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Sample records for alpha affects cell

  1. Recombinant human erythropoietin alpha improves the efficacy of radiotherapy of a human tumor xenograft, affecting tumor cells and microvessels

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    Loevey, J. [Dept. of Radiotherapy, National Inst. of Oncology, Budapest (Hungary); Bereczky, B.; Gilly, R.; Kenessey, I.; Raso, E.; Simon, E.; Timar, J. [Dept. of Tumor Progression, National Inst. of Oncology, Budapest (Hungary); Dobos, J. [Dept. of Tumor Progression, National Inst. of Oncology, Budapest (Hungary); National Koranyi Inst. of TBC and Pulmonology, Budapest (Hungary); Vago, A. [Central Lab., National Inst. of Oncology, Budapest (Hungary); Kasler, M. [Head and Neck Surgery, National Inst. of Oncology, Budapest (Hungary); Doeme, B. [National Koranyi Inst. of TBC and Pulmonology, Budapest (Hungary); Tovari, J. [National Koranyi Inst. of TBC and Pulmonology, Budapest (Hungary); 1. Inst. of Pathology and Experimental Cancer Research, Semmelweis Univ., Budapest (Hungary)

    2008-01-15

    Background and purpose: tumor-induced anemia often occurs in cancer patients, and is corrected by recombinant human erythropoietins (rHuEPOs). Recent studies indicated that, besides erythroid progenitor cells, tumor and endothelial cells express erythropoietin receptor (EPOR) as well; therefore, rHuEPO may affect their functions. Here, the effect of rHuEPO{alpha} on irradiation in EPOR-positive human squamous cell carcinoma xenograft was tested. Material and methods: A431 tumor-bearing SCID mice were treated from the tumor implantation with rHuEPO{alpha} at human-equivalent dose. Xenografts were irradiated (5 Gy) on day 14, and the final tumor mass was measured on day 22. The systemic effects of rHuEPO{alpha} on the hemoglobin level, on tumor-associated blood vessels and on hypoxia-inducible factor-(HIF-)1{alpha} expression of the tumor xenografts were monitored. The proliferation, apoptosis and clonogenic capacity of A431 cancer cells treated with rHuEPO{alpha} and irradiation were also tested in vitro. Results: in vitro, rHuEPO{alpha} treatment alone did not modify the proliferation of EPOR-positive A431 tumor cells but enhanced the effect of irradiation on proliferation, apoptosis and clonogenic capacity. In vivo, rHuEPO{alpha} administration compensated the tumor-induced anemia in SCID mice and decreased tumoral HIF-1{alpha} expression but had no effect on tumor growth. At the same time rHuEPO{alpha} treatment significantly increased the efficacy of radiotherapy in vivo (tumor weight of 23.9 {+-} 4.7 mg and 34.9 {+-} 4.6 mg, respectively), mediated by increased tumoral blood vessel destruction. Conclusion: rHuEPO{alpha} treatment may modulate the efficacy of cancer radiotherapy not only by reducing systemic hypoxia and tumoral HIF-1{alpha} expression, but also by destroying tumoral vessels. (orig.)

  2. Prognostic value of alpha 6 beta 4 integrin expression in breast carcinomas is affected by laminin production from tumor cells.

    Science.gov (United States)

    Tagliabue, E; Ghirelli, C; Squicciarini, P; Aiello, P; Colnaghi, M I; Ménard, S

    1998-02-01

    Immunocytochemical analysis of breast carcinoma specimens for alpha 6 beta 4 integrin expression and other different pathobiological markers revealed beta 4 integrin subunit expression in 36 of 80 cases analyzed and a significant association only with alpha 6 integrin subunit expression (P laminin production (P = 0.01) by tumor cells. Survival analysis indicated that beta 4 and alpha 6 expression are associated with poor prognosis (P = 0.02), whereas laminin production showed only borderline association (P = 0.06). However, analysis of disease outcome in relation to expression of both alpha 6 beta 4 and laminin indicated best outcomes for patients with tumors producing laminin but not expressing alpha 6 beta 4 integrin, whereas worst outcomes were observed for alpha 6 beta 4- and laminin-positive tumors, indicating that alpha 6 beta 4 expression was associated with prognosis, mainly in the laminin-producing tumor subset. These data indicate that the prognostic value of alpha 6 beta 4 integrin expression is affected by laminin production from tumor cells and suggest that interaction between these two molecules mediates distinct signals that are important for tumor progression.

  3. Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells.

    Science.gov (United States)

    Abraham, I; Marcus, M; Cabral, F; Gottesman, M M

    1983-10-01

    Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.

  4. Inducible alpha-synuclein overexpression affects human Neural Stem Cells behavior

    OpenAIRE

    Conti, Luciano; Zasso, Jacopo; Cutarelli, Alessandro; Ahmed, Mastad

    2018-01-01

    Converging evidence suggest that levels of alpha-Synuclein (aSyn) expression play a critical role in Parkinson's disease (PD). Several mutations of the SNCA gene, encoding for aSyn have been associated to either the familial or the sporadic forms of PD. Nonetheless, the mechanism underlying wild type aSyn-mediated neurotoxicity in neuronal cells as well as its specific driving role in PD pathogenesis has yet to be fully clarified. In this view, the development of proper in vitro cellular syst...

  5. Inducible alpha-synuclein expression affects human Neural Stem Cell behavior.

    Science.gov (United States)

    Zasso, Jacopo; Mastad, Ahmed; Cutarelli, Alessandro; Conti, Luciano

    2018-04-19

    Converging evidence suggest that levels of alpha-Synuclein (aSyn) expression play a critical role in Parkinson's disease (PD). Several mutations of the SNCA gene, encoding for aSyn have been associated to either the familial or the sporadic forms of PD. Nonetheless, the mechanism underlying wild type aSyn-mediated neurotoxicity in neuronal cells as well as its specific driving role in PD pathogenesis has yet to be fully clarified. In this view, the development of proper in vitro cellular systems is a crucial step. Here we present a novel human Tet-on hNSC cell line, in which aSyn timing and level of expression can be tightly experimentally tuned. Induction of aSyn in self-renewing hNSCs leads to progressive formation of aSyn aggregates and impairs their proliferation and cell survival. Furthermore, aSyn induction during the neuronal differentiation process results in reduced neuronal differentiation and increased number astrocytes and undifferentiated cells in culture. Finally, acute aSyn induction in hNSC-derived dopaminergic neuronal cultures results in cell toxicity. This novel conditional in vitro cell model system may be a valuable tool for dissecting of aSyn pathogenic effects in hNSCs and neurons and in developing new potential therapeutic strategies.

  6. alpha7 nicotinic receptor gene promoter polymorphisms in inbred mice affect expression in a cell type-specific fashion.

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    Mexal, Sharon; Jenkins, Paul M; Lautner, Meeghan A; Iacob, Eli; Crouch, Eric L; Stitzel, Jerry A

    2007-05-04

    Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.

  7. alpha-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption.

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    Torielli, Lucia; Tivodar, Simona; Montella, Rosa Chiara; Iacone, Roberto; Padoani, Gloria; Tarsini, Paolo; Russo, Ornella; Sarnataro, Daniela; Strazzullo, Pasquale; Ferrari, Patrizia; Bianchi, Giuseppe; Zurzolo, Chiara

    2008-08-01

    Genetic variation in alpha-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of alpha-adducin in controlling Na/K pump activity, wild-type or mutated rat and human alpha-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, alpha-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension.

  8. B-Cell-Activating Factor Affects the Occurrence of Thyroid Autoimmunity in Chronic Hepatitis C Patients Treated with Interferon Alpha

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    Yusuke Kajiyama

    2012-01-01

    Full Text Available Chronic hepatitis C (CHC patients frequently suffer from thyroid disorders during interferon therapy. However, the mechanism remains unclear. In this study, we investigated the association between serum B-cell-activating factor belonging to the TNF family (BAFF levels and the presence of antithyroid peroxidase antibody (anti-TPO in CHC patients treated with pegylated interferon alpha and ribavirin combination therapy. Six months after the therapy, anti-TPO antibody was detected in 10 (males, 1; females, 9 of 50 patients. The mean age of these patients was higher than that of the anti-TPO-negative patients (61 yr versus 55 yr. Before treatment, the serum BAFF levels of the anti-TPO-positive patients were higher than those of the anti-TPO-negative patients. After starting therapy, the serum BAFF levels of both the anti-TPO-positive and -negative patient groups were elevated. Our findings suggest that the serum BAFF concentration before therapy can predict the risk of thyroid autoimmunity in elderly female patients with CHC.

  9. Pentoxifylline inhibits tumor necrosis factor-alpha (TNF alpha)-induced T-lymphoma cell adhesion to endothelioma cells.

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    Weiss, J M; Vanscheidt, W; Pilarski, K A; Weyl, A; Peschen, M; Schöpf, E; Vestweber, D; Simon, J C

    1995-05-01

    Pentoxifylline, a methylxanthine derivative, has been shown to inhibit T-cell-mediated cutaneous immune response by yet ill-understood mechanisms. Because cell adhesion to endothelial cells is a critical step in the initiation of such immune responses, we analyzed whether pentoxifylline would affect this process. To address this issue, adhesion of mouse T-lymphoma cells (TK-1) to mouse endothelioma cells (eEnd.2), either untreated or stimulated with tumor necrosis factor-alpha (TNF alpha), was studied. Pentoxifylline reduced the ability of endothelioma cells stimulated with different concentrations of TNF alpha, but not of untreated endothelioma cells, to bind T-lymphoma cells in dose-dependent (10(-5)-10(-3) M) fashion. Selective incubation of either endothelioma cells or T-lymphoma cells revealed that pentoxifylline acted exclusively on the endothelioma cells, even when added after TNF alpha stimulation. We questioned whether pentoxifylline suppressed T-lymphoma cell/endothelioma cell interactions by interfering with adhesion molecules expressed by either cell. However, as determined by flow cytometry, pentoxifylline did not alter TNF alpha-induced upregulation of intercellular adhesion molecule-1 or vascular cellular adhesion molecule-1 on endothelioma cells nor did it affect constitutive CD11a, CD18, or alpha 4-integrin expression on T-lymphoma cells, suggesting that rather than affecting quantitative expression of these adhesion molecules, pentoxifylline might modulate their avidity. We conclude that pentoxifylline in therapeutically achievable concentrations is a potent inhibitor of TNF alpha-induced T-lymphoma cell adhesion to endothelioma cells. This finding may account, at least in part, for the recently discovered anti-inflammatory action of pentoxifylline.

  10. Factors affecting the solubility of Bacillus halmapalus alpha-amylase

    DEFF Research Database (Denmark)

    Faber, Cornelius; Hobley, Timothy John; Mollerup, Jørgen

    2008-01-01

    A detailed study of the solubility of recombinant Bacillus halmapalus alpha-amylase has been conducted. A semi-purified preparation from a bulk crystallisation was chos en that contained six isoforms with pI-values of between 5.5 and 6.1. The solubility was strongly affected by pH and could...

  11. Alternative splicing of T cell receptor (TCR) alpha chain transcripts containing V alpha 1 or V alpha 14 elements.

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    Mahotka, C; Hansen-Hagge, T E; Bartram, C R

    1995-10-01

    Human acute lymphoblastic leukemia cell lines represent valuable tools to investigate distinct steps of the complex regulatory pathways underlying T cell receptor recombination and expression. A case in point are V delta 2D delta 3 and subsequent V delta 2D delta 3J alpha rearrangements observed in human leukemic pre-B cells as well as in normal lymphopoiesis. The functional expression of these unusual (VD) delta (JC) alpha hybrids is almost exclusively prevented by alternative splicing events. In this report we show that alternative splicing at cryptic splice donor sites within V elements is not a unique feature of hybrid TCR delta/alpha transcripts. Among seven V alpha families analyzed by RT-PCR, alternatively spliced products were observed in TCR alpha recombinations containing V alpha 1 or V alpha 14 elements. In contrast to normal peripheral blood cells and thymocytes, the leukemia cell line JM expressing functional V alpha 1J alpha 3C alpha transcripts lacked evidence of aberrant TCR alpha RNA species.

  12. A DEFICIENCY OF ALPHA-SYNTROPHIN, THE ANCHORING PROTEIN OF AQUAPORIN-4 CHANNELS, AFFECTS CELL SWELLING IN CORTICAL TISSUE DURING HYPOOSMOTIC AND HYPERKALEMIC STRESS

    Czech Academy of Sciences Publication Activity Database

    Cicanič, Michal; Vargová, Lýdia; Syková, Eva

    2011-01-01

    Roč. 59, Supplement: 1 (2011), S102-S103 ISSN 0894-1491. [European meeting on Glial Cells in Health and Disease /10./. 13.09.2011-17.09.2011, Prague] Institutional research plan: CEZ:AV0Z50390703 Keywords : cell swelling * aquaporin-4 * diffusion Subject RIV: FH - Neurology

  13. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

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    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  14. Muramidase, alpha-1 antitrypsin, alpha-1 antichymotrypsin, and S-100 protein immunoreactivity in giant cell lesions.

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    Regezi, J A; Zarbo, R J; Lloyd, R V

    1987-01-01

    A spectrum of giant cell lesions was evaluated for muramidase, alpha-1 antitrypsin, alpha-1 antichymotrypsin, and S-100 protein immunoreactivity using an avidin-biotin-complex immunoperoxidase method. Peripheral giant cell granuloma, central giant cell granuloma, giant cell tumor, osteitis fibrosa cystica, cherubism, and giant cell tumor of tendon sheath showed similar patterns of reactivity. Granulomatous inflammatory lesions stained more intensely for muramidase than did noninflammatory lesions. Alpha-1-antichymotrypsin was a slightly better marker of giant cell lesions than was alpha-1-antitrypsin. Positive S-100 protein staining in half the lesions was thought to be due to the presence of Langerhans cells. The results supported the belief that giant cell lesions of bone and tendon sheath are differentiated toward cells of the mononuclear-phagocyte system and that multinucleated giant cells are derived from macrophages.

  15. Frontal alpha asymmetry neurofeedback for the reduction of negative affect and anxiety.

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    Mennella, Rocco; Patron, Elisabetta; Palomba, Daniela

    2017-05-01

    Frontal alpha asymmetry has been proposed to underlie the balance between approach and withdrawal motivation associated to each individual's affective style. Neurofeedback of EEG frontal alpha asymmetry represents a promising tool to reduce negative affect, although its specific effects on left/right frontal activity and approach/withdrawal motivation are still unclear. The present study employed a neurofeedback training to increase frontal alpha asymmetry (right - left), in order to evaluate discrete changes in alpha power at left and right sites, as well as in positive and negative affect, anxiety and depression. Thirty-two right-handed females were randomly assigned to receive either the neurofeedback on frontal alpha asymmetry, or an active control training (N = 16 in each group). The asymmetry group showed an increase in alpha asymmetry driven by higher alpha at the right site (p neurofeedback for the reduction of negative affect and anxiety in clinical settings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells

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    Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe

    2009-01-01

    We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell......-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However......, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell...

  17. Preferential target is mitochondria in alpha-mangostin-induced apoptosis in human leukemia HL60 cells.

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    Matsumoto, Kenji; Akao, Yukihiro; Yi, Hong; Ohguchi, Kenji; Ito, Tetsuro; Tanaka, Toshiyuki; Kobayashi, Emi; Iinuma, Munekazu; Nozawa, Yoshinori

    2004-11-15

    Our previous study has shown that alpha-mangostin, a xanthone from the pericarps of mangosteen, induces caspase-3-dependent apoptosis in HL60 cells. In the current study, we investigated the mechanism of apoptosis induced by alpha-mangostin in HL60 cells. Alpha-mangostin-treated HL60 cells demonstrated caspase-9 and -3 activation but not -8, which leads us to assume that alpha-mangostin may mediate the mitochondrial pathway in the apoptosis. Parameters of mitochondrial dysfunction including swelling, loss of membrane potential (deltapsim), decrease in intracellular ATP, ROS accumulation, and cytochrome c/AIF release, were observed within 1 or 2 h after the treatment. On the other hand, alpha-mangostin-treatment did not affect expression of bcl-2 family proteins and activation of MAP kinases. These findings indicate that alpha-mangostin preferentially targets mitochondria in the early phase, resulting in indication of apoptosis in HL60 cells. Furthermore, we examined the structure-activity relationship between xanthone derivatives including alpha-mangostin and the potency of deltapsim-loss in HL60 cells. Interestingly, replacement of hydroxyl group by methoxy group remarkably decreased its potency. It was also shown that the cytotoxicity substantially correlated with deltapsim decrease. These results indicate that alpha-mangostin and its analogs would be candidates for preventive and therapeutic application for cancer treatment.

  18. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    International Nuclear Information System (INIS)

    Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

    2011-01-01

    Highlights: ► Doxazocin directly up-regulated bone metabolism at a low dose. ► Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. ► This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor γ, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and according to our data doxazosin might be useful for application in the field of bone

  19. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

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    Choi, Yoon Jung [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Chung, Chong-Pyoung [Department of Periodontology, School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and

  20. Increased sensitivity to interferon-alpha in psoriatic T cells

    DEFF Research Database (Denmark)

    Eriksen, Karsten Wessel; Lovato, Paola; Skov, Lone

    2005-01-01

    Psoriasis is a chronic inflammatory skin disease characterized by abnormal epidermal proliferation. Several studies have shown that skin-infiltrating activated T cells and cytokines play a pivotal role during the initiation and maintenance of the disease. Interferon (IFN)-alpha plays an important...... disease characterized by CD8(+)-infiltrating T cells. In this study, we therefore investigate IFN-alpha signaling in T cells isolated from involved skin of psoriatic patients. We show that psoriatic T cells have increased and prolonged responses to IFN-alpha, on the level of signal transducers...... and activators of transcription (STAT) activation, compared with infiltrating T cells from skin of non-psoriatic donors. Functionally, the increased IFN-alpha signaling leads to an increased binding of STAT4 to the IFN-gamma promotor, IFN-gamma production, and inhibition of T cell growth. In contrast, to STAT...

  1. Involvement of the clock gene Rev-erb alpha in the regulation of glucagon secretion in pancreatic alpha-cells.

    Directory of Open Access Journals (Sweden)

    Elaine Vieira

    Full Text Available Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60-70% inhibition in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05 and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001. High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05. AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01 and glucagon release (p<0.05. These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.

  2. Curcumin inhibition of integrin (alpha6beta4)-dependent breast cancer cell motility and invasion.

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    Kim, Hong Im; Huang, Huang; Cheepala, Satish; Huang, Shile; Chung, Jun

    2008-10-01

    Curcumin, a polyphenol natural product isolated from the rhizome of the plant Curcuma longa, has emerged as a promising anticancer therapeutic agent. However, the mechanism by which curcumin inhibits cancer cell functions such as cell growth, survival, and cell motility is largely unknown. We explored whether curcumin affects the function of integrin alpha(6)beta(4), a laminin adhesion receptor with an established role in invasion and migration of cancer cells. Here we show that curcumin significantly reduced alpha(6)beta(4)-dependent breast cancer cell motility and invasion in a concentration-dependent manner without affecting apoptosis in MDA-MB-435/beta4 (beta(4)-integrin transfectants) and MDA-MB-231 breast cancer cell lines. Further, curcumin selectively reduced the basal phosphorylation of beta(4) integrin (Y1494), which has been reported to be essential in mediating alpha(6)beta(4)-dependent phosphatidylinositol 3-kinase activation and cell motility. Consistent with this finding, curcumin also blocked alpha(6)beta(4)-dependent Akt activation and expression of the cell motility-promoting factor ENPP2 in MDA-MB-435/beta4 cell line. A multimodality approach using curcumin in combination with other pharmacologic inhibitors of alpha(6)beta(4) signaling pathways showed an additive effect to block breast cancer cell motility and invasion. Taken together, these findings show that curcumin inhibits breast cancer cell motility and invasion by directly inhibiting the function of alpha(6)beta(4) integrin, and suggest that curcumin can serve as an effective therapeutic agent in tumors that overexpress alpha(6)beta(4).

  3. Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells.

    Science.gov (United States)

    Cui, W; Li, L X; Sun, C M; Wen, Y; Zhou, Y; Dong, Y L; Liu, P

    2010-04-01

    The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-alpha) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell microporous filters and treated with TNF-alpha (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-alpha treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-alpha decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-alpha did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-alpha increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

  4. Effects of zearalenone and alpha-Zearalenol in comparison with Raloxifene on T47D cells.

    Science.gov (United States)

    Khosrokhavar, Roya; Rahimifard, Nahid; Shoeibi, Shahram; Hamedani, Morteza Pirali; Hosseini, Mir-Jamal

    2009-03-01

    Zearalenone (Zen) is a mycotoxin with estrogenic effect which contaminates cereals. In cell culture, Zen and its metabolite, alpha-Zearalenol (alpha-Zel), stimulate breast cancer cells growth. Today hormone-dependent cancers are important because of high incidence and death rate. Previous studies showed that Zen and alpha-Zel have an effect on hormone-dependent cancers. This study explains the effects of the mentioned compounds in comparison with Raloxifene as an anti-estrogen. Cell culture technique was used with MDA-MB-231 and T47D cells for evaluation of compounds. MDA-MB-231 cells were used as negative control and also for proving that treatment compounds merely affect, due to their proliferation activity in the applied doses. According to the Resazurine-based method, for toxicity assay, none of the test compounds have an effect on MDA-MB-231 cells but do effect the growth of T47D cells. Zen and alpha-Zel at low concentrations (10-8-10-9 M) stimulated T47D cell growth and Raloxifene strongly inhibited cell growth induced by Zen and alpha-Zel. There is a noticeable result in controlling diet of hormonal carcinogenic compounds and applying novel anti-estrogens for prevention and treatment of hormone-dependent cancers.

