells and tissues. Of interest, cannabinoids have displayed great potency in reducing the growth of glioma tumors, one of the most aggressive CNS tumors, either in vitro or in animal experimental models curbing the growth of xenografts generated by subcutaneous or intrathecal injection of glioma cells in immune-deficient mice. Cannabinoids appear to be selective antitumoral agents as they kill glioma cells without affecting the viability of non-transformed cells. This review will summarize the anti-cancer properties that cannabinoids exert on gliomas and discuss their potential action mechanisms that appear complex, involving modulation of multiple key cell signaling pathways and induction of oxidative stress in glioma cells

  9. Molecular Mechanisms Involved in the Antitumor Activity of Cannabinoids on Gliomas: Role for Oxidative Stress

    Energy Technology Data Exchange (ETDEWEB)

    Massi, Paola [Department of Pharmacology, Chemotherapy and Toxicology, University of Milan, Via Vanvitelli 32, 20129 Milan (Italy); Valenti, Marta; Solinas, Marta; Parolaro, Daniela [Department of Structural and Functional Biology, Section of Pharmacology, Center of Neuroscience, University of Insubria, Via A. da Giussano 10, 20152 Busto Arsizio, Varese (Italy)

    2010-05-26

    Cannabinoids, the active components of Cannabis sativa, have been shown to exert antiproliferative and proapoptotic effects on a wide spectrum of tumor cells and tissues. Of interest, cannabinoids have displayed great potency in reducing the growth of glioma tumors, one of the most aggressive CNS tumors, either in vitro or in animal experimental models curbing the growth of xenografts generated by subcutaneous or intrathecal injection of glioma cells in immune-deficient mice. Cannabinoids appear to be selective antitumoral agents as they kill glioma cells without affecting the viability of non-transformed cells. This review will summarize the anti-cancer properties that cannabinoids exert on gliomas and discuss their potential action mechanisms that appear complex, involving modulation of multiple key cell signaling pathways and induction of oxidative stress in glioma cells.

  10. Molecular Mechanisms Involved in the Antitumor Activity of Cannabinoids on Gliomas: Role for Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Paola Massi

    2010-05-01

    Full Text Available Cannabinoids, the active components of Cannabis sativa, have been shown to exert antiproliferative and proapoptotic effects on a wide spectrum of tumor cells and tissues. Of interest, cannabinoids have displayed great potency in reducing the growth of glioma tumors, one of the most aggressive CNS tumors, either in vitro or in animal experimental models curbing the growth of xenografts generated by subcutaneous or intrathecal injection of glioma cells in immune-deficient mice. Cannabinoids appear to be selective antitumoral agents as they kill glioma cells without affecting the viability of non-transformed cells. This review will summarize the anti-cancer properties that cannabinoids exert on gliomas and discuss their potential action mechanisms that appear complex, involving modulation of multiple key cell signaling pathways and induction of oxidative stress in glioma cells.

  11. Dynamical analysis of uterine cell electrical activity model.

    Science.gov (United States)

    Rihana, S; Santos, J; Mondie, S; Marque, C

    2006-01-01

    The uterus is a physiological system consisting of a large number of interacting smooth muscle cells. The uterine excitability changes remarkably with time, generally quiescent during pregnancy, the uterus exhibits forceful synchronized contractions at term leading to fetus expulsion. These changes characterize thus a dynamical system susceptible of being studied through formal mathematical tools. Multiple physiological factors are involved in the regulation process of this complex system. Our aim is to relate the physiological factors to the uterine cell dynamic behaviors. Taking into account a previous work presented, in which the electrical activity of a uterine cell is described by a set of ordinary differential equations, we analyze the impact of physiological parameters on the response of the model, and identify the main subsystems generating the complex uterine electrical activity, with respect to physiological data.

  12. Membrane-associated IL 1-like activity on rat dendritic cells

    International Nuclear Information System (INIS)

    Nagelkerken, L.M.; van Breda Vriesman, P.J.C.

    1986-01-01

    The secretion of interleukin 1 (IL 1) by rat dendritic cells (DC) was studied in relation to their ability to induce the production interleukin 2 (IL 2 ) and to induce IL 2 responsiveness. IL 1 (or IL 1-like activity) was measured by its capacity to enhance IL 2 production by EL4 cells. In contrast to peritoneal exudate cells (PEC) or splenic adherent cells, DC from thoracic duct lymph (TD-DC) or from spleen did not secrete detectable amounts of IL 1 on stimulation with LPS/Silica. However, TD-DC and splenic DC were able to enhance IL 2 production by EL4 cells directly, and were only two times less effective than PEC. By preventing cell-to-cell contact between stimulator cells and EL4 cells, it was demonstrated that most of the IL 2-inducing activity of TD-DC and PEC was associated with the cell membrane. Treatment with 1% paraformaldehyde (PFA) to abolish metabolic activity resulted in a 50% decrease (or inactivation) of IL 2-inducing activity of TD-DC in the EL4 assay. Moreover, UVB-irradiation (300 mJ/cm 2 ) of TD-DC, which has been described to inhibit the release of IL 1 by macrophages, caused a 70% decrease in IL 2-inducing activity. These results suggest that membrane-associated structures, that are identical to or mimic Il 1, are involved in the activation of T cells by DC

  13. Multifocal Langerhans cell sarcoma involving epidermis: a case report and review

    Directory of Open Access Journals (Sweden)

    Wang Changsong

    2012-08-01

    Full Text Available Abstract Objective To study the clinico-pathological characteristics of Langerhans cell sarcoma (LCS which involving epidermis. Methods A case of primary multifocal LCS was analyzed in histopathology and immunophenotype. Results A 41-year-old man with multifocal cutaneous LCS involving the inguina and waist was reported. Clinical and pathology data were available. Neoplastic cells with markedly malignant cytological features were observed. Tumor cells exhibited irregular shape with abundant and eosinophilic red staining cytoplasm; large, irregular-shaped, showing lobulated or dented nucleus and some cells with a longitudinal nuclear groove and prominent nucleoli. The tumor cells expressed CD1a, Langerin (CD207, S-100 protein, CD68 and vimentin, and did not express pan-T or B cell markers and epithelial markers. The patient died less than 1 year after diagnosis due to local recurrence and metastasis to the lung, despite the administration of local radiation and chemotherapy. Conclusions LCS is a tumor with markedly malignant cytological features that originates from Langerhans cells. Primary multifocal neoplasms involving epidermis is even rare. Accurate diagnosis is based on the histopathological and immunohistochemical of the tumor cells. Virtual slide The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1182345104754765.

  14. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Lam, R.K.K. [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); Han, Wei [Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); State Key Laboratory in Marine Pollution, City University of Hong Kong (Hong Kong)

    2015-12-15

    Highlights: • Rescue effect was observed in both irradiated and HeLa and NIH/3T3 cells. • Novel setup and procedures to separate the rescue signals and the bystander signals. • Confirmed activation of NF-κB pathway in rescue effect using activation inhibitor. • Confirmed activation of NF-κB pathway in rescue effect using anti-NF-κB p65 antibody. - Abstract: We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased

  15. Detection of protease activity in cells and animals.

    Science.gov (United States)

    Verdoes, Martijn; Verhelst, Steven H L

    2016-01-01

    Proteases are involved in a wide variety of biologically and medically important events. They are entangled in a complex network of processes that regulate their activity, which makes their study intriguing, but challenging. For comprehensive understanding of protease biology and effective drug discovery, it is therefore essential to study proteases in models that are close to their complex native environments such as live cells or whole organisms. Protease activity can be detected by reporter substrates and activity-based probes, but not all of these reagents are suitable for intracellular or in vivo use. This review focuses on the detection of proteases in cells and in vivo. We summarize the use of probes and substrates as molecular tools, discuss strategies to deliver these tools inside cells, and describe sophisticated read-out techniques such as mass spectrometry and various imaging applications. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Streptomyces sporulation - Genes and regulators involved in bacterial cell differentiation

    OpenAIRE

    Larsson, Jessica

    2010-01-01

    Streptomycetes are Gram-positive bacteria with a complex developmental life cycle. They form spores on specialized cells called aerial hyphae, and this sporulation involves alterations in growth, morphogenesis and cell cycle processes like cell division and chromosome segregation. Understanding the developmental mechanisms that streptomycetes have evolved for regulating for example cell division is of general interest in bacterial cell biology. It can also be valuable in the design of new dru...

  17. Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

    OpenAIRE

    Esteves Mabel B.; Marques-Santos Luis F.; Affonso-Mitidieri Ottília R.; Rumjanek Vivian M.

    2005-01-01

    Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase) suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100nM ouabain to cultures of peripheral b...

  18. Langerhans cell histiocytosis with involvement of the pons: case report

    Energy Technology Data Exchange (ETDEWEB)

    Vourtsi, A. [Xatzopoulou, Athens (Greece)]|[Department of Radiology, University of Athens Medical School, Athens (Greece); Papadopoulos, A.; Moulopoulos, L.A.; Vlahos, L. [Department of Radiology, University of Athens Medical School, Athens (Greece); Xenellis, J. [Department of Otorhinolaryngology, University of Athens Medical School, Athens (Greece)

    1998-03-01

    Central nervous system involvement is uncommon in Langerhans cell histiocytosis. The suprasellar region is more frequently affected. There have been few reports of involvement of the brain parenchyma shown on CT or MRI. We present a case of involvement of the pons, showing marked contrast enhancement on MRI. (orig.) With 2 figs., 17 refs.

  19. Syncytin is involved in breast cancer-endothelial cell fusions

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Holck, S.; Christensen, I.J.

    2006-01-01

    Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

  20. HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein

    International Nuclear Information System (INIS)

    Yu Qingsheng; Minoda, Yasumasa; Yoshida, Ryoko; Yoshida, Hideyuki; Iha, Hidekatsu; Kobayashi, Takashi; Yoshimura, Akihiko; Takaesu, Giichi

    2008-01-01

    Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-κB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-κB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax

  1. Vav3 oncogene activates estrogen receptor and its overexpression may be involved in human breast cancer

    International Nuclear Information System (INIS)

    Lee, Kiwon; Liu, Yin; Mo, Jun Qin; Zhang, Jinsong; Dong, Zhongyun; Lu, Shan

    2008-01-01

    Our previous study revealed that Vav3 oncogene is overexpressed in human prostate cancer, activates androgen receptor, and stimulates growth in prostate cancer cells. The current study is to determine a potential role of Vav3 oncogene in human breast cancer and impact on estrogen receptor a (ERα)-mediated signaling axis. Immunohistochemistry analysis was performed in 43 breast cancer specimens and western blot analysis was used for human breast cancer cell lines to determine the expression level of Vav3 protein. The impact of Vav3 on breast cancer cell growth was determined by siRNA knockdown of Vav3 expression. The role of Vav3 in ERα activation was examined in luciferase reporter assays. Deletion mutation analysis of Vav3 protein was performed to localize the functional domain involved in ERα activation. Finally, the interaction of Vav3 and ERα was assessed by GST pull-down analysis. We found that Vav3 was overexpressed in 81% of human breast cancer specimens, particularly in poorly differentiated lesions. Vav3 activated ERα partially via PI3K-Akt signaling and stimulated growth of breast cancer cells. Vav3 also potentiated EGF activity for cell growth and ERα activation in breast cancer cells. More interestingly, we found that Vav3 complexed with ERα. Consistent with its function for AR, the DH domain of Vav3 was essential for ERα activation. Vav3 oncogene is overexpressed in human breast cancer. Vav3 complexes with ERα and enhances ERα activity. These findings suggest that Vav3 overexpression may aberrantly enhance ERα-mediated signaling axis and play a role in breast cancer development and/or progression

  2. PEA3activates CXCL12transcription in MCF-7breast cancer cells%PEA3 activates CXCL12 transcription in MCF-7 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Li; CHEN Bo-bin; LI Jun-jie; JIN Wei; SHAO Zhi-min

    2011-01-01

    Objective To explore the activity of PEA3 ( polyomavirus enhancer activator 3 ) on CXCL12 (Chemokine CXC motif ligand 12) transcription and to reveal the role of PEA3 involved in CXCL12-mediated metastasis and angiogenesis in breast cancer. Methods Methods such as cell transfection, ChIP assay (chromatin immunoprecipitation ), and siRNA (small interfering RNA) were applied to demonstrate and confirm the interaction between PEA3 and CXCL12. Results Over-expression of PEA3 could increase the CXCL12 mRNA level and the CXCL12 promoter activity in human MCF-7 breast cancer cells. ChIP assay demonstrated that PEA3 could bind to the CXCL12 promoter in the cells transfected with PEA3 expression vector. PEA3 siRNA decreased CXCL12 promoter activity and the binding of PEA3 to the CXCL12 promoter in MCF-7 cells. Conclusions PEA3 could activate CXCL12 promoter transcription. It may be a potential mechanism of tumor angiogenesis and metastasis regarding of PEA3 and CXCL12.

  3. Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts*

    Science.gov (United States)

    Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

    2016-01-01

    Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. PMID:26670608

  4. Involvement in Physical Activity

    Directory of Open Access Journals (Sweden)

    James Gavin

    2013-04-01

    Full Text Available A total of 1,096 adolescents participated in 123 focus groups regarding the perceived outcomes of their involvement in sports and physical activity (PA. The groups, segmented by grade level, sex, and school types, were conducted in both public and private high schools in Montreal, Quebec. We sought to understand, through the participants’ own words, their perception of the outcome matrix of involvement in sports and PA. Focus group questions emphasized changes that adolescents associated with such engagement. In particular, participants were asked how sports and PA might influence behaviors, emotional states, personal characteristics, and other outcomes. Twelve themes were identified in the responses: Positive Health and Physical Changes (18.5%, Activity-Related Positive Emotions (15.6%, and Personal Learning (11.3% were most prevalent in the discussions. A cluster of deeper personal changes thematically described as Self-Identity, Autonomy, and Positive Character Development accounted for another 16.5% of the responses. Relatively few commentaries emphasized negative effects (7.1%. Converting the proportions of qualitative data into a quantitative index allowed us to analyze potential differences in emphasis according to sex, age, and school type. Though a few significant findings emerged, the larger pattern was of a uniform perceptual map across the variables for this adolescent sample. Implications drawn from this investigation highlight the need to clearly articulate concrete pathways to positive nonphysical changes (e.g., mood states, autonomy, positive character development from engagements in sports and PA.

  5. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Directory of Open Access Journals (Sweden)

    Tongfang Tang

    Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  6. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Science.gov (United States)

    Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

    2013-01-01

    Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  7. Involvement of c-Met- and phosphatidylinositol 3-kinase dependent pathways in arsenite-induced downregulation of catalase in hepatoma cells.

    Science.gov (United States)

    Kim, Soohee; Lee, Seung Heon; Kang, Sukmo; Lee, Lyon; Park, Jung-Duck; Ryu, Doug-Young

    2011-01-01

    Catalase protects cells from reactive oxygen species-induced damage by catalyzing the breakdown of hydrogen peroxide to oxygen and water. Arsenite decreases catalase activity; it activates phosphatidylinositol 3-kinase (PI3K) and its key downstream effector Akt in a variety of cells. The PI3K pathway is known to inhibit catalase expression. c-Met, an upstream regulator of PI3K and Akt, is also involved in the regulation of catalase expression. To examine the involvement of c-Met and PI3K pathways in the arsenite-induced downregulation of catalase, catalase mRNA and protein expression were analyzed in the human hepatoma cell line HepG2 treated with arsenite and either an inhibitor of c-Met (PHA665752 (PHA)) or of PI3K (LY294002 (LY)). Arsenite treatment markedly activated Akt and decreased the levels of both catalase mRNA and protein. Both PHA and LY attenuated arsenite-induced activation of Akt. PHA and LY treatment also prevented the inhibitory effect of arsenite on catalase protein expression but did not affect the level of catalase mRNA. These findings suggest that arsenite-induced inhibition of catalase expression is regulated at the mRNA and post-transcriptional levels in HepG2 cells, and that the post-transcriptional regulation is mediated via c-Met- and PI3K-dependent mechanisms.

  8. Organelle-Specific Activity-Based Protein Profiling in Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wiedner, Susan D.; Anderson, Lindsey N.; Sadler, Natalie C.; Chrisler, William B.; Kodali, Vamsi K.; Smith, Richard D.; Wright, Aaron T.

    2014-02-06

    A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.

  9. The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

    Science.gov (United States)

    Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

    2009-01-01

    Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

  10. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

    Science.gov (United States)

    Ku, H; Meier, K E

    2000-04-14

    Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

  11. Modeling interdisciplinary activities involving Mathematics

    DEFF Research Database (Denmark)

    Iversen, Steffen Møllegaard

    2006-01-01

    In this paper a didactical model is presented. The goal of the model is to work as a didactical tool, or conceptual frame, for developing, carrying through and evaluating interdisciplinary activities involving the subject of mathematics and philosophy in the high schools. Through the terms...... of Horizontal Intertwining, Vertical Structuring and Horizontal Propagation the model consists of three phases, each considering different aspects of the nature of interdisciplinary activities. The theoretical modelling is inspired by work which focuses on the students abilities to concept formation in expanded...... domains (Michelsen, 2001, 2005a, 2005b). Furthermore the theoretical description rest on a series of qualitative interviews with teachers from the Danish high school (grades 9-11) conducted recently. The special case of concrete interdisciplinary activities between mathematics and philosophy is also...

  12. T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1

    Directory of Open Access Journals (Sweden)

    Malika Trad

    2015-01-01

    Full Text Available T lymphocytes activated by dendritic cells (DC which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.

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

    International Nuclear Information System (INIS)

    Yu, Teng; Ji, Jiang; Guo, Yong-li

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-08

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

  15. Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.

    Science.gov (United States)

    Lin, C S; Boltz, R C; Blake, J T; Nguyen, M; Talento, A; Fischer, P A; Springer, M S; Sigal, N H; Slaughter, R S; Garcia, M L

    1993-03-01

    The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

  16. T-cell activation and early gene response in dogs.

    Directory of Open Access Journals (Sweden)

    Sally-Anne Mortlock

    Full Text Available T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR, and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA (5μg/ml, including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2, early growth response 1 (EGR1, growth arrest and DNA damage-inducible gene (GADD45B, phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS, early growth response 2 (EGR2, hemogen (HEMGN, polo-like kinase 2 (PLK2 and polo-like kinase 3 (PLK3. Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in

  17. Involvement of HIF-1α activation in the doxorubicin resistance of human osteosarcoma cells.

    Science.gov (United States)

    Roncuzzi, Laura; Pancotti, Fabia; Baldini, Nicola

    2014-07-01

    Osteosarcoma is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, survival outcomes remain unsatisfactory, particularly in patients with metastatic and/or recurrent disease. Unfortunately, treatment failure is commonly due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. The aim of the present study was to investigate the role of hypoxia-inducible factor 1α (HIF‑1α) and its signalling pathways as mediators of drug-resistance in human osteosarcoma. Toward this aim, we established two osteosarcoma cell lines selected for resistance to doxorubicin, a drug of choice in the treatment of this tumour. Our results showed that the multidrug resistance (MDR) phenotype was also mediated by HIF-1α, the most important regulator of cell adaptation to hypoxia. Our data showed that this transcription factor promoted the outward transport of intracellular doxorubicin by activating the P-glycoprotein (P-gp) expression in osteosarcoma cells maintained in normoxic conditions. In addition, it hindered doxorubicin-induced apoptosis by regulating the expression of c-Myc and p21. Finally, we observed that the doxorubicin-resistant cells maintained for 2 months of continuous culture in a drug-free medium, lost their drug-resistance and this effect was associated with the absence of HIF-1α expression. The emerging role of HIF-1α in osteosarcoma biology indicates its use as a valuable therapeutic target.

  18. A teleost CD46 is involved in the regulation of complement activation and pathogen infection.

    Science.gov (United States)

    Li, Mo-Fei; Sui, Zhi-Hai; Sun, Li

    2017-11-03

    In mammals, CD46 is involved in the inactivation of complement by factor I (FI). In teleost, study on the function of CD46 is very limited. In this study, we examined the immunological property of a CD46 molecule (CsCD46) from tongue sole, a teleost species with important economic value. We found that recombinant CsCD46 (rCsCD46) interacted with FI and inhibited complement activation in an FI-dependent manner. rCsCD46 also interacted with bacterial pathogens via a different mechanism to that responsible for the FI interaction, involving different rCsCD46 sites. Cellular study showed that CsCD46 was expressed on peripheral blood leukocytes (PBL) and protected the cells against the killing effect of complement. When the CsCD46 on PBL was blocked by antibody before incubation of the cells with bacterial pathogens, cellular infection was significantly reduced. Consistently, when tongue sole were infected with bacterial pathogens in the presence of rCsCD46, tissue dissemination and survival of the pathogens were significantly inhibited. These results provide the first evidence to indicate that CD46 in teleosts negatively regulates complement activation via FI and protects host cells from complement-induced damage, and that CD46 is required for optimal bacterial infection probably by serving as a receptor for the bacteria.

  19. Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

    Science.gov (United States)

    Lee, Jin-Sun; Wang, Tsu-Shing; Lin, Ming Cheng; Lin, Wei-Wen; Yang, Jaw-Ji

    2017-10-31

    Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

  20. NMDA modulates oligodendrocyte differentiation of subventricular zone cells through PKC activation

    Directory of Open Access Journals (Sweden)

    Fabio eCavaliere

    2013-12-01

    Full Text Available Multipotent cells from the juvenile subventricular zone (SVZ possess the ability to differentiate into new neural cells. Depending on local signals, SVZ can generate new neurons, astrocytes or oligodendrocytes. We previously demonstrated that activation of NMDA receptors in SVZ progenitors increases the rate of oligodendrocyte differentiation. Here we investigated the mechanisms involved in NMDA receptor-dependent differentiation. Using functional studies performed with the reporter gene luciferase we found that activation of NMDA receptor stimulates PKC. In turn, stimulation of PKC precedes the activation of NADPH oxidase (NOX as demonstrated by translocation of the p67phox subunit to the cellular membrane. We propose that NOX2 is involved in the transduction of the signal from NMDA receptors through PKC activation as the inhibitor gp91 reduced their pro-differentiation effect. In addition, our data and that from other groups suggest that signaling through the NMDA receptor/PKC/NOX2 cascade generates ROS that activate the PI3/mTOR pathway and finally leads to the generation of new oligodendrocytes.

  1. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    International Nuclear Information System (INIS)

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-01-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK α subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21 waf/cip as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21 waf/cip and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21 waf/cip pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

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

    Directory of Open Access Journals (Sweden)

    Karimian H

    2014-09-01

    Full Text Available Hamed Karimian,1 Soheil Zorofchian Moghadamtousi,2 Mehran Fadaeinasab,3 Shahram Golbabapour,2 Mahboubeh Razavi,1 Maryam Hajrezaie,2 Aditya Arya,1 Mahmood Ameen Abdulla,4 Syam Mohan,5 Hapipah Mohd Ali,2 Mohamad Ibrahim Noordin1 1Department of Pharmacy, Faculty of Medicine, 2Institute of Biological Sciences, Faculty of Science, 3Department of Chemistry, 4Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 5Medical Research Centre, Jazan University, Jazan, Saudi Arabia Abstract: 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

  3. Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

    Science.gov (United States)

    Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

    2013-07-15

    ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. DMPD: Signals and receptors involved in recruitment of inflammatory cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 7744810 Signals and receptors involved in recruitment of inflammatory cells. Ben-Ba...ow Signals and receptors involved in recruitment of inflammatory cells. PubmedID 7744810 Title Signals and receptors involved in recr...uitment of inflammatory cells. Authors Ben-Baruch A, Mic

  5. AMP-activated kinase mediates adipose stem cell-stimulated neuritogenesis of PC12 cells.

    Science.gov (United States)

    Tan, B; Luan, Z; Wei, X; He, Y; Wei, G; Johnstone, B H; Farlow, M; Du, Y

    2011-05-05

    Adipose tissue stroma contains a population of mesenchymal stem cells, which support repair of damaged tissues through the protective effects of secreted trophic factors. Neurotrophic factors, including nerve growth factor (NGF) have been identified in media collected from cultured adipose-derived stem cells (ASC). We previously demonstrated that administration of cell-free ASC conditioned medium (ASC-CM) at 24 h after injury reduced lesion volume and promoted functional recovery in a rat model of neonatal brain hypoxic-ischemic (HI) injury. The timing of administration well after the peak in neural cell apoptosis in the affected region suggests that regeneration of lost neurons is promoted by factors in ASC-CM. In this study, we determined which of the factors in ASC-CM could induce neurogenesis by testing the ability of the mixture, either whole or after inactivating specific components, to stimulate neurite outgrowth in vitro using the neurogenic cell line PC12. Neuritogenesis in PC12 cells treated with ASC-CM was observed at a level comparable to that observed with purified recombinant NGF. It was observed that NGF in ASC-CM was mainly responsible for inducing PC12 cell neuritogenesis. Interestingly, both ASC-CM and NGF induced PC12 cell neuritogenesis through activation of the AMP-activated kinase (AMPK) pathway which is the central protein involved in controlling many critical functions in response to changes in the cellular energy status. Pharmacological and genetic inhibition of AMPK activity greatly reduced neuritogenesis in PC12 cells. These results suggest that, in addition to possessing neuroprotective properties, ASC-CM mediates repair of damaged tissues through inducing neuronal differentiation via NGF-induced AMPK activation. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Cellular processes involved in human epidermal cells exposed to extremely low frequency electric fields.

    Science.gov (United States)

    Collard, J-F; Hinsenkamp, M

    2015-05-01

    We observed on different tissues and organisms a biological response after exposure to pulsed low frequency and low amplitude electric or electromagnetic fields but the precise mechanism of cell response remains unknown. The aim of this publication is to understand, using bioinformatics, the biological relevance of processes involved in the modification of gene expression. The list of genes analyzed was obtained after microarray protocol realized on cultures of human epidermal explants growing on deepidermized human skin exposed to a pulsed low frequency electric field. The directed acyclic graph on a WebGestalt Gene Ontology module shows six categories under the biological process root: "biological regulation", "cellular process", "cell proliferation", "death", "metabolic process" and "response to stimulus". Enriched derived categories are coherent with the type of in vitro culture, the stimulation protocol or with the previous results showing a decrease of cell proliferation and an increase of differentiation. The Kegg module on WebGestalt has highlighted "cell cycle" and "p53 signaling pathway" as significantly involved. The Kegg website brings out interactions between FoxO, MAPK, JNK, p53, p38, PI3K/Akt, Wnt, mTor or NF-KappaB. Some genes expressed by the stimulation are known to have an exclusive function on these pathways. Analyses performed with Pathway Studio linked cell proliferation, cell differentiation, apoptosis, cell cycle, mitosis, cell death etc. with our microarrays results. Medline citation generated by the software and the fold change variation confirms a diminution of the proliferation, activation of the differentiation and a less well-defined role of apoptosis or wound healing. Wnt and DKK functional classes, DKK1, MACF1, ATF3, MME, TXNRD1, and BMP-2 genes proposed in previous publications after a manual analysis are also highlighted with other genes after Pathway Studio automatic procedure. Finally, an analysis conducted on a list of genes

  7. Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

    Science.gov (United States)

    Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

    2016-02-12

    Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. The exhausted CD4+CXCR5+ T cells involve the pathogenesis of human tuberculosis disease.

    Science.gov (United States)

    Bosco, Munyemana Jean; Wei, Ming; Hou, Hongyan; Yu, Jing; Lin, Qun; Luo, Ying; Sun, Ziyong; Wang, Feng

    2018-06-21

    The CD4 + CXCR5 + T cells have been previously established. However, their decreased frequency during tuberculosis (TB) disease is partially understood. The aim of this study was to explore the depletion of CD4 + CXCR5 + T cells in human TB. The frequency and function of CD4 + CXCR5 + T cells were evaluated in active TB (ATB) patients and healthy control (HC) individuals. The function of CD4 + CXCR5 + T cells was determined after blockade of inhibitory receptors. The frequency of CD4 + CXCR5 + T cells was decreased in ATB patients. The expression of activation markers (HLA-DR and ICOS) and inhibitory receptors (Tim-3 and PD-1) on CD4 + CXCR5 + T cells was increased in ATB group. TB-specific antigen stimulation induced higher expression of inhibitory receptors than phytohemagglutinin stimulation in ATB group. In contrast, TB antigen stimulation did not induce a significantly increased expression of IL-21 and Ki-67 on CD4 + CXCR5 + T cells. However, blockade of inhibitory receptors Tim-3 and PD-1 not only increased the frequency of CD4 + CXCR5 + T cells, but also restored their proliferation and cytokine secretion potential. An increased expression of inhibitory receptors involves the depletion of CD4 + CXCR5 + T cells, and blockade of inhibitory receptors can restore the function of CD4 + CXCR5 + T cells in ATB patients. Copyright © 2018. Published by Elsevier Ltd.

  9. High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

    Directory of Open Access Journals (Sweden)

    Gregory Kohn

    Full Text Available BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i, which could be cytotoxic because of its high affinity for Ca(2+, thereby interfering with Ca(2+ signaling.

  10. Isolated cutaneous involvement in a child with nodal anaplastic large cell lymphoma

    Directory of Open Access Journals (Sweden)

    Vibhu Mendiratta

    2016-01-01

    Full Text Available Non-Hodgkin lymphoma is a common childhood T-cell and B-cell neoplasm that originates primarily from lymphoid tissue. Cutaneous involvement can be in the form of a primary extranodal lymphoma, or secondary to metastasis from a non-cutaneous location. The latter is uncommon, and isolated cutaneous involvement is rarely reported. We report a case of isolated secondary cutaneous involvement from nodal anaplastic large cell lymphoma (CD30 + and ALK + in a 7-year-old boy who was on chemotherapy. This case is reported for its unusual clinical presentation as an acute febrile, generalized papulonodular eruption that mimicked deep fungal infection, with the absence of other foci of systemic metastasis.

  11. Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

    Science.gov (United States)

    Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

    2017-06-15

    Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate

  12. Cerebellar and basal ganglion involvement in Langerhans cell histiocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Saatci, I.; Baskan, O.; Haliloglu, M.; Aydingoz, U. [Department of Radiology, Hacettepe University Hospital, Sihhiye 06100, Ankara (Turkey)

    1999-06-01

    Langerhans cell histiocytosis (LCH) is a disease of unknown cause characterised by proliferation of histiocytic granulomas in tissues; the primary cerebral manifestation is diabetes insipidus caused by hypothalamic infiltration. We present a patient in whom, except for the absence of high signal on T 1 weighting in the posterior pituitary, consistent with central diabetes insipidus, MRI showed no evidence of hypothalamic involvement by histiocytosis, despite the long duration of the disease. However, there was bilateral, symmetrical involvement of the cerebellum and globus pallidus in addition to a calvarial lesion. High signal in the cerebellar white matter on T 2-weighted images may represent demyelination, gliosis and cell loss, as previously reported on pathologic examination. (orig.) With 5 figs., 22 refs.

  13. PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

    Science.gov (United States)

    Miranda, Dante; Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra; Montoya, Margarita

    2016-01-01

    Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

  14. Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease.

    Science.gov (United States)

    Akiyama, Mitsuhiro; Yasuoka, Hidekata; Yamaoka, Kunihiro; Suzuki, Katsuya; Kaneko, Yuko; Kondo, Harumi; Kassai, Yoshiaki; Koga, Keiko; Miyazaki, Takahiro; Morita, Rimpei; Yoshimura, Akihiko; Takeuchi, Tsutomu

    2016-07-13

    naïve B cells into plasmablasts and enhanced production of IgG4 in patients with active, untreated IgG4-RD. Furthermore, activated Tfh2 cells reflect disease activity, suggesting the involvement of this T cell subset in the pathogenesis of IgG4-RD. Interestingly, the number of activated Tfh1 cells was also increased in IgG4-RD, correlating with disease activity but not with serum IgG4 level, suggesting the involvement of Tfh1 cells but not in the process of IgG4 production in patients with IgG4-RD.

  15. Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics.

    Science.gov (United States)

    Mravec, Jozef; Guo, Xiaoyuan; Hansen, Aleksander Riise; Schückel, Julia; Kračun, Stjepan Krešimir; Mikkelsen, Maria Dalgaard; Mouille, Grégory; Johansen, Ida Elisabeth; Ulvskov, Peter; Domozych, David S; Willats, William George Tycho

    2017-06-01

    The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea ( Pisum sativum ) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. Caspase Activation of p21-Activated Kinase 2 Occurs During Cisplatin-Induced Apoptosis of SH-SY5Y Neuroblastoma Cells and in SH-SY5Y Cell Culture Models of Alzheimer’s and Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Jerry W. Marlin

    2010-04-01

    Full Text Available p21-activated kinase 2 (PAK-2 appears to have a dual function in the regulation of cell survival and cell death. Activation of full-length PAK-2 by the p21 G-proteins Rac or Cdc42 stimulates cell survival. However, PAK-2 is unique among the PAK family because it is also activated through proteolytic cleavage by caspase 3 or similar caspases to generate the constitutively active PAK-2p34 fragment. Caspase activation of PAK-2 correlates with the induction of apoptosis in response to many stimuli and recombinant expression of PAK-2p34 has been shown to stimulate apoptosis in several human cell lines. Here, we show that caspase activation of PAK-2 also occurs during cisplatin-induced apoptosis of SH-SY5Y neuroblastoma cells as well as in SH-SY5Y cell culture models for Alzheimer’s and Parkinson’s disease. Inhibition of mitochondrial complex I or of ubiquitin/proteasome-mediated protein degradation, which both appear to be involved in Parkinson’s disease, induce apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Overexpression of the amyloid precursor protein, which results in accumulation and aggregation of β-amyloid peptide, the main component of β-amyloid plaques in Alzheimer’s disease, also induces apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Expression of the PAK-2 regulatory domain inhibits caspase-activated PAK-2p34 and prevents apoptosis in 293T human embryonic kidney cells, indicating that caspase activation of PAK-2 is directly involved in the apoptotic response. This is the first evidence that caspase activation of PAK-2 correlates with apoptosis in cell culture models of Alzheimer’s and Parkinson’s disease and that selective inhibition of caspase-activated PAK-2p34 could prevent apoptosis.

  17. 1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase.

    Science.gov (United States)

    Kolesnick, R N; Clegg, S

    1988-05-15

    It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C

  18. Protective effect of the poly(ADP-ribose polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation

    Directory of Open Access Journals (Sweden)

    Radnai Balazs

    2012-05-01

    Full Text Available Abstract Background 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11 was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose-polymerase (PARP, protein kinase B/Akt and mitogen activated protein kinase (MAPK activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. Results We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2, and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. Conclusions These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

  19. Telomerase Activity Impacts on Epstein-Barr Virus Infection of AGS Cells

    Science.gov (United States)

    Rac, Jürgen; Haas, Florian; Schumacher, Andrina; Middeldorp, Jaap M.; Delecluse, Henri-Jacques; Speck, Roberto F.

    2015-01-01

    The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva. PMID:25856387

  20. Microtubule array reorientation in response to hormones does not involve changes in microtubule nucleation modes at the periclinal cell surface

    Science.gov (United States)

    Atkinson, Samantha; Kirik, Angela; Kirik, Viktor

    2014-01-01

    Aligned microtubule arrays spatially organize cell division, trafficking, and determine the direction of cell expansion in plant cells. In response to changes in environmental and developmental signals, cells reorganize their microtubule arrays into new configurations. Here, we tested the role of microtubule nucleation during hormone-induced microtubule array reorientation. We have found that in the process of microtubule array reorientation the ratios between branching, parallel, and de-novo nucleations remained constant, suggesting that the microtubule reorientation mechanism does not involve changes in nucleation modes. In the ton2/fass mutant, which has reduced microtubule branching nucleation frequency and decreased nucleation activity of the γ-tubulin complexes, microtubule arrays were able to reorient. Presented data suggest that reorientation of microtubules into transverse arrays in response to hormones does not involve changes in microtubule nucleation at the periclinal cell surface PMID:25135522

  1. Metabolic activity is necessary for activation of T suppressor cells by B cells

    International Nuclear Information System (INIS)

    Elkins, K.L.; Stashak, P.W.; Baker, P.J.

    1990-01-01

    Ag-primed B cells must express cell-surface IgM, but not IgD or Ia Ag, and must remain metabolically active, in order to activate suppressor T cells (Ts) specific for type III pneumococcal polysaccharide. Ag-primed B cells that were gamma-irradiated with 1000r, or less, retained the ability to activate Ts; however, Ag-primed B cells exposed to UV light were not able to do so. gamma-Irradiated and UV-treated Ag-primed B cells both expressed comparable levels of cell-surface IgM, and both localized to the spleen after in vivo transfer; neither could proliferate in vitro in response to mitogens. By contrast, gamma-irradiated primed B cells were still able to synthesize proteins, whereas UV-treated primed B cells could not. These findings suggest that in order for Ag-primed B cells to activate Ts, they must (a) express cell-associated IgM (sIgM) antibody bearing the idiotypic determinants of antibody specific for type III pneumococcal polysaccharide, and (b) be able to synthesize protein for either the continued expression of sIgM after cell transfer, or for the elaboration of another protein molecule that is also required for the activation of Ts; this molecule does not appear to be Ia Ag

  2. Langerhans cell histiocytosis involving central nervous system: a case report

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Won Jin; Park, Dong Woo; Lee, Seung Ro; Hahm, Chang Kok; Ju, Kyung Bin [Hanyang University College of Medicine, Seoul (Korea, Republic of); Kim, Sung Tae [Ulsan University College of Medicine, Seoul (Korea, Republic of)

    1997-01-01

    Langerhans cell histiocytosis(LCH) is a systemic disorder characterized by idiopathic proliferation of histiocytes in the reticuloendothelial system; CNS involvement outside the hypothalamus or pituitary gland is uncommon. We present a case of LCH involving the brainstem, cerebellum, and temporal lobes, and also showing hypothalamic involvement. The lesions were isointense or hypointense on T1WI and hyperintense on T2WI, and showed multifocal enhancing nodules on post-contrast CT and Gd-enhanced MRI.

  3. Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205

    International Nuclear Information System (INIS)

    Gupta, Dyuti Datta; Saha, Subhrajit; Chakrabarti, Manoj K.

    2005-01-01

    The present study was undertaken to determine the involvement of calcium-protein kinase C pathway in the mechanism of action of Escherichia coli heat stable enterotoxin (STa) apart from STa-induced activation of guanylate cyclase in human colonic carcinoma cell line COLO-205, which was used as a model cultured cell line to study the mechanism of action of E. coli STa. In response to E. coli STa, protein kinase C (PKC) activity was increased in a time-dependent manner with its physical translocation from cytosol to membrane. Inhibition of the PKC activity in membrane fraction and inhibition of its physical translocation in response to IP 3 -mediated calcium release inhibitor dantrolene suggested the involvement of intracellular store depletion in the regulation of PKC activity. Among different PKC isoforms, predominant involvement of calcium-dependent protein kinase C (PKCα) was specified using isotype-specific pseudosubstrate, which showed pronounce enzyme activity. Inhibition of enzyme activity by PKCα-specific inhibitor Goe6976 and immunoblott study employing isotype-specific antibody further demonstrated the involvement of calcium-dependent isoform of PKC in the mechanism of action of E. coli STa. Moreover, inhibition of guanylate cyclase activity by PKCα-specific inhibitor Goe6976 suggested the involvement of PKCα in the regulation of guanylate cyclase activity

  4. Neuroimmune regulation of microglial activity involved in neuroinflammation and neurodegenerative diseases.

    Science.gov (United States)

    González, Hugo; Elgueta, Daniela; Montoya, Andro; Pacheco, Rodrigo

    2014-09-15

    Neuroinflammation constitutes a fundamental process involved in the progression of several neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and multiple sclerosis. Microglial cells play a central role in neuroinflammation, promoting neuroprotective or neurotoxic microenvironments, thus controlling neuronal fate. Acquisition of different microglial functions is regulated by intercellular interactions with neurons, astrocytes, the blood-brain barrier, and T-cells infiltrating the central nervous system. In this study, an overview of the regulation of microglial function mediated by different intercellular communications is summarised and discussed. Afterward, we focus in T-cell-mediated regulation of neuroinflammation involved in neurodegenerative disorders. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Epithelial-mesenchymal transition and cancer stem cells, mediated by a long non-coding RNA, HOTAIR, are involved in cell malignant transformation induced by cigarette smoke extract

    International Nuclear Information System (INIS)

    Liu, Yi; Luo, Fei; Xu, Yuan; Wang, Bairu; Zhao, Yue; Xu, Wenchao; Shi, Le; Lu, Xiaolin; Liu, Qizhan

    2015-01-01

    The incidence of lung diseases, including cancer, caused by cigarette smoke is increasing, but the molecular mechanisms of gene regulation induced by cigarette smoke remain unclear. This report describes a long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) and experiments utilizing lncRNAs to integrate inflammation with the epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells. The present study shows that, induced by CSE, IL-6, a pro-inflammatory cytokine, leads to activation of STAT3, a transcription activator. A ChIP assay determined that the interaction of STAT3 with the promoter regions of HOX transcript antisense RNA (HOTAIR) increased levels of HOTAIR. Blocking of IL-6 with anti-IL-6 antibody, decreasing STAT3, and inhibiting STAT3 activation reduced HOTAIR expression. Moreover, for HBE cells cultured in the presence of HOTAIR siRNA for 24 h, the CSE-induced EMT, formation of cancer stem cells (CSCs), and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates HOTAIR in an autocrine manner, contributes to the EMT and to CSCs induced by CSE. These data define a link between inflammation and EMT, processes involved in the malignant transformation of cells caused by CSE. This link, mediated through lncRNAs, establishes a mechanism for CSE-induced lung carcinogenesis. - Highlights: • STAT3 directly regulates the levels of LncRNA HOTAIR. • LncRNA HOTAIR mediates the link between inflammation and EMT. • LncRNA HOTAIR is involved in the malignant transformation of cells caused by CSE

  6. Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival

    Directory of Open Access Journals (Sweden)

    Hsieh Fu-Chuan

    2008-10-01

    Full Text Available Abstract Background Constitutive activation of signal transducer and activator of transcription 3 (Stat3 signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer. Results We found that elevated Stat3 phosphorylation in 19 of 100 (19% bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC. The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin and a cell cycle regulating gene (cyclin D1 was associated with the cell growth inhibition and apoptosis. Conclusion These results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

  7. PKR Activation Favors Infectious Pancreatic Necrosis Virus Replication in Infected Cells

    Directory of Open Access Journals (Sweden)

    Amr A.A. Gamil

    2016-06-01

    Full Text Available The double-stranded RNA-activated protein kinase R (PKR is a Type I interferon (IFN stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells.

  8. Lipid raft involvement in yeast cell growth and death

    Energy Technology Data Exchange (ETDEWEB)

    Mollinedo, Faustino, E-mail: fmollin@usal.es [Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas - Universidad de Salamanca, Salamanca (Spain)

    2012-10-10

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na{sup +}, K{sup +}, and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  9. Lipid raft involvement in yeast cell growth and death

    International Nuclear Information System (INIS)

    Mollinedo, Faustino

    2012-01-01

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na + , K + , and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases.