  5. Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.

    Science.gov (United States)

    Zia, S; Ndoye, A; Nguyen, V T; Grando, S A

    1997-09-01

    The purpose of this study was to investigate the possibility of direct toxic effects of nicotine (Nic) on human bronchial epithelial cells (BEC) suggested by our previous findings of functional nicotinic acetylcholine receptors (nAChRs) in the epithelial cells lining mucocutaneous membranes. We now demonstrate for the first time that human and murine BEC both in vivo and in vitro express functional nAChRs, and that classic alpha 3, alpha 4, alpha 5 and alpha 7 subunits can contribute to formation of these acetylcholine-gated ion channels. In human bronchial and mouse lung tissues, and in cultures of human BEC, the nAChRs were visualized by subunit-specific antibodies on the cell membranes, particularly at the sites of cell-to-cell contacts. The epithelial cells of submucosal glands abundantly expressed alpha 7 nAChRs. Smoking significantly (p epithelial nAChRs apparently involve regulation of cell-to-cell communications, adhesion and motility, because Mec caused rapid and profound changes in these cell functions which were reversible by Nic. An over exposure of BEC to Nic, however, produced an antagonist-like effect, suggesting that the pathobiological effects of Nic toxicity might result from both activation of nAChR channels and nAChR desensitization. We conclude that medical consequences of smoking can be mediated by direct toxic effects of inhaled Nic on the respiratory tissues wherein Nic specifically binds to and activates the nicotinic ion channels present on the cell surfaces of BEC. We believe that outside the neural system Nic interferes with functioning of non-neuronal cholinergic networks by displacing from nAChR its natural ligand acetylcholine which acts as a local hormone or cytokine in a variety of non-neuronal locations.

  6. Protective role of gamma/delta T cells and alpha/beta T cells in tuberculosis.

    Science.gov (United States)

    Ladel, C H; Blum, C; Dreher, A; Reifenberg, K; Kaufmann, S H

    1995-10-01

    Tuberculosis is a chronic infectious disease which causes major health problems globally. Although acquired resistance crucially depends on alpha/beta lymphocytes, circumstantial evidence suggests that, in addition, gamma/delta T lymphocytes contribute to protection against tuberculosis. We have studied Mycobacterium tuberculosis infection in TcR-delta-/- or TcR-beta-/- gene deletion mutants which completely lack gamma/delta T cells or alpha/beta T cells, respectively. Low inocula of M. tuberculosis led to death of TcR-beta-/- mice and transient disease exacerbation in TcR-delta-/- mutants. Infection with higher inocula caused rapid death of TcR-delta-/- mice. The development of and bacterial containment in granulomatous lesions was markedly impaired in TcR-beta-/-, and less severely affected in TcR-delta-/- mutants. Mycobacteria-induced IFN-gamma production by spleen cells in vitro was almost abolished in TcR-beta-/- and virtually unaffected in TcR-delta-/- mice. Our data confirm the crucial role of alpha/beta T cells in protection against established tuberculosis and formally prove a protective role of gamma/delta T cells in early tuberculosis.

  7. Deficient repair of chemical adducts in alpha DNA of monkey cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-01-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

  8. Sensitivity of EEG upper alpha activity to cognitive and affective creativity interventions.

    Science.gov (United States)

    Fink, Andreas; Schwab, Daniela; Papousek, Ilona

    2011-12-01

    We investigated whether creative cognition can be improved by means of cognitive and affective stimulation and whether these interventions are associated with changes of EEG alpha activity. Participants were required to generate original uses of conventional objects (Alternative Uses task, AU) while the EEG was recorded. In the cognitive stimulation condition, participants worked on the AU task subsequent to the exposure to other people's ideas. In the affective stimulation condition, they had to think creatively in positive affective states, induced via emotionally contagious sound clips. Creative cognition generally elicited alpha synchronization, most prominent in the prefrontal cortex and in the right hemisphere. The interventions were associated with stronger prefrontal alpha activity in the upper alpha band (10-12 Hz) than the control condition (no intervention), possibly indicating a state of heightened internal awareness, which might have a beneficial impact on creativity. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Alpha particles induce expression of immunogenic markers on tumour cells

    International Nuclear Information System (INIS)

    Gorin, J.B.; Gouard, S.; Cherel, M.; Davodeau, F.; Gaschet, J.; Morgenstern, A.; Bruchertseifer, F.

    2013-01-01

    The full text of the publication follows. Radioimmunotherapy (RIT) is an approach aiming at targeting the radioelements to tumours, usually through the use of antibodies specific for tumour antigens. The radiations emitted by the radioelements then induce direct killing of the targeted cells as well as indirect killing through bystander effect. Interestingly, it has been shown that ionizing radiations, in some settings of external radiotherapy, can foster an immune response directed against tumour cells. Our research team is dedicated to the development of alpha RIT, i.e RIT using alpha particle emitters, we therefore decided to study the effects of such particles on tumour cells in regards to their immunogenicity. First, we studied the effects of bismuth 213, an alpha emitter, on cellular death and autophagy in six different tumour cell lines. Then, we measured the expression of 'danger' signals and MHC molecules at the cell surface to determine whether irradiation with 213 Bi could cause the tumour cells to be recognized by the immune system. Finally a co-culture of dendritic cells with irradiated tumour cells was performed to test whether it would induce dendritic cells to mature. No apoptosis was detected within 48 hours after irradiation in any cell line, however half of them exhibited signs of autophagy. No increase in membrane expression of 'danger' signals was observed after treatment with 213 Bi, but we showed an increase in expression of MHC class I and II for some cell lines. Moreover, the co-culture experiment indicated that the immunogenicity of a human adenocarcinoma cell line (LS 174T) was enhanced in vitro after irradiation with alpha rays. These preliminary data suggest that alpha particles could be of interest in raising an immune response associated to RIT. (authors)

  10. Opposing actions of the progesterone metabolites, 5alpha-dihydroprogesterone (5alphaP) and 3alpha-dihydroprogesterone (3alphaHP) on mitosis, apoptosis, and expression of Bcl-2, Bax and p21 in human breast cell lines.

    Science.gov (United States)

    Wiebe, John P; Beausoleil, Michel; Zhang, Guihua; Cialacu, Valentin

    2010-01-01

    Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5alpha-dihydroprogesterone (5alphaP) and 3alpha-dihydroprogesterone (3alphaHP) and that 3alphaHP suppresses, whereas 5alphaP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5alphaP- and 3alphaHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3alphaHP and 5alphaP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5alphaP increased, whereas 3alphaHP decreased cell numbers, [(3)H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3alphaHP and suppressed by 5alphaP. 5alphaP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3alphaHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3alphaHP or 5alphaP on cell numbers, [(3)H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3alphaHP, and was unaffected by 5alphaP. The results provide the first evidence that 5alphaP stimulates mitosis and suppresses apoptosis, whereas 3alphaHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5alphaP and 3alphaHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5alphaP formation and/or increasing 3alphaHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes

  11. Uptake of neutral alpha- and beta-amino acids by human proximal tubular cells

    DEFF Research Database (Denmark)

    Jessen, H; Røigaard, H; Jacobsen, Christian

    1996-01-01

    experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, L-aspartic acid, and L-arginine. By contrast, the influx of beta-alanine was only drastically reduced by beta-amino acids, whereas the inhibition by neutral alpha-amino acids...... was relatively low. Nor did L-arginine and L-aspartic acid affect the uptake of beta-alanine into AHKE cells. Comparison with the results obtained for normal (NHKE) and immortalized (IHKE) embryonic cells suggested an unaltered expression of the types of transport carriers for neutral alpha- and beta-amino acids...

  12. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

    Directory of Open Access Journals (Sweden)

    Romina Baaske

    2016-12-01

    Full Text Available Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla. This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L, which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin.

  13. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    DEFF Research Database (Denmark)

    Goldfinger, L E; Hopkinson, S B; deHart, G W

    1999-01-01

    . In these cells, integrin alpha3beta1 occasionally colocalizes with the staining generated by the 12C4 antibody but alpha6beta4 integrin does not. In wounded MCF-10A cell cultures, the 12C4 antibody stains the extracellular matrix beneath those cells at the very edge of the cellular sheet that moves to cover......Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found...... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...

  14. Role of macrophage inflammatory protein-1alpha in T-cell-mediated immunity to viral infection

    DEFF Research Database (Denmark)

    Madsen, Andreas N; Nansen, Anneline; Christensen, Jan P

    2003-01-01

    The immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1alpha (MIP-1alpha) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1alpha-deficient mice. Furthermore, MIP-1alpha is not required for T-cell-mediated virus...... control or virus-induced T-cell-dependent inflammation. Thus, MIP-1alpha is not mandatory for T-cell-mediated antiviral immunity....

  15. NADPH oxidase 1 controls the persistence of directed cell migration by a Rho-dependent switch of alpha2/alpha3 integrins.

    Science.gov (United States)

    Sadok, Amine; Pierres, Anne; Dahan, Laetitia; Prévôt, Charles; Lehmann, Maxime; Kovacic, Hervé

    2009-07-01

    NADPH oxidase 1 (Nox1) is expressed mainly in colon epithelial cells and produces superoxide ions as a primary function. We showed that Nox1 knockdown inhibits directional persistence of migration on collagen I. This paper dissects the mechanism by which Nox1 affects the direction of colonic epithelial cell migration in a two-dimensional model. Transient activation of Nox1 during cell spreading on collagen 1 temporarily inactivated RhoA and led to efficient exportation of alpha2beta1 integrin to the cell surface, which supported persistent directed migration. Nox1 knockdown led to a loss of directional migration which takes place through a RhoA-dependent alpha2/alpha3 integrin switch. Transient RhoA overactivation upon Nox1 inhibition led to transient cytoskeletal reorganization and increased cell-matrix contact associated with a stable increase in alpha3 integrin cell surface expression. Blocking of alpha3 integrin completely reversed the loss of directional persistence of migration. In this model, Nox1 would represent a switch between random and directional migration through RhoA-dependent integrin cell surface expression modulation.

  16. Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

    Science.gov (United States)

    Koo, Hyun-Na; Hong, Seung-Heon; Song, Bong-Keun; Kim, Cheorl-Ho; Yoo, Young-Hyun; Kim, Hyung-Min

    2004-01-16

    Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

  17. Evidence of a liver-alpha cell axis in humans

    DEFF Research Database (Denmark)

    Wewer Albrechtsen, Nicolai J; Færch, Kristine; Jensen, Troels M

    2018-01-01

    in plasma γ-glutamyltransferase levels. CONCLUSIONS/INTERPRETATION: This cross-sectional study supports the existence of a liver-alpha cell axis in humans: glucagon regulates plasma levels of amino acids, which in turn feedback to regulate the secretion of glucagon. With hepatic insulin resistance...

  18. Alpha thalassemia among sickle cell anaemia patients in Kampala ...

    African Journals Online (AJOL)

    The New England Journal of Medicine. 1983;309(3):189-90. Epub 1983/07/21. 4. Embury SH, Clark MR, Monroy G, Mohandas N. Concurrent sickle cell anemia and alpha-thalassemia. Effect on pathological properties of sickle erythrocytes. The Journal of Clinical Investigation. 1984;73(1):116-23. Epub 1984/01/01. 5.

  19. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    DEFF Research Database (Denmark)

    Goldfinger, L E; Hopkinson, S B; deHart, G W

    1999-01-01

    Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

  20. D-Glucosamine down-regulates HIF-1{alpha} through inhibition of protein translation in DU145 prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jee-Young; Park, Jong-Wook; Suh, Seong-Il [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of); Baek, Won-Ki, E-mail: wonki@dsmc.or.kr [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of)

    2009-04-24

    D-Glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. In this study we report a novel response to D-glucosamine involving the translation regulation of hypoxia inducible factor (HIF)-1{alpha} expression. D-Glucosamine caused a decreased expression of HIF-1{alpha} under normoxic and hypoxic conditions without affecting HIF-1{alpha} mRNA expression in DU145 prostate cancer cells. D-Glucosamine inhibited HIF-1{alpha} accumulation induced by proteasome inhibitor MG132 and prolyl hydroxylase inhibitor DMOG suggesting D-glucosamine reduces HIF-1{alpha} protein expression through proteasome-independent pathway. Metabolic labeling assays indicated that D-glucosamine inhibits translation of HIF-1{alpha} protein. In addition, D-glucosamine inhibited HIF-1{alpha} expression induced by serum stimulation in parallel with inhibition of p70S6K suggesting D-glucosamine inhibits growth factor-induced HIF-1{alpha} expression, at least in part, through p70S6K inhibition. Taken together, these results suggest that D-glucosamine inhibits HIF-1{alpha} expression through inhibiting protein translation and provide new insight into a potential mechanism of the anticancer properties of D-glucosamine.

  1. Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4

    DEFF Research Database (Denmark)

    Jinquan, T; Jacobi, H H; Jing, C

    2000-01-01

    The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1alpha (SDF-1alpha) is the only known ligand for CXCR4. To date, the CXCR4 expression and function...... of SDF-1alpha in basophils are unknown....

  2. Mechanisms of cell sensitization to alpha radioimmunotherapy by doxorubicin or paclitaxel in multiple myeloma cell lines.

    Science.gov (United States)

    Supiot, Stephane; Gouard, Sebastien; Charrier, Josiane; Apostolidis, Christos; Chatal, Jean-Francois; Barbet, Jacques; Davodeau, François; Cherel, Michel

    2005-10-01

    The purpose of this study was to analyze different mechanisms (cell cycle synchronization, DNA damage, and apoptosis) that might underlie potential synergy between chemotherapy (paclitaxel or doxorubicin) and radioimmunotherapy with alpha radionuclides. Three multiple myeloma cell lines (LP1, RMI 8226, and U266) were treated with 213Bi-radiolabeled B-B4, a monoclonal antibody that recognizes syndecan-1 (CD138) 24 hours after paclitaxel (1 nmol/L) or doxorubicin (10 nmol/L) treatment. Cell survival was assessed using a clonogenic survival assay. Cell cycle modifications were assessed by propidium iodide staining and DNA strand breaks by the comet assay. Level of apoptosis was determined by Apo 2.7 staining. Radiation enhancement ratio showed that paclitaxel and doxorubicin were synergistic with alpha radioimmunotherapy. After a 24-hour incubation, paclitaxel and doxorubicin arrested all cell lines in the G2-M phase of the cell cycle. Doxorubicin combined with alpha radioimmunotherapy increased tail DNA in the RPMI 8226 cell line but not the LP1 or U266 cell lines compared with doxorubicin alone or alpha radioimmunotherapy alone. Neither doxorubicin nor paclitaxel combined with alpha radioimmunotherapy increased the level of apoptosis induced by either drug alone or alpha radioimmunotherapy alone. Both cell cycle arrest in the G2-M phase and an increase in DNA double-strand breaks could lead to radiosensitization of cells by doxorubicin or paclitaxel, but apoptosis would not be involved in radiosensitization mechanisms.

  3. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

    Directory of Open Access Journals (Sweden)

    Freya M Mowat

    2010-06-01

    Full Text Available Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

  4. Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

    DEFF Research Database (Denmark)

    Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E

    2003-01-01

    decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...

  5. Stromal cell-derived factor-1 alpha (SDF-1 alpha) improves neural recovery after spinal cord contusion in rats

    NARCIS (Netherlands)

    Zendedel, A.; Nobakht, M.; Bakhtiyari, M.; Beyer, C.; Kipp, M.; Baazm, M.; Joghataie, M.T.

    2012-01-01

    Stromal cell-derived factor-1 alpha (SDF-1α) is an important cytokine, implicated in the control of stem cell trafficking and bone marrow-derived stem cell mobilization. Generally, SDF-1α regulates multiple physiological processes such as embryonic development and organ homeostasis. There is also

  6. Acute moderate elevation of TNF-{alpha} does not affect systemic and skeletal muscle protein turnover in healthy humans

    DEFF Research Database (Denmark)

    Petersen, Anne Marie; Plomgaard, Peter; Fischer, Christian P

    2009-01-01

    Context: Skeletal muscle wasting has been associated with elevations in circulating inflammatory cytokines, in particular TNF-alpha. Objective: In this study, we investigated whether TNF-alpha affects human systemic and skeletal muscle protein turnover, via a 4 hours recombinant human TNF......-alpha infusion (rhTNF-alpha). We hypothesize that TNF-alpha increases human muscle protein breakdown and/or inhibit synthesis. Subjects and Methods: Using a randomized controlled, crossover design post-absorptive healthy young males (n=8) were studied 2 hours under basal conditions followed by 4 hours infusion...... of either rhTNF-alpha (700 ng.m(-2).h(-1)) or 20% human albumin (Control) which was the vehicle of rhTNF-alpha. Systemic and skeletal muscle protein turnover were estimated by a combination of tracer dilution methodology (primed continuous infusion of L-[ring-(2)H5]phenylalanine and L-[(15)N...

  7. Tracking cell surface GABAB receptors using an alpha-bungarotoxin tag.

    Science.gov (United States)

    Wilkins, Megan E; Li, Xinyan; Smart, Trevor G

    2008-12-12

    GABA(B) receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABA(B) receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABA(B) receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, alpha-bungarotoxin. By using the alpha-bungarotoxin binding site-tagged GABA(B) R1a subunit (R1a(BBS)), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, alpha-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABA(B) receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors.

  8. The Alpha-defensin Test for Periprosthetic Joint Infections Is Not Affected by Prior Antibiotic Administration.

    Science.gov (United States)

    Shahi, Alisina; Parvizi, Javad; Kazarian, Gregory S; Higuera, Carlos; Frangiamore, Salvatore; Bingham, Joshua; Beauchamp, Christopher; Valle, Craig Della; Deirmengian, Carl

    2016-07-01

    Previous studies have demonstrated that the administration of antibiotics to patients before performing diagnostic testing for periprosthetic joint infection (PJI) can interfere with the accuracy of test results. Although a single-institution study has suggested that alpha-defensin maintains its concentration and sensitivity even after antibiotic treatment, this has not yet been demonstrated in a larger multiinstitutional study. (1) For the evaluation of PJI, is prior antibiotic administration associated with decreased alpha-defensin levels? (2) When prior antibiotics are given, is alpha-defensin a better screening test for PJI than the traditional tests (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP], fluid white blood cells, fluid polymorphonuclear cells [PMNs], and fluid culture)? This retrospective study included data from 106 hip and knee arthroplasties with Musculoskeletal Infection Society-defined PJI from four centers. Of the 106 patients in this study, 30 (28%) were treated with antibiotics for PJI before diagnostic workup (ABX group), and 76 (72%) were not treated before the diagnostic workup (NO-ABX group). There were no differences in age, sex, joint, culture-negative rate, or bacteriology between groups. The patients in the ABX group had antibiotics initiated by physicians who commenced care before assessment for PJI by the treating surgeon's service. We compared the alpha-defensin levels and sensitivity between the ABX and NO-ABX groups. Additionally, the sensitivity of the alpha-defensin test was compared to that of traditional tests for PJI among patients on antibiotics. The administration of antibiotics before performing the alpha-defensin test for PJI was not associated with a decreased median alpha-defensin level (ABX group, median 4.2 [range, 1.79-12.8 S/CO] versus NO-ABX, median 4.9 [range, 0.5-16.8 S/CO], difference of medians: 0.68 S/CO [95% confidence interval {CI}, -0.98 to 1.26], p = 0.451). Furthermore, the alpha

  9. Failure of isolated rat tibial periosteal cells to 5 alpha reduce testosterone to 5 alpha-dihydrotestosterone

    International Nuclear Information System (INIS)

    Turner, R.T.; Bleiberg, B.; Colvard, D.S.; Keeting, P.E.; Evans, G.; Spelsberg, T.C.