  10. Activation of human B lymphocytes. 8. Differential radiosensitivity of subpopulations of lymphoid cells involved in the polyclonally-induced PFC responses of peripheral blood B lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Fauci, A S; Pratt, K R; Whalen, G [National Inst. of Allergy and Infectious Diseases, Bethesda, MD (USA)

    1978-11-01

    The differential effect of various doses of irradiation on subpopulations of human peripheral blood lymphoid cells involved in the pokeweed mitogen induced PFC response against sheep red blood cells was studied. The plaque forming B cells were quite sensitive to low doses of irradiation with complete suppression of responses at 300 to 500 rad. On the contrary, helper T-cell function was resistant to 2000 rad. Co-culture of irradiated T cells with autologous or allogeneic B cells resulted in marked enhancement of PFC responses consistent with the suppression of naturally occurring suppressor cells with a resulting pure helper effect. Irradiated T-cell-depleted suspensions failed to produce this effect as did heat killed T cells, whereas mitomycin C treated T cells gave effects similar to irradiated T cells. These findings are consistent with a lack of requirement of cell division for a T-cell helper effect and a requirement of mitosis or another irradiation sensitive, mitomycin C sensitive process for a T-suppressor cell effect. These studies have potential relevance in the evaluation of subpopulations of human lymphoid cells involved in antibody production in normal individuals and in disease states.

  11. Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

    Science.gov (United States)

    Hultsch, T; Brand, P; Lohmann, S; Saloga, J; Kincaid, R L; Knop, J

    1998-05-01

    FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.

  12. Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

    International Nuclear Information System (INIS)

    Wang, Wei; Zhang, Yuan; Xu, Ming; Zhang, You-Yi; He, Bei

    2015-01-01

    The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β 2 -adrenergic receptor (β 2 -AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β 2 -AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β 2 -AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β 2 -AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production

  13. Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei [Department of Respiratory Medicine, Peking University Third Hospital, Beijing (China); Department of Infectious Diseases, Peking University Third Hospital, Beijing (China); Zhang, Yuan [Department of Respiratory Medicine, Peking University Third Hospital, Beijing (China); Xu, Ming; Zhang, You-Yi [Department of Institute of Vascular Medicine and Beijing Key Laboratory of Cardiovascular Receptors Research, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Peking University Third Hospital, Beijing (China); He, Bei, E-mail: puh3_hb@bjmu.edu.cn [Department of Respiratory Medicine, Peking University Third Hospital, Beijing (China)

    2015-06-26

    The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.

  14. Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

    Science.gov (United States)

    Pahl, Jens H W; Kwappenberg, Kitty M C; Varypataki, Eleni M; Santos, Susy J; Kuijjer, Marieke L; Mohamed, Susan; Wijnen, Juul T; van Tol, Maarten J D; Cleton-Jansen, Anne-Marie; Egeler, R Maarten; Jiskoot, Wim; Lankester, Arjan C; Schilham, Marco W

    2014-03-10

    In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our

  15. Egr-1 activation by cancer-derived extracellular vesicles promotes endothelial cell migration via ERK1/2 and JNK signaling pathways.

    Directory of Open Access Journals (Sweden)

    Yae Jin Yoon

    Full Text Available Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs, also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1 activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference-mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases.

  16. Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

    Science.gov (United States)

    Pirger, Zsolt; Rácz, Boglárka; Kiss, Tibor

    2009-02-01

    PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

  17. Antitumor potential of crown ethers: structure-activity relationships, cell cycle disturbances, and cell death studies of a series of ionophores.

    Science.gov (United States)

    Marjanović, Marko; Kralj, Marijeta; Supek, Fran; Frkanec, Leo; Piantanida, Ivo; Smuc, Tomislav; Tusek-Bozić, Ljerka

    2007-03-08

    The present paper demonstrates the antiproliferative ability and structure-activity relationships (SAR) of 14 crown and aza-crown ether analogues on five tumor-cell types. The most active compounds were di-tert-butyldicyclohexano-18-crown-6 (3), which exhibited cytotoxicity in the submicromolar range, and di-tert-butyldibenzo-18-crown-6 (5) (IC50 values of approximately 2 microM). Also, 3 and 5 induced marked influence on the cell cycle phase distribution--strong G1 arrest, followed by the induction of apoptosis. A computational SAR modeling effort offers insight into possible mechanisms of crown ether biological activity, presumably involving penetration into cell membranes, and points out structural features of molecules important for this activity. The results reveal that crown ethers possess marked tumor-cell growth inhibitory activity, the extent of which depends on the characteristics of the hydrophilic macrocylic cavity and the surrounding hydrophobic ring. Our work supports the hypothesis that crown ether compounds inhibit tumor-cell growth by disrupting potassium ion homeostasis, which in turn leads to cell cycle perturbations and apoptosis.

  18. Mast cells enhance T cell activation: Importance of mast cell-derived TNF

    Science.gov (United States)

    Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

    2005-05-01

    Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

  19. Abnormal expression of leiomyoma cytoskeletal proteins involved in cell migration.

    Science.gov (United States)

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Athanasakis, Emmanouil; Aloisio, Michelangelo; Monasta, Lorenzo; Ricci, Giuseppe

    2016-05-01

    Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two‑dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.

  20. Ubiquitin-activating enzyme is necessary for 17β-estradiol-induced breast cancer cell proliferation and migration.

    Science.gov (United States)

    Pesiri, Valeria; Totta, Pierangela; Marino, Maria; Acconcia, Filippo

    2014-08-01

    The sex steroid hormone 17β-estradiol (E2) regulates breast cancer (BC) cell proliferation and migration through the activation of a plethora of signal transduction cascades (e.g., PI3K/AKT activation) starting after E2 binding to the estrogen receptor alpha (ERα). The activity of the ubiquitin (Ub)-system modulates many physiological processes (e.g., cell proliferation and migration), and recently, a specific inhibitor (Pyr-41) of the Ub-activating enzyme (E1), which works as the activator of the Ub-based signaling, has been identified to prevent the functions of the Ub-system. Here, by using Pyr-41, we studied the involvement of the Ub-system in E2-induced signaling to proliferation and migration of BC cells. Our data indicate that E1 activity is involved in the E2:ERα signaling important for cell proliferation and migration through the modulation of the E2-evoked activation of the PI3K/AKT and the p38/MAPK pathways. These discoveries indicate a new molecular circuitry that can be further explored to define new opportunities for BC treatment. © 2014 International Union of Biochemistry and Molecular Biology.

  1. AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation.

    Science.gov (United States)

    Han, Xiaojuan; Tai, Haoran; Wang, Xiaobo; Wang, Zhe; Zhou, Jiao; Wei, Xiawei; Ding, Yi; Gong, Hui; Mo, Chunfen; Zhang, Jie; Qin, Jianqiong; Ma, Yuanji; Huang, Ning; Xiang, Rong; Xiao, Hengyi

    2016-06-01

    AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress-induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide-induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP-RFP-LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD(+) levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD(+) synthesis. In addition, the mechanistic relationship of autophagic flux and NAD(+) synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress-induced senescence by improving autophagic flux and NAD(+) homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD(+) homeostasis, and it is also valuable in the development of innovative strategies to combat aging. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  2. Downregulation of Endogenous Hydrogen Sulfide Pathway Is Involved in Mitochondrion-Related Endothelial Cell Apoptosis Induced by High Salt

    Directory of Open Access Journals (Sweden)

    Yanfang Zong

    2015-01-01

    Full Text Available Background. The study aimed to investigate whether endogenous H2S pathway was involved in high-salt-stimulated mitochondria-related vascular endothelial cell (VEC apoptosis. Methods. Cultured human umbilical vein endothelial cells (HUVECs were used in the study. H2S content in the supernatant was detected. Western blot was used to detect expression of cystathionine gamma-lyase (CSE, cleaved-caspase-3, and mitochondrial and cytosolic cytochrome c (cytc. Fluorescent probes were used to quantitatively detect superoxide anion generation and measure the in situ superoxide anion generation in HUVEC. Mitochondrial membrane pore opening, mitochondrial membrane potential, and caspase-9 activities were measured. The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL methods. Results. High-salt treatment downregulated the endogenous VEC H2S/CSE pathway, in association with increased generation of oxygen free radicals, decreased mitochondrial membrane potential, enhanced the opening of mitochondrial membrane permeability transition pore and leakage of mitochondrial cytc, activated cytoplasmic caspase-9 and caspase-3 and subsequently induced VEC apoptosis. However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt. Conclusion. H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult. The protective mechanisms for VEC damage might involve inhibiting oxidative stress and protecting mitochondrial injury.

  3. The Potential Mechanism of ZFX Involvement in the Cell Growth

    Directory of Open Access Journals (Sweden)

    Mahboube Ganji arjenaki

    2016-04-01

    Full Text Available Background:The zinc-finger X linked (ZFX gene encodes a transcription factor that acts as a regulator of self-renewal of stem cells. Due to the role of ZFX in cell growth, understanding ZFX protein-protein interactions helps to clarify its proper biological functions in signaling pathways. The aim of this study is to define ZFX protein-protein interactions and the role of ZFX in cell growth. Materials and Methods: The PIPs output includes three interacting proteins with ZFX: eukaryotic translation initiation factor 3 subunit I(EIF3I, eukaryotic translation initiation factor 3 subunit G(EIF3G and protein nuclear pore and COPII coat complex component homolog isoform 3 (SEC13L1. Results: As a cargo and transmembrane protein interacting with Sec13,eIF3I and eIF3G, ZFX mediates cargo sorting in COPII vesicles at ER exit sites. While traveling to cis-Golgi, eIF3I is phosphorylated by the mechanistic target of rapamycin (mTOR. Proteins transport by COPI vesicles to the nucleusouter site layer containing SEC13 via the contribution of microtubules. EIF3G and eIF3I interact with coatomer protein complex subunit beta 2 (COPB2 that helps to enclose ZFX in COPI vesicle. ZFX and eIF3G enter nucleolus where activation of transcription from pre rDNA genes occurs. Conclusion:We proposed a model in which ZFX is involved in cell growth by promoting the transcription of rDNA genes.

  4. Interacting active elastic dimers: Two cells moving on a rigid track

    Science.gov (United States)

    Das, Moumita; Mayett, David; Schwarz, J. M.

    2015-03-01

    Cell migration in morphogenesis and cancer metastasis typically involves an interplay between different cell types. The rules governing such interplay remain largely unknown, however, a recent experiment studying the interaction between neural crest (NC) cells and placodal cells reveals an example of such rules. The study found that NC cells chase the placodal cells by chemotaxis, while placodal cells run away from NC cells when contacted by them. Motivated by this observation, we construct and study a minimal one-dimensional cell-cell model comprised of two cells with each cell represented by two-beads-connected-by-an-active spring. The active spring for each moving cell models the stress fibers with their myosin-driven contractility (and alpha-actinin extendability), while the friction coefficients of the beads describe the catch/slip bond behavior of the integrins in focal adhesions. We also include a dynamic contact interaction between the two cells, as well as a chemotactic potential, to decipher the chase-and-run dynamics observed in the experiment. We then use our modeling to further generalize the rules governing the interplay between different cell types during collective cell migration.

  5. Active Involvement of Software Developers in Usability Engineering

    DEFF Research Database (Denmark)

    Bornoe, Nis; Stage, Jan

    2017-01-01

    The essence of usability evaluations is to produce feedback that supports the downstream utility so the interaction design can be improved and problems can be fixed. In practice, software development organizations experience several obstacles for conducting usability engineering. One suggested...... approach is to train and involve developers in all phases of usability activities from evaluations, to problem reporting, and making redesign proposals. Only limited work has previously investigated the impact of actively involving developers in usability engineering. In this paper, we present two small......, and problem fixing. At the organizational level, we found that the attitude towards and understanding of the role of usability engineering improved....

  6. Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

    Science.gov (United States)

    Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

    2009-01-01

    High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226

  7. Protein tyrosine phosphatase-PEST (PTP-PEST) regulates mast cell-activating signals in PTP activity-dependent and -independent manners.

    Science.gov (United States)

    Motohashi, Satoru; Koizumi, Karen; Honda, Reika; Maruyama, Atsuko; Palmer, Helen E F; Mashima, Keisuke

    2014-01-01

    Aggregation of the high-affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Tyrosine phosphorylation of proteins in response to FcεRI aggregation has been implicated in mast cell activation. Here, we determined the role of PTP-PEST (encoded by PTPN12) in the regulation of mast cell activation using the RBL-2H3 rat basophilic leukemia cell line as a model. PTP-PEST expression was significantly induced upon FcεRI-crosslinking, and aggregation of FcεRI induced the phosphorylation of PTP-PEST at Ser39, thus resulting in the suppression of PTP activity. By overexpressing a phosphatase-dead mutant (PTP-PEST CS) and a constitutively active mutant (PTP-PEST SA) in RBL-2H3 cells, we showed that PTP-PEST decreased degranulation and enhanced IL-4 and IL-13 transcription in FcεRI-crosslinked RBL-2H3 cells, but PTP activity of PTP-PEST was not necessary for this regulation. However, FcεRI-induced TNF-α transcription was increased by the overexpression of PTP-PEST SA and suppressed by the overexpression of PTP-PEST CS. Taken together, these results suggest that PTP-PEST is involved in the regulation of FcεRI-mediated mast cell activation through at least two different processes represented by PTP activity-dependent and -independent pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

    Directory of Open Access Journals (Sweden)

    Claire M Perks

    2011-05-01

    Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

  9. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    International Nuclear Information System (INIS)

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8 + T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract SEB

  10. Antihepatic Fibrosis Effect of Active Components Isolated from Green Asparagus (Asparagus officinalis L.) Involves the Inactivation of Hepatic Stellate Cells.

    Science.gov (United States)

    Zhong, Chunge; Jiang, Chunyu; Xia, Xichun; Mu, Teng; Wei, Lige; Lou, Yuntian; Zhang, Xiaoshu; Zhao, Yuqing; Bi, Xiuli

    2015-07-08

    Green asparagus (Asparagus officinalis L.) is a vegetable with numerous nutritional properties. In the current study, a total of 23 compounds were isolated from green asparagus, and 9 of these compounds were obtained from this genus for the first time. Preliminary data showed that the ethyl acetate (EtOAc)-extracted fraction of green asparagus exerted a stronger inhibitory effect on the growth of t-HSC/Cl-6 cells, giving an IC50 value of 45.52 μg/mL. The biological activities of the different compounds isolated from the EtOAc-extracted fraction with respect to antihepatic fibrosis were investigated further. Four compounds, C3, C4, C10, and C12, exhibited profound inhibitory effect on the activation of t-HSC/Cl-6 cells induced by TNF-α. The activation t-HSC/Cl-6 cells, which led to the production of fibrotic matrix (TGF-β1, activin C) and accumulation of TNF-α, was dramatically decreased by these compounds. The mechanisms by which these compounds inhibited the activation of hepatic stellate cells appeared to be associated with the inactivation of TGF-β1/Smad signaling and c-Jun N-terminal kinases, as well as the ERK phosphorylation cascade.

  11. Activated alveolar macrophage and lymphocyte alveolitis in extrathoracic sarcoidosis without radiological mediastinopulmonary involvement

    International Nuclear Information System (INIS)

    Wallaert, B.; Ramon, P.; Fournier, E.C.; Prin, L.; Tonnel, A.B.; Voisin, C.

    1986-01-01

    Cellular characteristics of BAL were investigated in 18 patients with proved extrathoracic sarcoidosis (that is, sarcoidosis that affected the skin, eyes, parotid glands, stomach, nose, kidneys, or meninges) without clinical or radiological mediastinopulmonary involvement. Computed tomography of the thorax was performed on five patients: four patients were normal, and one had enlarged lymph nodes (these enlargements were not detectable on the patient's chest roentgenogram). The results of pulmonary function tests were normal in all patients. The total BAL cell count did not differ significantly between controls and patients. Abnormal percentages of alveolar lymphocytes (from 18 to 87%) were noted in 15 out of 18 patients. SACE levels were normal in 15 patients. No pulmonary gallium uptake was detected. The chemiluminescence of AM's, whether spontaneous or PMA induced, was increased in five out of seven patients. The percentages of T3+ lymphocytes in sarcoidosis patients did not significantly differ from those in controls. The T4+:T8+ ratio was normal in four patients and slightly increased in one. Follow-up of patients showed that alveolar lymphocytosis is as lasting as extrathoracic involvement. Our data demonstrate increased percentages of lymphocytes and activated AM's in the BAL of patients with extrathoracic sarcoidosis. This may be due to the initial involvement of the respiratory tract in extrathoracic sarcoidosis or to the diffusion of activated macrophages and lymphocytes from an extrathoracic site into the lung

  12. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    International Nuclear Information System (INIS)

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-01-01

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47 phox , p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-α induces

  13. Molecular mechanisms involved in the inhibition of tumor cells proliferation exposed to elevated concentrations of the epidermal growth factor

    International Nuclear Information System (INIS)

    Guillen, Isabel A; Berlanga, Jorge; Camacho, Hanlet

    2013-01-01

    The EGF promotes inhibition of cell proliferation in vitro and in vivo models depending on its concentration, application schema and the type of tumor cells on which it acts. Our research hypothesis was based on the fact that the EGF varies the expression of genes involved in a negative regulation of tumor cell lines proliferation carrying high levels of its receptor (EGFR). Our objectives were, to obtain information about the effect of EGF on tumor cell proliferation in vitro and in vivo models and, know the gene expression patterns of a group of genes involved in cancer signaling pathways and EGFR. The results showed that EGF at nanomolar concentrations inhibits the tumor cells proliferation bearing high levels of EGFR and, promotes the survival of treated animals, establishing a direct relationship between the inhibition of cell proliferation, high concentrations of EGF and, high amount of EGFR in the cells. The differential gene expression profile showed a variation in a group of genes which exert a powerful control over the cell cycle progression, gene transcription and apoptosis. It was concluded that the inhibition of tumor cell proliferation by the action of EGF is due to activation of molecular mechanisms controlling cell cycle progression. This work won the Annual Award of the Cuban Academy of Sciences in 2012

  14. ERK1/2 signalling pathway is involved in CD147-mediated gastric cancer cell line SGC7901 proliferation and invasion.

    Science.gov (United States)

    Chen, Liping; Pan, Yuqin; Gu, Ling; Nie, Zhenlin; He, Bangshun; Song, Guoqi; Li, Rui; Xu, Yeqiong; Gao, Tianyi; Wang, Shukui

    2013-08-01

    This study aimed to investigate the role of CD147 in the progression of gastric cancer and the signalling pathway involved in CD147-mediated gastric cancer cell line SGC7901 proliferation and invasion. Short hairpin RNA (shRNA) expression vectors targeting CD147 were constructed to silence CD147, and the expression of CD147 was monitored by quantitative realtime reverse transcriptase polymerase chain reaction and Western blot and further confirmed by immunohistochemistry in vivo. Cell proliferation was determined by Cell Counting Kit-8 assay, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were determined by gelatin zymography, and the invasion of SGC7901 was determined by invasion assay. The phosphorylation and non-phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase1/2 (ERK1/2), P38 and c-Jun NH2-terminal kinase were examined by Western blot. Additionally, the ERK1/2 inhibitor U0126 were used to confirm the signalling pathway involved in CD147-mediated SGC7901 progression. The BALB/c nude mice were used to study tumour progression in vivo. The results revealed that CD147 silencing inhibited the proliferation and invasion of SGC7901 cells, and down-regulated the activities of MMP-2 and MMP-9 and the phosphorylation of the ERK1/2 in SGC7901 cells. ERK1/2 inhibitor U0126 decreased the proliferation, and invasion of SGC7901 cells, and down-regulated the MMP-2 and MMP-9 activities. In a nude mouse model of subcutaneous xenografts, the tumour volume was significantly smaller in the SGC7901/shRNA group compared to the SGC7901 and SGC7901/snc-RNA group. Immunohistochemistry analysis showed that CD147 and p-ERK1/2 protein expressions were down-regulated in the SGC7901/shRNA2 group compared to the SGC7901 and SGC7901/snc-RNA group. These results suggest that ERK1/2 pathway involves in CD147-mediated gastric cancer growth and invasion. These findings further highlight the importance of CD147 in cancer progression

  15. Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis.

    Science.gov (United States)

    Ceotto-Vigoder, H; Marques, S L S; Santos, I N S; Alves, M D B; Barrias, E S; Potter, A; Alviano, D S; Bastos, M C F

    2016-07-01

    The biofilm produced by Staphylococcus aureus isolates involved in clinical or subclinical bovine mastitis and the activity of nisin and lysostaphin against the preformed biofilm produced by these strains were investigated. Eighteen strains were tested and all produced biofilm. Eight strains with distinct biofilm composition were selected for the antimicrobial activity assays. The minimal inhibitory concentration of each bacteriocin was determined against the planktonic cells and ranged from 15·6 to 500 μg ml(-1) for nisin, and from 3·9 to 50 μg ml(-1) , for lysostaphin. Lysostaphin treatment (0·4 μg ml(-1) ) for 4 h caused a strong Staph. aureus 4181 biofilm detachment and death of the majority of the sessile cells, while nisin treatment (100 μg ml(-1) ) for the same time caused only a great reduction in cell viability. Additionally, combination of both bacteriocins for 4 h resulted in significant death of the sessile cells but no biofilm detachment. The treatment with lysostaphin alone or in combination with nisin was effective in killing most biofilm sessile cells. The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved. © 2016 The Society for Applied Microbiology.

  16. DCD – a novel plant specific domain in proteins involved in development and programmed cell death

    Directory of Open Access Journals (Sweden)

    Doerks Tobias

    2005-07-01

    Full Text Available Abstract Background Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins. Results The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in development and cell death. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone. Conclusion It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes.