    1990-01-01

    Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-alpha 2(I) chain of type 1 precollagen. The isolated periosteal cells were incubated with 1 nM [3H]testosterone [( 3 H]T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. [ 3 H]5 alpha-dihydrotestosterone [( 3 H]DHT) was not detected in extracts of periosteal cell incubations. In contrast, [ 3 H]DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5 alpha-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT

  10. Environmentally relevant exposure to 17{alpha}-ethinylestradiol affects the telencephalic proteome of male fathead minnows

    Energy Technology Data Exchange (ETDEWEB)

    Martyniuk, Christopher J., E-mail: cmartyn@unb.ca [Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, 32611 (United States); Kroll, Kevin J.; Doperalski, Nicholas J.; Barber, David S.; Denslow, Nancy D. [Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, 32611 (United States)

    2010-07-15

    Estrogens are key mediators of neuronal processes in vertebrates. As such, xenoestrogens present in the environment have the potential to alter normal central nervous system (CNS) function. The objectives of the present study were (1) to identify proteins with altered abundance in the male fathead minnow telencephalon as a result of low-level exposure to17{alpha}-ethinylestradiol (EE{sub 2}), and (2) to better understand the underlying mechanisms of 17{beta}-estradiol (E{sub 2}) feedback in this important neuroendocrine tissue. Male fathead minnows exposed to a measured concentration of 5.4 ng EE{sub 2}/L for 48 h showed decreased plasma E{sub 2} levels of approximately 2-fold. Of 77 proteins that were quantified statistically, 14 proteins were down-regulated after EE{sub 2} exposure, including four histone proteins, ATP synthase, H+ transporting subunits, and metabolic proteins (lactate dehydrogenase B4, malate dehydrogenase 1b). Twelve proteins were significantly induced by EE{sub 2} including microtubule-associated protein tau (Mapt), astrocytic phosphoprotein, ependymin precursor, and calmodulin. Mapt showed an increase in protein abundance but a decrease in mRNA expression after EE{sub 2} exposure{sub ,} suggesting there may be a negative feedback response in the telencephalon to decreased mRNA transcription with increasing Mapt protein abundance. These results demonstrate that a low, environmentally relevant exposure to EE{sub 2} can rapidly alter the abundance of proteins involved in cell differentiation and proliferation, neuron network morphology, and long-term synaptic potentiation. Together, these findings provide a better understanding of the molecular responses underlying E{sub 2} feedback in the brain and demonstrate that quantitative proteomics can be successfully used in ecotoxicology to characterize affected cellular pathways and endocrine physiology.

  11. The role of p38alpha in Schwann cells in regulating peripheral nerve myelination and repair.

    Science.gov (United States)

    Roberts, Sheridan L; Dun, Xin-Peng; Dee, Gemma; Gray, Bethany; Mindos, Thomas; Parkinson, David B

    2017-04-01

    Myelination in the peripheral nervous system (PNS) is controlled by both positive and negative regulators within Schwann cells to ensure timely onset and correct myelin thickness for saltatory conduction by neurons. Transcription factors such as Sox10, octamer-binding transcription factor 6 (Oct6) and Krox20 form a positive regulatory network, whereas negative regulators such as cJun and Sox2 oppose myelination in Schwann cells. The role of the p38 MAPK pathway has been studied in PNS myelination, but its precise function remains unclear, with both positive and negative effects of p38 activity reported upon both myelination and processes of nerve repair. To clarify the role of p38 MAPK in the PNS, we have analysed mice with a Schwann cell-specific ablation of the major p38 isoform, p38alpha. In line with previous findings of an inhibitory role for p38 MAPK, we observe acceleration of post-natal myelination in p38alpha null nerves, a delay in myelin down-regulation following injury, together with a small increase in levels of re-myelination following injury. Finally we explored roles for p38alpha in controlling axonal regeneration and functional repair following PNS injury and observe that loss of p38alpha function in Schwann cells does not appear to affect these processes as previously reported. These studies therefore provide further proof for a role of p38 MAPK signalling in the control of myelination by Schwann cells in the PNS, but do not show an apparent role for signalling by this MAP kinase in Schwann cells controlling other elements of Wallerian degeneration and functional repair following injury. Cover Image for this issue: doi: 10.1111/jnc.13793. © 2016 International Society for Neurochemistry.

  12. Alpha thalassemia among sickle cell anaemia patients in Kampala, Uganda.

    Science.gov (United States)

    Lubega, Irene; Ndugwa, Christopher M; Mworozi, Edison A; Tumwine, James K

    2015-06-01

    Sickle cell anaemia is prevalent in sub Saharan Africa. While α+-thalassaemia is known to modulate sickle cell anaemia, its magnitude and significance in Uganda have hitherto not been described. To determine the prevalence of α+thalassaemia among sickle cell anaemia patients in Mulago Hospital and to describe the clinical and laboratory findings in these patients. A cross sectional study was carried out on patients with sickle cell anaemia in Kampala. Dried blood spots were used to analyze for the deletional α+ thalassaemia using multiplex polymerase chain reaction. Of the 142 patients with sickle cell anaemia, 110 (77.5%) had the αα+thalassaemia deletion. The gene frequency of (-α) was 0.425. Ninety one percent (100/110) of those with α+thalassaemia were heterozygous (αα/α-). Amongst the patients older than 60 months, 15 (83.3%) of those without αα+thalassaemia had significant hepatomegaly of greater than 4 cm compared to 36 (45.6%) of those with α+thalassaemia (p=0.003). The gene frequency of (-α) of 0.425 noted in this study is higher than that reported from many places in Africa. Concurrent alpha thalassemia might be a protective trait against significant hepatomegaly in sickle cell anaemia patients more than 60 months of age at Mulago hospital.

  13. Extinction of alpha1-antitrypsin expression in cell hybrids is independent of HNF1alpha and HNF4 and involves both promoter and internal DNA sequences.

    OpenAIRE

    Bulla, G A

    1999-01-01

    In rat hepatoma x fibroblast somatic cell hybrids, extinction of rat alpha1-antitrypsin (alpha1AT) gene expression is accompanied by the loss of liver-enriched transcription factors hepatocyte nuclear factor 1 (HNF1alpha) and hepatocyte nuclear factor 4 (HNF4). Previous analysis showed that forced expression of functional HNF1alpha failed to prevent extinction of the rat alpha1AT locus in cell hybrids. Here I show that ectopic co-expression of HNF1alpha plus HNF4 fails to prevent extinction o...

  14. Alpha-ketoglutarate (AKG) lowers body weight and affects intestinal innate immunity through influencing intestinal microbiota.

    Science.gov (United States)

    Chen, Shuai; Bin, Peng; Ren, Wenkai; Gao, Wei; Liu, Gang; Yin, Jie; Duan, Jielin; Li, Yinghui; Yao, Kang; Huang, Ruilin; Tan, Bie; Yin, Yulong

    2017-06-13

    Alpha-ketoglutarate (AKG), a precursor of glutamate and a critical intermediate in the tricarboxylic acid cycle, shows beneficial effects on intestinal function. However, the influence of AKG on the intestinal innate immune system and intestinal microbiota is unknown. This study explores the effect of oral AKG administration in drinking water (10 g/L) on intestinal innate immunity and intestinal microbiota in a mouse model. Mouse water intake, feed intake and body weight were recorded throughout the entire experiment. The ileum was collected for detecting the expression of intestinal proinflammatory cytokines and innate immune factors by Real-time Polymerase Chain Reaction. Additionally, the ileal luminal contents and feces were collected for 16S rDNA sequencing to analyze the microbial composition. The intestinal microbiota in mice was disrupted with an antibiotic cocktail. The results revealed that AKG supplementation lowered body weight, promoted ileal expression of mammalian defensins of the alpha subfamily (such as cryptdins-1, cryptdins-4, and cryptdins-5) while influencing the intestinal microbial composition (i.e., lowering the Firmicutes to Bacteroidetes ratio). In the antibiotic-treated mouse model, AKG supplementation failed to affect mouse body weight and inhibited the expression of cryptdins-1 and cryptdins-5 in the ileum. We concluded that AKG might affect body weight and intestinal innate immunity through influencing intestinal microbiota.

  15. Mapping of HNF4alpha target genes in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Boyd, Mette; Bressendorff, Simon; Moller, Jette

    2009-01-01

    not previously been described as being regulated by HNF4alpha. The 1,541 genes contributed significantly to gene ontology (GO) pathways categorized by lipid and amino acid transport and metabolism. An analysis of the homeodomain transcription factor Cdx-2 (CDX2), the disaccharidase trehalase (TREH......ABSTRACT: BACKGROUND: The role of HNF4alpha has been extensively studied in hepatocytes and pancreatic beta-cells, and HNF4alpha is also regarded as key regulator of intestinal epithelial cell differentiation as well. The aim of the present work is to identify novel HNF4alpha target genes...... in the human intestinal epithelial cells in order to elucidate the role of HNF4alpha in the intestinal differentiation progress. METHODS: We have performed a ChIP-chip analysis of the human intestinal cell line Caco-2 in order to make a genome-wide identification of HNF4alpha binding to promoter regions...

  16. Tumor necrosis factor alpha selectively sensitizes human immunodeficiency virus-infected cells to heat and radiation

    International Nuclear Information System (INIS)

    Wong, G.H.; McHugh, T.; Weber, R.; Goeddel, D.V.

    1991-01-01

    We report here that infection of the human T-cell line HUT-78 with human immunodeficiency virus (HIV) increases its sensitivity to heat and radiation toxicity. A possible explanation for this result may be the reduced expression of manganous superoxide dismutase (MnSOD) in HIV-infected cells compared to uninfected cells. Tumor necrosis factor alpha (TNF-alpha) further sensitizes HIV-infected cells but not uninfected cells to heat and radiation. This is consistent with the ability of TNF-alpha to induce the expression of MnSOD in uninfected but not in HIV-infected cells. HIV-infected HUT-78 cell lines engineered to overexpress MnSOD are more resistant to heat and radiation than HIV-infected cells that do not overexpress MnSOD. However, treatment with TNF-alpha still sensitizes these cells to heat and radiation

  17. Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

    2011-04-15

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  18. Genetic evidence that HNF-1alpha-dependent transcriptional control of HNF-4alpha is essential for human pancreatic beta cell function

    DEFF Research Database (Denmark)

    Hansen, Sara K; Párrizas, Marcelina; Jensen, Maria L

    2002-01-01

    , and consequently in reduced HNF-1alpha-dependent activation. These findings provide genetic evidence that HNF-1alpha serves as an upstream regulator of HNF-4alpha and interacts directly with the P2 promoter in human pancreatic cells. Furthermore, they indicate that this regulation is essential to maintain normal...... in human islets and exocrine cells is primarily mediated by the P2 promoter. Furthermore, we describe a G --> A mutation in a conserved nucleotide position of the HNF-1alpha binding site of the P2 promoter, which cosegregates with MODY. The mutation results in decreased affinity for HNF-1alpha...

  19. The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522 (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.

  20. Immunoreactive transforming growth factor alpha and epidermal growth factor in oral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Therkildsen, M H; Poulsen, Steen Seier; Bretlau, P

    1993-01-01

    , the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus......Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected...... previous results confirms the existence of TGF-alpha, EGF, and EGF-receptors in the majority of oral squamous cell carcinomas and their metastases....

  1. Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond

    Energy Technology Data Exchange (ETDEWEB)

    Liu, K-K; Chen, P-Y; Lee, Tony J F; Chao, J-I [Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien 970, Taiwan (China); Chen, M-F [Neuro-Medical Scientific Center, Tzu Chi General Hospital, Hualien 970, Taiwan (China); Cheng, C-L [Department of Physics, National Dong Hwa University, Hualien 974, Taiwan (China); Chang, C-C [Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu 300, Taiwan (China); Ho, Y-P [Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan (China)], E-mail: chaoji@mail.tcu.edu.tw

    2008-05-21

    Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin ({alpha}-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor ({alpha}7-nAChR). The electrostatic binding of cND-{alpha}-BTX was mediated by the negative charge of the cND and the positive charge of the {alpha}-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that {alpha}-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled {alpha}-BTX presented a yellow color at the same location, which indicated that {alpha}-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human {alpha}7-nAChR proteins by microinjection with {alpha}7-nAChR mRNA. The cND-{alpha}-BTX complexes were bound to {alpha}7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked {alpha}7-nAChR-mediated inward currents of the oocytes were blocked by cND-{alpha}-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-{alpha}-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated {alpha}-BTX still preserves its biological activity in blocking the function of {alpha}7-nAChR, and provide a visual

  2. Bioactive interleukin-1alpha is cytolytically released from Candida albicans-infected oral epithelial cells.

    Science.gov (United States)

    Dongari-Bagtzoglou, A; Kashleva, H; Villar, C Cunha

    2004-12-01

    Oral epithelial cells are primary targets of Candida albicans in the oropharynx and may regulate the inflammatory host response to this pathogen. This investigation studied the mechanisms underlying interleukin-1alpha (IL-1alpha) release by oral epithelial cells and the role of IL-1alpha in regulating the mucosal inflammatory response to C. albicans. Infected oral epithelial cells released processed IL-1alpha protein in culture supernatants. The IL-1alpha generated was stored intracellularly and was released upon cell lysis. This was further supported by the fact that different C. albicans strains induced variable IL-1alpha release, depending on their cytolytic activity. IL-1alpha from C. albicans-infected oral epithelial cells upregulated proinflammatory cytokine secretion (IL-8 and GM-CSF) in uninfected oral epithelial or stromal cells. Our studies suggest that production of IL-1alpha, IL-8 and GM-CSF may take place in the oral mucosa in response to lytic infection of epithelial cells with C. albicans. This process can act as an early innate immune surveillance system and may contribute to the clinicopathologic signs of infection in the oral mucosa.

  3. Modifications of the alpha,beta-double bond in chalcones only marginally affect the antiprotozoal activities

    DEFF Research Database (Denmark)

    Nielsen, S F; Kharazmi, A; Christensen, S B

    1998-01-01

    Methods for selective alkylation of chalcones in the alpha- or beta-position and for selective reduction of the alpha,beta-double bond have been developed. The antiparasitic potencies of the alpha,beta-double bond modified chalcones only differ marginally from the potencies of the parent chalcones...

  4. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

    International Nuclear Information System (INIS)

    Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

    2017-01-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

  5. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

    Science.gov (United States)

    Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr; Sirma-Ekmekci, Sema, E-mail: semasirma@gmail.com; Yildirim, Ozlem, E-mail: ozlm-yildirim@hotmail.com; Erginel-Unaltuna, Nihan, E-mail: nihanerginel@yahoo.com

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

  7. Failure of isolated rat tibial periosteal cells to 5 alpha reduce testosterone to 5 alpha-dihydrotestosterone

    Energy Technology Data Exchange (ETDEWEB)

    Turner, R.T.; Bleiberg, B.; Colvard, D.S.; Keeting, P.E.; Evans, G.; Spelsberg, T.C. (Mayo Clinic, Rochester, MN (USA))

    1990-07-01

    Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-alpha 2(I) chain of type 1 precollagen. The isolated periosteal cells were incubated with 1 nM (3H)testosterone (({sup 3}H)T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. ({sup 3}H)5 alpha-dihydrotestosterone (({sup 3}H)DHT) was not detected in extracts of periosteal cell incubations. In contrast, ({sup 3}H)DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5 alpha-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT.

  8. Ric-8A and Gi alpha recruit LGN, NuMA, and dynein to the cell cortex to help orient the mitotic spindle.

    Science.gov (United States)

    Woodard, Geoffrey E; Huang, Ning-Na; Cho, Hyeseon; Miki, Toru; Tall, Gregory G; Kehrl, John H

    2010-07-01

    In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein alpha (G alpha) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G alpha(i) function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G alpha(i), thus preventing its GEF activity for G alpha(i). Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G alpha(i) expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.

  9. Phosphorylation of the von Hippel-Lindau protein (VHL) by protein kinase CK2 reduces its protein stability and affects p53 and HIF-1alpha mediated transcription.

    Science.gov (United States)

    Ampofo, Emmanuel; Kietzmann, Thomas; Zimmer, Andreas; Jakupovic, Mirza; Montenarh, Mathias; Götz, Claudia

    2010-10-01

    The von Hippel-Lindau tumour suppressor gene encodes a protein with 213 amino acids, which is known to be part of an E3-ubiquitin ligase targeting the HIF-1alpha transcription factor as well as to form a complex with p53. The VHL protein can be phosphorylated by protein kinase CK2 at serines 33, 38 and 43. However, the role of VHL phosphorylation in the context of p53 and HIF-1alpha regulation remained so far unknown. In the present study we investigated whether phosphorylation of VHL by CK2 might affect the function of p53 and HIF-1alpha. By using 4,5,6,7-tetrabromobenzotriazole (TBB), a CK2-specific inhibitor, as well as a mutant VHL where serines 33, 38 and 43 were replaced by alanines we found that CK2 phosphorylation affected the VHL protein half-life and increased VHL protein stability. Further, we found that inhibition of VHL phosphorylation by CK2 reduced p53 function. In addition, the enhanced levels of VHL due to CK2 inhibition contributed to the down-regulation of HIF-activity and degradation of HIF-1alpha. Thus, these results demonstrate that phosphorylation of VHL by CK2 plays an important role in the regulation of VHL protein stability and may contribute to the survival of tumour cells. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. The Alpha-Melanocyte Stimulating Hormone Induces Conversion of Effector T Cells into Treg Cells

    Directory of Open Access Journals (Sweden)

    Andrew W. Taylor

    2011-01-01

    Full Text Available The neuropeptide alpha-melanocyte stimulating hormone (α-MSH has an important role in modulating immunity and homeostasis. The production of IFN-γ by effector T cells is suppressed by α-MSH, while TGF-β production is promoted in the same cells. Such α-MSH-treated T cells have immune regulatory activity and suppress hypersensitivity, autoimmune diseases, and graft rejection. Previous characterizations of the α-MSH-induced Treg cells showed that the cells are CD4+ T cells expressing the same levels of CD25 as effector T cells. Therefore, we further analyzed the α-MSH-induced Treg cells for expression of effector and regulatory T-cell markers. Also, we examined the potential for α-MSH-induced Treg cells to be from the effector T-cell population. We found that the α-MSH-induced Treg cells are CD25+  CD4+ T cells that share similar surface markers as effector T cells, except that they express on their surface LAP. Also, the α-MSH treatment augments FoxP3 message in the effector T cells, and α-MSH induction of regulatory activity was limited to the effector CD25+ T-cell population. Therefore, α-MSH converts effector T cells into Treg cells, which suppress immunity targeting specific antigens and tissues.

  11. Characterization of a new cell-bound alpha-amylase in Bacillus subtilis 168 Marburg that is only immunologically related to the exocellular alpha-amylase.

    OpenAIRE

    Haddaoui, E; Petit-Glatron, M F; Chambert, R

    1995-01-01

    Immunoblot analysis of Bacillus subtilis cell extracts with polyclonal antibodies, raised against purified exocellular alpha-amylase, revealed one protein species of 82,000 Da. This protein was found even in cells in which the amyE gene, encoding exocellular alpha-amylase, was disrupted. Isolated from the membrane fraction, the 82,000-M(r) protein displayed an alpha-amylase activity in vitro.

  12. Characterization of a new cell-bound alpha-amylase in Bacillus subtilis 168 Marburg that is only immunologically related to the exocellular alpha-amylase.

    Science.gov (United States)

    Haddaoui, E; Petit-Glatron, M F; Chambert, R

    1995-01-01

    Immunoblot analysis of Bacillus subtilis cell extracts with polyclonal antibodies, raised against purified exocellular alpha-amylase, revealed one protein species of 82,000 Da. This protein was found even in cells in which the amyE gene, encoding exocellular alpha-amylase, was disrupted. Isolated from the membrane fraction, the 82,000-M(r) protein displayed an alpha-amylase activity in vitro. PMID:7665495

  13. In whole blood, LPS, TNF-alpha and GM-CSF increase monocyte uptake of 99mtechnetium stannous colloid but do not affect neutrophil uptake

    International Nuclear Information System (INIS)

    Ramsay, Stuart C.; Maggs, Jacqueline; Powell, Kellie; Barnes, Jodie; Ketheesan, Natkunam

    2006-01-01

    Introduction: 99m Technetium stannous colloid (TcSnC) is used in white cell scanning. It labels neutrophils and monocytes via phagocytosis, with uptake mediated by the phagocytic receptor CD11b/CD18 in neutrophils. Uptake of TcSnC is altered by gram-negative infection, possibly due to the endotoxin component lipopolysaccharide (LPS) or to cytokines released during infection (e.g., TNF-alpha and IFN-gamma). Endotoxemia and increased TNF-alpha levels also occur in inflammatory bowel disease. Another potential confounder in cell labeling is that sepsis patients may be treated with GM-CSF and G-CSF, which alter phagocytic cell function. This study aimed to determine how these factors affect TcSnC cellular uptake. Methods: Whole blood from six healthy volunteers was incubated with LPS, TNF-alpha, IFN-gamma, GM-CSF or G-CSF. Samples were then mixed with TcSnC. Blood was separated across density gradients and imaged using a gamma camera. Three radioactive count peaks were observed in each tube: free plasma activity, mononuclear cell uptake and neutrophil uptake. Results: Compared with controls, significant increases in mononuclear cell uptake were induced by LPS, TNF-alpha and GM-CSF stimulation. It was incidentally noted that exogenous estrogens appear to affect TcSnC labeling and may influence the neutrophil response to stimulation. Neutrophil uptake and plasma activity were not significantly affected. IFN-gamma and G-CSF had no significant effect. Conclusions: In whole blood, the effect of LPS on TcSnC monocyte uptake is different to its effect on neutrophils, consistent with previously reported differences in CD11b/CD18 expression. TNF-alpha response parallels LPS response. GM-CSF also increases TcSnC uptake by monocytes. These effects should be considered when using TcSnC for imaging purposes, as they will tend to increase monocyte labeling. Estrogens may also affect TcSnC labeling. Responses to IFN-gamma and G-CSF are consistent with previously reported effects of

  14. In whole blood, LPS, TNF-alpha and GM-CSF increase monocyte uptake of {sup 99m}technetium stannous colloid but do not affect neutrophil uptake

    Energy Technology Data Exchange (ETDEWEB)

    Ramsay, Stuart C. [Townsville Nuclear Medicine, Mater Hospital, Pimlico, Queensland 4812 (Australia) and School of Medicine, James Cook University, Townsville, Queensland 4811 (Australia)]. E-mail: stuart.ramsay1@jcu.edu.au; Maggs, Jacqueline [Department of Nuclear Medicine, Townsville Hospital, Townsville, Queensland 4814 (Australia); Powell, Kellie [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); School of Medicine, James Cook University, Townsville, Queensland 4811 (Australia); Barnes, Jodie [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); Ketheesan, Natkunam [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); School of Medicine, James Cook University, Townsville, Queensland 4811 (Australia)

    2006-07-15

    Introduction: {sup 99m}Technetium stannous colloid (TcSnC) is used in white cell scanning. It labels neutrophils and monocytes via phagocytosis, with uptake mediated by the phagocytic receptor CD11b/CD18 in neutrophils. Uptake of TcSnC is altered by gram-negative infection, possibly due to the endotoxin component lipopolysaccharide (LPS) or to cytokines released during infection (e.g., TNF-alpha and IFN-gamma). Endotoxemia and increased TNF-alpha levels also occur in inflammatory bowel disease. Another potential confounder in cell labeling is that sepsis patients may be treated with GM-CSF and G-CSF, which alter phagocytic cell function. This study aimed to determine how these factors affect TcSnC cellular uptake. Methods: Whole blood from six healthy volunteers was incubated with LPS, TNF-alpha, IFN-gamma, GM-CSF or G-CSF. Samples were then mixed with TcSnC. Blood was separated across density gradients and imaged using a gamma camera. Three radioactive count peaks were observed in each tube: free plasma activity, mononuclear cell uptake and neutrophil uptake. Results: Compared with controls, significant increases in mononuclear cell uptake were induced by LPS, TNF-alpha and GM-CSF stimulation. It was incidentally noted that exogenous estrogens appear to affect TcSnC labeling and may influence the neutrophil response to stimulation. Neutrophil uptake and plasma activity were not significantly affected. IFN-gamma and G-CSF had no significant effect. Conclusions: In whole blood, the effect of LPS on TcSnC monocyte uptake is different to its effect on neutrophils, consistent with previously reported differences in CD11b/CD18 expression. TNF-alpha response parallels LPS response. GM-CSF also increases TcSnC uptake by monocytes. These effects should be considered when using TcSnC for imaging purposes, as they will tend to increase monocyte labeling. Estrogens may also affect TcSnC labeling. Responses to IFN-gamma and G-CSF are consistent with previously reported effects

  15. Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yu [Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006 (China); Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4 (Canada); Cheng, Jung-Chien [Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4 (Canada); Huang, He-Feng, E-mail: huanghefg@hotmail.com [Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006 (China); Leung, Peter C.K., E-mail: peter.leung@ubc.ca [Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006 (China); Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4 (Canada)

    2013-11-01

    Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.

  16. Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

    International Nuclear Information System (INIS)

    Zhang, Yu; Cheng, Jung-Chien; Huang, He-Feng; Leung, Peter C.K.

    2013-01-01

    Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells

  17. Alpha-synuclein accumulates in the brain of scrapie-affected sheep and goats.

    Science.gov (United States)

    Adjou, K T; Allix, S; Ouidja, M O; Backer, S; Couquet, C; Cornuejols, M-J; Deslys, J-P; Brugère, H; Brugère-Picoux, J; El-Hachimi, K H

    2007-07-01

    Immunohistochemical examination demonstrated widespread granular deposits of alpha-synuclein (alphaSN) in the brains of sheep and goats with natural scrapie, especially in the cornu ammonis and subiculum of the hippocampus; this contrasted with the diffuse and non-granular immunolabelling seen in healthy controls. There was non-regular "co-localization" of PrP(Sc) and alphaSN. The findings resembled those reported in Creutzfeldt-Jakob disease and in experimental prion disease in hamsters and mice. The results suggest that perturbation of alphaSN metabolism plays a role in human and animal prion diseases.

  18. Delirium after interleukin-2 and alpha-interferon therapy for renal cell carcinoma

    NARCIS (Netherlands)

    Van Steijn, JHM; Nieboer, P; Hospers, GAP; De Vries, EGE; Mulder, NH

    2001-01-01

    A 55-year-old man receiving alpha-interferon and interieukin-2 therapy for renal cell carcinoma presented with seizures and delirium. A CT-scan of the cerebrum did not reveal any disorder. Both alpha-interferon and interleukin-2 were stopped Treatment with steroids led to complete regression of

  19. Use of Peltier cells in high resolution alpha spectrometry

    International Nuclear Information System (INIS)

    Bueno, C.C.; Santos, M.D.S.; Goncalves, J.A.C.

    1994-01-01

    The experiments with low-cost commercial silicon PIN photodiodes have shown the possibility of their transformation for use as alpha detectors with performance comparable with surface barrier detectors which are more expensive. Utilizing the silicon photodiode with reverse bias, an energy resolution for 241 Am alpha particles of 28 KeV and 23 KeV were obtained at room temperature and at -30 0 C respectively. (author). 4 refs, 4 figs

  20. Structure of the T cell receptor in a Ti alpha V beta 2, alpha V beta 8-positive T cell line

    DEFF Research Database (Denmark)

    Hou, X; Dietrich, J; Kuhlmann, J

    1994-01-01

    not known; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a Ti alpha V beta 2, alpha V beta 8-positive T cell line which expressed the endogenous human TiV beta 8 and the transfected mouse TiV beta 2 both in association......The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Ti alpha beta or Ti gamma delta) and the noncovalently associated CD3 chains (CD3 gamma delta epsilon zeta). The exact number of subunits constituting the TcR is still...... with the endogenous Ti alpha and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of Ti alpha V beta 8 by immunoprecipitation with anti V beta 8 monoclonal antibody (mAb) did not reduce the amount of Ti...

  1. Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line.

    Science.gov (United States)

    Varadi, G; Orlowski, J; Schwartz, A

    1989-07-03

    The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.

  2. Expression and ligand binding of alpha 2 beta 1 integrin on breast carcinoma cells.

    Science.gov (United States)

    Maemura, M; Akiyama, S K; Woods, V L; Dickson, R B

    1995-07-01

    We examined the expression and ligand specificity of the alpha 2 beta 1 integrin on human mammary epithelial cells (HMEC) and a panel of breast carcinoma cell lines in vitro. We found that the alpha 2 beta 1 integrin was universally, but quite variably expressed on these cells by FACS analysis. No significant correlation was observed between its expression and other known cellular phenotypes. Substrate attachment assays using blocking antibodies demonstrated that alpha 2 beta 1 integrin served as a receptor for collagen on HMEC and almost all breast carcinoma cells. However, its contribution to laminin binding of these cells appeared to be related to cellular differentiation as evaluated by sex steroid receptor status and by markers of epithelial-mesenchymal transition, i.e. loss of E-cadherin and expression of vimentin. Two different populations of non-malignant immortalized HMEC (184A1N4 and MCF-10A) contained cells capable of using alpha 2 beta 1 integrin as a laminin receptor. Breast cancer cell lines positive for estrogen receptor (ER) and E-cadherin (MCF-7, T47D, ZR75-1) could also use alpha 2 beta 1 integrin as a laminin receptor. Conversely, alpha 2 beta 1 integrin appeared to be incapable of binding to laminin or to be a very minor receptor for laminin on metastatic ER-negative breast carcinoma cells that expressed vimentin (MDA-MB 231, MDA-MB 435, and MDA-MB 436). These findings suggest that the ligand specificity of alpha 2 beta 1 integrin, i.e. its function as a laminin receptor, may be regulated during the malignant progression of breast carcinoma cells. A reduced contribution of alpha 2 beta 1 integrin to the cellular laminin binding appears to be associated with an increased malignant phenotype and with an epithelial-mesenchymal transition of breast carcinoma cells.

  3. Characterization of the hemin-sensitive eukaryotic initiation factor 2alpha kinase from mouse nonerythroid cells.

    Science.gov (United States)

    Berlanga, J J; Herrero, S; de Haro, C

    1998-11-27

    The heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (heme-regulated inhibitor (HRI)) is activated by heme deficiency in reticulocytes and plays an important role in translational control in these cells. Previously, HRI was cloned from rabbit reticulocytes and rat brain, but a heme-regulated eIF2alpha kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme-sensitive eIF2alpha kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse liver eIF2alpha kinase (mHRI), which exhibits 83 and 94% identities to rabbit and rat HRIs, respectively. Both the purified enzyme and recombinant mHRI exhibited an autokinase and an eIF2alpha kinase activity, and both activities were inhibited in vitro by hemin. In addition, wild-type mHRI, but not the inactive mHRI-K196R mutant, was autophosphorylated in vivo when it was expressed in 293 cells. Quantitation of mHRI mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction revealed relatively high levels in liver, kidney, and testis. These results provide strong evidence that mHRI is a ubiquitous eIF2alpha kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.

  4. Bisphenol A and Its Derivatives Induce Degradation of HIF-1alpha via the Lysosomal Pathway in Human Hepatocarcinoma Cell Line, Hep3B.

    Science.gov (United States)

    Kobayashi, Yukino; Oguro, Ami; Imaoka, Susumu

    2018-01-01

    Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane), one of the phenolic compounds widely used in the manufacture of plastic and epoxy resins, is known as an endocrine disruptor. In a previous study, we found that BPA induced hypoxia inducible factor-1alpha (HIF-1alpha) degradation by dissociation from heat shock protein 90 (Hsp90). In this study, to investigate the structural requirements for degradation of HIF-1alpha, we estimated the effect of BPA derivatives (BPE, BPF, BPB, Dimethyl butylidene diphenol (DMBDP), Ethyl hexylidene diphenol (EHDP), Bishydroxyphenyl cyclohexane (BHCH), and Methyl benzylidene bisphenol (MBBP)) on HIF-1alpha protein degradation, using human hepatocarcinoma cell line, Hep3B. BPB, DMBDP, BHCH, and MBBP decreased HIF-1alpha protein levels more efficiently than BPA, but BPE, BPF, and EHDP did not affect HIF-1alpha protein levels. BPA degraded HIF-1alpha even in the presence of MG132, a proteasome inhibitor. In this study, we found that ammonium chloride (NH 4 Cl), a lysosomal enzyme inhibitor, efficiently restored the decrease in HIF-1alpha protein levels by BPA. Recent studies indicated that HIF-1alpha is degraded by the lysosomal pathway as well as the proteasomal pathway. Therefore, we investigated the levels of heat shock cognate 70 kDa protein (HSC70) protein after treatment with BPA. We found that BPA induced HSC70 protein and overexpression of HSC70 enhanced HIF-1alpha degradation in Hep3B cells. These results suggested that BPA causes the degradation of HIF-1alpha by induction of HSC70, leading lysosomal degradation of HIF-1alpha.

  5. Characterization of alpha-latrotoxin interaction with rat brain synaptosomes and PC12 cells.

    Science.gov (United States)

    Grasso, A; Pelliccia, M; Alemà, S

    1982-01-01

    alpha-latrotoxin, a polypeptide neurotoxin purified from the venom of the spider Latrodectus mactans tredecimguttatus, induces a massive release of a variety of neurotransmitters from rat brain synaptosomes and a clonal pheochromocytoma cell line (PC12 cells). In both systems secretion of catecholamines is dose- and calcium-dependent. Efflux of catecholamines is coupled with a substantial release of intracellular ATP. Independent of alpha-latrotoxin with PC12 cells is followed by a rapid influx of calcium and sodium ions, the rate being dependent on toxin and calcium concentrations. By reductive methylation it is possible to radioactively label alpha-latrotoxin without appreciable loss of neurotoxicity. A sensitive binding assay in vitro allows the identification of a limited number of specific binding sites in central nervous system synaptic membranes and PC12 cells, for which tritiated alpha-latrotoxin displays nanomolar affinity.

  6. Stevioside counteracts the alpha-cell hypersecretion caused by long-term palmitate exposure

    DEFF Research Database (Denmark)

    Hong, J; Chen, L; Jeppesen, P B

    2006-01-01

    Long-term exposure to fatty acids impairs beta-cell function in type 2 diabetes, but little is known about the chronic effects of fatty acids on alpha-cells. We therefore studied the prolonged impact of palmitate on alpha-cell function and on the expression of genes related to fuel metabolism. We......-activated receptor-gamma, and stearoyl-CoA desaturase gene expressions in the presence of palmitate (Pterm exposure to elevated fatty acids leads to a hypersecretion of glucagon and an accumulation of TG content in clonal alpha-TC1-6 cells. Stevioside was able to counteract the alpha......, and changes in gene expression. Glucagon secretion was dose-dependently increased after 72-h culture, with palmitate at concentrations >or=0.25 mM (P

  7. Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

    LENUS (Irish Health Repository)

    Greene, C M

    2010-05-01

    alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

  8. The anti-aromatase effect of progesterone and of its natural metabolites 20alpha- and 5alpha-dihydroprogesterone in the MCF-7aro breast cancer cell line.

    Science.gov (United States)

    Pasqualini, J R; Chetrite, G

    2008-01-01

    Progesterone is metabolized in the normal breast mainly into 4-ene-pregnenes (e.g. 20alpha-dihydroprogesterone, 20alphaDHP) but, in contrast, in breast cancer tissue the 5alpha-dihydropregnanes (e.g. 5alpha-dihydroprogesterone, 5alphaDHP) are prevalent. In the present study the effect of progesterone and its main metabolites 20alphaDHP and 5alphaDHP on the aromatase activity in a stable aromatase-expressing estrogen receptor-positive human breast cancer cell line, MCF-7aro, was explored. The MCF-7aro cells were stripped of endogenous steroids and incubated with physiological concentrations of [3H]-testosterone ([3H]-testos: 5 x 10(-9)M) alone or in the presence of progesterone, 20alphaDHP or 5alphaDHP (5 x 10(-6) or 5 x 10(-8)M) for 24 h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [3H]-Estradiol (E2), [3H]-estrone (E1) and [3H]-testos were characterized by thin layer chromatography and quantified using the corresponding standard. Aromatase activity was present at a high level in the MCF-7aro cells after incubation with [3H]-testos when the concentration of [3H]-E2 was 3.70 pmol/mg DNA; 20alphaDHP at concentrations of 5 x 10(-6)M or 5 x 10(-8)M significantly inhibited this conversion by 50.3% and 36.5%, respectively. No significant effect was found with the metabolite 5alphaDHP or the parent hormone, progesterone. The MCF-7aro cell line shows high detectable aromatase activity. The present data indicate that the progesterone metabolite 20alphaDHP, found mainly in normal breast tissue, can act as an anti-aromatase agent.

  9. Identification of alpha beta and gamma delta T cell receptor-positive cells

    DEFF Research Database (Denmark)

    Geisler, C; Larsen, J K; Plesner, T

    1988-01-01

    distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study...... was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR...

  10. Alpha7 nicotinic receptor mediated protection against ethanol-induced cytotoxicity in PC12 cells.

    Science.gov (United States)

    Li, Y; King, M A; Grimes, J; Smith, N; de Fiebre, C M; Meyer, E M

    1999-01-16

    Ethanol caused a concentration-dependent loss of PC12 cells over a 24 h interval, accompanied by an increase in intracellular calcium. The specific alpha7 nicotinic receptor partial agonist DMXB attenuated both of these ethanol-induced actions at a concentration (3 microM) found previously to protect against apoptotic and necrotic cell loss. The alpha7 nicotinic receptor antagonist methylylaconitine blocked the neuroprotective action of DMXB when applied with but not 30 min after the agonist. These results indicate that activation of alpha7 nicotinic receptors may be therapeutically useful in preventing ethanol-neurotoxicity. Copyright 1999 Elsevier Science B.V.

  11. Development of a transmission alpha particle dosimetry technique using A549 cells and a Ra-223 source for targeted alpha therapy.

    Science.gov (United States)

    Al Darwish, R; Staudacher, A H; Li, Y; Brown, M P; Bezak, E

    2016-11-01

    In targeted radionuclide therapy, regional tumors are targeted with radionuclides delivering therapeutic radiation doses. Targeted alpha therapy (TAT) is of particular interest due to its ability to deliver alpha particles of high linear energy transfer within the confines of the tumor. However, there is a lack of data related to alpha particle distribution in TAT. These data are required to more accurately estimate the absorbed dose on a cellular level. As a result, there is a need for a dosimeter that can estimate, or better yet determine the absorbed dose deposited by alpha particles in cells. In this study, as an initial step, the authors present a transmission dosimetry design for alpha particles using A549 lung carcinoma cells, an external alpha particle emitting source (radium 223; Ra-223) and a Timepix pixelated semiconductor detector. The dose delivery to the A549 lung carcinoma cell line from a Ra-223 source, considered to be an attractive radionuclide for alpha therapy, was investigated in the current work. A549 cells were either unirradiated (control) or irradiated for 12, 1, 2, or 3 h with alpha particles emitted from a Ra-223 source positioned below a monolayer of A549 cells. The Timepix detector was used to determine the number of transmitted alpha particles passing through the A549 cells and DNA double strand breaks (DSBs) in the form of γ-H2AX foci were examined by fluorescence microscopy. The number of transmitted alpha particles was correlated with the observed DNA DSBs and the delivered radiation dose was estimated. Additionally, the dose deposited was calculated using Monte Carlo code SRIM. Approximately 20% of alpha particles were transmitted and detected by Timepix. The frequency and number of γ-H2AX foci increased significantly following alpha particle irradiation as compared to unirradiated controls. The equivalent dose delivered to A549 cells was estimated to be approximately 0.66, 1.32, 2.53, and 3.96 Gy after 12, 1, 2, and 3 h

  12. Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

    Directory of Open Access Journals (Sweden)

    W. Cui

    2010-04-01

    Full Text Available The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

  13. Protective effect of alpha-mangostin against oxidative stress induced-retinal cell death.

    Science.gov (United States)

    Fang, Yuan; Su, Tu; Qiu, Xiaorong; Mao, Pingan; Xu, Yidan; Hu, Zizhong; Zhang, Yi; Zheng, Xinhua; Xie, Ping; Liu, Qinghuai

    2016-02-18

    It is known that oxidative stress plays a pivotal role in age-related macular degeneration (AMD) pathogenesis. Alpha-mangostin is the main xanthone purified from mangosteen known as anti-oxidative properties. The aim of the study was to test the protective effect of alpha-mangostin against oxidative stress both in retina of light-damaged mice model and in hydrogen peroxide (H2O2)-stressed RPE cells. We observed that alpha-mangostin significantly inhibited light-induced degeneration of photoreceptors and 200 μM H2O2-induced apoptosis of RPE cells. 200 μM H2O2-induced generation of reactive oxygen species (ROS) and light-induced generation of malondialdehyde (MDA) were suppressed by alpha-mangostin. Alpha-mangostin stimulation resulted in an increase of superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity and glutathione (GSH) content both in vivo and vitro. Furthermore, the mechanism of retinal protection against oxidative stress by alpha-mangostin involves accumulation and the nuclear translocation of the NF-E2-related factor (Nrf2) along with up-regulation the expression of heme oxygenas-1 (HO-1). Meanwhile, alpha-mangostin can activate the expression of PKC-δ and down-regulate the expression of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, P38. The results suggest that alpha-mangostin could be a new approach to suspend the onset and development of AMD.

  14. Apoptotic effect of alpha-mangostin on head and neck squamous carcinoma cells.

    Science.gov (United States)

    Kaomongkolgit, Ruchadaporn; Chaisomboon, Niratcha; Pavasant, Prasit

    2011-05-01

    The purposes of this study were to measure the cytotoxic effect of alpha-mangostin on head and neck squamous cell carcinoma (HNSCC) cell lines, to evaluate the apoptotic aspect of dead cells, and to identify the molecular mechanism involved in apoptosis. The human HNSCC cell lines HN-22, HN-30 and HN-31 were treated with alpha-mangostin. The apoptotic effects of alpha-mangostin on HNSCC cells were determined by observation the morphological changes of cells, immunofluorescence for single-stranded DNA (ssDNA), and DNA fragmentation analysis. The expression of bax, bcl-2, and p53 were detected by RT-PCR and Western blot analysis. Alpha-mangostin showed excellent apoptotic effects on HNSCC cell lines, which induced the down-regulation of bcl-2, but up-regulation of bax and p53 in HN-22, HN-30 and HN-31. The present study suggests that the induction of apoptosis by alpha-mangostin seemed to be modulated by bcl-2, bax and p53 level in HNSCC cell lines. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. Asthma induction in mice leads to appearance of alpha2-3- and alpha2-6-linked sialic acid residues in respiratory goblet-like cells

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Jensen, Niels-Erik Viby; Mandel, Ulla

    2008-01-01

    incubation with the sialic acid detecting agents, while the goblet-like cells expressed both alpha2-3- and alpha2-6-linked sialic acid residues in the asthmatic animals. The lectins but not the antibodies reacted with intestinal goblet cells. Instead, an antibody recognizing a disialoganglioside, stained......Allergic asthmatic inflammation in mice was induced by sensitization with ovalbumin and lipopolysaccharide from Escherichia coli and visualized in the airways of asthmatic mice by spatial and temporal changes of carbohydrates containing sialic acid residues. Immunohistochemistry was used...... to demonstrate binding of lectins and antibodies that detect alpha2-3- and alpha2-6-linked sialic acid residues. After sensitization and challenge, the histology of the lung changed markedly, and goblet-like cells appeared, most likely caused by Clara cell metaplasia. Normal Clara cells showed no reaction after...