  17. Elevated p21-Activated Kinase 2 Activity Results in Anchorage-Independent Growth and Resistance to Anticancer Drug–Induced Cell Death

    Directory of Open Access Journals (Sweden)

    Jerry W. Marlin

    2009-03-01

    Full Text Available p21-Activated kinase 2 (PAK-2 seems to be a regulatory switch between cell survival and cell death signaling. We have shown previously that activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival, whereas caspase activation of PAK-2 to the proapoptotic PAK-2p34 fragment is involved in the cell death response. In this study, we present a role of elevated activity of full-length PAK-2 in anchorage-independent growth and resistance to anticancer drug–induced apoptosis of cancer cells. Hs578T human breast cancer cells that have low levels of PAK-2 activity were more sensitive to anticancer drug–induced apoptosis and showed higher levels of caspase activation of PAK-2 than MDA-MB435 and MCF-7 human breast cancer cells that have high levels of PAK-2 activity. To examine the role of elevated PAK-2 activity in breast cancer, we have introduced a conditionally active PAK-2 into Hs578T human breast cells. Conditional activation of PAK-2 causes loss of contact inhibition and anchorage-independent growth of Hs578T cells. Furthermore, conditional activation of PAK-2 suppresses activation of caspase 3, caspase activation of PAK-2, and apoptosis of Hs578T cells in response to the anticancer drug cisplatin. Our data suggest a novel mechanism by which full-length PAK-2 activity controls the apoptotic response by regulating levels of activated caspase 3 and thereby its own cleavage to the proapoptotic PAK-2p34 fragment. As a result, elevated PAK-2 activity interrupts the apoptotic response and thereby causes anchorage-independent survival and growth and resistance to anticancer drug–induced apoptosis.

  18. Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion

    Directory of Open Access Journals (Sweden)

    Pei-Wen Tsai

    2017-12-01

    Full Text Available Background/Purpose: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. Methods: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. Results: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Conclusion: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs. Keywords: Candida albicans, cell adhesion, Hom6, homoserine dehydrogenase, protein synthesis

  19. Learning models of activities involving interacting objects

    DEFF Research Database (Denmark)

    Manfredotti, Cristina; Pedersen, Kim Steenstrup; Hamilton, Howard J.

    2013-01-01

    We propose the LEMAIO multi-layer framework, which makes use of hierarchical abstraction to learn models for activities involving multiple interacting objects from time sequences of data concerning the individual objects. Experiments in the sea navigation domain yielded learned models that were t...

  20. Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants.

    Science.gov (United States)

    Tatsumi, H; Furuichi, T; Nakano, M; Toyota, M; Hayakawa, K; Sokabe, M; Iida, H

    2014-01-01

    Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca(2+) concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca(2+) -permeable channels, which can be the molecular mechanism of a Ca(2+) concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  1. Natural killer activity and suppressor cells in irradiated mice repopulated with a mixture of cells from normal and 89Sr-treated donors

    International Nuclear Information System (INIS)

    Levy, E.M.; Kumar, V.; Bennett, M.

    1981-01-01

    Mice that have been injected with 89 Sr have fairly normal B and T cell function, but are abnormal in that they lack natural killer (NK) activity and other functions that require an intact bone marrow. These mice also have an increased potential for suppressor cell activity. We had previously shown that spleen cells from 89 Sr-treated mice could transfer low NK activity and increased suppressor cell function to lethally irradiated syngeneic recipients. To investigate the mechanisms involved in perpetuating these defects, groups of normal spleen or bone marrow cells. Recipients were assayed for their NK activity and suppressor cell function 5 to 14 wk later. it was found that the addition of normal cells in the donor inoculum resulted in normal NK activity. This indicates that low NK activity in 89 Sr-treated mice was not due to the presence of a suppressor cell that prevented NK cell generation. It was additionally found that low NK activity in recipient mice could be boosted by interferon inducers. This would indicate that NK activity in the recipients was not due to a lack of interferon-sensitive pre-NK cells. Suppressor cell function in recipient mice depended on the type and number of normal cells in the donor inoculum. Bone marrow cells were very efficient in overcoming the tendency to produce suppressor cells. It took approximately 20 times more normal spleen cells to produce the same results. The implications of these findings are discussed

  2. NOD1 cooperates with TLR2 to enhance T cell receptor-mediated activation in CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Blandine C Mercier

    Full Text Available Pattern recognition receptors (PRR, like Toll-like receptors (TLR and NOD-like receptors (NLR, are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR. This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions.

  3. CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

    International Nuclear Information System (INIS)

    Oue, Erika; Lee, Ji-Won; Sakamoto, Kei; Iimura, Tadahiro; Aoki, Kazuhiro; Kayamori, Kou; Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo; Yamaguchi, Akira

    2012-01-01

    Highlights: ► Oral cancer cells synthesize CXCL2. ► CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. ► CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. ► We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction.

  4. CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

    Energy Technology Data Exchange (ETDEWEB)

    Oue, Erika [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Lee, Ji-Won; Sakamoto, Kei [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Iimura, Tadahiro [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Aoki, Kazuhiro [Section of Pharmacology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Kayamori, Kou [Section of Diagnostic Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Department of Pathology, Ome Municipal General Hospital, Ome, Tokyo (Japan); Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo [Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Yamaguchi, Akira, E-mail: akira.mpa@tmd.ac.jp [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Oral cancer cells synthesize CXCL2. Black-Right-Pointing-Pointer CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. Black-Right-Pointing-Pointer CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. Black-Right-Pointing-Pointer We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first

  5. Involvement of epigenetic modifiers in the pathogenesis of testicular dysgenesis and germ cell cancer

    DEFF Research Database (Denmark)

    Lawaetz, Andreas C.; Almstrup, Kristian

    2015-01-01

    Testicular germ cell cancer manifests mainly in young adults as a seminoma or non-seminoma. The solid tumors are preceded by the presence of a non-invasive precursor cell, the carcinoma in situ cell (CIS), which shows great similarity to fetal germ cells. It is therefore hypothesized that the CIS...... of epigenetic modifiers with a focus on jumonji C enzymes in the development of testicular dysgenesis and germ cell cancer in men....... cell is a fetal germ cell that has been arrested during development due to testicular dysgenesis. CIS cells retain a fetal and open chromatin structure, and recently several epigenetic modifiers have been suggested to be involved in testicular dysgenesis in mice. We here review the possible involvement...

  6. Modulation of Telomerase Activity in Cancer Cells by Dietary Compounds: A Review

    Directory of Open Access Journals (Sweden)

    Takahiro Eitsuka

    2018-02-01

    Full Text Available Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol, and the other downregulates the expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol. In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.

  7. TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Benedicte Stavik

    Full Text Available Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and mi

  8. TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

    Science.gov (United States)

    Stavik, Benedicte; Skretting, Grethe; Olstad, Ole Kristoffer; Sletten, Marit; Dehli Vigeland, Magnus; Sandset, Per Morten; Iversen, Nina

    2012-01-01

    Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes

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

    Science.gov (United States)

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

    2010-02-01

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

  10. Role of signaling lymphocytic activation molecule in T helper cell responses

    Directory of Open Access Journals (Sweden)

    Jan E. de Vries

    1998-01-01

    Full Text Available Signaling lymphocytic activation molecule (SLAM; CDw150 is a 70 kDa glycoprotein. Signaling lymphocytic activation molecule is constitutively expressed on memory T cells, CD56+ T cells, a subset of T cell receptor γδ+ cells, immature thymocytes and, at low levels, on a proportion of peripheral blood B cells. Signaling lymphocytic activation molecule is rapidly upregulated on all T and B cells after activation. Engagement of SLAM by F(ab’2 fragments of an anti-SLAM monoclonal antibody (mAb A12 enhances antigen-specific T cell proliferation. In addition, mAb A12 was directly mitogenic for T cell clones and activated T cells. T cell proliferation induced by mAb A12 is independent of interleukin (IL-2, IL-4, IL-12 and IL-15, but is cyclosporin A sensitive. Ligation of SLAM during antigen-specific T cell proliferation resulted in upregulation of interferon (IFN-γ production, even by allergen-specific T helper cell (Th 2 clones, whereas the levels of IL-4 and IL-5 production were only marginally affected. The mAb A12 was unable to induce IL-4 and IL-5 production by Th1 clones. Co-stimulation of skin-derived Der P1-specific Th2 cells from patients with atopic dermatitis via SLAM resulted in the generation of a population of IFN-γ-producing cells, thereby reverting their phenotype to a Th0 pattern. Signaling lymphocytic activation molecule is a high-affinity self ligand mediating homophilic cell interaction. In addition, soluble SLAM enhances both T and B cell proliferation. Collectively, these data indicate that SLAM molecules act both as receptors and ligands that are not only involved in T cell expansion but also drive the expanding T cells during immune responses into the Th0/Th1 pathway. This suggests that signaling through SLAM plays a role in directing Th0/Th1 development.

  11. The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host-Cell Interaction.

    Science.gov (United States)

    Gil-Bona, Ana; Reales-Calderon, Jose A; Parra-Giraldo, Claudia M; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

    2016-01-01

    Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a "veil growth," never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain.

  12. Neuroprotective effect of lurasidone via antagonist activities on histamine in a rat model of cranial nerve involvement.

    Science.gov (United States)

    He, Baoming; Yu, Liang; Li, Suping; Xu, Fei; Yang, Lili; Ma, Shuai; Guo, Yi

    2018-04-01

    Cranial nerve involvement frequently involves neuron damage and often leads to psychiatric disorder caused by multiple inducements. Lurasidone is a novel antipsychotic agent approved for the treatment of cranial nerve involvement and a number of mental health conditions in several countries. In the present study, the neuroprotective effect of lurasidone by antagonist activities on histamine was investigated in a rat model of cranial nerve involvement. The antagonist activities of lurasidone on serotonin 5‑HT7, serotonin 5‑HT2A, serotonin 5‑HT1A and serotonin 5‑HT6 were analyzed, and the preclinical therapeutic effects of lurasidone were examined in a rat model of cranial nerve involvement. The safety, maximum tolerated dose (MTD) and preliminary antitumor activity of lurasidone were also assessed in the cranial nerve involvement model. The therapeutic dose of lurasidone was 0.32 mg once daily, administered continuously in 14‑day cycles. The results of the present study found that the preclinical prescriptions induced positive behavioral responses following treatment with lurasidone. The MTD was identified as a once daily administration of 0.32 mg lurasidone. Long‑term treatment with lurasidone for cranial nerve involvement was shown to improve the therapeutic effects and reduce anxiety in the experimental rats. In addition, treatment with lurasidone did not affect body weight. The expression of the language competence protein, Forkhead‑BOX P2, was increased, and the levels of neuroprotective SxIP motif and microtubule end‑binding protein were increased in the hippocampal cells of rats with cranial nerve involvement treated with lurasidone. Lurasidone therapy reinforced memory capability and decreased anxiety. Taken together, lurasidone treatment appeared to protect against language disturbances associated with negative and cognitive impairment in the rat model of cranial nerve involvement, providing a basis for its use in the clinical treatment of

  13. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    Energy Technology Data Exchange (ETDEWEB)

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S., E-mail: prakash@mailbox.sc.edu

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8{sup +} T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract

  14. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Directory of Open Access Journals (Sweden)

    Shweta Jain

    Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  15. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Science.gov (United States)

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  16. Myeloid-Derived Suppressor Cells Specifically Suppress IFN-γ Production and Antitumor Cytotoxic Activity of Vδ2 T Cells

    Directory of Open Access Journals (Sweden)

    Alessandra Sacchi

    2018-06-01

    Full Text Available γδ T cells represent less than 5% of circulating T cells; they exert a potent cytotoxic function against tumor or infected cells and secrete cytokines like conventional αβ T cells. As αβ T cells γδ T cells reside in the typical T cell compartments (the lymph nodes and spleen, but are more widely distributed in tissues throughout the body. For these reasons, some investigators are exploring the possibility of immunotherapies aimed to expand and activate Vδ2 T cells, or using them as Chimeric Antigen Receptor carriers. However, the role of immunosuppressive microenvironment on Vδ2 T cells during infections and cancers has not been completely elucidated. In particular, the effects of myeloid-derived suppressor cells (MDSC, largely expanded in such pathologies, were not explored. In the present work, we demonstrated that MDSC may inhibit IFN-γ production and degranulation of phosphoantigen-activated Vδ2 T cells. Moreover, the Vδ2 T cells cytotoxic activity against the Burkitt lymphoma cell line Daudi and Jurkat cell line were impaired by MDSC. The Arginase I seems to be involved in the impairment of Vδ2 T cell function induced by both tumor cells and MDSC. These data open a key issue in the context of Vδ2-targeted immunoteraphy, suggesting the need of combined strategies aimed to boost Vδ2 T cells circumventing tumor- and MDSC-induced Vδ2 T cells suppression.

  17. Active patient involvement in the education of health professionals.

    Science.gov (United States)

    Towle, Angela; Bainbridge, Lesley; Godolphin, William; Katz, Arlene; Kline, Cathy; Lown, Beth; Madularu, Ioana; Solomon, Patricia; Thistlethwaite, Jill

    2010-01-01

    Patients as educators (teaching intimate physical examination) first appeared in the 1960s. Since then, rationales for the active involvement of patients as educators have been well articulated. There is great potential to promote the learning of patient-centred practice, interprofessional collaboration, community involvement, shared decision making and how to support self-care. We reviewed and summarised the literature on active patient involvement in health professional education. A synthesis of the literature reveals increasing diversity in the ways in which patients are involved in education, but also the movement's weaknesses. Most initiatives are 'one-off' events and are reported as basic descriptions. There is little rigorous research or theory of practice or investigation of behavioural outcomes. The literature is scattered and uses terms (such as 'patient'!) that are contentious and confusing. We propose future directions for research and development, including a taxonomy to facilitate dialogue, an outline of a research strategy and reference to a comprehensive bibliography covering all health and human services.

  18. What Are the Molecules Involved in Regulatory T-Cells Induction by Dendritic Cells in Cancer?

    Directory of Open Access Journals (Sweden)

    Rodrigo Nalio Ramos

    2013-01-01

    Full Text Available Dendritic cells (DCs are essential for the maintenance of homeostasis in the organism, and they do that by modulating lymphocyte priming, expansion, and response patterns according to signals they receive from the environment. The induction of suppressive lymphocytes by DCs is essential to hinder the development of autoimmune diseases but can be reverted against homeostasis when in the context of neoplasia. In this setting, the induction of suppressive or regulatory T cells contributes to the establishment of a state of tolerance towards the tumor, allowing it to grow unchecked by an otherwise functional immune system. Besides affecting its local environment, tumor also has been described as potent sources of anti-inflammatory/suppressive factors, which may act systemically, generating defects in the differentiation and maturation of immune cells, far beyond the immediate vicinity of the tumor mass. Cytokines, as IL-10 and TGF-beta, as well as cell surface molecules like PD-L1 and ICOS seem to be significantly involved in the redirection of DCs towards tolerance induction, and recent data suggest that tumor cells may, indeed, modulate distinct DCs subpopulations through the involvement of these molecules. It is to be expected that the identification of such molecules should provide molecular targets for more effective immunotherapeutic approaches to cancer.

  19. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  20. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    International Nuclear Information System (INIS)

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

    2012-01-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H 2 O 2 ) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H 2 O 2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

  1. Apoptosis Signal-Regulating Kinase 1 Is Involved in Brain-Derived Neurotrophic Factor (BDNF)-Enhanced Cell Motility and Matrix Metalloproteinase 1 Expression in Human Chondrosarcoma Cells

    Science.gov (United States)

    Lin, Chih-Yang; Chang, Sunny Li-Yun; Fong, Yi-Chin; Hsu, Chin-Jung; Tang, Chih-Hsin

    2013-01-01

    Chondrosarcoma is the primary malignancy of bone that is characterized by a potent capacity to invade locally and cause distant metastasis, and is therefore associated with poor prognoses. Chondrosarcoma further shows a predilection for metastasis to the lungs. The brain-derived neurotrophic factor (BDNF) is a small molecule in the neurotrophin family of growth factors that is associated with the disease status and outcome of cancers. However, the effect of BDNF on cell motility in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma cell lines had significantly higher cell motility and BDNF expression compared to normal chondrocytes. We also found that BDNF increased cell motility and expression of matrix metalloproteinase-1 (MMP-1) in human chondrosarcoma cells. BDNF-mediated cell motility and MMP-1 up-regulation were attenuated by Trk inhibitor (K252a), ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), and p38 inhibitor (SB203580). Furthermore, BDNF also promoted Sp1 activation. Our results indicate that BDNF enhances the migration and invasion activity of chondrosarcoma cells by increasing MMP-1 expression through a signal transduction pathway that involves the TrkB receptor, ASK1, JNK/p38, and Sp1. BDNF thus represents a promising new target for treating chondrosarcoma metastasis. PMID:23892595

  2. Motivation and involvement toward physical activity among ...

    African Journals Online (AJOL)

    This study investigates intrinsic and extrinsic factors that influence involvement of university students to participate in physical activity. 400 students comprising 200 men and 200 women were used as the main respondents were respond to the adapted Exercise Motivations Inventory questionnaire. It revealed that highest ...

  3. Proteasome inhibitors activate autophagy involving inhibition of PI3K-Akt-mTOR pathway as an anti-oxidation defense in human RPE cells.

    Directory of Open Access Journals (Sweden)

    Bingrong Tang

    Full Text Available The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE, ARPE-19 cells, proteasome inhibitors, clasto-lactacystinβ-lactone (LA or epoxomicin (Epo, at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3 from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf. Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt, a downstream target of phosphatidylinositol-3-kinases (PI3K, and mammalian target of rapamycin (mTOR in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3 or 4-hydroxynonenal (4-HNE. Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy.

  4. Role of Bioavailable Iron in Coal Dust-Induced Activation of Activator Protein-1 and Nuclear Factor of Activated T Cells

    Science.gov (United States)

    Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi

    2010-01-01

    Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers’ pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH2-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions. PMID:12397016

  5. Comparison of the effect of activated or non-activated PRP in various concentrations on osteoblast and fibroblast cell line proliferation.

    Science.gov (United States)

    Vahabi, Surena; Yadegari, Zahra; Mohammad-Rahimi, Hossein

    2017-09-01

    Platelet-rich plasma (PRP) contains growth factors which positively affect cell proliferation, cell differentiation, chemotaxis and intracellular matrix synthesis. All these processes are involved in wound healing and tissue regeneration; thus, PRP as a source of growth factors can be used in periodontal regenerative therapies. The purpose of the present study was to assess the effect of various concentrations of activated and non-activated PRP on proliferation of osteoblasts and fibroblasts in vitro. PRP was obtained from three healthy volunteers. 75, 50, 25, and 10% concentrations of f PRP were prepared by dilution in Dulbecco's modified Eagle's medium. In activated PRP groups, PRP concentrations were activated by adding calcium gluconate. Human gingival fibroblast (HGF) cell line and MG-63 (osteosarcoma) human osteoblast-like cell line were used in the study. The MTT proliferation assay was used to assess the effect of different types of PRP concentrates on proliferation of HGF and MG-63 cells, in 24, 48 and 72 h. After 24, 48, and 72 h, the proliferation rate of both cell lines was higher in the positive control group, except in 72 h in HGF cell lines, that 10% non-activated PRP group and 10 and 25% activated PRP groups has higher proliferation rate than the positive control group, which it was not significant. Proliferation rate in cells with 10% activated PRP was highest among samples containing PRP. The current study failed to show the significant effect of activated or non-activated PRP on proliferation of HGFs or MG-63 osteoblast-like cells. However, our results showed that activated PRP had a greater effect than non-activated PRP.

  6. Involvement of major components from Sporothrix schenckii cell wall in the caspase-1 activation, nitric oxide and cytokines production during experimental sporotrichosis.

    Science.gov (United States)

    Gonçalves, Amanda Costa; Maia, Danielle Cardoso Geraldo; Ferreira, Lucas Souza; Monnazzi, Luis Gustavo Silva; Alegranci, Pâmela; Placeres, Marisa Campos Polesi; Batista-Duharte, Alexander; Carlos, Iracilda Zeppone

    2015-02-01

    Sporotrichosis is a chronic infection caused by the dimorphic fungus Sporothrix schenckii, involving all layers of skin and the subcutaneous tissue. The role of innate immune toll-like receptors 2 and 4 in the defense against this fungus has been reported, but so far, there were no studies on the effect of cell wall major components over the cytosolic oligo-merization domain (NOD)-like receptors, important regulators of inflammation and responsible for the maturation of IL-1β and IL-18, whose functions are dependents of the caspase-1 activation, that can participate of inflammasome. It was evaluated the percentage of activation of caspase-1, the production of IL-1β, IL-18, IL-17, IFN-γ and nitric oxide in a Balb/c model of S. schenckii infection. It was observed a decreased activity of caspase-1 during the fourth and sixth weeks of infection accompanied by reduced secretion of the cytokines IL-1β, IL-18 and IL-17 and high production of nitric oxide. IFN-γ levels were elevated during the entire time course of infection. This temporal reduction in caspase-1 activity coincides exactly with the reported period of fungal burden associated with a transitory immunosuppression induced by this fungus and detected in similar infection models. These results indicate the importance of interaction between caspase-1, cytokines IL-1β and IL-18 in the host defense against S. schenckii infection, suggesting a participation the inflammasome in this response.

  7. Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

    Science.gov (United States)

    Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

    2016-01-01

    High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

  8. Lifestyles and mental health status are associated with natural killer cell and lymphokine-activated killer cell activities.