  16. Overexpression of protein tyrosine phosphatase-alpha (PTP-alpha) but not PTP-kappa inhibits translocation of GLUT4 in rat adipose cells

    DEFF Research Database (Denmark)

    Cong, L N; Chen, H; Li, Y

    1999-01-01

    Protein tyrosine phosphatases (PTPases) are likely to play important roles in insulin action. We recently demonstrated that the nontransmembrane PTPase PTP1B can act as a negative modulator of insulin-stimulated translocation of GLUT4. We now examine the role of PTP-alpha and PTP-kappa (two...... transmembrane PTPases) in this metabolic action of insulin. Rat adipose cells were transfected with either PTP-alpha or PTP-kappa and effects of these PTPases on the translocation of a cotransfected epitope-tagged GLUT4 were studied. Cells overexpressing wild-type PTP-alpha had significantly lower levels...... of cell surface GLUT4 in response to insulin and a threefold decrease in insulin sensitivity when compared with control cells expressing only tagged GLUT4. Co-overexpression of PTP-alpha and PTP1B did not have additive effects, suggesting that these PTPases share common substrates. Cells overexpressing...

  17. Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.

    Science.gov (United States)

    Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A

    2004-03-15

    Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.

  18. Suppression of saccharin-induced mutagenicity by interferon-alpha in human RSa cells.

    Science.gov (United States)

    Suzuki, N; Suzuki, H

    1995-10-01

    Saccharin is an artificial sweetener commonly used in the formulation of foods and beverages. Sodium saccharin-induced mutagenicity is detectable in human RSa cells by estimation of cloning efficiency of ouabain-resistant mutant cells and determination of K-ras codon 12 mutation in genomic DNA, analyzed by PCR and differential dot-blot hybridization. However, in this study no phenotypic or genetic mutations were detected in RSa cells cultured with human IFN (HuIFN)-alpha before sodium saccharin treatment. The suppressive effect was lessened by transient treatment with antipain immediately after sodium saccharin treatment. Elevation of antipain-sensitive protease activity was found, furthermore, in RSa cells cultured with HuIFN-alpha and subsequently treated with sodium saccharin. Thus, antipain-sensitive protease induction in cells tested here may be involved in suppression of the mutagenicity of saccharin by HuIFN-alpha.

  19. Cell survival following alpha particle irradiation: critical sites and implications for carcinogenesis

    International Nuclear Information System (INIS)

    Lloyd, E.L.; Gemmell, M.A.; Henning, C.B.; Gemmell, D.S.; Zabransky, B.J.

    1976-01-01

    In experiments in which mammalian cells were irradiated with 5.6 MeV alpha particles from a Tandem Van de Graaff machine we have confirmed the finding of others that the mean lethal dose (D 0 ) is about 100 rad, but by measurements of the area of the cell nuclei as irradiated we found that this mean lethal dose corresponds not to 1, as expected, but to about 27 alpha particles per cell nucleus. (The exact number appears to change slightly with cell passage number.) This allows for the possibility that the direct action of alpha particles on the nucleus may be the important event in carcinogenesis, a theory which was previously difficult to accept if a single particle hitting the nucleus anywhere was considered to be lethal. Evidence is presented to implicate the nucleolus as a possible critical site for the inhibition of reproductive integrity of the cell

  20. Alpha-mangostin inhibits intracellular fatty acid synthase and induces apoptosis in breast cancer cells

    OpenAIRE

    Li, Ping; Tian, Weixi; Ma, Xiaofeng

    2014-01-01

    Background Fatty acid synthase (FAS) has been proven over-expressed in human breast cancer cells and consequently, has been recognized as a target for breast cancer treatment. Alpha-mangostin, a natural xanthone found in mangosteen pericarp, has a variety of biological activities, including anti-cancer effect. In our previous study, alpha-mangostin had been found both fast-binding and slow-binding inhibitions to FAS in vitro. This study was designed to investigate the activity of alpha-mangos...

  1. Protective effect of alpha-mangostin against oxidative stress induced-retinal cell death

    OpenAIRE

    Fang, Yuan; Su, Tu; Qiu, Xiaorong; Mao, Pingan; Xu, Yidan; Hu, Zizhong; Zhang, Yi; Zheng, Xinhua; Xie, Ping; Liu, Qinghuai

    2016-01-01

    It is known that oxidative stress plays a pivotal role in age-related macular degeneration (AMD) pathogenesis. Alpha-mangostin is the main xanthone purified from mangosteen known as anti-oxidative properties. The aim of the study was to test the protective effect of alpha-mangostin against oxidative stress both in retina of light-damaged mice model and in hydrogen peroxide (H2O2)-stressed RPE cells. We observed that alpha-mangostin significantly inhibited light-induced degeneration of photore...

  2. Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells.

    Directory of Open Access Journals (Sweden)

    Ilker Kudret Sariyer

    Full Text Available PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV, which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs. We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF. SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen.Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins.Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral reactivation leading to

  3. Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells.

    Directory of Open Access Journals (Sweden)

    Robin R Craven

    Full Text Available Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal alpha-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus alpha-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant alpha-hemolysin, we now demonstrate that alpha-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant alpha-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to alpha-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by alpha-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs to induce secretion of pro-inflammatory cytokines IL-1beta and IL-18. Additionally, alpha-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1. These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

  4. Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells.

    Science.gov (United States)

    Craven, Robin R; Gao, Xi; Allen, Irving C; Gris, Denis; Bubeck Wardenburg, Juliane; McElvania-Tekippe, Erin; Ting, Jenny P; Duncan, Joseph A

    2009-10-14

    Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal alpha-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus alpha-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant alpha-hemolysin, we now demonstrate that alpha-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant alpha-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to alpha-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by alpha-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1beta and IL-18. Additionally, alpha-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

  5. Immunomodulatory activities of alpha-mangostin on peripheral blood mononuclear cells.

    Science.gov (United States)

    Kasemwattanaroj, Pimolkan; Moongkarndi, Primchanien; Pattanapanyasat, Kovit; Mangmool, Supachoke; Rodpai, Ekkarat; Samer, Jutima; Konlata, Julaporn; Sukapirom, Kasama

    2013-09-01

    Mangosteen (Garcinia mangostana L.) a tropical fruit, has been used in traditional medicine. A frequently used part of mangosteen is the pericarp, containing a high content of xanthones. alpha-Mangostin, one of the major xanthone derivatives, exhibits a variety of actions, including antimicrobial, antioxidant, cytotoxic and antitumor; however, its function on the immune system is still equivocal. This study aimed to examine the immunomodulatory activities of alpha-mangostin on lymphocyte lineage and cytokine production in human peripheral blood mononuclear cells (PBMCs). The cytotoxic activity of alpha-mangostin was measured by MTT assay. The concentration of alpha-mangostin at 5.55 microg/mL resulted in a 50% survival of PBMCs, which was as potent a cytotoxic activity as that of paclitaxel. After 24 h of PBMCs culture, the percentages of T cells (CD3+), B cells (CD19+) and NK cells (CD3-CD16+CD56+) were not significantly changed by treatment with 1, 2 and 4 microg/mL of alpha-mangostin compared with untreated-PBMCs; in addition, the percentages of these lymphocytes treated with the combination of alpha-mangostin (1, 2 and 4 microg/mL) and the mitogen concanavalin A (ConA) was not significantly different from that of ConA-treated PBMCs. For cytokine secretion, alpha-mangostin (1, 2 and 4 microg/mL) did not significantly induce either proinflammatory cytokines (i.e., TNF-alpha and IL-1beta) or cytokine of adaptive immunity (i.e., IL-2). The combination of alpha-mangostin (1, 2 and 4 microg/mL) and ConA did not significantly alter the relative difference of TNF-alpha and IL-1beta compared with ConA-treated PBMCs; however, these combinations could significantly decrease the relative difference of IL-2 compared with ConA-treated PBMCs. These data indicated that alpha-mangostin was able to inhibit IL-2 release without interfering with human immune cells; therefore, further studies are necessary to investigate its effect on IL-2 production.

  6. Disruption of glucagon receptor signaling causes hyperaminoacidemia exposing a possible liver - alpha-cell axis

    DEFF Research Database (Denmark)

    Galsgaard, Katrine D; Winther-Sørensen, Marie; Ørskov, Cathrine

    2018-01-01

    Glucagon secreted from the pancreatic alpha-cells is essential for regulation of blood glucose levels. However, glucagon may play an equally important role in the regulation of amino acid metabolism by promoting ureagenesis. We hypothesized that disruption of glucagon receptor signaling would lead...... to an increased plasma concentration of amino acids, which in a feedback manner stimulates the secretion of glucagon, eventually associated with compensatory proliferation of the pancreatic alpha-cells. To address this, we performed plasma profiling of glucagon receptor knockout (Gcgr-/-) mice and wild-type (WT...... component distinguishing the two groups of mice. Apart from their hyperaminoacidemia, Gcgr-/- mice display hyperglucagonemia, increased pancreatic content of glucagon and somatostatin (but not insulin), and alpha-cell hyperplasia and hypertrophy compared to WT littermates. Incubating cultured α-TC1.9 cells...

  7. HNF1 alpha activates the aminopeptidase N promoter in intestinal (Caco-2) cells

    DEFF Research Database (Denmark)

    Olsen, Jørgen; Laustsen, Lotte; Troelsen, J

    1994-01-01

    The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates...... a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation....

  8. Alpha-mangostin inhibits intracellular fatty acid synthase and induces apoptosis in breast cancer cells.

    Science.gov (United States)

    Li, Ping; Tian, Weixi; Ma, Xiaofeng

    2014-06-03

    Fatty acid synthase (FAS) has been proven over-expressed in human breast cancer cells and consequently, has been recognized as a target for breast cancer treatment. Alpha-mangostin, a natural xanthone found in mangosteen pericarp, has a variety of biological activities, including anti-cancer effect. In our previous study, alpha-mangostin had been found both fast-binding and slow-binding inhibitions to FAS in vitro. This study was designed to investigate the activity of alpha-mangostin on intracellular FAS activity in FAS over-expressed human breast cancer cells, and to testify whether the anti-cancer activity of alpha-mangostin may be related to its inhibitory effect on FAS. We evaluated the cytotoxicity of alpha-mangostin in human breast cancer MCF-7 and MDA-MB-231 cells. Intracellular FAS activity was measured by a spectrophotometer at 340 nm of NADPH absorption. Cell Counting Kit assay was used to test the cell viability. Immunoblot analysis was performed to detect FAS expression level, intracellular fatty acid accumulation and cell signaling (FAK, ERK1/2 and AKT). Apoptotic effects were detected by flow cytometry and immunoblot analysis of PARP, Bax and Bcl-2. Small interfering RNA was used to down-regulate FAS expression and/or activity. Alpha-mangostin could effectively suppress FAS expression and inhibit intracellular FAS activity, and result in decrease of intracellular fatty acid accumulation. It could also reduce cell viability, induce apoptosis in human breast cancer cells, increase in the levels of the PARP cleavage product, and attenuate the balance between anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. Moreover, alpha-mangostin inhibited the phosphorylation of FAK. However, the active forms of AKT, and ERK1/2 proteins were not involved in the changes of FAS expression induced by alpha-mangostin. Alpha-mangostin induced breast cancer cell apoptosis by inhibiting FAS, which provide a basis for the development of xanthone as an agent for

  9. Alpha-mangostin suppresses MMP-2 and MMP-9 expression in head and neck squamous carcinoma cells.

    Science.gov (United States)

    Kaomongkolgit, Ruchadaporn

    2013-07-01

    The purpose of this study was to investigate the effect of alpha-mangostin on matrix metalloproteinase (MMP)-2 and MMP-9 expression in head and neck squamous cell carcinoma (HNSCC). The human HNSCC cell lines were treated with alpha-mangostin and the cytotoxicity of alpha-mangostin in HNSCC was determined using the MTS assay. To determine the effect of alpha-mangostin on the expression of MMP-2 and MMP-9 in HNSCC, gelatin zymography and RT-PCR were performed. The results showed that alpha-mangostin increased in growth inhibition of HNSCC cell lines in a concentration-dependent manner. Treatment with alpha-mangostin decreased MMP-2 and MMP-9 expression in a concentration-dependent manner in all cell lines. These findings suggested that alpha-mangostin might be a potential therapeutic agent for HNSCC.

  10. The PI3K isoforms p110alpha and p110delta are essential for pre-B cell receptor signaling and B cell development.

    Science.gov (United States)

    Ramadani, Faruk; Bolland, Daniel J; Garcon, Fabien; Emery, Juliet L; Vanhaesebroeck, Bart; Corcoran, Anne E; Okkenhaug, Klaus

    2010-08-10

    B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes produce a functional B cell receptor (BCR) and antibodies. As part of this process, recombination-activating gene (Rag) proteins regulate the in-frame assembly of the Ig-encoding genes. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igalpha and Igbeta chains. Whereas the activation of the tyrosine kinases Src and Syk is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110delta isoform of phosphatidylinositol 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on agonist-independent or "tonic" BCR signaling, are not substantially affected by a deficiency in p110delta. Here, we show that p110alpha, but not p110beta, compensated in the absence of p110delta to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110alpha and p110delta activities, pre-BCR signaling failed to suppress the production of Rag proteins and to promote developmental progression of B cell progenitors. Unlike p110delta, however, p110alpha did not contribute to agonist-induced BCR signaling. These studies indicate that either p110alpha or p110delta can mediate tonic signaling from the BCR, but only p110delta can contribute to antigen-dependent activation of B cells.

  11. The structure of cell wall alpha-glucan from fission yeast

    NARCIS (Netherlands)

    Grün, Christian H.; Hochstenbach, Frans; Humbel, Bruno M.; Verkleij, Arie J.; Sietsma, J. Hans; Klis, Frans M.; Kamerling, Johannis P.; Vliegenthart, Johannes F. G.

    2005-01-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the

  12. The structure of cell wall alpha-glucan from fission yeast.

    NARCIS (Netherlands)

    Grün, C.H.; Hochstenbach, F.; Humbel, B.M.; Verkleij, A.J.; Sietsma, J.H.; Klis, F.M.; Kamerling, J.P.; Vliegenthart, J.F.G.

    2005-01-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1rarr3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the

  13. Induction of regulatory dendritic cells by dexamethasone and 1alpha,25-Dihydroxyvitamin D(3)

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Gad, Monika; Walter, Mark R

    2004-01-01

    Dendritic cells (DC) modulated to induce T cell hyporesponsiveness have promising potential in immunotherapy of autoimmune disorders and for the prevention of allograft rejection. While studying the effect of immunosuppressive agents on the maturation of DC we found that 1alpha,25-Dihydroxyvitamin...

  14. Aerobic Fitness Level Affects Cardiovascular and Salivary Alpha Amylase Responses to Acute Psychosocial Stress.

    Science.gov (United States)

    Wyss, Thomas; Boesch, Maria; Roos, Lilian; Tschopp, Céline; Frei, Klaus M; Annen, Hubert; La Marca, Roberto

    2016-12-01

    Good physical fitness seems to help the individual to buffer the potential harmful impact of psychosocial stress on somatic and mental health. The aim of the present study is to investigate the role of physical fitness levels on the autonomic nervous system (ANS; i.e. heart rate and salivary alpha amylase) responses to acute psychosocial stress, while controlling for established factors influencing individual stress reactions. The Trier Social Stress Test for Groups (TSST-G) was executed with 302 male recruits during their first week of Swiss Army basic training. Heart rate was measured continuously, and salivary alpha amylase was measured twice, before and after the stress intervention. In the same week, all volunteers participated in a physical fitness test and they responded to questionnaires on lifestyle factors and personal traits. A multiple linear regression analysis was conducted to determine ANS responses to acute psychosocial stress from physical fitness test performances, controlling for personal traits, behavioural factors, and socioeconomic data. Multiple linear regression revealed three variables predicting 15 % of the variance in heart rate response (area under the individual heart rate response curve during TSST-G) and four variables predicting 12 % of the variance in salivary alpha amylase response (salivary alpha amylase level immediately after the TSST-G) to acute psychosocial stress. A strong performance at the progressive endurance run (high maximal oxygen consumption) was a significant predictor of ANS response in both models: low area under the heart rate response curve during TSST-G as well as low salivary alpha amylase level after TSST-G. Further, high muscle power, non-smoking, high extraversion, and low agreeableness were predictors of a favourable ANS response in either one of the two dependent variables. Good physical fitness, especially good aerobic endurance capacity, is an important protective factor against health

  15. Alpha9beta1 integrin in melanoma cells can signal different adhesion states for migration and anchorage

    DEFF Research Database (Denmark)

    Lydolph, Magnus C; Morgan-Fisher, Marie; Høye, Anette M

    2009-01-01

    Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The alpha9 integrin subunit, which pairs only with beta1, has specific roles in the immune system and may regulate cell...... migration. Melanoma cells express abundant alpha9beta1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported alpha9beta1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced alpha9......beta1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of alpha9beta1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by beta1 integrin activating antibody. The alpha9beta1...

  16. Basal cell carcinoma is associated with high TNF-alpha release but nor with TNF-alpha polymorphism at position--308

    DEFF Research Database (Denmark)

    Skov, Lone; Allen, Michael H; Bang, Bo

    2003-01-01

    secretion of TNF-alpha has been identified in humans. We have therefore investigated the association of the --308 polymorphism with the risk of basal cell carcinoma (BCC) in humans. The frequency of TNF G and TNF A alleles among Caucasian patients with a previous BCC (n=191) and health adults (n-107) were...... compared. For the TNF--308 polymorphism there was significant association between the genotype or allele frequencies and having BCC. To determine whether patients with a previous BCC had an increased capacity to secrete TNF-alpha, mononuclear cells were stimulated with lipopolysaccharide. Mononuclear cells...... from patients with a previous BCC (n=15) demonstrated a significantly increased release of TNF-alpha upon stimulation with lipopolysaccharide (Pcells age-matched control subjects (n=16). Further studies of other polymorphisms of the TNF-alpha gene associated...

  17. Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

    Science.gov (United States)

    Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

  18. The effect of alpha-thalassemia on cord blood red cell indices and interaction with sickle cell gene

    International Nuclear Information System (INIS)

    Quadri, Mohammad I.; Islam, Sherief I.A.M.; Nasserullah, Z.

    2000-01-01

    Alpha-thalassemia is known to be prevalent in the Eastern region of Saudi Arabia. There are no large scale reports regarding the effect of alpha-thalassemia on red cell indices of cord blood from Saudi Arabia. Similarly, there are reports regarding the interaction of alpha-thalassemia and the sickle-cell gene in relation to red cell indices in cord blood. To address these issues, we undertook a study on neonatal cold blood samples. In a prospective study, cord blood samples from 504 neonates from the Qatif area of the Eastern Province of Saudi Arabia were analyzed for complete blood counts (CBC) and cellulose acetate Hb electrophoresis. Hb S was confirmed by citrate agar Hb electrophoresis. There were 243 case samples with normal Hb electrophoresis (Hb A 27.2+- 7% and Hb F 72.6+-7.7%). Their mean Hb (g/dL), RBC (x10/L), Hct (%), MCV (pg), MCHC (g/dL), RDW-SD (fl) and RDW-CV (%) were 15.05+-1.6, 4.5+-0.5, 47.4+-5.3, 106+-8, 33.6+-2.3, 31.8+-1.7, 69.2+-9.5 and 17.9+-1.7, respectively. There were 136 cases with alpha-thalassemia trait (alphaTT), 57 cases with sickle cell trait (SCT) and 50 cases of sickle cell trait with alplha-thalassemia trait (SCT/ alphaTT). There were ten cases of Hb H disease (6 definite), including one with sickle cell disease (SCD) and two with SCT, Hb Bart's 23.9%-43.6%; four probable with Hb Bart's 10.9%-16.1% and one with SCT. The effect on red cell parameters in Hb H disease were most pronounced. In addition, there seven cases of SCD, four of whom had coexistent alpha-thalassemia trait (SCD/alphaTT). The prevalence of alpha-thalassemia in this cohort of Saudi population was 39.99%. Hb H disease appeared as common as SCD. Sickle cell gene was seen in 23.4% of neonatal samples. Apha-thalassemia gene significantly reduces MCH, MCV, RDW-SD, Hct, Hb and increase RBC count in both normal or sickle cell trait neonates. Generally, the variation of red cell parameters is directly proportional to the amount of Hb Bart's in the cord blood. Sickle cell

  19. Survival of human osteosarcoma cells and normal human fibroblasts following alpha particle irradiation

    International Nuclear Information System (INIS)

    Lloyd, E.L.; Gemmell, M.A.