    Science.gov (United States)

    Morimoto, K; Takeshita, T; Inoue-Sakurai, C; Maruyama, S

    2001-04-10

    We investigated the association of lifestyle and mental health status with natural killer (NK) cell and lymphokine-activated killer (LAK) cell activities in healthy males. NK cell activity was determined in 105 male workers and LAK cell activity was determined in 54 male workers. Peripheral blood was obtained from each subject and peripheral blood mononuclear cells (PBMC) were isolated from the blood. These PBMC were used as effector cells. LAK cells were generated by incubation of PBMC with interleukin-2 for 72 h. NK cell activity against NK-sensitive K562 cells and LAK cell activity against NK-resistant Raji cells were examined by 51Cr release assay. Overall lifestyles were evaluated according to the answers on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, eating breakfast, hours of sleep, hours of work, physical exercise, nutritional balance, mental stress). Subjects with a good overall lifestyle showed significantly higher NK cell (P mental status had significantly lower NK cell activity than those who reported stable mental status. When subjects were divided into four groups by lifestyle and mental health status, subjects who had poor or moderate lifestyle and reported unstable mental status showed the lowest NK cell activity and subjects who had good lifestyle and reported stable mental status showed the highest NK cell activity among four groups.

  9. Ion channels involved in cell volume regulation: effects on migration, proliferation, and programmed cell death in non adherent EAT cells and adherent ELA cells.

    Science.gov (United States)

    Hoffmann, Else Kay

    2011-01-01

    This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation, and programmed cell death. Copyright © 2011 S. Karger AG, Basel.

  10. Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

    Science.gov (United States)

    Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko

    2014-01-01

    Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

  11. Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

    Directory of Open Access Journals (Sweden)

    Masahiro Ito

    Full Text Available Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

  12. Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

    Directory of Open Access Journals (Sweden)

    Linghui Kong

    Full Text Available The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

  13. The mast cell degranulator compound 48/80 directly activates neurons.

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    Michael Schemann

    Full Text Available BACKGROUND: Compound 48/80 is widely used in animal and tissue models as a "selective" mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents. METHODOLOGY/PRINCIPAL FINDINGS: We used in vivo recordings from extrinsic intestinal afferents together with Ca(++ imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca(++ and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca(++ transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H(1 and H(2 antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca(++ transients in mast cell-free enteric neuron cultures. CONCLUSIONS/SIGNIFICANCE: The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn.

  14. Interaction of Mycoplasma hominis PG21 with Human Dendritic Cells: Interleukin-23-Inducing Mycoplasmal Lipoproteins and Inflammasome Activation of the Cell.

    Science.gov (United States)

    Goret, J; Béven, L; Faustin, B; Contin-Bordes, C; Le Roy, C; Claverol, S; Renaudin, H; Bébéar, C; Pereyre, S

    2017-08-01

    Mycoplasma hominis lacks a cell wall, and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of M. hominis enriched with lipoproteins was shown to stimulate the production of interleukin-23 (IL-23) by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon M. hominis infection. We studied here the interaction between M. hominis PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell. IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two nondenaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and enzyme-linked immunosorbent assay (ELISA). We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular mass between 20 and 35 kDa and 19 with a molecular mass between 40 and 100 kDa. Among them, lipoprotein MHO_4720 was identified as potentially bioactive, and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1β was observed. After 24 h of coincubation of hDCs with M. homini s, downregulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction of M. hominis and hDCs. IMPORTANCE Mycoplasma hominis is a human urogenital pathogen involved in gynecologic and opportunistic infections. M. hominis lacks a cell wall, and its membrane contains many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on

  15. The cell wall protein Ecm33 of Candida albicans is involved in chronological life span, morphogenesis, cell wall regeneration, stress tolerance and host-cell interaction

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    Ana eGil-Bona

    2016-02-01

    Full Text Available Ecm33 is a glycosylphosphatidylinositol (GPI-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the cell wall integrity pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a veil growth, never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain.

  16. NAC selectively inhibit cancer telomerase activity: A higher redox homeostasis threshold exists in cancer cells

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    Pengying Li

    2016-08-01

    Full Text Available Telomerase activity controls telomere length, and this plays an important role in stem cells, aging and tumors. Antioxidant was shown to protect telomerase activity in normal cells but inhibit that in cancer cells, but the underlying mechanism is elusive. Here we found that 7721 hepatoma cells held a higher redox homeostasis threshold than L02 normal liver cells which caused 7721 cells to have a higher demand for ROS; MnSOD over-expression in 7721 decreased endogenous reactive oxygen species (ROS and inhibited telomerase activity; Akt phosphorylation inhibitor and NAC both inhibited 7721 telomerase activity. The over-elimination of ROS by NAC resulted in the inhibition of Akt pathway. Our results suggest that ROS is involved in the regulation of cancer telomerase activity through Akt pathway. The different intracellular redox homeostasis and antioxidant system in normal cells and tumor cells may be the cause of the opposite effect on telomerase activity in response to NAC treatment. Our results provide a theoretical base of using antioxidants selectively inhibit cancer telomerase activity. Findings of the present study may provide insights into novel approaches for cancer treatment.

  17. Multiple dendritic cell populations activate CD4+ T cells after viral stimulation.

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    Adele M Mount

    2008-02-01

    Full Text Available Dendritic cells (DC are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+ and CD8(+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+ and CD4(+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+ T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.

  18. Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics1[OPEN

    Science.gov (United States)

    2017-01-01

    The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes. PMID:28400496

  19. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: Involvement of interleukin-2 system

    Energy Technology Data Exchange (ETDEWEB)

    Cervia, Davide, E-mail: d.cervia@unitus.it [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Catalani, Elisabetta; Belardinelli, Maria Cristina [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Perrotta, Cristiana [Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Picchietti, Simona [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Alimenti, Claudio [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy); Casini, Giovanni; Fausto, Anna Maria [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Vallesi, Adriana [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ► Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ► Er-1 increases the T-cell production of specific cytokines. ► Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ► The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.

  20. Thioridazine affects transcription of genes involved in cell wall biosynthesis in methicillin-resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Bonde, Mette; Højland, Dorte Heidi; Kolmos, Hans Jørn

    2011-01-01

    have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis...... in response to thioridazine in combination with oxacillin. We observed that the oxacillin-induced expression of genes belonging to the VraSR regulon is reduced by the addition of thioridazine. The exclusion of such key factors involved in cell wall biosynthesis will most likely lead to a weakened cell wall...... reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis....

  1. CMEIAS-Aided Microscopy of the Spatial Ecology of Individual Bacterial Interactions Involving Cell-to-Cell Communication within Biofilms

    Directory of Open Access Journals (Sweden)

    Frank B. Dazzo

    2012-05-01

    Full Text Available This paper describes how the quantitative analytical tools of CMEIAS image analysis software can be used to investigate in situ microbial interactions involving cell-to-cell communication within biofilms. Various spatial pattern analyses applied to the data extracted from the 2-dimensional coordinate positioning of individual bacterial cells at single-cell resolution indicate that microbial colonization within natural biofilms is not a spatially random process, but rather involves strong positive interactions between communicating cells that influence their neighbors’ aggregated colonization behavior. Geostatistical analysis of the data provide statistically defendable estimates of the micrometer scale and interpolation maps of the spatial heterogeneity and local intensity at which these microbial interactions autocorrelate with their spatial patterns of distribution. Including in situ image analysis in cell communication studies fills an important gap in understanding the spatially dependent microbial ecophysiology that governs the intensity of biofilm colonization and its unique architecture.

  2. 3-bromopyruvate enhanced daunorubicin-induced cytotoxicity involved in monocarboxylate transporter 1 in breast cancer cells.

    Science.gov (United States)

    Liu, Zhe; Sun, Yiming; Hong, Haiyu; Zhao, Surong; Zou, Xue; Ma, Renqiang; Jiang, Chenchen; Wang, Zhiwei; Li, Huabin; Liu, Hao

    2015-01-01

    Increasing evidence demonstrates that the hexokinase inhibitor 3-bromopyruvate (3-BrPA) induces the cell apoptotic death by inhibiting ATP generation in human cancer cells. Interestingly, some tumor cell lines are less sensitive to 3-BrPA-induced apoptosis than others. Moreover, the molecular mechanism of 3-BrPA-trigged apoptosis is unclear. In the present study, we examined the effects of 3-BrPA on the viability of the breast cancer cell lines MDA-MB-231 and MCF-7. We further investigated the potential roles of monocarboxylate transporter 1 (MCT1) in drug accumulation and efflux of breast cancer cells. Finally, we explored whether 3-BrPA enhanced daunorubicin (DNR)-induced cytotoxicity through regulation of MCT1 in breast cancer cells. MTT and colony formation assays were used to measure cell viability. Western blot analysis, flow cytometric analysis and fluorescent microscopy were used to determine the molecular mechanism of actions of MCT1 in different breast cancer cell lines. Whole-body bioluminescence imaging was used to investigate the effect of 3-BrPA in vivo. We found that 3-BrPA significantly inhibited cell growth and induced apoptosis in MCF-7 cell line, but not in MDA-MB-231 cells. Moreover, we observed that 3-BrPA efficiently enhanced DNR-induced cytotoxicity in MCF-7 cells by inhibiting the activity of ATP-dependent efflux pumps. We also found that MCT1 overexpression increased the efficacy of 3-BrPA in MDA-MB-231 cells. 3-BrPA markedly suppressed subcutaneous tumor growth in combination with DNR in nude mice implanted with MCF-7 cells. Lastly, our whole-body bioluminescence imaging data indicated that 3-BrPA promoted DNR accumulation in tumors. These findings collectively suggest that 3-BrPA enhanced DNR antitumor activity in breast cancer cells involved MCT-1, suggesting that inhibition of glycolysis could be an effective therapeutic approach for breast cancer treatment.

  3. Involvement of dendritic cells in allograft rejection new implications of dendritic cell-endothelial cell interactions.

    Science.gov (United States)

    Schlichting, C L; Schareck, W D; Kofler, S; Weis, M

    2007-04-01

    For almost half a century immunologists have tried to tear down the MHC barrier, which separates two unrelated individuals during transplantation. Latest experimental data suggest that a breakthrough in vitro is imminent. Dendritic cells (DCs), which activate naïve allo-reactive T-cells (TCs), play a central role in the establishment of allo-antigen-specific immunity. Allograft solid organ rejection is initiated at the foreign endothelial cell (EC) layer, which forms an immunogenic barrier for migrating DCs. Thus, DC/EC interactions might play a crucial role in antigen-specific allograft rejection. Organ rejection is mediated by host allo-reactive TCs, which are activated by donor DCs (direct activation) or host DCs (indirect activation). Direct allo-antigen presentation by regulatory dendritic cells (DCreg) can play an instructive role towards tolerance induction. Several groups established that, DCregs, if transplanted beforehand, enter host thymus, spleen, or bone marrow where they might eventually establish allo-antigen-specific tolerance. A fundamental aspect of DC function is migration throughout the entire organism. After solid organ transplantation, host DCs bind to ECs, invade allograft tissues, and finally transmigrate into lymphoid vessels and secondary lymphoid organs, where they present allo-antigens to naïve host TCs. Recent data suggest that in vitro manipulated DCregs may mediate allo-transplantation tolerance induction. However, the fundamental mechanisms on how such DCregs cause host TCs in the periphery towards tolerance remain unclear. One very promising experimental concept is the simultaneous manipulation of DC direct and indirect TC activation/suppression, towards donor antigen-specific allo-transplantation tolerance. The allo-antigen-specific long-term tolerance induction mediated by DCreg pre-transplantation (with simultaneous short-term immunosuppression) has become reproducible in the laboratory animal setting. Despite the shortcomings

  4. Involvment of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP/MPP-treated neurons.

    Directory of Open Access Journals (Sweden)

    Agnès Petit-Paitel

    Full Text Available Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson's disease (PD. 1-methyl-4-phenylpyridinium iodide (MPP(+, the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3beta (GSK-3beta, a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3beta in modulating MPP(+-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP(+ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3beta, evidenced by the increased level of the active form of the kinase, i.e. GSK-3beta phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3beta partially localized within mitochondria in both neuronal cell models. Moreover, MPP(+ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3beta labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP(+ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3beta activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP(+-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3beta is a critical mediator of MPTP/MPP(+-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3beta activity might provide protection against

  5. Characterization of early events involved in human dendritic cell maturation induced by sensitizers: Cross talk between MAPK signalling pathways

    International Nuclear Information System (INIS)

    Trompezinski, Sandra; Migdal, Camille; Tailhardat, Magalie; Le Varlet, Beatrice; Courtellemont, Pascal; Haftek, Marek; Serres, Mireille

    2008-01-01

    Dendritic cells (DCs), efficient-antigen presenting cells play an important role in initiating and regulating immune responses. DC maturation following exposure to nickel or DNCB induced an up-regulation of phenotypic markers and inflammatory cytokine secretion. Early intracellular mechanisms involved in DC maturation required to be precise. To address this purpose, DCs derived from human monocytes were treated with sensitizers (nickel, DNCB or thimerosal) in comparison with an irritant (SDS). Our data confirming the up-regulation of CD86, CD54 and cytokine secretion (IL-8 and TNFα) induced by sensitizers but not by SDS, signalling transduction involved in DC maturation was investigated using these chemicals. Kinase activity measurement was assessed using two new sensitive procedures (Face TM and CBA) requiring few cells. SDS did not induce changes in signalling pathways whereas NiSO 4 , DNCB and thimerosal markedly activated p38 MAPK and JNK, in contrast Erk1/2 phosphorylation was completely inhibited by DNCB or thimerosal and only activated by nickel. A pre-treatment with p38 MAPK inhibitor (SB203580) suppressed Erk1/2 inhibition induced by DNCB or thimerosal demonstrating a direct interaction between p38 MAPK and Erk1/2. A pre-treatment with an antioxidant, N-acetyl-L-cysteine (NAC) markedly reduced Erk1/2 inhibition and p38 MAPK phosphorylation induced by DNCB and thimerosal, suggesting a direct activation of p38 MAPK via an oxidative stress and a regulation of MAPK signalling pathways depending on chemicals. Because of a high sensitivity of kinase activity measurements, these procedures will be suitable for weak or moderate sensitizer screening

  6. Imaging proteolytic activity in live cells and animal models.

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    Stefanie Galbán

    Full Text Available In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors; their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

  7. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed

    2012-01-01

    ) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence......Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA...... results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells....

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

    Science.gov (United States)

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

    2009-11-30

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

  9. The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host–Cell Interaction

    Science.gov (United States)

    Gil-Bona, Ana; Reales-Calderon, Jose A.; Parra-Giraldo, Claudia M.; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

    2016-01-01

    Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a “veil growth,” never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain. PMID:26870022

  10. Bifidobacterium breve MCC-117 Induces Tolerance in Porcine Intestinal Epithelial Cells: Study of the Mechanisms Involved in the Immunoregulatory Effect

    Science.gov (United States)

    MURATA, Kozue; TOMOSADA, Yohsuke; VILLENA, Julio; CHIBA, Eriko; SHIMAZU, Tomoyuki; ASO, Hisashi; IWABUCHI, Noriyuki; XIAO, Jin-zhong; SAITO, Tadao; KITAZAWA, Haruki

    2014-01-01

    Bifidobacterium breve MCC-117 is able to significantly reduce the expression of inflammatory cytokines in porcine intestinal epithelial (PIE) cells and to improve IL-10 levels in CD4+CD25high Foxp3+ lymphocytes in response to heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs), while the immunoregulatory effect of B. adolescentis ATCC15705 was significantly lower than that observed for the MCC-117 strain. Considering the different capacities of the two bifidobacterium strains to activate toll-like receptor (TLR)-2 and their differential immunoregulatory activities in PIE and immune cells, we hypothesized that comparative studies with both strains could provide important information regarding the molecular mechanism(s) involved in the anti-inflammatory activity of bifidobacteria. In this work, we demonstrated that the anti-inflammatory effect of B. breve MCC-117 was achieved by a complex interaction of multiple negative regulators of TLRs as well as inhibition of multiple signaling pathways. We showed that B. breve MCC-117 reduced heat-stable ETEC PAMP-induced NF-κB, p38 MAPK and PI3 K activation and expression of pro-inflammatory cytokines in PIE cells. In addition, we demonstrated that B. breve MCC-117 may activate TLR2 synergistically and cooperatively with one or more other pattern recognition receptors (PRRs), and that interactions may result in a coordinated sum of signals that induce the upregulation of A20, Bcl-3, Tollip and SIGIRR. Upregulation of these negative regulators could have an important physiological impact on maintaining or reestablishing homeostatic TLR signals in PIE cells. Therefore, in the present study, we gained insight into the molecular mechanisms involved in the immunoregulatory effect of B. breve MCC-117. PMID:24936377

  11. Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

    Directory of Open Access Journals (Sweden)

    Alexander Zhyvoloup

    2017-07-01

    Full Text Available HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

  12. Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

    Science.gov (United States)

    Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

    2017-07-01

    HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

  13. Glucose activates prenyltransferases in pancreatic islet {beta}-cells

    Energy Technology Data Exchange (ETDEWEB)

    Goalstone, Marc [Department of Medicine, University of Colorado, VA Medical Center, Denver, CO 80220 (United States); Kamath, Vasudeva [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States); Kowluru, Anjaneyulu, E-mail: akowluru@med.wayne.edu [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States)

    2010-01-01

    A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet {beta}-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 {beta}-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the {alpha}-subunits of FTase/GGTase-1, but not the {beta}-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

  14. Glucose activates prenyltransferases in pancreatic islet β-cells

    International Nuclear Information System (INIS)

    Goalstone, Marc; Kamath, Vasudeva; Kowluru, Anjaneyulu

    2010-01-01

    A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet β-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 β-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the α-subunits of FTase/GGTase-1, but not the β-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

  15. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.

    2011-10-01

    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  16. The role of adrenergic activation on murine luteal cell viability and progesterone production.

    Science.gov (United States)

    Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

    2016-09-15

    Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Lysophosphatidylcholine-induced taurine release in HeLa cells involves protein kinase activity

    DEFF Research Database (Denmark)

    Lambert, Ian H.; Falktoft, Birgitte

    2001-01-01

    Calmodulin; CaMKII; Ehrlich ascites tumor cells; Ischemia; Lysophospholipids; NIH/3T3; Chelerythrine; Reactive oxygen species......Calmodulin; CaMKII; Ehrlich ascites tumor cells; Ischemia; Lysophospholipids; NIH/3T3; Chelerythrine; Reactive oxygen species...

  18. Active-involvement principle in dental health education

    DEFF Research Database (Denmark)

    Schou, L

    1985-01-01

    A basic problem in dental health education (DHE) is that the effect usually disappears shortly after the termination of a program. The purpose of the present study was to obtain long-term effect of a DHE-program by emphasizing the active involvement of the participants. The sample comprised...... an experimental and a control group, each of 68 unskilled workers, aged 18-64. Active participation was obtained by various means: Teaching was carried out in pre-existing peer groups, the participants' own goals and needs were included, the traditional dentist-patient barriers were excluded, the traditional...

  19. Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells

    Science.gov (United States)

    Huang, Lei; Kondo, Fumio; Harato, Misako; Feng, Guo-Gang; Ishikawa, Naoshisa; Fujiwara, Yoshihiro; Okada, Shoshiro

    2014-01-01

    The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression. PMID:24466034

  20. Efficacy of Vismodegib (Erivedge) for Basal Cell Carcinoma Involving the Orbit and Periocular Area.

    Science.gov (United States)

    Demirci, Hakan; Worden, Francis; Nelson, Christine C; Elner, Victor M; Kahana, Alon

    2015-01-01

    Evaluate the effectiveness of vismodegib in the management of basal cell carcinoma with orbital extension and/or extensive periocular involvement. Retrospective chart review of 6 consecutive patients with biopsy-proven orbital basal cell carcinoma and 2 additional patients with extensive periocular basal cell carcinoma who were treated with oral vismodegib (150 mg/day) was performed. Basal cell carcinoma extended in the orbit in 6 of 8 patients (involving orbital bones in 1 patient), and 2 of 8 patients had extensive periocular involvement (1 with basal cell nevus syndrome). Vismodegib therapy was the only treatment in 6 patients, off-label neoadjuvant in 1 patient, and adjuvant treatment in 1 patient. Orbital tumors in all 4 patients who received vismodegib as sole treatment showed partial response with a mean 83% shrinkage in tumor size after a median of 7 months of therapy. In the 2 patients receiving vismodegib as neoadjuvant or adjuvant therapies, there was complete response after a median of 7 months of therapy and no evidence of clinical recurrence after discontinuing therapy for a median of 15 months. The 2 patients with extensive periocular involvement experienced complete clinical response after a median 14 months of treatment. During treatment, the most common side effects were muscle spasm (75%) followed by alopecia (50%), dysgeusia (25%), dysosmia, and episodes of diarrhea and constipation (13%). Basal cell carcinoma with orbital extension and extensive periocular involvement responds to vismodegib therapy. The long-term prognosis remains unknown, and additional prospective studies are indicated.

  1. Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

    Science.gov (United States)

    Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

    2010-11-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

  2. An intermediate level of CD161 expression defines a novel activated, inflammatory, and pathogenic subset of CD8+ T cells involved in multiple sclerosis.