    1981-01-01

    Cell survival of human osteosarcoma cells in culture following alpha particle irradiation is reported here for the first time. The osteosarcoma cell line (TE-85) is found to be less sensitive to inactivation by 5.6 MeV alpha particles (LET 86 keV/μm) than normal diploid human fibroblasts (NFS). Values for the mean lethal doses were estimated to be 103 rads for the TE-85 cells compared with 68 rads for the NFS cultures irradiated under identical conditions. It is postulated that the aneuploidy of the tumor cells with increased DNA chromosomal material may confer a selective advantage for the survival of tumor cells relative to normal cells with diploid chromosomes

  20. Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

    Energy Technology Data Exchange (ETDEWEB)

    Kazmi, S.M.; Mishra, R.K.

    1989-02-15

    The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. (/sup 3/H)Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, (/sup 3/H)Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.

  1. PDGFBB promotes PDGFR{alpha}-positive cell migration into artificial bone in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Shigeyuki [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Iwasaki, Ryotaro; Kawana, Hiromasa [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Miyauchi, Yoshiteru [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Integrated Bone Metabolism and Immunology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Hoshi, Hiroko; Miyamoto, Hiroya; Mori, Tomoaki [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Kanagawa, Hiroya [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Katsuyama, Eri; Fujie, Atsuhiro [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Hao, Wu [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); and others

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We examined effects of PDGFBB in PDGFR{alpha} positive cell migration in artificial bones. Black-Right-Pointing-Pointer PDGFBB was not expressed in osteoblastic cells but was expressed in peripheral blood cells. Black-Right-Pointing-Pointer PDGFBB promoted PDGFR{alpha} positive cell migration into artificial bones but not osteoblast proliferation. Black-Right-Pointing-Pointer PDGFBB did not inhibit osteoblastogenesis. -- Abstract: Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor {alpha} (PDGFR{alpha})-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGF{beta}) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.

  2. Alpha thalassemia among sickle cell anaemia patients in Kampala ...

    African Journals Online (AJOL)

    thalassaemia is known to modulate sickle cell anaemia, its magnitude and significance in Uganda have hitherto not been described. Objectives: To determine the prevalence of α+thalassaemia among sickle cell anaemia patients in Mulago ...

  3. Investigation of the genes for RET and its ligand complex, GDNF/GFR alpha-1, in small cell lung carcinoma

    NARCIS (Netherlands)

    Mulligan, LM; Timmer, T; Ivanchuk, SM; Campling, BG; Young, LC; Rabbitts, PH; Sundaresan, [No Value; Hofstra, RMW; Eng, C

    RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFR alpha-1). Activating RET mutations are found

  4. Factors affecting the energy resolution in alpha particle spectrometry with silicon diodes

    International Nuclear Information System (INIS)

    Camargo, Fabio de.

    2005-01-01

    In this work are presented the studies about the response of a multi-structure guard rings silicon diode for detection and spectrometry of alpha particles. This ion-implanted diode (Al/p + /n/n + /Al) was processed out of 300 μm thick, n type substrate with a resistivity of 3 kΩ·cm and an active area of 4 mm 2 . In order to use this diode as a detector, the bias voltage was applied on the n + side, the first guard ring was grounded and the electrical signals were readout from the p + side. These signals were directly sent to a tailor made preamplifier, based on the hybrid circuit A250 (Amptek), followed by a conventional nuclear electronic. The results obtained with this system for the direct detection of alpha particles from 241 Am showed an excellent response stability with a high detection efficiency (≅ 100 %). The performance of this diode for alpha particle spectrometry was studied and it was prioritized the influence of the polarization voltage, the electronic noise, the temperature and the source-diode distance on the energy resolution. The results showed that the major contribution for the deterioration of this parameter is due to the diode dead layer thickness (1 μm). However, even at room temperature, the energy resolution (FWHM = 18.8 keV) measured for the 5485.6 MeV alpha particles ( 241 Am) is comparable to those obtained with ordinary silicon barrier detectors frequently used for these particles spectrometry. (author)

  5. Influence of the mycotoxins alpha- and beta-zearalenol and deoxynivalenol on the cell cycle of cultured porcine endometrial cells.

    Science.gov (United States)

    Tiemann, U; Viergutz, T; Jonas, L; Schneider, F

    2003-01-01

    The present study investigated the effects of the mycotoxins alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) at concentrations of 7.5, 15, and 30 microM, and deoxynivalenol (DON) at concentrations of 0.94, 1.88, and 3.76 microM on cell cycle distribution (propidium iodide, PI staining) in combination with the proliferating cell nuclear antigen (PCNA) by flow cytometry. The viability of porcine uterine cells was not impaired at 30 microM alpha-ZOL, whereas beta-ZOL at this concentration and 3.76 microM DON significantly decreased cell number. Some cells showed ultrastructural features of cell death indicated by swollen mitochondria, disrupted cell membranes, and many vacuoles. After 24 and 48h of exposure to alpha-ZOL (7.5, 15, or 30 microM), the cell cycle distribution was still comparable to the control groups. An anti-proliferative effect of beta-ZOL and DON was detected by a significant reduction in the S-phase together with arrest of cells in the G(0)/G(1)-phase. The results show that beta-ZOL (7.5, 15, or 30 microM) and DON (0.94, 1.88, or 3.76 microM) control the progression of cells through the cycle by decreasing S-phase and arresting cells in the G(0)/G(1)-phase of the cell cycle. A significant decrease in the expression of the proliferation marker PCNA amounts indicates that beta-ZOL and DON disengaged cells from active cycling. We confirm that alpha-ZOL possesses a relative binding affinity to porcine uterine cytoplasmic estrogen receptor.

  6. Modulation of the nicotinic alpha-bungarotoxin site in chromaffin cells in culture by a factor(s) endogenous to neuronal tissue.

    Science.gov (United States)

    Quik, M; Fournier, S; Trifaró, J M

    1986-04-30

    An endogenous factor(s) which affects the in vitro binding of (alpha-BGT) to rat brain membranes has previously been found in brain supernatant. This fraction, as well as a partially purified preparation of this material from bovine brain, is here shown to affect the binding of alpha-BGT to chromaffin cell membranes. To study possible long term effects, the supernatant extract was added to adrenal medullary chromaffin cells in culture. The cells were incubated for several days and at the end of this time, the medium bathing the cells, which contained the endogenous factor(s), was removed and alpha-BGT binding to the cells measured. Binding to control cultures had shown that alpha-BGT bound to the chromaffin cells in a saturable manner, with high affinity (Kd = 1.5 nM) and the specificity of a nicotinic receptor ligand. After incubation of the cells with supernatant factor, a marked decline in the number of alpha-BGT binding sites was observed with no change in affinity. This does not appear to be due to a detrimental effect on the cells as cell number did not appear to be decreased in the cultures preincubated with the supernatant extract and the DNA and protein content were similar in the control and treated cultures. The possibility that there was some non-specific detrimental effect to the chromaffin cell membrane was considered; however, the stimulated release of noradrenaline from the cells was not affected by treatment of the cultures in the presence of the supernatant fractions. In addition, tyrosine hydroxylase activity was significantly increased in the treated cultures. D-Tubo-curarine, an antagonist at the acetylcholine receptor, caused an increase in alpha-BGT binding after 7 days of treatment, while the agonist nicotine and choline had no effect. These results suggest that in brain supernatant there may exist an endogenous factor(s), which may function in the regulation of the nicotinic-like alpha-BGT receptors in neuronal cell.

  7. PANCREATIC ALPHA CELL DYSFUNCTION IN DIABETES MELLITUS AND ITS MANAGEMENT STRATEGIES

    Directory of Open Access Journals (Sweden)

    Butungeshwar Pradhan

    2017-06-01

    Full Text Available BACKGROUND Diabetes mellitus has primarily centered around the insulin deficiency owing to pancreatic beta-cell dysfunction or loss and associated insulin resistance. Recently, numerous findings indicate that defect of glucagon secreting alpha-cell get involved with development and exacerbation of hyperglycaemia in both type 1 and type 2 DM. Aberrant α-cell responses exhibit both fasting and postprandial hyperglucagonaemia contributes to fasting and postprandial hyperglycaemia caused by inappropriate hepatic glucose production owing to blunted α-cell suppression. Thus, blockade of glucagon receptor or suppression of glucagon secretion from α-cell would be novel therapeutic target for control of hyperglycaemia. There have not been remarkable advances in developing new class of drugs, currently glucagon-like peptide-1 and dipeptidyl peptidase-4 inhibitors and amylin agonist are available targeting alpha-cell dysfunction for the treatment of diabetes mellitus.

  8. Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Ng, Stanley K.L. [Singapore Immunology Network A-STAR (Singapore); Neo, Soek-Ying, E-mail: neo_soek_ying@sics.a-star.edu.sg [Singapore Immunology Network A-STAR (Singapore); Yap, Yann-Wan [Singapore Immunology Network A-STAR (Singapore); Karuturi, R. Krishna Murthy; Loh, Evelyn S.L. [Genome Institute of Singapore A-STAR (Singapore); Liau, Kui-Hin [Department of General Surgery, Tan Tock Seng Hospital (Singapore); Ren, Ee-Chee, E-mail: ren_ee_chee@immunol.a-star.edu.sg [Singapore Immunology Network A-STAR (Singapore); Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore (Singapore)

    2009-09-18

    Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2{alpha}) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2{alpha} mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2{alpha} was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2{alpha} can modulate HCC cell growth.

  9. The diversity and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis

    Directory of Open Access Journals (Sweden)

    Mengoni Alessio

    2010-04-01

    Full Text Available Abstract Background In the bacterium Caulobacter crescentus, CtrA coordinates DNA replication, cell division, and polar morphogenesis and is considered the cell cycle master regulator. CtrA activity varies during cell cycle progression and is modulated by phosphorylation, proteolysis and transcriptional control. In a phosphorylated state, CtrA binds specific DNA sequences, regulates the expression of genes involved in cell cycle progression and silences the origin of replication. Although the circuitry regulating CtrA is known in molecular detail in Caulobacter, its conservation and functionality in the other alpha-proteobacteria are still poorly understood. Results Orthologs of Caulobacter factors involved in the regulation of CtrA were systematically scanned in genomes of alpha-proteobacteria. In particular, orthologous genes of the divL-cckA-chpT-ctrA phosphorelay, the divJ-pleC-divK two-component system, the cpdR-rcdA-clpPX proteolysis system, the methyltransferase ccrM and transcriptional regulators dnaA and gcrA were identified in representative genomes of alpha-proteobacteria. CtrA, DnaA and GcrA binding sites and CcrM putative methylation sites were predicted in promoter regions of all these factors and functions controlled by CtrA in all alphas were predicted. Conclusions The regulatory cell cycle architecture was identified in all representative alpha-proteobacteria, revealing a high diversification of circuits but also a conservation of logical features. An evolutionary model was proposed where ancient alphas already possessed all modules found in Caulobacter arranged in a variety of connections. Two schemes appeared to evolve: a complex circuit in Caulobacterales and Rhizobiales and a simpler one found in Rhodobacterales.

  10. TNF-alpha induced junctional modulation enhances response to radiation in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Yeonjoo; Ahn, Hyein; Park, Jina; Lee, Byungryong; Chung, Eunkyung [Hallym University, Seoul (Korea, Republic of); Yi, Jaeoun [KIRAMS, Seoul (Korea, Republic of)

    2007-10-15

    The Adhesion molecules mediated cell-cell and cell matrix interactions are essential for variety of physiological and pathological processes including maintenance of normal tissues integrity as well as tumor development and progression. Cell-cell interaction is initiated by interactions of tight junctional proteins with neighboring cells. Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction formation are an early and key event during vascular spread cancer and inflammation. TNF-alpha plays an important role in the intestinal inflammation by increase of intestinal epithelial tight junction permeability. It has been reported that TNF alpha- modulated intestinal epithelial tight junction barrier is mediated by myosin light-chain kinase protein expression through NFk-B activation. However, the alterations of tight junctional proteins involved in the TNF-alpha-induced increase of intestinal TJ permeability remain unclear. Claudin is essential to the formation and maintenance of tight junction (TJ) and has been identified 24 members so far. Claudin-1, 3, 4, 6, 10 and 16 have been shown altered in various cancers and they may have important roles in cell survival, motility, and invasion of cancer cells. However, the functions of these proteins in tumorigenesis and inflammation are still being elucidated.

  11. Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Pan Zhongzong

    2009-01-01

    Full Text Available Abstract Background Estrogen receptor-α (ERα is essential for mammary gland development and is a major oncogene in breast cancer. Since ERα is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERα mediated cell proliferation. In the paracrine model, ERα-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERα does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERα in some primary breast tumors. Methods Colocalization of ERα with Ki-67 in ERα-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1 was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERα was determined by co-immunofluorescent staining of ERα and the major cyclins (D, E, A, B, and by flow cytometry analysis of ERαhigh cells. To further confirm the autocrine action of ERα, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERα and cell cycle progression. Results Colocalization of ERα with Ki-67 was present in all three ERα-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERα is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERα and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERα. Conclusion Our data indicate

  12. The glomerular parietal epithelial cell's responses are influenced by SM22 alpha levels.

    Science.gov (United States)

    Naito, Shokichi; Pippin, Jeffrey W; Shankland, Stuart J

    2014-11-06

    Studies have shown in several diseases initially affecting podocytes, that the neighboring glomerular parietal epithelial cells (PECs) are secondarily involved. The PEC response might be reparative under certain circumstances, yet injurious under others. The factors governing these are not well understood. We have shown that SM22α, an actin-binding protein considered a marker of smooth muscle differentiation, is upregulated in podocytes and PECs in several models of podocyte disease. However, the impact of SM22α levels on PECs is not known. Experimental glomerular disease, characterized by primary podocyte injury, was induced in aged-matched SM22α+/+ and SM22α-/-mice by intraperitoneal injection of sheep anti-rabbit glomeruli antibody. Immunostaining methods were employed on days 7 and 14 of disease. The number of PEC transition cells, defined as cells co-expressing a PEC protein (PAX2) and podocyte protein (Synaptopodin) was higher in diseased SM22α-/-mice compared with SM22α+/+mice. WT1 staining along Bowman's capsule is higher in diseased SM22α-/-mice. This was accompanied by increased PEC proliferation (measured by ki-67 staining), and an increase in immunostaining for the progenitor marker NCAM, in a subpopulation of PECs in diseased SM22α-/-mice. In addition, immunostaining for vimentin and alpha smooth muscle actin, markers of epithelial-to-mesenchymal transition (EMT), was lower in diseased SM22α-/-mice compared to diseased SM22α+/+mice. SM22α levels may impact how PECs respond following a primary podocyte injury in experimental glomerular disease. Absent/lower levels favor an increase in PEC transition cells and PECs expressing a progenitor marker, and a lower EMT rate compared to SM22α+/+mice, where SM22 levels are markedly increased in PECs.

  13. Word class and context affect alpha-band oscillatory dynamics in an older population

    Directory of Open Access Journals (Sweden)

    Monika eMellem

    2012-04-01

    Full Text Available Differences in the oscillatory EEG dynamics of reading open class and closed class words have previously been found (Bastiaansen et al., 2005 and are thought to reflect differences in lexical-semantic content between these word classes. In particular, the theta band (4–7 Hz seems to play a prominent role in lexical-semantic retrieval. We tested whether this theta effect is robust in an older population of subjects. Additionally, we examined how the context of a word can modulate the oscillatory dynamics underlying retrieval for the two different classes of words. Older participants (mean age 55 read words presented in either syntactically-correct sentences or in a scrambled order (scrambled sentence while their EEG was recorded. We performed time-frequency analysis to examine how power varied based on the context or class of the word. We observed larger power decreases in the alpha (8–12Hz band between 200–700 ms for the open class compared to closed class words, but this was true only for the scrambled sentence context. We did not observe differences in theta power between these conditions. Context exerted an effect on the alpha and low beta (13–18 Hz bands between 0–700 ms. These results suggest that the previously observed word class effects on theta power changes in a younger participant sample do not seem to be a robust effect in this older population. Though this is an indirect comparison between studies, it may suggest the existence of aging effects on word retrieval dynamics for different populations. Additionally, the interaction between word class and context suggests that word retrieval mechanisms interact with sentence-level comprehension mechanisms in the alpha band.

  14. Upregulation of alpha cell glucagon-like peptide 1 (GLP-1) in Psammomys obesus--an adaptive response to hyperglycaemia?

    DEFF Research Database (Denmark)

    Hansen, A M K; Bödvarsdottir, T B; Nordestgaard, D N E

    2011-01-01

    of the study was to evaluate if, during the development of diabetes, alpha cells produce GLP-1 that, in turn, might trigger beta cell growth. Methods Beta cell mass, GLP-1 and insulin levels were measured in the gerbil Psammomys obesus (P. obesus), a rodent model of nutritionally induced diabetes. Furthermore...... from alpha cells is upregulated in P. obesus during the development of diabetes. A similar response is seen in islets exposed to high glucose, which supports the hypothesis that GLP-1 released from alpha cells promotes an increase in beta cell mass and function during metabolic challenge...

  15. Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion

    DEFF Research Database (Denmark)

    Iba, K; Albrechtsen, R; Gilpin, B J

    1999-01-01

    The ADAMs (A disintegrin and metalloprotease) comprise a family of membrane-anchored cell surface proteins with a putative role in cell-cell and/or cell-matrix interactions. By immunostaining, ADAM 12 (meltrin alpha) was up-regulated in several human carcinomas and could be detected along the tumor...... tumor cell adhesion. We found that the disintegrin-like domain of human ADAM 15 supported adhesion of alphavbeta3-expressing A375 melanoma cells. In the case of human ADAM 12, however, recombinant polypeptides of the cysteine-rich domain but not the disintegrin-like domain supported cell adhesion...... of a panel of carcinoma cell lines. On attachment to recombinant polypeptides from the cysteine-rich domain of human ADAM 12, most tumor cell lines, such as MDA-MB-231 breast carcinoma cells, were rounded and associated with numerous actin-containing filopodia and used a cell surface heparan sulfate...

  16. Individual Amino Acid Supplementation Can Improve Energy Metabolism and Decrease ROS Production in Neuronal Cells Overexpressing Alpha-Synuclein.

    Science.gov (United States)

    Delic, Vedad; Griffin, Jeddidiah W D; Zivkovic, Sandra; Zhang, Yumeng; Phan, Tam-Anh; Gong, Henry; Chaput, Dale; Reynes, Christian; Dinh, Vinh B; Cruz, Josean; Cvitkovic, Eni; Placides, Devon; Frederic, Ernide; Mirzaei, Hamed; Stevens, Stanley M; Jinwal, Umesh; Lee, Daniel C; Bradshaw, Patrick C

    2017-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein accumulation and loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Increased levels of alpha-synuclein have been shown to result in loss of mitochondrial electron transport chain complex I activity leading to increased reactive oxygen species (ROS) production. WT alpha-synuclein was stably overexpressed in human BE(2)-M17 neuroblastoma cells resulting in increased levels of an alpha-synuclein multimer, but no increase in alpha-synuclein monomer levels. Oxygen consumption was decreased by alpha-synuclein overexpression, but ATP levels did not decrease and ROS levels did not increase. Treatment with ferrous sulfate, a ROS generator, resulted in decreased oxygen consumption in both control and alpha-synuclein overexpressing cells. However, this treatment only decreased ATP levels and increased ROS production in the cells overexpressing alpha-synuclein. Similarly, paraquat, another ROS generator, decreased ATP levels in the alpha-synuclein overexpressing cells, but not in the control cells, further demonstrating how alpha-synuclein sensitized the cells to oxidative insult. Proteomic analysis yielded molecular insights into the cellular adaptations to alpha-synuclein overexpression, such as the increased abundance of many mitochondrial proteins. Many amino acids and citric acid cycle intermediates and their ester forms were individually supplemented to the cells with L-serine, L-proline, L-aspartate, or L-glutamine decreasing ROS production in oxidatively stressed alpha-synuclein overexpressing cells, while diethyl oxaloacetate or L-valine supplementation increased ATP levels. These results suggest that dietary supplementation with individual metabolites could yield bioenergetic improvements in PD patients to delay loss of dopaminergic neurons.

  17. Transport of alpha- and beta-D-glucose by the intact human red cell

    Energy Technology Data Exchange (ETDEWEB)

    Carruthers, A.; Melchior, D.L.

    1985-07-16

    The kinetics of alpha- and beta-D-glucose mutarotation and the transport of these anomers by intact human red cells were determined at 0.6 and 36.6 degrees C. The mutarotation coefficients for alpha- and beta-D-glucose in cell-free tris(hydroxymethyl)aminomethane medium (pH 7.4) at 0.6 degrees C are (2.25 +/- 0.2) and (1.73 +/- 0.42) X 10(-3) min-1, respectively, and at 36.6 degrees C are (69 +/- 12) and (75 +/- 5) X 10(-3) min-1, respectively. These values are in good agreement with previous estimates. At 0.6 degrees C, the red cell contains no detectable mutarotase activity. Initial rates of sugar uptake were measured by using radiolabeled D-glucose and time courses of uptake by turbidimetry. The time courses of alpha- and beta-D-glucose and an equilibrium mixture of alpha- and beta-D-glucose infinite-cis entry are identical at 0.66 degrees C (n = 41) where negligible mutarotation is observed. The apparent Ki values for inhibition of radiolabeled D-glucose initial uptake by unlabeled alpha- or beta-D-glucose at 0.6 degrees C are identical (1.6 mM). The calculated Vmax parameters for uptake of the radiolabeled anomers at this temperature are also indistinguishable. The time courses of infinite-cis alpha- and beta-D-glucose uptake at 36.66 degrees C are identical (n = 40). While D-glucose mutarotation is more rapid at this temperature, the anomers of D-glucose are not transported differently by the red cell hexose transfer system.

  18. Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells : role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway

    NARCIS (Netherlands)

    Haslinger, Bettina; Strangfeld, Katrin; Peters, Georg; Schulze-Osthoff, Klaus; Sinha, Bhanu

    2003-01-01

    Staphylococcus aureus infections can result in septic and toxic shock with depletion of immune cells and massive cytokine production. Recently, we showed that, in S. aureus-infected Jurkat T cells, alpha-toxin is the major mediator of caspase activation and apoptosis. Here, we investigated the

  19. Tumour necrosis factor-alpha (TNF-alpha) transcription and translation in the CD4+ T cell-transplanted scid mouse model of colitis

    DEFF Research Database (Denmark)

    Williams, A M; Whiting, C V; Bonhagen, K

    1999-01-01

    The adoptive transfer of activated CD4+ alpha/beta T cell blasts from the spleens of immunocompetent C.B-17+/+ or BALB/cdm2 mice into C.B-17scid/scid (scid) mice induces a colitis in the scid recipient within 8 weeks, which progresses to severe disease within 16 weeks. T cells isolated from...

  20. Alpha 4 beta 1 and alpha 5 beta 1 are differentially expressed during myelopoiesis and mediate the adherence of human CD34+ cells to fibronectin in an activation-dependent way

    NARCIS (Netherlands)

    Kerst, J. M.; Sanders, J. B.; Slaper-Cortenbach, I. C.; Doorakkers, M. C.; Hooibrink, B.; van Oers, R. H.; von dem Borne, A. E.; van der Schoot, C. E.

    1993-01-01

    To study the receptors involved in the interaction between extracellular matrix proteins and hematopoietic progenitor cells, we analyzed the expression of beta 1 integrins on CD34+ bone marrow cells by means of immunoflowcytometry. Alpha 4 beta 1 and alpha 5 beta 1 were expressed, whereas alpha 1

  1. Influence of catechins on bystander responses in CHO cells induced by alpha-particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Law, Y.L.; Wong, T.P.W. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Yu, K.N. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong)], E-mail: peter.yu@cityu.edu.hk

    2010-04-15

    In this work, we studied alpha-particle induced and medium-mediated bystander effects in Chinese hamster ovary (CHO) cells through micronucleus (MN) assay. We showed that signal transduction from irradiated cells to bystander cells occur within a short time after irradiation. We then studied the effects of ROS (reactive oxygen species)-scavenging catechins in the medium before irradiation. We observed decreases in the percentage of bystander cells with MN formation and thus proved the protection effect of catechins on bystander cells from radiation.

  2. Rasagiline protects against alpha-synuclein induced sensitivity to oxidative stress in dopaminergic cells.

    Science.gov (United States)

    Chau, K Y; Cooper, J M; Schapira, A H V

    2010-11-01

    Rasagiline is a propargylamine and irreversible monoamine oxidase (MAO) B inhibitor used for the treatment of Parkinson's disease (PD). It has demonstrated neuroprotective properties in laboratory studies. Current concepts of PD aetiopathogenesis include the role of alpha-synuclein, protein aggregation, free radical metabolism and mitochondrial dysfunction in contributing to cell death. We have used a combination of alpha-synuclein and free radical mediated toxicity in a dopaminergic cell line to provide a model of nigral toxicity in order to investigate the potential molecular mechanisms that mediate rasagiline protection. We demonstrate that rasagiline protects against cell death induced by the combination of free radicals generated by paraquat and either wild-type or A53T mutant alpha-synuclein over-expression. This protection was associated with a reduction in caspase 3 activation, a reduction in superoxide generation and a trend to ameliorate the fall in mitochondrial membrane potential. Rasagiline induced an increase in cellular glutathione levels. The results support a role for rasagiline in protecting dopaminergic cells against free radical mediated damage and apoptosis in the presence of alpha-synuclein over-expression. The data are of relevance to the interpretation of the potential mechanisms of action of rasagiline in explaining the results of disease modification trials in PD. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. Aggressive giant cell granuloma of the jaws treated with interferon alpha: a report of two cases.

    Science.gov (United States)

    O'Connell, J E; Kearns, G J

    2013-06-01

    Giant cell granulomas (GCGs) are benign tumours of the jaws of unknown aetiology. Aggressive lesions are difficult to manage and demonstrate a tendency to recur after surgical curettage. In the early 1980s, interferon alpha-2a was found to inhibit angiogenesis through a series of laboratory experiments and was subsequently used to treat a child with pulmonary haemangiomatosis. It has been hypothesised that GCGs are proliferative vascular lesions and would, therefor, be expected to respond to antiangiogenic therapy. The purpose of this study is to report a treatment protocol consisting of enucleation, followed by subcutaneous interferon alpha. Patients with a biopsy-confirmed giant cell lesion satisfying criteria for "aggressive" giant cell tumours were included. All lesions were enucleated, and the patients commenced interferon alpha-2a (3,000,000 units/m(2)) 48-72 h post-operatively. Two patients satisfied the criteria for aggressive giant cell lesions. All tumours were enucleated. There were no post-operative complications, and all patients tolerated the interferon therapy well. To date, there has been no evidence of tumour recurrence. The follow-up periods were 144 and 81 months, respectively. Antiangiogenic therapy, in combination with curettage, has proven to be a useful strategy for the management of these tumours. The use of interferon alpha-2a, following enucleation of these lesions, resulted in complete remission of all lesions, and decreased operative morbidity compared with conventional treatment.

  4. Specific alpha v integrin receptors modulate K1735 murine melanoma cell behavior.

    Science.gov (United States)

    Yang, Yongjian; Dang, Dongmin; Atakilit, Amha; Schmidt, Brian; Regezi, Joseph; Li, Xiaowu; Eisele, David; Ellis, Duncan; Ramos, Daniel M

    2003-09-05

    Expression of beta 3 integrins is increased in invasive melanoma. In this study we show that K1735 cell proliferation is enhanced by the expression of either beta 3 or a constitutively active Src. We investigated possible modulators of FN matrix assembly and found that matrix metalloproteinase 2 (MMP2) was activated by alpha v beta 3. alpha v beta 3 integrin was localized to focal contacts whereas alpha v beta 5 was peripherally distributed. MMP2 was also activated by expression of CASrc. MMP2 activation inversely correlated with FN matrix assembly, in that it dramatically reduced the organization of a FN matrix. K1735 cell migration on VN and invasion through a reconstituted basement membrane were decreased in the presence of anti-MMP2 antibodies. These results demonstrate that the expression of the alpha v beta 3 complex modulates melanoma cell behavior including activation of Src, organization of the cytoskeleton, assembly of the extracellular matrix, cell motility, and activation of MMP2.

  5. The alpha-smooth muscle actin-positive cells in healing human myocardial scars

    NARCIS (Netherlands)

    Willems, I. E.; Havenith, M. G.; de Mey, J. G.; Daemen, M. J.

    1994-01-01

    Interstitial cells in the scars of human myocardial infarctions of different postinfarction times (6 hours to 17 years old) were characterized by antibodies to alpha-smooth muscle actin (ASMA), vimentin, and desmin. Basal lamina deposition was studied with antibodies to the basal lamina protein type

  6. Genomic organization of the human T-cell antigen-receptor alpha/delta locus

    NARCIS (Netherlands)

    Satyanarayana, K.; Hata, S.; Devlin, P.; Roncarolo, M. G.; de Vries, J. E.; Spits, H.; Strominger, J. L.; Krangel, M. S.

    1988-01-01

    Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V

  7. Erythropoietin protects myocardin-expressing cardiac stem cells against cytotoxicity of tumor necrosis factor-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Madonna, Rosalinda [The Center for Cardiovascular Biology and Atherosclerosis Research, The University of Texas Health Science Center at Houston, Texas (United States); Institute of Cardiology, and Center of Excellence on Aging, ' G. d' Annunzio' University, Chieti (Italy); Shelat, Harnath; Xue, Qun; Willerson, James T. [The Center for Cardiovascular Biology and Atherosclerosis Research, The University of Texas Health Science Center at Houston, Texas (United States); The Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, Texas (United States); De Caterina, Raffaele [Institute of Cardiology, and Center of Excellence on Aging, ' G. d' Annunzio' University, Chieti (Italy); Geng, Yong-Jian, E-mail: yong-jian.geng@uth.tmc.edu [The Center for Cardiovascular Biology and Atherosclerosis Research, The University of Texas Health Science Center at Houston, Texas (United States); The Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, Texas (United States)

    2009-10-15

    Cardiac stem cells are vulnerable to inflammation caused by infarction or ischemic injury. The growth factor, erythropoietin (Epo), ameliorates the inflammatory response of the myocardium to ischemic injury. This study was designed to assess the role of Epo in regulation of expression and activation of the cell death-associated intracellular signaling components in cardiac myoblasts stimulated with the proinflammatory cytokine tumor necrosis factor (TNF)-{alpha}. Cardiac myoblasts isolated from canine embryonic hearts characterized by expression of myocardin A, a promyogenic transcription factor for cardiovascular muscle development were pretreated with Epo and then exposed to TNF-{alpha}. Compared to untreated cells, the Epo-treated cardiac myoblasts exhibited better morphology and viability. Immunoblotting revealed lower levels of active caspase-3 and reductions in iNOS expression and NO production in Epo-treated cells. Furthermore, Epo pretreatment reduced nuclear translocation of NF-{kappa}B and inhibited phosphorylation of inhibitor of kappa B (I{kappa}B) in TNF-{alpha}-stimulated cardiac myoblasts. Thus, Epo protects cardiac myocyte progenitors or myoblasts against the cytotoxic effects of TNF-{alpha} by inhibiting NF-{kappa}B-mediated iNOS expression and NO production and by preventing caspase-3 activation.

  8. Allele-specific expression of the IL-1 alpha gene in human CD4+ T cell clones

    NARCIS (Netherlands)

    Bayley, Jean-Pierre; van Rietschoten, Johanna G. I.; Bakker, Aleida M.; van Baarsen, Lisa; Kaijzel, Eric L.; Wierenga, Eddy A.; van der Pouw Kraan, Tineke C. T. M.; Huizinga, Tom W. J.; Verweij, Cornelis L.

    2003-01-01

    A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1alpha gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1alpha gene shows

  9. Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

    International Nuclear Information System (INIS)

    Le Marchand-Brustel, Y.; Ballotti, R.; Gremeaux, T.; Tanti, J.F.; Brandenburg, D.; Van Obberghen, E.

    1989-01-01

    Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form

  10. Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Alexandra M Fajardo

    Full Text Available Tocopherylquinone (TQ, the oxidation product of alpha-tocopherol (AT, is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells, whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.

  11. Cell Cycle Checkpoint Proteins p21 and Hus1 Regulating Intercellular Signaling Induced By Alpha Particle Irradiation

    Science.gov (United States)

    Wu, Lijun; Zhao, Ye; Wang, Jun; Hang, Haiying

    In recent years, the attentions for radiation induced bystander effects (RIBE) have been paid on the intercellular signaling events connecting the irradiated and non-irradiated cells. p21 is a member of the Cip/Kip family and plays essential roles in cell cycle progression arrest after cellular irradiation. DNA damage checkpoint protein Hus1 is a member of the Rad9-Rad1-Hus1 complex and functions as scaffold at the damage sites to facilitate the activation of downstream effectors. Using the medium trasfer method and the cells of MEF, MEF (p21-/-), MEF (p21-/-Hus1-/-) as either medium donor or receptor cells, it was found that with 5cGy alpha particle irradiation, the bystander cells showed a significant induction of -H2AX for normal MEFs (p¡0.05). However, the absence of p21 resulted in deficiency in inducing bystander effects. Further results indicated p21 affected the intercellular DNA damage signaling mainly through disrupting the production or release of the damage signals from irradiated cells. When Hus1 and p21 were both knocked out, an obvious induction of -H2AX recurred in bystander cells and the induction of -H2AX was GJIC (gap junction-mediated intercellular communication) dependent, indicating the interrelationship between p21 and Hus1 regulated the production and relay of DNA damage signals from irradiated cells to non-irradiated bystander cells.

  12. Diverse inflammatory cytokines induce selectin ligand expression on murine CD4 T cells via p38 alpha MAP kinase1

    Science.gov (United States)

    Ebel, Mark E.; Awe, Olufolakemi; Kaplan, Mark H.; Kansas, Geoffrey S.

    2015-01-01

    Selectins are glycan-binding adhesion molecules which mediate the initial steps of leukocyte recognition of endothelium. Cytokines control numerous aspects of CD4 T helper differentiation, but how cytokines control induction of ligands for E- and P-selectin on T helper subsets remains poorly understood. Among 20 cytokines that affect T helper cell differentiation, we identified six, IL-12, IL-18, IL-27, IL-9, IL-25 and TGFβ1, that induce expression of selectin ligands on murine CD4 T cells above the low levels associated with TCR engagement. Collectively, these six cytokines could potentially account for selectin ligand expression on all of the currently defined non-sessile T helper lineages, including Th1, Th2, Th9, Th17 and Treg. Induction of selectin ligand expression by each of these six cytokines was almost completely inhibited by pharmacologic inhibition of p38 MAPK, but not other MAPKs, or by conditional genetic deletion of p38 alpha MAPK. Analysis of the expression of key glycosyltransferase genes revealed that p38 alpha signaling was selectively required for induction of Fut7 and Gcnt1, but not for induction of St3gal4 or St3gal6. Constitutively active MKK6, an immediate upstream activator of p38 MAPK, induced selectin ligand expression equivalent to that of cytokines, and this induction was completely dependent on expression of p38 alpha. Our results identify the repertoire of cytokines responsible for selectin ligand induction on CD4 T cells and provide a mechanistic link between T helper development and T cell migration. PMID:25941329

  13. Cytotoxicity effects induced by Zearalenone metabolites, alpha Zearalenol and beta Zearalenol, on cultured Vero cells.

    Science.gov (United States)

    Othmen, Zouhour Ouanes-Ben; Golli, Emna El; Abid-Essefi, Salwa; Bacha, Hassen

    2008-10-30

    Zearalenone (Zen) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium. It has been implicated in several mycotoxicosis in farm animals and in humans. The major metabolites of this mycotoxin in various species are alpha and beta Zearalenol. In vivo, Zen is mainly reduced to these alcoholic metabolites which cause reproductive tract disorders and impaired fertility due to their estrogenic activities. In this study, we examined the cytotoxicity of alpha and beta Zearalenol in cultured cells. For this purpose, the MTT assay was carried out and the influence of alpha and beta Zearalenol on protein and DNA syntheses was assessed. To evaluate the cell stress caused by these two metabolites, oxidative stress measured by MDA induction and stress protein induction (Hsp 70, Hsp 27) were tested. Results showed that alpha and beta Zearalenol were metabolites that caused cytotoxicity by inhibiting cell viability, protein and DNA syntheses and inducing oxidative damage and over-expression of stress proteins. However, the Zen metabolites exhibited lower toxicity than Zen, with beta zearalenol being the more active of the two metabolites.

  14. The influence of nonuniform micro-distribution of alpha emitter on microdosimetry in cells

    International Nuclear Information System (INIS)

    Tian Yuan; Zhang Liang'an; Dai Guangfu

    2007-01-01

    Objective: To study the influence of nonuniform micro-distribution of alpha emitter on cellular S values(in the radioimmunotherapy). Methods: Emission of alpha particles is randomly simulated by Monte Carlo method; the incident energy and exit energy are calculated with interpolation technique based on the relationship between range and energy of alpha particle and the analytical Continuous Slowing Down Approximation (CSDA) model. So energy deposited in the target area can be obtained. To take 213 Po as an example, cellular S values with various cell dimensions and possible micro-distributions of radioactivity are calculated, such as linear increase, linear decrease, exponential increase and exponential decrease. Results: S values from cell to cell of uniform distribution showed no difference with the Hamacher's results. S values of different micro-distributions are distinguishing with each other. It is indicated that different micro-distributions of radioactivity will result in significant change of average chord length of alpha particles traveling in the target area, as well as the change of average stopping power over the chord, which is primary reason for differences of S values. Conclusions: The nonuniform micro-distributions show remarkable influence on cellular S values and hence should be taken consideration in cellular absorbed dose estimation, especially in microdosimetry. (authors)

  15. 1alpha-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines.

    Science.gov (United States)

    Lagishetty, Venu; Chun, Rene F; Liu, Nancy Q; Lisse, Thomas S; Adams, John S; Hewison, Martin

    2010-07-01

    Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). In cells such as macrophages, expression of 1alpha-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and beta-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1alpha-hydroxylase. Alternative activators of 1alpha-hydroxylase such as transforming growth factor beta were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  16. Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

    Science.gov (United States)

    Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

    1996-11-01

    The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3

  17. Retinoic acid dramatically enhances the arsenic trioxide-induced cell cycle arrest and apoptosis in retinoic acid receptor alpha-positive human T-cell lymphotropic virus type-I-transformed cells.

    Science.gov (United States)

    Darwiche, N; El-Sabban, M; Bazzi, R; Nasr, R; Al-Hashimi, S; Hermine, O; de Thé, H; Bazarbachi, A

    2001-01-01

    Adult T-cell leukemia/lymphoma, caused by the human T-cell lymphotropic virus type I, is an aggressive neoplasm of mature activated T cells that is generally resistant to conventional therapy. While arsenic trioxide (As) inhibits the growth and induces apoptosis in HTLV-I-infected T cells, synergistically, when combined with interferon-alpha, variable effects on growth with all trans retinoic acid treatment have been reported in ATL-derived cell lines and fresh ATL cells. In this study, we investigate the effects of ATRA alone or in combination with As in HTLV-I-transformed cells. Four HTLV-I-transformed cell lines (HuT-102, MT2, C8166 and C91PL) were treated with different doses of ATRA alone or in combination with As for one to three days. Cell growth was assessed by cell count with 3H-thymidine incorporation. Cell cycle distribution was assessed by propidium iodine-labeled DNA content by flow cytometry. Apoptosis was evaluated by Hoechst nuclear staining and annexin-V binding assays. Expression of retinoid receptors, the viral transactivator Tax, and the proteins bcl-2 and IkappaB-alpha proteins, was analysed by Western blot. Only C8166 cells were sensitive to the ATRA-induced growth inhibitory effect while HuT-102, MT2, and C91PL were resistant to ATRA treatment (up to 10(-5) M). The retinoid X receptor alpha and the retinoic acid receptor gamma (RARgamma) proteins were expressed in all four cell lines, while RARalpha protein was only detected in the HuT-102 and C8166 cells. The combination ATRA/As showed a highly synergistic effect on HuT-102 cells, and, to a lesser extent, on C8166 cells and resulted in a dramatic inhibition of cell proliferation and induction of massive apoptosis in HuT-102 cells, associated with caspase activation. While ATRA alone had no effect on Tax and IkappaB-alpha protein levels, ATRA increased the As-induced Tax degradation and the up-regulation of IkappaB-alpha protein. In contrast, the expression of bcl-2 protein was not

  18. Alpha-Gamma Hot-Cell Facility at Argonne National Laboratory East

    International Nuclear Information System (INIS)

    Neimark, L.A.; Jackson, W.D.; Donahue, D.A.

    1979-01-01

    The Alpha-Gamma Hot-Cell Facility has been in operation at Argonne National Laboratory East (ANL-E) for 15 years. The facility was designed for plutonium research in support of ANL's LMFBR program. The facility consists of a kilocurie, nitrogen-atmosphere alpha-gamma hot cell and supporting laboratories. Modifications to the facility and its equipment have been made over the years as the workload and nature of the work changed. These modifications included inerting the entire hot cell, adding four work stations, modifying in-loading procedures and examination equipment to handle longer test articles, and changing to a new sodium-vapor lighting system. Future upgrading includes the addition of a decontamination and repair facility, use of radio-controlled transfer carts, refurbishment of the zinc bromide windows, and the installation of an Auger microprobe

  19. [Expression of interferon alpha family gene of Chinese marmot in eukaryotic and prokaryotic cells].

    Science.gov (United States)

    Lu, Yin-ping; Wang, Bao-ju; Huang, Hong-ping; Tian, Yong-jun; Yang, Yan; Dong, Ji-hua; Lu, Meng-ji; Yang, Dong-liang

    2006-02-01

    To investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells. Eukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity. The Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction. The IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.

  20. Induction of a bystander mutagenic effect of alpha particles in mammalian cells

    Science.gov (United States)

    Zhou, H.; Randers-Pehrson, G.; Waldren, C. A.; Vannais, D.; Hall, E. J.; Hei, T. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    Ever since the discovery of X-rays was made by Rontgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear/extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected A(L) cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 microM dose of lindane, which inhibits cell-cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5-10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.