    Science.gov (United States)

    Nicol, Bryan; Salou, Marion; Vogel, Isabel; Garcia, Alexandra; Dugast, Emilie; Morille, Jeremy; Kilens, Stéphanie; Charpentier, Eric; Donnart, Audrey; Nedellec, Steven; Jacq-Foucher, Marylène; Le Frère, Fabienne; Wiertlewski, Sandrine; Bourreille, Arnaud; Brouard, Sophie; Michel, Laure; David, Laurent; Gourraud, Pierre-Antoine; Degauque, Nicolas; Nicot, Arnaud B; Berthelot, Laureline; Laplaud, David-Axel

    2018-03-01

    Several lines of evidence support a key role for CD8 + T cells in central nervous system tissue damage of patients with multiple sclerosis. However, the precise phenotype of the circulating CD8 + T cells that may be recruited from the peripheral blood to invade the CNS remains largely undefined to date. It has been suggested that IL-17 secreting CD8 (Tc17) T cells may be involved, and in humans these cells are characterized by the expression of CD161. We focused our study on a unique and recently described subset of CD8 T cells characterized by an intermediate expression of CD161 as its role in neuroinflammation has not been investigated to date. The frequency, phenotype, and function of CD8 + T cells with an intermediate CD161 expression level were characterized ex-vivo, in vitro, and in situ using RNAseq, RT-PCR, flow cytometry, TCR sequencing, and immunohistofluorescence of cells derived from healthy volunteers (n = 61), MS subjects (n = 90), as well as inflammatory (n = 15) and non-inflammatory controls (n = 6). We report here that CD8 + CD161 int T cells present characteristics of effector cells, up-regulate cell-adhesion molecules and have an increased ability to cross the blood-brain barrier and to secrete IL-17, IFNγ, GM-CSF, and IL-22. We further demonstrate that these cells are recruited and enriched in the CNS of MS subjects where they produce IL-17. In the peripheral blood, RNAseq, RT-PCR, high-throughput TCR repertoire analyses, and flow cytometry confirmed an increased effector and transmigration pattern of these cells in MS patients, with the presence of supernumerary clones compared to healthy controls. Our data demonstrate that intermediate levels of CD161 expression identifies activated and effector CD8 + T cells with pathogenic properties that are recruited to MS lesions. This suggests that CD161 may represent a biomarker and a valid target for the treatment of neuroinflammation. Copyright © 2017 The Authors. Published by Elsevier Ltd

  3. Involvement of serotonin 2A receptor activation in modulating medial prefrontal cortex and amygdala neuronal activation during novelty-exposure.

    Science.gov (United States)

    Hervig, Mona El-Sayed; Jensen, Nadja Cecilie Hvid; Rasmussen, Nadja Bredo; Rydbirk, Rasmus; Olesen, Mikkel Vestergaard; Hay-Schmidt, Anders; Pakkenberg, Bente; Aznar, Susana

    2017-05-30

    The medial prefrontal cortex (PFC) plays a major role in executive function by exerting a top-down control onto subcortical areas. Novelty-induced frontal cortex activation is 5-HT 2A receptor (5-HT 2A R) dependent. Here, we further investigated how blockade of 5-HT 2A Rs in mice exposed to a novel open-field arena affects medial PFC activation and basolateral amygdala (BLA) reactivity. We used c-Fos immunoreactivity (IR) as a marker of neuronal activation and stereological quantification for obtaining the total number of c-Fos-IR neurons as a measure of regional activation. We further examined the impact of 5-HT 2A R blockade on the striatal-projecting BLA neurons. Systemic administration of ketanserin (0.5mg/kg) prior to novel open-field exposure resulted in reduced total numbers of c-Fos-IR cells in dorsomedial PFC areas and the BLA. Moreover, there was a positive correlation between the relative time spent in the centre of the open-field and BLA c-Fos-IR in the ketanserin-treated animals. Unilateral medial PFC lesions blocked this effect, ascertaining an involvement of this frontal cortex area. On the other hand, medial PFC lesioning exacerbated the more anxiogenic-like behaviour of the ketanserin-treated animals, upholding its involvement in modulating averseness. Ketanserin did not affect the number of activated striatal-projecting BLA neurons (measured by number of Cholera Toxin b (CTb) retrograde labelled neurons also being c-Fos-IR) following CTb injection in the ventral striatum. These results support a role of 5-HT 2A R activation in modulating mPFC and BLA activation during exposure to a novel environment, which may be interrelated. Conversely, 5-HT 2A R blockade does not seem to affect the amygdala-striatal projection. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Acoustic input and efferent activity regulate the expression of molecules involved in cochlear micromechanics

    Science.gov (United States)

    Lamas, Veronica; Arévalo, Juan C.; Juiz, José M.; Merchán, Miguel A.

    2015-01-01

    Electromotile activity in auditory outer hair cells (OHCs) is essential for sound amplification. It relies on the highly specialized membrane motor protein prestin, and its interactions with the cytoskeleton. It is believed that the expression of prestin and related molecules involved in OHC electromotility may be dynamically regulated by signals from the acoustic environment. However little is known about the nature of such signals and how they affect the expression of molecules involved in electromotility in OHCs. We show evidence that prestin oligomerization is regulated, both at short and relatively long term, by acoustic input and descending efferent activity originating in the cortex, likely acting in concert. Unilateral removal of the middle ear ossicular chain reduces levels of trimeric prestin, particularly in the cochlea from the side of the lesion, whereas monomeric and dimeric forms are maintained or even increased in particular in the contralateral side, as shown in Western blots. Unilateral removal of the auditory cortex (AC), which likely causes an imbalance in descending efferent activity on the cochlea, also reduces levels of trimeric and tetrameric forms of prestin in the side ipsilateral to the lesion, whereas in the contralateral side prestin remains unaffected, or even increased in the case of trimeric and tetrameric forms. As far as efferent inputs are concerned, unilateral ablation of the AC up-regulates the expression of α10 nicotinic Ach receptor (nAChR) transcripts in the cochlea, as shown by RT-Quantitative real-time PCR (qPCR). This suggests that homeostatic synaptic scaling mechanisms may be involved in dynamically regulating OHC electromotility by medial olivocochlear efferents. Limited, unbalanced efferent activity after unilateral AC removal, also affects prestin and β-actin mRNA levels. These findings support that the concerted action of acoustic and efferent inputs to the cochlea is needed to regulate the expression of major

  5. Involvement of immune cells in the pathogenesis of endometriosis.

    Science.gov (United States)

    Izumi, Gentaro; Koga, Kaori; Takamura, Masashi; Makabe, Tomoko; Satake, Erina; Takeuchi, Arisa; Taguchi, Ayumi; Urata, Yoko; Fujii, Tomoyuki; Osuga, Yutaka

    2018-02-01

    Endometriosis is characterized by the implantation and growth of endometriotic tissues outside the uterus. It is widely accepted the theory that endometriosis is caused by the implantation of endometrial tissue from retrograde menstruation; however, retrograde menstruation occurs in almost all women and other factors are required for the establishment of endometriosis, such as cell survival, cell invasion, angiogenesis, and cell growth. Immune factors in the local environment may, therefore, contribute to the formation and progression of endometriosis. Current evidence supports the involvement of immune cells in the pathogenesis of endometriosis. Peritoneal neutrophils and macrophages secrete biochemical factors that help endometriotic cell growth and invasion, and angiogenesis. Peritoneal macrophages and NK cells in endometriosis have limited capability of eliminating endometrial cells in the peritoneal cavity. An imbalance of T cell subsets leads to aberrant cytokine secretions and inflammation that results in the growth of endometriosis lesions. It is still uncertain whether these immune cells have a role in the initial cause and/or stimulate actions that enhance disease; however, in either case, modulating the actions of these cells may prevent initiation or disease progression. Further studies are needed to deepen the understanding of the pathology of endometriosis and to develop novel management approaches of benefit to women suffering from this disease. © 2018 Japan Society of Obstetrics and Gynecology.

  6. Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species.

    Science.gov (United States)

    De Milito, Angelo; Iessi, Elisabetta; Logozzi, Mariantonia; Lozupone, Francesco; Spada, Massimo; Marino, Maria Lucia; Federici, Cristina; Perdicchio, Maurizio; Matarrese, Paola; Lugini, Luana; Nilsson, Anna; Fais, Stefano

    2007-06-01

    Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose- and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI.

  7. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  8. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  9. Involvement of Arabidopsis Hexokinase1 in Cell Death Mediated by Myo -Inositol Accumulation

    KAUST Repository

    Bruggeman, Quentin

    2015-06-05

    Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants. © 2015 American Society of Plant Biologists. All rights reserved.

  10. Sulfatide-activated type II NKT cells prevent allergic airway inflammation by inhibiting type I NKT cell function in a mouse model of asthma.

    Science.gov (United States)

    Zhang, Guqin; Nie, Hanxiang; Yang, Jiong; Ding, Xuhong; Huang, Yi; Yu, Hongying; Li, Ruyou; Yuan, Zhuqing; Hu, Suping

    2011-12-01

    Asthma is a common chronic inflammatory disease involving many different cell types. Recently, type I natural killer T (NKT) cells have been demonstrated to play a crucial role in the development of asthma. However, the roles of type II NKT cells in asthma have not been investigated before. Interestingly, type I and type II NKT cells have been shown to have opposing roles in antitumor immunity, antiparasite immunity, and autoimmunity. We hypothesized that sulfatide-activated type II NKT cells could prevent allergic airway inflammation by inhibiting type I NKT cell function in asthma. Strikingly, in our mouse model, activation of type II NKT cells by sulfatide administration and adoptive transfer of sulfatide-activated type II NKT cells result in reduced-inflammation cell infiltration in the lung and bronchoalveolar lavage fluid, decreased levels of IL-4 and IL-5 in the BALF; and decreased serum levels of ovalbumin-specific IgE and IgG1. Furthermore, it is found that the activation of sulfatide-reactive type II NKT cells leads to the functional inactivation of type I NKT cells, including the proliferation and cytokine secretion. Our data reveal that type II NKT cells activated by glycolipids, such as sulfatide, may serve as a novel approach to treat allergic diseases and other disorders characterized by inappropriate type I NKT cell activation.

  11. Increased p21ras activity in human fibroblasts transduced with survivin enhances cell proliferation

    International Nuclear Information System (INIS)

    Temme, Achim; Diestelkoetter-Bachert, Petra; Schmitz, Marc; Morgenroth, Agnieszka; Weigle, Bernd; Rieger, Michael A.; Kiessling, Andrea; Rieber, E. Peter

    2005-01-01

    Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21 ras mRNA and protein expression and concomitant rise in levels of activated p21 ras were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21 ras , is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21 ras activity

  12. Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

    Directory of Open Access Journals (Sweden)

    Leoh Lai

    2009-08-01

    Full Text Available Abstract Background Hormone-refractory prostate cancer (HRPC is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality. Results We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75, a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL

  13. N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism.

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    Budhaditya Mazumdar

    Full Text Available Activated hepatic stellate cells (HSCs are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM collected from immortalized human hepatocytes (IHH have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.

  14. Fear conditioning-related changes in cerebellar Purkinje cell activities in goldfish

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    Yoshida Masayuki

    2012-10-01

    Full Text Available Abstract Background Fear conditioning-induced changes in cerebellar Purkinje cell responses to a conditioned stimulus have been reported in rabbits. It has been suggested that synaptic long-term potentiation and the resulting increases in firing rates of Purkinje cells are related to the acquisition of conditioned fear in mammals. However, Purkinje cell activities during acquisition of conditioned fear have not been analysed, and changes in Purkinje cell activities throughout the development of conditioned fear have not yet been investigated. In the present study, we tracked Purkinje cell activities throughout a fear conditioning procedure and aimed to elucidate further how cerebellar circuits function during the acquisition and expression of conditioned fear. Methods Activities of single Purkinje cells in the corpus cerebelli were tracked throughout a classical fear conditioning procedure in goldfish. A delayed conditioning paradigm was used with cardiac deceleration as the conditioned response. Conditioning-related changes of Purkinje cell responses to a conditioned stimulus and unconditioned stimulus were examined. Results The majority of Purkinje cells sampled responded to the conditioned stimulus by either increasing or decreasing their firing rates before training. Although there were various types of conditioning-related changes in Purkinje cells, more than half of the cells showed suppressed activities in response to the conditioned stimulus after acquisition of conditioned fear. Purkinje cells that showed unconditioned stimulus-coupled complex-spike firings also exhibited conditioning-related suppression of simple-spike responses to the conditioned stimulus. A small number of Purkinje cells showed increased excitatory responses in the acquisition sessions. We found that the magnitudes of changes in the firing frequencies of some Purkinje cells in response to the conditioned stimulus correlated with the magnitudes of the conditioned

  15. Predictors of activity involvement in dementia care homes: a cross-sectional study.

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    Smit, Dieneke; de Lange, Jacomine; Willemse, Bernadette; Pot, Anne Margriet

    2017-08-04

    Despite the finding that involvement in activities is one of the most important needs of residents with dementia living in care homes, care facilities struggle to fulfill this need. Over the years, various factors are suggested which may contribute to or disable activity provision in dementia care homes. These include limited financial resources, task oriented staff and disease-related characteristics of residents. This study aims to further clarify which of these factors predict higher activity involvement. Data were derived from the second measurement (2011) of the Living Arrangements for people with Dementia study. One thousand two hundred eighteen people residing in 139 dementia care homes were involved. Forty predictors of higher involvement were studied. Multilevel backward regression analyses were performed. The most important predictors of higher involvement were: absence of agitation, less ADL dependency, and a higher cognitive status of the residents, higher staff educational level, lower experienced job demands by care staff and a smaller number of residents living in the dementia care wards of a facility. More social supervisor support as perceived by staff was found to predict less activity involvement. To increase the activity involvement of care home residents with dementia it seems vital to: 1) reduce staff's experienced job demands; 2) elevate their overall educational level; 3) train staff to provide suitable activities, taking account of the behavior and preserved capabilities of residents; and 4) foster transition towards small-scale care. In order to achieve these aims, care organizations might need to evaluate the use of their financial means.

  16. Sprouty regulates cell migration by inhibiting the activation of Rac1 GTPase

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    Poppleton, Helen M.; Edwin, Francis; Jaggar, Laura; Ray, Ramesh; Johnson, Leonard R.; Patel, Tarun B.

    2004-01-01

    Sprouty (SPRY) protein negatively modulates fibroblast growth factor and epidermal growth factor actions. We showed that human SPRY2 inhibits cell growth and migration in response to serum and several growth factors. Using rat intestinal epithelial (IEC-6) cells, we investigated the involvement of the Rho family of GTPases, RhoA, Rac1, and cdc42 in SPRY2-mediated inhibition of cell migration and proliferation. The ability of TAT-tagged SPRY2 to inhibit proliferation and migration of IEC-6 cells transfected with constitutively active mutants of RhoA(G14V), Rac1(G12V), and cdc42 (F28L) was determined. Constitutively active RhoA(G14V), Rac1(G12V), or cdc42(F28L) did not protect cells from the anti-proliferative actions of TAT-SPRY2. The ability of TAT-hSPRY2 to inhibit migration was not altered by of RhoA(G14V) and cdc42(F28L). However, Rac1(G12V) obliterated the ability of SPRY2 to inhibit cell autonomous or serum-induced migration. Also, the activation of endogenous Rac1 was attenuated by TAT-SPRY2. Thus, SPRY2 mediates its anti-migratory actions by inhibiting Rac1 activation

  17. Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling

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    Sujeong Jang

    2015-01-01

    Full Text Available Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs.

  18. Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation

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    Yoon, Jaemin; Ham, Hyeonmi; Sung, Jeehye; Kim, Younghwa; Choi, Youngmin; Lee, Jeom-Sig; Jeong, Heon-Sang; Lee, Junsoo

    2014-01-01

    BACKGROUND/OBJECTIVES The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress. PMID:24741394

  19. Estrogen receptor and PI3K/Akt signaling pathway involvement in S-(-equol-induced activation of Nrf2/ARE in endothelial cells.

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    Ting Zhang

    Full Text Available S-(-equol, a natural product of the isoflavone daidzein, has been reported to offer cytoprotective effects with respect to the cardiovascular system, but how this occurs is unclear. Interestingly, S-(-equol is produced by the human gut, suggesting a role in physiological processes. We report that treatment of human umbilical vein endothelial cells and EA.hy926 cells with S-(-equol induces ARE-luciferase reporter gene activity that is dose and time dependent. S-(-equol (10-250 nM increases nuclear factor-erythroid 2-related factor 2 (Nrf2 as well as gene products of Nrf2 target genes heme oxygenase-1 (HO-1 and NAD(PH (nicotinamide-adenine-dinucleotide-phosphate quinone oxidoreductase 1 (NQO1. Endothelial cells transfected with an HA-Nrf2 expression plasmid had elevated HA-Nrf2, HO-1, and NQO1 in response to S-(-equol exposure. S-(-equol treatment affected Nrf2 mRNA only slightly but significantly increased HO-1 and NQO1 mRNA. The pretreatment of cells with specific ER inhibitors or PI3K/Akt (ICI182,780 and LY294002 increased Nrf2, HO-1, and NQO1 protein, impaired nuclear translocation of HA-Nrf2, and decreased ARE-luciferase activity. Identical experiments were conducted with daidzein, which had effects similar to S-(-equol. In addition, DPN treatment (an ERβ agonist induced the ARE-luciferase reporter gene, promoting Nrf2 nuclear translocation. Cell pretreatment with an ERβ antagonist (PHTPP impaired S-(-equol-induced Nrf2 activation. Pre-incubation of cells followed by co-treatment with S-(-equol significantly improved cell survival in response to H2O2 or tBHP and reduced apoptotic and TUNEL-positively-stained cells. Notably, the ability of S-(-equol to protect against H2O2-induced cell apoptosis was attenuated in cells transfected with an siRNA against Nrf2. Thus, beneficial effects of S-(-equol with respect to cytoprotective antioxidant gene activation may represent a novel strategy to prevent and treat cardiovascular diseases.

  20. Camptothecin and khat (Catha edulis Forsk. induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines

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    Fossan Kjell O

    2009-11-01

    Full Text Available Abstract Background An organic extract of the recreational herb khat (Catha edulis Forsk. triggers cell death in various leukemia cell lines in vitro. The chemotherapeutics camptothecin, a plant alkaloid topoisomerase I inhibitor, was tested side-by-side with khat in a panel of acute myeloid leukemia cell lines to elucidate mechanisms of toxicity. Results Khat had a profound effect on MOLM-13 cells inducing mitochondrial damage, chromatin margination and morphological features of autophagy. The effects of khat on mitochondrial ultrastructure in MOLM-13 correlated with strongly impaired routine respiration, an effect neither found in the khat-resistant MV-4-11 cells nor in camptothecin treated cells. Enforced expression of anti-apoptotic Bcl-2 protein provided protection against camptothecin-induced cell death and partly against khat toxicity. Khat-induced cell death in MOLM-13 cells included reduced levels of anti-apoptotic Mcl-1 protein, while both khat and camptothecin induced c-FLIPL cleavage and procaspase-8 activation. Conclusion Khat activated a distinct cell death pathway in sensitive leukemic cells as compared to camptothecin, involving mitochondrial damage and morphological features of autophagy. This suggests that khat should be further explored in the search for novel experimental therapeutics.

  1. Involvement of miR-30c in resistance to doxorubicin by regulating YWHAZ in breast cancer cells

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    Fang, Y. [Department of Central Laboratory, The First Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Shen, H. [Department of Oncology, The First Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Cao, Y. [Department of Central Laboratory, The First Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Li, H. [Department of Central Laboratory, The Fourth Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Qin, R. [Department of Oncology, The First Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Chen, Q. [Department of Central Laboratory, The First Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Long, L. [Department of Oncology, The First Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Zhu, X.L. [Department of Central Laboratory, The Fourth Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Xie, C.J. [Department of Central Laboratory, The First Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China); Xu, W.L. [Department of Central Laboratory, The Fourth Affiliated People’s Hospital, Jiangsu University, Zhenjiang, Jiangsu (China)

    2014-01-10

    MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression implicated in cancer, which play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. The aim of this study was to investigate whether miR-30c mediated the resistance of breast cancer cells to the chemotherapeutic agent doxorubicin (ADR) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). miR-30c was downregulated in the doxorubicin-resistant human breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR compared with their parental MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, we observed that transfection of an miR-30c mimic significantly suppressed the ability of MCF-7/ADR to resist doxorubicin. Moreover, the anti-apoptotic gene YWHAZ was confirmed as a target of miR-30c by luciferase reporter assay, and further studies indicated that the mechanism for miR-30c on the sensitivity of breast cancer cells involved YWHAZ and its downstream p38 mitogen-activated protein kinase (p38MAPK) pathway. Together, our findings provided evidence that miR-30c was one of the important miRNAs in doxorubicin resistance by regulating YWHAZ in the breast cancer cell line MCF-7/ADR.

  2. Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity.

    Science.gov (United States)

    Evans, D J; Frank, D W; Finck-Barbançon, V; Wu, C; Fleiszig, S M

    1998-04-01

    Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.

  3. The negative cell cycle regulator, Tob (transducer of ErbB-2), is involved in motor skill learning

    International Nuclear Information System (INIS)

    Wang Xinming; Gao Xiang; Zhang Xuehan; Tu Yanyang; Jin Meilei; Zhao Guoping; Yu Lei; Jing Naihe; Li Baoming

    2006-01-01

    Tob (transducer of ErbB-2) is a negative cell cycle regulator with anti-proliferative activity in peripheral tissues. Our previous study identified Tob as a protein involved in hippocampus-dependent memory consolidation (M.L. Jin, X.M. Wang, Y.Y. Tu, X.H. Zhang, X. Gao, N. Guo, Z.Q. Xie, G.P. Zhao, N.H. Jing, B.M. Li, Y.Yu, The negative cell cycle regulator, Tob (Transducer of ErbB-2), is a multifunctional protein involved in hippocampus-dependent learning and memory, Neuroscience 131 (2005) 647-659). Here, we provide evidence that Tob in the central nervous system is engaged in acquisition of motor skill. Tob has a relatively high expression in the cerebellum. Tob expression is up-regulated in the cerebellum after rats receive training on a rotarod-running task. Rats infused with Tob antisense oligonucleotides into the 4th ventricle exhibit a severe deficit in running on a rotating rod or walking across a horizontally elevated beam

  4. Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours.

    Science.gov (United States)

    Rudolph, Christiane; Melau, Cecilie; Nielsen, John E; Vile Jensen, Kristina; Liu, Dekang; Pena-Diaz, Javier; Rajpert-De Meyts, Ewa; Rasmussen, Lene Juel; Jørgensen, Anne

    2017-08-01

    Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p cisplatin sensitivity. This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in

  5. Enhanced Expression of WD Repeat-Containing Protein 35 via CaMKK/AMPK Activation in Bupivacaine-Treated Neuro2a Cells

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    Huang, Lei; Kondo, Fumio; Gosho, Masahiko; Feng, Guo-Gang; Harato, Misako; Xia, Zhong-yuan; Ishikawa, Naohisa; Fujiwara, Yoshihiro; Okada, Shoshiro

    2014-01-01

    We previously reported that bupivacaine induces reactive oxygen species (ROS) generation, p38 mitogen-activated protein kinase (MAPK) activation and nuclear factor-kappa B activation, resulting in an increase in expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. However, the identity of signaling upstream of p38 MAPK pathways to WDR35 expression remains unclear. It has been shown that AMP-activated protein kinase (AMPK) can activate p38 MAPK through diverse mechanisms. In addition, several kinases acting upstream of AMPK have been identified including Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Recent studies reported that AMPK may be involved in bupivacaine-induced cytotoxicity in Schwann cells and in human neuroblastoma SH-SY5Y cells. The present study was undertaken to test whether CaMKK and AMPK are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Our results showed that bupivacaine induced activation of AMPK and p38 MAPK in Neuro2a cells. The AMPK inhibitors, compound C and iodotubercidin, attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. Treatment with the CaMKK inhibitor STO-609 also attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. These results suggest that bupivacaine activates AMPK and p38 MAPK via CaMKK in Neuro2a cells, and that the CaMKK/AMPK/p38 MAPK pathway is involved in regulating WDR35 expression. PMID:24859235

  6. Fast gamma oscillations are generated intrinsically in CA1 without the involvement of fast-spiking basket cells.

    Science.gov (United States)

    Craig, Michael T; McBain, Chris J

    2015-02-25

    Information processing in neuronal networks relies on the precise synchronization of ensembles of neurons, coordinated by the diverse family of inhibitory interneurons. Cortical interneurons can be usefully parsed by embryonic origin, with the vast majority arising from either the caudal or medial ganglionic eminences (CGE and MGE). Here, we examine the activity of hippocampal interneurons during gamma oscillations in mouse CA1, using an in vitro model where brief epochs of rhythmic activity were evoked by local application of kainate. We found that this CA1 KA-evoked gamma oscillation was faster than that in CA3 and, crucially, did not appear to require the involvement of fast-spiking basket cells. In contrast to CA3, we also found that optogenetic inhibition of pyramidal cells in CA1 did not significantly affect the power of the oscillation, suggesting that excitation may not be essential for gamma genesis in this region. We found that MGE-derived interneurons were generally more active than CGE interneurons during CA1 gamma, although a group of CGE-derived interneurons, putative trilaminar cells, were strongly phase-locked with gamma oscillations and, together with MGE-derived axo-axonic and bistratified cells, provide attractive candidates for being the driver of this locally generated, predominantly interneuron-driven model of gamma oscillations. Copyright © 2015 the authors 0270-6474/15/353616-09$15.00/0.

  7. Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

    Science.gov (United States)

    Hauptmann, Peter; Lehle, Ludwig

    2008-07-04

    N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

  8. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

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    Lorena Olivares-González

    Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  9. Phagocytosis in Teleosts. Implications of the New Cells Involved

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    María Ángeles Esteban

    2015-12-01

    Full Text Available Phagocytosis is the process by which cells engulf some solid particles to form internal vesicles known as phagosomes. Phagocytosis is in fact a specific form of endocytosis involving the vesicular interiorization of particles. Phagocytosis is essentially a defensive reaction against infection and invasion of the body by foreign substances and, in the immune system, phagocytosis is a major mechanism used to remove pathogens and/or cell debris. For these reasons, phagocytosis in vertebrates has been recognized as a critical component of the innate and adaptive immune responses to pathogens. Furthermore, more recent studies have revealed that phagocytosis is also crucial for tissue homeostasis and remodeling. Professional phagocytes in teleosts are monocyte/macrophages, granulocytes and dendritic cells. Nevertheless, in recent years phagocytic properties have also been attributed to teleost lymphocytes and thrombocytes. The possible implications of such cells on this important biological process, new factors affecting phagocytosis, evasion of phagocytosis or new forms of phagocytosis will be considered and discussed.

  10. Boletus edulis biologically active biopolymers induce cell cycle arrest in human colon adenocarcinoma cells.

    Science.gov (United States)

    Lemieszek, Marta Kinga; Cardoso, Claudia; Ferreira Milheiro Nunes, Fernando Hermínio; Ramos Novo Amorim de Barros, Ana Isabel; Marques, Guilhermina; Pożarowski, Piotr; Rzeski, Wojciech

    2013-04-25

    The use of biologically active compounds isolated from edible mushrooms against cancer raises global interest. Anticancer properties are mainly attributed to biopolymers including mainly polysaccharides, polysaccharopeptides, polysaccharide proteins, glycoproteins and proteins. In spite of the fact that Boletus edulis is one of the widely occurring and most consumed edible mushrooms, antitumor biopolymers isolated from it have not been exactly defined and studied so far. The present study is an attempt to extend this knowledge on molecular mechanisms of their anticancer action. The mushroom biopolymers (polysaccharides and glycoproteins) were extracted with hot water and purified by anion-exchange chromatography. The antiproliferative activity in human colon adenocarcinoma cells (LS180) was screened by means of MTT and BrdU assays. At the same time fractions' cytotoxicity was examined on the human colon epithelial cells (CCD 841 CoTr) by means of the LDH assay. Flow cytometry and Western blotting were applied to cell cycle analysis and protein expression involved in anticancer activity of the selected biopolymer fraction. In vitro studies have shown that fractions isolated from Boletus edulis were not toxic against normal colon epithelial cells and in the same concentration range elicited a very prominent antiproliferative effect in colon cancer cells. The best results were obtained in the case of the fraction designated as BE3. The tested compound inhibited cancer cell proliferation which was accompanied by cell cycle arrest in the G0/G1-phase. Growth inhibition was associated with modulation of the p16/cyclin D1/CDK4-6/pRb pathway, an aberration of which is a critical step in the development of many human cancers including colon cancer. Our results indicate that a biopolymer BE3 from Boletus edulis possesses anticancer potential and may provide a new therapeutic/preventive option in colon cancer chemoprevention.

  11. Real-time monitoring of caspase cascade activation in living cells.

    Science.gov (United States)

    Zhu, Lei; Huang, Xinglu; Choi, Ki Young; Ma, Ying; Zhang, Fan; Liu, Gang; Lee, Seulki; Chen, Xiaoyuan

    2012-10-10

    We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery. Published by Elsevier B.V.

  12. Collagen cross-linking by adipose-derived mesenchymal stromal cells and scar-derived mesenchymal cells: Are mesenchymal stromal cells involved in scar formation?

    NARCIS (Netherlands)

    Bogaerdt, van den A.J.; Veen, van der A.G.; Zuijlen, van P.P.; Reijnen, L.; Verkerk, M.; Bank, R.A.; Middelkoop, E.; Ulrich, M.

    2009-01-01

    In this work, different fibroblast-like (mesenchymal) cell populations that might be involved in wound healing were characterized and their involvement in scar formation was studied by determining collagen synthesis and processing. Depending on the physical and mechanical properties of the tissues,

  13. Collagen cross-linking by adipose-derived mesenchymal stromal cells and scar-derived mesenchymal cells : Are mesenchymal stromal cells involved in scar formation?

    NARCIS (Netherlands)

    van den Bogaerdt, Antoon J.; van der Veen, Vincent C.; van Zuijlen, Paul P. M.; Reijnen, Linda; Verkerk, Michelle; Bank, Ruud A.; Middelkoop, Esther; Ulrich, Magda M. W.

    2009-01-01

    In this work, different fibroblast-like (mesenchymal) cell populations that might be involved in wound healing were characterized and their involvement in scar formation was studied by determining collagen synthesis and processing. Depending on the physical and mechanical properties of the tissues,

  14. Prostanoid-dependent bladder pain caused by proteinase-activated receptor-2 activation in mice: Involvement of TRPV1 and T-type Ca2+ channels

    Directory of Open Access Journals (Sweden)

    Maho Tsubota

    2018-01-01

    Full Text Available We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2 in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL, caused delayed mechanical hypersensitivity in the lower abdomen, namely ‘referred hyperalgesia’, 6–24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca2+ channels.

  15. Neuronal activity in the hub of extrasynaptic Schwann cell-axon interactions

    Directory of Open Access Journals (Sweden)

    Chrysanthi eSamara

    2013-11-01

    Full Text Available The integrity and function of neurons depend on their continuous interactions with glial cells. In the peripheral nervous system glial functions are exerted by Schwann cells (SCs. SCs sense synaptic and extrasynaptic manifestations of action potential propagation and adapt their physiology to support neuronal activity. We review here existing literature data on extrasynaptic bidirectional axon-SC communication, focusing particularly on neuronal activity implications. To shed light on underlying mechanisms, we conduct a thorough analysis of microarray data from SC-rich mouse sciatic nerve at different developmental stages and in neuropathic models. We identify molecules that are potentially involved in SC detection of neuronal activity signals inducing subsequent glial responses. We further suggest that alterations in the activity-dependent axon-SC crosstalk impact on peripheral neuropathies. Together with previously reported data, these observations open new perspectives for deciphering glial mechanisms of neuronal function support.

  16. Role of protein kinase C in the TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

    Science.gov (United States)

    Abraha, Abraham B.; Rana, Krupa; Whalen, Margaret M.

    2010-01-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposures of NK cells to tributyltin (TBT) greatly diminish their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in the NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. The TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in the inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposures. TBT caused a 2–3 fold activation of PKC at concentrations ranging from 50–300 nM (16–98 ng/mL), indicating that activation of PKC occurs in response to TBT exposures. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that in NK cells where PKC activation was blocked there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including the activation of p44/42 by TBT in NK cells. PMID:20390410

  17. Involvement of ERK-Nrf-2 signaling in ionizing radiation induced cell death in normal and tumor cells.

    Directory of Open Access Journals (Sweden)

    Raghavendra S Patwardhan

    Full Text Available Prolonged oxidative stress favors tumorigenic environment and inflammation. Oxidative stress may trigger redox adaptation mechanism(s in tumor cells but not normal cells. This may increase levels of intracellular antioxidants and establish a new redox homeostasis. Nrf-2, a master regulator of battery of antioxidant genes is constitutively activated in many tumor cells. Here we show that, murine T cell lymphoma EL-4 cells show constitutive and inducible radioresistance via activation of Nrf-2/ERK pathway. EL-4 cells contained lower levels of ROS than their normal counterpart murine splenic lymphocytes. In response to radiation, the thiol redox circuits, GSH and thioredoxin were modified in EL-4 cells. Pharmacological inhibitors of ERK and Nrf-2 significantly enhanced radiosensitivity and reduced clonogenic potential of EL-4 cells. Unirradiated lymphoma cells showed nuclear accumulation of Nrf-2, upregulation of its dependent genes and protein levels. Interestingly, MEK inhibitor abrogated its nuclear translocation suggesting role of ERK in basal and radiation induced Nrf-2 activation in tumor cells. Double knockdown of ERK and Nrf-2 resulted in higher sensitivity to radiation induced cell death as compared to individual knockdown cells. Importantly, NF-kB which is reported to be constitutively active in many tumors was not present at basal levels in EL-4 cells and its inhibition did not influence radiosensitivity of EL-4 cells. Thus our results reveal that, tumor cells which are subjected to heightened oxidative stress employ master regulator cellular redox homeostasis Nrf-2 for prevention of radiation induced cell death. Our study reveals the molecular basis of tumor radioresistance and highlights role of Nrf-2 and ERK.

  18. Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

    Directory of Open Access Journals (Sweden)

    Shepherd Trevor G

    2010-02-01

    Full Text Available Abstract Background Activation of bone morphogenetic protein (BMP4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer. Methods To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT and cell adhesion. Results Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and

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

    Science.gov (United States)

    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.

  20. 5-Lipoxygenase contributes to PPARγ activation in macrophages in response to apoptotic cells.

    Science.gov (United States)

    von Knethen, Andreas; Sha, Lisa K; Kuchler, Laura; Heeg, Annika K; Fuhrmann, Dominik; Heide, Heinrich; Wittig, Ilka; Maier, Thorsten J; Steinhilber, Dieter; Brüne, Bernhard

    2013-12-01

    Macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells is a contributing hallmark to immune suppression during the late phase of sepsis. Although the peroxisome proliferator-activated receptor γ (PPARγ) supports this macrophage phenotype switch, it remains elusive how apoptotic cells activate PPARγ. Assuming that a molecule causing PPARγ activation in macrophages originates in the cell membrane of apoptotic cells we analyzed lipid rafts from apoptotic, necrotic, and living human Jurkat T cells which showed the presence of 5-lipoxygenase (5-LO) in lipid rafts of apoptotic cells only. Incubating macrophages with lipid rafts of apoptotic, but not necrotic or living cells, induced PPAR responsive element (PPRE)-driven mRuby reporter gene expression in RAW 264.7 macrophages stably transduced with a 4xPPRE containing vector. Experiments with lipid rafts of apoptotic murine EL4 T cells revealed similar results. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated with the 5-LO inhibitor MK-866 prior to induction of apoptosis, which failed to induce mRuby expression. Similar results were obtained with lipid rafts of apoptotic EL4 T cells preexposed to the 5-LO inhibitors zileuton and CJ-13610. Interestingly, Jurkat T cells overexpressing 5-LO failed to activate PPARγ in macrophages, while their 5-LO overexpressing apoptotic counterparts did. Our results suggest that during apoptosis 5-LO gets associated with lipid rafts and synthesizes ligands that in turn stimulate PPARγ in macrophages. © 2013.

  1. Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control

    DEFF Research Database (Denmark)

    Ozaki, Shogo; Schalch-Moser, Annina; Zumthor, Ludwig

    2014-01-01

    that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal......A to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit...

  2. Maternal involvement in children's leisure activities in rural China: Relations with adjustment outcomes.

    Science.gov (United States)

    Zhao, Siman; Chen, Xinyin

    2018-02-01

    This 1-year longitudinal study examined maternal involvement in children's leisure activities and its relations with children's adjustment in rural China. Participants included 184 children (93 boys and 91 girls) initially in third grade (mean age = 9.31 years). Children were asked to report the frequencies of mothers' involvement in leisure activities. Information on children's social, school, and psychological adjustment were collected from multiple sources including peer evaluations, teacher ratings, self-reports, and school records. The results showed that children's perceptions of maternal involvement in leisure activities positively predicted later social and school adjustment, particularly in boys. Furthermore, child initial adjustment status moderated the relations between maternal leisure activity involvement and child outcomes. The results suggest that maternal involvement in children's leisure activities, which has traditionally been neglected in the society, is a significant factor in contributing to child development in today's rural China. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

  3. Intrinsically active and pacemaker neurons in pluripotent stem cell-derived neuronal populations.

    Science.gov (United States)

    Illes, Sebastian; Jakab, Martin; Beyer, Felix; Gelfert, Renate; Couillard-Despres, Sébastien; Schnitzler, Alfons; Ritter, Markus; Aigner, Ludwig

    2014-03-11

    Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks.

  4. Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway

    International Nuclear Information System (INIS)

    Zhan, Meixiao; Sun, Xiaohan; Liu, Jinxiao; Li, Yan; Li, Yong; He, Xu; Zhou, Zizhang; Lu, Ligong

    2017-01-01

    The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system and further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs. - Highlights: • Loss of usp7 blocks the proliferation and metastasis of MB cells. • Usp7 regulates MB cell growth and migration through stimulating Shh pathway. • Usp7 inhibitors hamper MB cell proliferation and migration. • Usp7 inhibitors could attenuate Shh pathway activity.

  5. Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data

    Science.gov (United States)

    Schrader, Alexandra; Meyer, Katharina; Walther, Neele; Stolz, Ailine; Feist, Maren; Hand, Elisabeth; von Bonin, Frederike; Evers, Maurits; Kohler, Christian; Shirneshan, Katayoon; Vockerodt, Martina; Klapper, Wolfram; Szczepanowski, Monika; Murray, Paul G.; Bastians, Holger; Trümper, Lorenz; Spang, Rainer; Kube, Dieter

    2016-01-01

    To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery. PMID:27166259

  6. Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

    Directory of Open Access Journals (Sweden)

    Yannan Qin

    2014-11-01

    Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

  7. Versatile strategy for controlling the specificity and activity of engineered T cells

    Science.gov (United States)

    Ma, Jennifer S. Y.; Kim, Ji Young; Kazane, Stephanie A.; Choi, Sei-hyun; Yun, Hwa Young; Kim, Min Soo; Rodgers, David T.; Pugh, Holly M.; Singer, Oded; Sun, Sophie B.; Fonslow, Bryan R.; Kochenderfer, James N.; Wright, Timothy M.; Schultz, Peter G.; Young, Travis S.; Kim, Chan Hyuk; Cao, Yu

    2016-01-01

    The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR–T-cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as “switch” molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a “universal” anti-FITC–directed CAR-T cell. Optimization of this CAR–switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR–T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy. PMID:26759368

  8. Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

    Science.gov (United States)

    Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

    2018-05-01

    Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Possible Involvement of Human Mast Cells in the Establishment of Pregnancy via Killer Cell Ig-Like Receptor 2DL4.

    Science.gov (United States)

    Ueshima, Chiyuki; Kataoka, Tatsuki R; Hirata, Masahiro; Sugimoto, Akihiko; Iemura, Yoshiki; Minamiguchi, Sachiko; Nomura, Takashi; Haga, Hironori

    2018-06-01

    The involvement of mast cells in the establishment of pregnancy is unclear. Herein, we found that human mast cells are present in the decidual tissues of parous women and expressed a human-specific protein killer cell Ig-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen G expressed on human trophoblasts. In contrast, decreased numbers of decidual mast cells and reduced KIR2DL4 expression were observed in these cells of infertile women who had undergone long-term corticosteroid treatment. Co-culture of the human mast cell line, LAD2, and human trophoblast cell line, HTR-8/SVneo, accelerated the migration and tube formation of HTR-8/SVneo cells in a KIR2DL4-dependent manner. These observations suggest the possible involvement of human mast cells in the establishment of pregnancy via KIR2DL4 and that long-term corticosteroid treatment may cause infertility by influencing the phenotypes of decidual mast cells. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  10. Mast cells in the colon of Trypanosoma cruzi-infected patients: are they involved in the recruitment, survival and/or activation of eosinophils?

    Science.gov (United States)

    Martins, Patrícia Rocha; Nascimento, Rodolfo Duarte; Lopes, Júlia Guimarães; Santos, Mônica Morais; de Oliveira, Cleida Aparecida; de Oliveira, Enio Chaves; Martinelli, Patrícia Massara; d'Ávila Reis, Débora

    2015-05-01

    Megacolon is frequently observed in patients who develop the digestive form of Chagas disease. It is characterized by dilation of the rectum-sigmoid portion and thickening of the colon wall. Microscopically, the affected organ presents denervation, which has been considered as consequence of an inflammatory process that begins at the acute phase and persists in the chronic phase of infection. Inflammatory infiltrates are composed of lymphocytes, macrophages, natural killer cells, mast cells, and eosinophils. In this study, we hypothesized that mast cells producing tryptase could influence the migration and the activation of eosinophils at the site, thereby contributing to the immunopathology of the chronic phase. We seek evidence of interactions between mast cells and eosinophils through (1) evaluation of eosinophils, regarding the expression of PAR2, a tryptase receptor; (2) correlation analysis between densities of mast cells and eosinophils; and (3) ultrastructural studies. The electron microscopy studies revealed signs of activation of mast cells and eosinophils, as well as physical interaction between these cells. Immunohistochemistry and correlation analyses point to the participation of tryptase immunoreactive mast cells in the migration and/or survival of eosinophils at the affected organ.

  11. Deregulated microRNAs in CD4+ T cells from individuals with latent tuberculosis versus active tuberculosis.

    Science.gov (United States)

    Fu, Yurong; Yi, Zhengjun; Li, Jianhua; Li, Ruifang

    2014-03-01

    The mechanisms of latent tuberculosis (TB) infection remain elusive. Roles of microRNA (miRNA) have been highlighted in pathogen-host interactions recently. To identify miRNAs involved in the immune response to TB, expression profiles of miRNAs in CD4(+) T cells from patients with latent TB, active TB and healthy controls were investigated by microarray assay and validated by RT-qPCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyse the significant functions and involvement in signalling pathways of the differentially expressed miRNAs. To identify potential target genes for miR-29, interferon-γ (IFN-γ) mRNA expression was measured by RT-qPCR. Our results showed that 27 miRNAs were deregulated among the three groups. RT-qPCR results were generally consistent with the microarray data. We observed an inverse correlation between miR-29 level and IFN-γ mRNA expression in CD4(+) T cells. GO and KEGG pathway analysis showed that the possible target genes of deregulated miRNAs were significantly enriched in mitogen-activated protein kinase signalling pathway, focal adhesion and extracellular matrix receptor interaction, which might be involved in the transition from latent to active TB. In all, for the first time, our study revealed that some miRNAs in CD4(+) T cells were altered in latent and active TB. Function and pathway analysis highlighted the possible involvement of miRNA-deregulated mRNAs in TB. The study might help to improve understanding of the relationship between miRNAs in CD4(+) T cells and TB, and laid an important foundation for further identification of the underlying mechanisms of latent TB infection and its reactivation. © 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  12. The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

    Directory of Open Access Journals (Sweden)

    Soriano-Carot María

    2012-02-01

    Full Text Available Abstract Background The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. Results This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU, methylmetanosulfonate (MMS, phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. Conclusions Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt

  13. Fine-tuning of mast cell activation by FceRIbeta chain

    Directory of Open Access Journals (Sweden)

    Chisei eRa

    2012-05-01

    Full Text Available Mast cells play the key role in allergic reaction and disorders, being activated by the high affinity receptor for IgE, FceRI. There are two types of FceRI expressed on the cell surface of human mast cells, abg2 type and ag2 type (without b chain, while in mouse mast cells only the tetrameric abg2 type is expressed. In the lesion of allergic inflammation such as atopic conjunctivitis and atopic dermatitis, mast cells increase in number and exclusively express the abg2 type FceRI, in contrast in healthy conjunctiva and skin most mast cells express the ag2 type. The human and mouse FceRI genes contain seven exons and in the human gene we found a repressor element locates in the forth intron. Through the repressor element HDACs are recruited to the FceRIb gene by MZF-1/FHL3/NFY complex and repress b transcription by deacetylation of histones in the presence of GM-CSF. It has been long recognized that the function of the b chain ITAM is a signal amplifier, but we have recently revealed bidirectional (positive and negative functions of the b chain ITAM in the regulation of the mast cell activation and effector functions. Namely, the b chain enhances the mast cell activation signal triggered with low intensity stimulation such as lower dose antigen than threshold while it suppresses the signal of high intensity stimulation. Employing mouse model of CHS induced by oxazolone, we have revealed that IgE-mediated mast cell activation is required for CHS and that the b chain is crucially involved in this model. On the other hand diverse immune receptors including TLRs, SCF receptor and GPCRs are known to mediate signals which modulate FceRI・adenosine receptors, one of GPCRs, trigger synergistic degranulation response in mast cells even when the FceRI stimulation is of ‘lower intensity’ than the threshold. We have recently elucidated, in this synergistic degranulation response, b chain ITAM plays positive role, possibly reflecting in vivo allergic

  14. The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Francesca Carlini

    Full Text Available BACKGROUND: Transposable Elements (TEs comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1 and Human Endogenous Retroviruses (HERVs that code for their own endogenous reverse transcriptase (RT. Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC, a nucleoside reverse transcription inhibitor (NRTI, on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

  15. Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

    Science.gov (United States)

    Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

    2008-02-01

    Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

  16. Tissue transglutaminase (TG2) is involved in the resistance of cancer cells to the histone deacetylase (HDAC) inhibitor vorinostat.

    Science.gov (United States)

    Carbone, Carmine; Di Gennaro, Elena; Piro, Geny; Milone, Maria Rita; Pucci, Biagio; Caraglia, Michele; Budillon, Alfredo

    2017-03-01

    Vorinostat demonstrated preclinical and clinical efficacy in human cancers and is the first histone deacetylase inhibitor (HDACi) approved for cancer treatment. Tissue transglutaminase (TG2) is a multifunctional enzyme that catalyzes a Ca 2+ dependent transamidating reaction resulting in covalent cross-links between proteins. TG2 acts also as G-protein in trans-membrane signaling and as a cell surface adhesion mediator. TG2 up-regulation has been demonstrated in several cancers and its expression levels correlate with resistance to chemotherapy and metastatic potential. We demonstrated that the anti-proliferative effect of the HDACi vorinostat is paralleled by the induction of TG2 mRNA and protein expression in cancer cells but not in ex vivo treated peripheral blood lymphocytes. This effect was also shared by other pan-HDACi and resulted in increased TG2 transamidating activity. Notably, high TG2 basal levels in a panel of cancer cell lines correlated with lower vorinostat antiproliferative activity. Notably, in TG2-knockdown cancer cells vorinostat anti-proliferative and pro-apoptotic effects were enhanced, whereas in TG2-full-length transfected cells were impaired, suggesting that TG2 could represent a mechanism of intrinsic or acquired resistance to vorinostat. In fact, co-treatment of tumor cells with inhibitors of TG2 transamidating activity potentiated the antitumor effect of vorinostat. Moreover, vorinostat-resistant MCF7 cells selected by stepwise increasing concentrations of the drug, significantly overexpressed TG2 protein compared to parental cells, and co-treatment of these cells with TG2 inhibitors reversed vorinostat-resistance. Taken together, our data demonstrated that TG2 is involved in the resistance of cancer cells to vorinostat, as well as to other HDACi.

  17. Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells.

    Science.gov (United States)

    Soundara Rajan, Thangavelu; Giacoppo, Sabrina; Scionti, Domenico; Diomede, Francesca; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2017-06-01

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 118: 1531-1546, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

    Science.gov (United States)

    2013-01-01

    Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

  19. Involvement of bone marrow stem cells in periodontal wound healing.

    Science.gov (United States)

    Zhou, Li Li; Liu, Hong Wei; Wen, Xin Xin; Xie, Han

    2014-01-01

    To test the hypothesis whether bone marrow stem cells (BMSCs) could migrate into the periodontium as the precursor available for the repair of tissue injury. A chimeric mouse model was established by transplanting BMSCs derived from red fluorescent protein mouse into irradiated BALB/c mice. Subsequently, a periodontal defect was created beside the maxillary first molar and filled with ceramic bovine bone. Finally, the chimeric mice were divided into three groups and were observed 3, 14 and 28 days later respectively. The involvement of BMSCs in periodontal defects was analysed using an in vivo imaging system and immunohistochemical staining of CD45, CD105 and CD31. Cell surface marker expression in injured tissue was also compared with that in normal tissue. Increasing numbers of BMSCs migrated into the periodontal defect with time. The distribution was initially limited to ceramic bovine bone and then around blood vessels and near alveolar bone. Furthermore, expression of CD105 and CD31 was much higher in injured periodontal tissue than that in healthy periodontium, although CD45 was not expressed in either of these tissues. BMSCs, but not haemopoietic stem cells, were involved in periodontal defect; they entered the periodontium probably via blood vessels.

  20. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

    Directory of Open Access Journals (Sweden)

    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

  1. Immune activation induces immortalization of HTLV-1 LTR-Tax transgenic CD4+ T cells

    OpenAIRE

    Swaims, Alison Y.; Khani, Francesca; Zhang, Yingyu; Roberts, Arthur I.; Devadas, Satish; Shi, Yufang; Rabson, Arnold B.

    2010-01-01

    Infection with the human T-cell leukemia virus-1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia/lymphoma (ATL). Although the pathogenesis of these disorders is poorly understood, it involves complex interactions with the host immune system. Activation of infected T cells may play an important role in disease pathogenesis through induction of the oncogenic HTLV-1 Tax transactivator protein. To test this hypothesis, we employed transgenic mice in which Tax is regulate...

  2. Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Wada, Hiromichi; Abe, Mitsuru; Ono, Koh; Morimoto, Tatsuya; Kawamura, Teruhisa; Takaya, Tomohide; Satoh, Noriko; Fujita, Masatoshi; Kita, Toru; Shimatsu, Akira; Hasegawa, Koji

    2008-01-01

    The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 μM of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-α-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 μM), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs

  3. Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Wada, Hiromichi [Division of Translational Research, National Hospital Organization Kyoto Medical Center, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto 612-8555 (Japan); Abe, Mitsuru; Ono, Koh [Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Morimoto, Tatsuya; Kawamura, Teruhisa; Takaya, Tomohide [Division of Translational Research, National Hospital Organization Kyoto Medical Center, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto 612-8555 (Japan); Satoh, Noriko [Division of Metabolic Research, National Hospital Organization Kyoto Medical Center, Kyoto (Japan); Fujita, Masatoshi [Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Kita, Toru [Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Shimatsu, Akira [Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto (Japan); Hasegawa, Koji [Division of Translational Research, National Hospital Organization Kyoto Medical Center, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto 612-8555 (Japan)

    2008-10-03

    The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 {mu}M of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-{alpha}-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 {mu}M), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.

  4. The mechanisms involved at the cell level

    International Nuclear Information System (INIS)

    Leblanc, G.; Pourcher, Th.; Perron, B.; Guillain, F.; Quemeneur, E.; Fritsch, P.

    2003-01-01

    The mechanisms responsible at the cell level for inducing toxic reactions after contamination are as yet only imperfectly known. Work still needs to be done for both contaminants that have a biological role, such as iodine, and those that do not, such as cadmium, uranium and plutonium. In particular, these mechanisms bring into play, in biological membranes, carriers which are the physiological partners responsible for material exchange with the environment or inside the body. As they lack absolute selectivity, these carriers, which are involved in the assimilation and accumulation of vital mineral elements, also have the ability to transport toxic elements and isotopes. (authors)

  5. Involvement of Nrf2-Mediated Upregulation of Heme Oxygenase-1 in Mollugin-Induced Growth Inhibition and Apoptosis in Human Oral Cancer Cells

    Directory of Open Access Journals (Sweden)

    Young-Man Lee

    2013-01-01

    Full Text Available Although previous studies have shown that mollugin, a bioactive phytochemical isolated from Rubia cordifolia L. (Rubiaceae, exhibits antitumor effects, its biological activity in oral cancer has not been reported. We thus investigated the effects and putative mechanism of apoptosis induced by mollugin in human oral squamous cell carcinoma cells (OSCCs. Results show that mollugin induces cell death in a dose-dependent manner in primary and metastatic OSCCs. Mollugin-induced cell death involved apoptosis, characterized by the appearance of nuclear shrinkage, flow cytometric analysis of sub-G1 phase arrest, and annexin V-FITC and propidium iodide staining. Western blot analysis and RT-PCR revealed that mollugin suppressed activation of NF-κB and NF-κB-dependent gene products involved in antiapoptosis (Bcl-2 and Bcl-xl, invasion (MMP-9 and ICAM-1, and angiogenesis (FGF-2 and VEGF. Furthermore, mollugin induced the activation of p38, ERK, and JNK and the expression of heme oxygenase-1 (HO-1 and nuclear factor E2–related factor 2 (Nrf2. Mollugin-induced growth inhibition and apoptosis of HO-1 were reversed by an HO-1 inhibitor and Nrf2 siRNA. Collectively, this is the first report to demonstrate the effectiveness of mollugin as a candidate for a chemotherapeutic agent in OSCCs via the upregulation of the HO-1 and Nrf2 pathways and the downregulation of NF-κB.

  6. Involvement of histone H3 phosphorylation via the activation of p38 MAPK pathway and intracellular redox status in cytotoxicity of HL-60 cells induced by Vitex agnus-castus fruit extract.

    Science.gov (United States)

    Kikuchi, Hidetomo; Yuan, Bo; Yuhara, Eisuke; Imai, Masahiko; Furutani, Ryota; Fukushima, Shin; Hazama, Shingo; Hirobe, Chieko; Ohyama, Kunio; Takagi, Norio; Toyoda, Hiroo

    2014-08-01

    We have demonstrated that an extract from the ripe fruit of Vitex angus-castus (Vitex), might be a promising anticancer candidate. In order to further provide a molecular rationale for clinical development in anticancer therapy, a detailed mechanism underlying the efficacy of Vitex against HL-60 cells was investigated. Vitex induced a dose- and time-dependent decrease in cell viability associated with induction of apoptosis and G(2)/M cell cycle arrest, both of which were suppressed by the addition of SB203580, an inhibitor for p38 MAPK. Furthermore, SB203580 significantly suppressed Vitex-induced phosphorylation of histone H3, a downstream molecule of p38 MAPK known to be involved in apoptosis induction in tumor cells. Notably, Vitex induced upregulation of intracellular ATP, known to bind its binding pocket inside activated p38 MAPK and to be required for the activation of p38 MAPK pathway. These results, thus, suggest that upregulation of intracellular ATP and phosphorylation of histone H3 are closely associated with the activation of p38 MAPK pathway, consequently contributing to Vitex-mediated cytotoxicity. Intriguingly, a significant decrease of intracellular ROS levels and downregulation of expression level of gp91(phox), an important component of NADPH oxidase, were observed in Vitex-treated cells. A greater decline in ROS levels along with enhanced apoptosis was observed after treatment with Vitex in combination with SnPP, an inhibitor specific for HO-1. Since NADPH oxidase and HO-1 are closely correlated to redox status associated with intracellular ROS levels, the two enzymes are suggested to be implicated in Vitex-mediated cytotoxicity in HL-60 cells by regulating ROS generation. We also suggest that activation of the p38 MAPK pathway may be dependent on the alterations of intracellular ATP levels, rather than that of intracellular ROS levels. These results may have important implications for appropriate clinical uses of Vitex and provide novel insights

  7. Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways.

    Science.gov (United States)

    Saleh, A M; Vijayasarathy, C; Masoud, L; Kumar, L; Shahin, A; Kambal, A

    2003-07-01

    The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide

  8. Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

    International Nuclear Information System (INIS)

    O'Grady, Michael; Raha, Debasish; Hanson, Bonnie J; Bunting, Michaeline; Hanson, George T

    2005-01-01

    The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a β-lactamase reporter gene, a 10–12 fold shift or 2.5–3 fold shift toward greater potency in the IC 50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. EGFR pathway components were qualified as

  9. Involvement of DNA methylation in the control of cell growth during heat stress in tobacco BY-2 cells.

    Science.gov (United States)

    Centomani, Isabella; Sgobba, Alessandra; D'Addabbo, Pietro; Dipierro, Nunzio; Paradiso, Annalisa; De Gara, Laura; Dipierro, Silvio; Viggiano, Luigi; de Pinto, Maria Concetta

    2015-11-01

    The alteration of growth patterns, through the adjustment of cell division and expansion, is a characteristic response of plants to environmental stress. In order to study this response in more depth, the effect of heat stress on growth was investigated in tobacco BY-2 cells. The results indicate that heat stress inhibited cell division, by slowing cell cycle progression. Cells were stopped in the pre-mitotic phases, as shown by the increased expression of CycD3-1 and by the decrease in the NtCycA13, NtCyc29 and CDKB1-1 transcripts. The decrease in cell length and the reduced expression of Nt-EXPA5 indicated that cell expansion was also inhibited. Since DNA methylation plays a key role in controlling gene expression, the possibility that the altered expression of genes involved in the control of cell growth, observed during heat stress, could be due to changes in the methylation state of their promoters was investigated. The results show that the altered expression of CycD3-1 and Nt-EXPA5 was consistent with changes in the methylation state of the upstream region of these genes. These results suggest that DNA methylation, controlling the expression of genes involved in plant development, contributes to growth alteration occurring in response to environmental changes.

  10. Promotion of haematopoietic activity in embryonic stem cells by the aorta-gonad-mesonephros microenvironment

    International Nuclear Information System (INIS)

    Krassowska, Anna; Gordon-Keylock, Sabrina; Samuel, Kay; Gilchrist, Derek; Dzierzak, Elaine; Oostendorp, Robert; Forrester, Lesley M.; Ansell, John D.

    2006-01-01

    We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We conclude that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance

  11. Cannabinoid-induced cell death in endometrial cancer cells: involvement of TRPV1 receptors in apoptosis.

    Science.gov (United States)

    Fonseca, B M; Correia-da-Silva, G; Teixeira, N A

    2018-05-01

    Among a variety of phytocannabinoids, Δ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most promising therapeutic compounds. Besides the well-known palliative effects in cancer patients, cannabinoids have been shown to inhibit in vitro growth of tumor cells. Likewise, the major endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), induce tumor cell death. The purpose of the present study was to characterize cannabinoid elements and evaluate the effect of cannabinoids in endometrial cancer cell viability. The presence of cannabinoid receptors, transient receptor potential vanilloid 1 (TRPV1), and endocannabinoid-metabolizing enzymes were determined by qRT-PCR and Western blot. We also examined the effects and the underlying mechanisms induced by eCBs and phytocannabinoids in endometrial cancer cell viability. Besides TRPV1, both EC cell lines express all the constituents of the endocannabinoid system. We observed that at concentrations higher than 5 μM, eCBs and CBD induced a significant reduction in cell viability in both Ishikawa and Hec50co cells, whereas THC did not cause any effect. In Ishikawa cells, contrary to Hec50co, treatment with AEA and CBD resulted in an increase in the levels of activated caspase -3/-7, in cleaved PARP, and in reactive oxygen species generation, confirming that the reduction in cell viability observed in the MTT assay was caused by the activation of the apoptotic pathway. Finally, these effects were dependent on TRPV1 activation and intracellular calcium levels. These data indicate that cannabinoids modulate endometrial cancer cell death. Selective targeting of TPRV1 by AEA, CBD, or other stable analogues may be an attractive research area for the treatment of estrogen-dependent endometrial carcinoma. Our data further support the evaluation of CBD and CBD-rich extracts for the potential treatment of endometrial cancer, particularly, that has become non-responsive to common therapies.

  12. Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

    Science.gov (United States)

    Guon, Tae Eun; Chung, Ha Sook

    2017-08-01

    The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

  13. Three-dimensional organoid culture reveals involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells.

    Science.gov (United States)

    Yoshida, Takahiro; Sopko, Nikolai A; Kates, Max; Liu, Xiaopu; Joice, Gregory; McConkey, David J; Bivalacqua, Trinity J

    2018-02-16

    There has been increasing awareness of the importance of three-dimensional culture of cancer cells. Tumor cells growing as multicellular spheroids in three-dimensional culture, alternatively called organoids, are widely believed to more closely mimic solid tumors in situ . Previous studies concluded that the Wnt/β-catenin pathway is required for regeneration of the normal urothelium after injury and that β-catenin is upregulated in human bladder cancers, but no clear evidence has been advanced to support the idea that the Wnt/β-catenin pathway is directly involved in deregulated proliferation and the other malignant characteristics of bladder cancer cells. Here we report that the Wnt/β-catenin pathway activator, CHIR99021, promoted proliferation of established human bladder cancer cell lines when they were grown in organoid culture but not when they were grown in conventional adherent cultures. CHIR99021 activated Wnt/β-catenin pathway in bladder cancer cell lines in organoid culture. CHIR99021 also stimulated proliferation and the Wnt/b-catenin pathway in primary human bladder cancer organoids. RNAi-mediated knockdown of β-catenin blocked growth of organoids. The effects of CHIR99021 were associated with decreased expression of the urothelial terminal differentiation marker, cytokeratin 20. Our data suggest that the Wnt/β-catenin pathway is required for the proliferation of bladder cancer cells in three-dimensional organoid culture and provide a concrete example of why organoid culture is important for cancer research.

  14. Interaction of translationally controlled tumor protein with Apaf-1 is involved in the development of chemoresistance in HeLa cells

    International Nuclear Information System (INIS)

    Jung, Jaehoon; Kim, Hyo Young; Maeng, Jeehye; Kim, Moonhee; Shin, Dong Hae; Lee, Kyunglim

    2014-01-01

    Translationally controlled tumor protein (TCTP), alternatively called fortilin, is believed to be involved in the development of the chemoresistance of tumor cells against anticancer drugs such as etoposide, taxol, and oxaliplatin, the underlying mechanisms of which still remain elusive. Cell death analysis of TCTP-overexpressing HeLa cells was performed following etoposide treatment to assess the mitochondria-dependent apoptosis. Apoptotic pathway was analyzed through measuring the cleavage of epidermal growth factor receptor (EGFR) and phospholipase C-γ (PLC-γ), caspase activation, mitochondrial membrane perturbation, and cytochrome c release by flow cytometry and western blotting. To clarify the role of TCTP in the inhibition of apoptosome, in vitro apoptosome reconstitution and immunoprecipitation was used. Pull-down assay and silver staining using the variants of Apaf-1 protein was applied to identify the domain that is responsible for its interaction with TCTP. In the present study, we confirmed that adenoviral overexpression of TCTP protects HeLa cells from cell death induced by cytotoxic drugs such as taxol and etoposide. TCTP antagonized the mitochondria-dependent apoptotic pathway following etoposide treatment, including mitochondrial membrane damage and resultant cytochrome c release, activation of caspase-9, and -3, and eventually, the cleavage of EGFR and PLC-γ. More importantly, TCTP interacts with the caspase recruitment domain (CARD) of Apaf-1 and is incorporated into the heptameric Apaf-1 complex, and that C-terminal cleaved TCTP specifically associates with Apaf-1 of apoptosome in apoptosome-forming condition thereby inhibiting the amplification of caspase cascade. TCTP protects the cancer cells from etoposide-induced cell death by inhibiting the mitochondria-mediated apoptotic pathway. Interaction of TCTP with Apaf-1 in apoptosome is involved in the molecular mechanism of TCTP-induced chemoresistance. These findings suggest that TCTP may serve

  15. Management of a Parturient with Mast Cell Activation Syndrome: An Anesthesiologist’s Experience

    Directory of Open Access Journals (Sweden)

    Sangeeta Kumaraswami

    2018-01-01

    Full Text Available Mast cell activation syndrome (MCAS is a disorder in which patients experience symptoms and signs attributable to inappropriate mast cell activation and mediator release. Multiorgan involvement in patients can result in significant morbidity and possible mortality. Limited literature exists regarding anesthetic management of patients with MCAS. We report a case of vaginal delivery with neuraxial labor analgesia in a parturient with this condition and highlight the importance of multidisciplinary planning for uneventful outcomes. Stress can trigger life-threatening symptoms, and counseling is important to allay patients’ fears. Optimum medical control, adequate premedication, avoidance of triggers, and preparedness to treat serious mediator effects are key. We review MCAS and discuss anesthetic considerations for patients with this mast cell disorder.

  16. Liver X receptor activation inhibits PC-3 prostate cancer cells via the beta-catenin pathway.

    Science.gov (United States)

    Youlin, Kuang; Li, Zhang; Weiyang, He; Jian, Kang; Siming, Liang; Xin, Gou

    2017-03-01

    Liver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved. Prostate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers. Data showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased. This study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. Actinomycete enzymes and activities involved in straw saccharification

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, A J; Ball, A S [Liverpool Univ. (UK). Dept. of Genetics and Microbiology

    1990-01-01

    This research programme has been directed towards the analysis of actinomycete enzyme systems involved in the degradation of plant biomass (lignocellulose.) The programme was innovative in that a novel source of enzymes was systematically screened and wheat straw saccharifying activity was the test criterion. Over 200 actinomycete strains representing a broad taxonomic range were screened. A range of specific enzyme activities were involved and included cellulase, xylanase, arabinofuranosidase, acetylesterase, {beta}-xylosidase and {beta}-glucosidase. S