  1. TNF-alpha is a principal cytokine involved in the recruitment of NK cells to liver parenchyma.

    Science.gov (United States)

    Pilaro, A M; Taub, D D; McCormick, K L; Williams, H M; Sayers, T J; Fogler, W E; Wiltrout, R H

    1994-07-01

    Isolated murine splenic NK cells and the cultured murine endothelioma cell line, eEND2, were used to study the effects of cytokines on NK cell/endothelial cell adhesion. Treatment of eEND2 cells with TNF-alpha induced a marked increase (four- to sevenfold) in adherence of NK cells, as compared with control cultures of endothelioma cells or eEND2 cells treated with IL-1 alpha or IL-6. TNF-alpha induction of NK cell adherence to eEND2 was dose dependent with rapid kinetics, reaching a maximum at concentrations between 10 and 1000 U/ml after a 2-h incubation. TNF-alpha treatment of L929 fibroblasts or CL-2 hepatoma cells did not result in increased NK cell adhesion. The concentration range of TNF-alpha that was found to maximally augment NK cell adhesion to eEND2 also induced NK cell chemokinetic activity. The relevance of these in vitro results was subsequently analyzed in vivo. Initial studies confirmed that a single dose of polyinosinic-polycytidylic acid and poly-L-lysine stabilized in carboxymethyl cellulose (poly-ICLC), augmented hepatic NK activity and resulted in a 2.2-fold increase in the number of liver-associated NK cells. Concomitant treatment of mice with a TNF-alpha neutralizing antisera eliminated both the hepatic influx of NK cells and the increase in poly-ICLC-induced liver NK activity. These results suggest that TNF-alpha is a principal cytokine involved in the in vivo recruitment and localization of parenchymal NK cells after treatment with a biological response modifier, and that this regulation seems to occur via alterations in NK cell/endothelial cell interactions.

  2. Two Bg1II RFLPs of the human. alpha. -globin gene cluster in the American sickle cell population

    Energy Technology Data Exchange (ETDEWEB)

    Embury, S.H.; Blachman, T.; Kroop, G.L.; Suzuki, J.K.; Boyle, M. (Univ. of California and Northern California Comprehensive Sickle Cell Center, San Fransicso (USA))

    1989-11-11

    Human {alpha}-globin cDNA cloned into plasmid pMB9(JW101) was used as a hybridization probe for assessing the {alpha}-globin genotypes of 2271 Americans with sickle cell anemia. The normal duplicated human {alpha}-globin genes, {alpha}2 and {alpha}1, residue on separate Bg1 II fragments, each of which is cleaved by Hin dIII. Both {alpha} loci reside on a single 14 kb Bam HI fragment. The authors performed single Bg1 II and BAM HI digests to detect {alpha}-globin gene deletions in 2271 subjects enrolled in the National Cooperative Study of Sickel Cell Disease (NCSSCD). In addition to gene deletions and duplications, two Bg1 II RFLP were found. The human {alpha}-globin genes reside on the short arm of chromosome 16. The {alpha}2-specific RFLP occurs in linkage dysequilibrium and the mother of one subject with the {alpha}1-specific RFLP had this RFLP, suggesting their Mendelian inheritance.

  3. Transmembrane signaling by interferon alpha involves diacylglycerol production and activation of the epsilon isoform of protein kinase C in Daudi cells.

    OpenAIRE

    Pfeffer, L M; Eisenkraft, B L; Reich, N C; Improta, T; Baxter, G; Daniel-Issakani, S; Strulovici, B

    1991-01-01

    The early events that occur after treatment of the highly interferon alpha (IFN-alpha)-sensitive human lymphoblastoid Daudi cell line with human leukocyte IFN-alpha have been examined. IFN-alpha treatment of Daudi cells results in a rapid and transient increase in the cellular content of diacylglycerol, which occurs in the absence of inositol phospholipid turnover, or an increase in intracellular calcium concentration. Furthermore, IFN-alpha treatment results in a selective, time-dependent ac...

  4. Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

    Energy Technology Data Exchange (ETDEWEB)

    Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.; Shyamala, G.; Moses, Harold L.; Barcellos-Hoff, Mary Helen

    2005-03-03

    Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha} co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.

  5. Interleukin-8 production in response to tumor necrosis factor-alpha by cholesteatoma keratinocytes in cell culture.

    Science.gov (United States)

    Hilton, Christopher W; Ondrey, Frank G; Wuertz, Beverley R; Levine, Samuel C

    2011-02-01

    Keratinocytes harvested from acquired cholesteatoma and grown in cell culture will demonstrate increased interleukin-8 (IL-8) production in response to tumor necrosis factor (TNF)-alpha as compared with a control keratinocyte cell line. Immunohistochemical studies have identified IL-8 and TNF-alpha, mediators of bony destruction, in tissue samples of cholesteatoma. TNF-alpha stimulates IL-8 production in healthy epidermal keratinocyte cell lines. It is not known whether TNF-alpha stimulates IL-8 production in cultured cholesteatoma keratinocytes. Prospective controlled tissue culture experiment. Tissue from an acquired cholesteatoma was dissociated and grown in keratinocyte serum-free media for 8 weeks. Cholesteatoma keratinocytes and a control cell line of skin epidermal keratinocytes were treated with TNF-alpha. Conditioned media were harvested; production of IL-8 was measured by enzyme-linked immunosorbent assay, and cell counts were performed. At a zero concentration of TNF-alpha, mean production of IL-8 by cholesteatoma keratinocytes was 39,809 pg/mL/24hr/1 × 10(6) cells versus 1,907 pg/mL/24hr/1 × 10(6) cells from skin epidermal keratinocytes, a statistically significant difference (P alpha and a 2.44-fold increase in response to 20 pg/mL of TNF-alpha. The skin epidermal keratinocyte cell line demonstrated a 1.07- and 1.13-fold increase to respective concentrations of TNF-alpha. Cholesteatoma keratinocytes appear to retain cell signaling characteristics in vitro that distinguish them from skin epidermal keratinocytes. This finding may indicate that cholesteatoma keratinocytes undergo a change in behavior in vivo that is preserved after the cells are removed from the inflammatory environment of the middle ear. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

  6. Metallothionein treatment reduces proinflammatory cytokines IL-6 and TNF-alpha and apoptotic cell death during experimental autoimmune encephalomyelitis (EAE)

    DEFF Research Database (Denmark)

    Penkowa, M; Hidalgo, J

    2001-01-01

    . However, it is not known how EAE progression is regulated nor how cytokine production and cell death can be reduced. We herewith demonstrate that treatment with Zn-MT-II significantly decreased the CNS expression of IL-6 and TNF-alpha during EAE. Zn-MT-II treatment could also significantly reduce...... apoptotic cell death of neurons and oligodendrocytes during EAE, as judged by using TUNEL and immunoreactivity for cytochrome c and caspases 1 and 3. In contrast, the number of apoptotic lymphocytes and macrophages was less affected by Zn-MT-II treatment. The Zn-MT-II-induced decrease in proinflammatory...... cytokines and apoptosis during EAE could contribute to the reported diminution of clinical symptoms and mortality in EAE-immunized rats receiving Zn-MT-II treatment. Our results demonstrate that MT-II reduces the CNS expression of proinflammatory cytokines and the number of apoptotic neurons during EAE...

  7. The regulation of catalase activity by PPAR gamma is affected by alpha-synuclein

    NARCIS (Netherlands)

    Yakunin, Eugenia; Kisos, Haya; Kulik, Willem; Grigoletto, Jessica; Wanders, Ronald J. A.; Sharon, Ronit

    2014-01-01

    Objective: While evidence for oxidative injury is frequently detected in brains of humans affected by Parkinson's disease (PD) and in relevant animal models, there is uncertainty regarding its cause. We tested the potential role of catalase in the oxidative injury that characterizes PD. Methods:

  8. Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

    Science.gov (United States)

    Graham, G J; Wilkinson, P C; Nibbs, R J; Lowe, S; Kolset, S O; Parker, A; Freshney, M G; Tsang, M L; Pragnell, I B

    1996-12-02

    We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.

  9. Collagen-IV and laminin-1 regulate estrogen receptor alpha expression and function in mouse mammary epithelial cells.

    Science.gov (United States)

    Novaro, Virginia; Roskelley, Calvin D; Bissell, Mina J

    2003-07-15

    The expression level and functional activity of estrogen receptor alpha is an important determinant of breast physiology and breast cancer treatment. However, it has been difficult to identify the signals that regulate estrogen receptor because cultured mammary epithelial cells generally do not respond to estrogenic signals. Here, we use a combination of two- and three-dimensional culture systems to dissect the extracellular signals that control endogenous estrogen receptor alpha. Its expression was greatly reduced when primary mammary epithelial cells were placed on tissue culture plastic; however, the presence of a reconstituted basement membrane in combination with lactogenic hormones partially prevented this decrease. Estrogen receptor alpha expression in primary mammary fibroblasts was not altered by these culture conditions, indicating that its regulation is cell type specific. Moreover, estrogen receptor-dependent reporter gene expression, as well as estrogen receptor alpha levels, were increased threefold in a functionally normal mammary epithelial cell line when reconstituted basement membrane was added to the medium. This regulatory effect of reconstituted basement membrane was reproduced by two of its components, collagen-IV and laminin-1, and it was blocked by antibodies against alpha2, alpha6 and beta1 integrin subunits. Our results indicate that integrin-mediated response to specific basement membrane components, rather than cell rounding or cell growth arrest induced by reconstituted basement membrane, is critical in the regulation of estrogen receptor alpha expression and function in mammary epithelial cells.

  10. In vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero TGEV coronavirus injection.

    Science.gov (United States)

    Splíchal, I; Reháková, Z; Sinkora, M; Sinkora, J; Trebichavský, I; Laude, H; Charley, B

    1997-05-01

    Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFN alpha) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFN alpha secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFN alpha-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFN alpha was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFN alpha in utero inducibility by mutant virus as compared with wild virus. Flow cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFN alpha secretion. The present data provided in vivo evidence of IFN alpha secretion at the cell level in foetal lymphohaematopoietic organs. Such IFN alpha-secreting cells in lymphohaematopoietic tissues may be the source of IFN alpha detected during foetal infections.

  11. Cells of the rectum of Bombyx mori affected by experimental inoculation with Alphabaculovirus.

    Science.gov (United States)

    Vessaro-Silva, S A; Brancalhão, R M C; Baggio, M P D; Ribeiro, L F C

    2014-08-29

    Bombyx mori is an insect whose cocoon is used in the sericulture industry, which is an important activity in parts of southern Brazil. When parasitized by Bombyx mori nucleopolyhedrovirus (BmNPV) of the genus Alphabaculovirus (alphaBV), it develops nuclear polyhedrosis disease. In Brazil, an alphaBV was isolated from larvae of B. mori and various target tissues were identified. However, how this geographic viral isolate affects the rectum of silk moths was unknown. The rectum, a component of the cryptonephric system, acts to absorb water and mineral salts, and its importance for the metabolic balance of insects provoked interest in analyzing how it is affected by BmMNPV infection. Fifth instar B. mori larvae were inoculated with a viral suspension, and from the second to the ninth day post-inoculation, segments of the rectum (anterior and anal canal) were examined using light microscopy. The cryptonephric epithelial cells in the anterior region revealed no evidence of infection. However, from the fifth day post-inoculation, cells of the anal canal showed cytopathologies characteristic of alphaBV. Infection of the anal canal and other surrounding tissues led to tissue disorganization, with accumulation of polyhedra in the perinephric space and compartmentalization of the cryptonephric system, promoting changes in the fecal pellets, signalling physiological changes. These observations contribute to our understanding of the infectious cycle of BmMNPV in B. mori.

  12. Emotion regulation and positive affect in the context of salivary alpha-amylase response to pain in children with cancer.

    Science.gov (United States)

    Jenkins, Brooke N; Granger, Douglas A; Roemer, Ryan J; Martinez, Ariana; Torres, Tara K; Fortier, Michelle A

    2018-01-19

    Children with cancer routinely undergo painful medical procedures invoking strong physiological stress responses. Resilience to this pain may be conferred through resources such as emotion regulation strategies and positive affect. This study measured dispositional positive affect in children with cancer (N = 73) and randomly assigned participants to one of three emotion regulation strategy conditions (distraction, reappraisal, or reassurance). Children applied their assigned strategy during an experimental pain procedure (the cold pressor task [CPT]) and provided saliva samples before, immediately after, and 15 min after the CPT. Saliva samples were later assayed for salivary alpha amylase (sAA)-a surrogate marker for autonomic/sympathetic nervous system activity and regulation. Children in the reassurance group had sAA levels that continued to rise after completion of the CPT compared to children in the distraction (b = -1.68, P = 0.021) and reappraisal conditions (b = -1.24, P = 0.084). Furthermore, dispositional positive affect moderated the effect of condition such that children in the reassurance group with lower levels of positive affect had sAA levels that continued to rise after completion of the CPT (dy/dx = 1.56, P = 0.027), whereas children in the reassurance condition with higher levels of positive affect did not exhibit this rise (P > 0.05). Specific emotion regulation strategies, such as distraction and reappraisal, may attenuate the stress response to pain in pediatric patients with cancer, and positive affect may confer resilience in response to pain even with use of less effective coping strategies such as reassurance. © 2018 Wiley Periodicals, Inc.

  13. Radio-induced inactivation and transformation of rat epithelial cell by alpha particles

    International Nuclear Information System (INIS)

    Poncy, Jean-Luc; Tourdes, Francoise; Bailly, Isabelle

    2003-01-01

    Following alpha- and gamma-irradiations, radio-induced cell inactivation and cellular transformation were measured in primary cultures of tracheal epithelial cells from two rat strains, Sprague Dawley (SD) and Wistar Furth/Fisher F344 (WF/Fi) rats. The survival ratio for each of the two rat strain cells appeared to be statistically different after alpha irradiation. WF/Fi rat cells were 1.7 times more radiosensitive than SD rat cells, whereas no difference is observed following low LET irradiation. Comparison of survival of the cells yielded an RBE of 2.8 and 4.5 for SD and WF/Fi rat cells respectively. The relative transformation frequency (RTF) for WF/Fi primary cells was very close to the level of the spontaneous incidence and independent on the two irradiation types used. On the contrary, the RTF for the SD primary cells, exposed to α-particles of γ-rays, is significantly enhanced when the survival of cells at risk decreases under 40%. For a SD cell survival of about 10%, RTF after low-LET irradiation was approximately 3-fold higher than after exposure to high-LET radiation. In order to determine if the radiosensitivity and the radio-induced cellular transformation of the cells collected from the two rat strains, could be related to their respective antioxidant defenses, activities of the main antioxidant enzymes (superoxide dismutase, catalase and Glutathione peroxidase) have been measured in the primary cell in culture. In our cellular model, the basal level of antoxidant enzymes is not associated with the radiosensitivity induced by high LET radiation. (author)

  14. HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Jana Kudová

    Full Text Available Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7 and with a decreasing percentage of myosin heavy chain α and β, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and β and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha. These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro.

  15. HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Kudová, Jana; Procházková, Jiřina; Vašiček, Ondřej; Perečko, Tomáš; Sedláčková, Miroslava; Pešl, Martin; Pacherník, Jiří; Kubala, Lukáš

    2016-01-01

    Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and β, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and β) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro.

  16. [Interaction of alpha-(125)latrocrustotoxin with nerve cell membranes from the river crab Astacus astacus].

    Science.gov (United States)

    Krasnoperov, V G; Shamotienko, O G; Grishin, E V

    1991-05-01

    alpha-Latrocrustatoxin, the crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus was radioactively labelled with Bolton-Hunter reagent to the specific activity of 160 Ci/mmol with retention of the biological activity. A highly specific binding of radioactive toxin on plasmatic membranes from the crayfish Astacus astacus nerve cells with Bmax = 0.04 pmol binding toxin/mg membrane protein and Kd = 0.7 x 10(-10) M was demonstrated.

  17. Receptor-mediated antigen delivery into macrophages. Complexing antigen to alpha 2-macroglobulin enhances presentation to T cells.

    Science.gov (United States)

    Chu, C T; Pizzo, S V

    1993-01-01

    Macrophages secrete alpha 2-macroglobulin (alpha 2M), a protein that may facilitate early Ag handling. alpha 2M is able to entrap and form covalent linkages with diverse proteins during a transient proteinase-activated state. The resulting complexes are rapidly endocytosed after binding to high affinity receptors. Such a system could be capable of efficiently delivering a multitude of proteins to macrophages. We have used T hybridoma clones that respond only to hen egg lysozyme, in a MHC-restricted manner, to probe the effect of complex formation on Ag uptake and processing by murine macrophages. Radiolabeled lysozyme was internalized more rapidly and to a greater extent when bound to alpha 2M than when unbound. Macrophages pulsed with lysozyme-alpha 2M-elastase complexes required 200 to 250 times less Ag than those pulsed with free lysozyme to achieve effective presentation to T cells. Adding equimolar amounts of alpha 2M-elastase complexes, or of alpha 2M-methylamine, to free lysozyme had no effect on basal lysozyme presentation. Receptor-recognized forms of alpha 2M, but not lysozyme or BSA, competed effectively for both uptake and presentation of lysozyme-alpha 2M-elastase complexes. These results indicate that proteinase-activated alpha 2M can enhance Ag processing by carrying Ag into macrophages through a receptor-mediated process.

  18. A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth

    DEFF Research Database (Denmark)

    Greenberger, Lee M; Horak, Ivan D; Filpula, David

    2008-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth factor...... the expression of HIF-1alpha mRNA. In vitro, in human prostate (15PC3, PC3, and DU145) and glioblastoma (U373) cells, EZN-2968 induced a potent, selective, and durable antagonism of HIF-1 mRNA and protein expression (IC(50), 1-5 nmol/L) under normoxic and hypoxic conditions associated with inhibition of tumor......-regulation of endogenous HIF-1alpha and vascular endothelial growth factor in the liver. The effect can last for days after administration of single dose of EZN-2968 and is associated with long residence time of locked nucleic acid in certain tissues. In efficacy studies, tumor reduction was found in nude mice implanted...

  19. Metronidazole affects breast cancer cell lines.

    Science.gov (United States)

    Sadowska, A; Prokopiuk, S; Miltyk, W; Surażyński, A; Konończuk, J; Sawicka, D; Car, H

    2013-01-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on cytotoxicity and DNA synthesis in MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) breast cancer cell lines. Toxicity of MTZ was determined by MTT test. MCF-7 and MDA-MB-231 cells were incubated with metronidazole used in different concentrations for 24, 48 and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. We showed that MTZ in concentration 250 μg/ml significantly increases the growth of MCF-7 cell lines after 24 hours of incubation, but it reduces cell viability in concentrations 1 and 10 μg/ml 72 hours after the drug application. Significant increase of MDA-MB-231 cell viability was obtained in MTZ concentration of 250 μg/ml after 24 and 72 hours. The increase of [3H]-thymidine incorporation in MCF-7 cell line treated with MTZ in concentration 250 μg/ml was statistically significant after 24 hours. Great suppression of cell proliferation was obtained in MDA-MB-231 breast cell line after application of the following concentrations of MTZ: 0.1 μg/ml (after 24 hours) and 0.1, 10, 50, 250 μg/ml (after 72h). We found that metronidazole exerts different dose- and time- dependent effects on human breast cancer cell lines characterized by presence or absence of estrogen receptors. We suggest that these discrepancies may be influenced by the estrogen signaling.

  20. Affective and cognitive behavior in the alpha-galactosidase A deficient mouse model of Fabry disease.

    Directory of Open Access Journals (Sweden)

    Lukas Hofmann

    Full Text Available Fabry disease is an X-linked inherited lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3 due to α-galactosidase A (α-Gal A deficiency. Fabry patients frequently report of anxiety, depression, and impaired cognitive function. We characterized affective and cognitive phenotype of male mice with α-Gal A deficiency (Fabry KO and compared results with those of age-matched male wildtype (WT littermates. Young (3 months and old (≥ 18 months mice were tested in the naïve state and after i.pl. injection of complete Freund`s adjuvant (CFA as an inflammatory pain model. We used the elevated plus maze (EPM, the light-dark box (LDB and the open field test (OF to investigate anxiety-like behavior. The forced swim test (FST and Morris water maze (MWM were applied to assess depressive-like and learning behavior. The EPM test revealed no intergroup difference for anxiety-like behavior in naïve young and old Fabry KO mice compared to WT littermates, except for longer time spent in open arms of the EPM for young WT mice compared to young Fabry KO mice (p<0.05. After CFA injection, young Fabry KO mice showed increased anxiety-like behavior compared to young WT littermates (p<0.05 and naïve young Fabry KO mice (p<0.05 in the EPM as reflected by shorter time spent in EPM open arms. There were no relevant differences in the LDB and the OF test, except for longer time spent in the center zone of the OF by young WT mice compared to young Fabry KO mice (p<0.05. Complementary to this, depression-like and learning behavior were not different between genotypes and age-groups, except for the expectedly lower memory performance in older age-groups compared to young mice. Our results indicate that genetic influences on affective and cognitive symptoms in FD may be of subordinate relevance, drawing attention to potential influences of environmental and epigenetic factors.

  1. Interaction of alpha-2-macroglobulin and HSV-1 during infection of neuronal cells.

    Science.gov (United States)

    Alonso, M; Dimitrijevic, A; Recuero, M; Serrano, E; Valdivieso, F; López-Guerrero, J A

    2001-12-01

    We describe the effect of pretreatment with alpha-2-macroglobulin (A2M) on the susceptibility of the human neuroblastoma SKNMC cell line to infection by herpes virus type 1 (HSV-1). ELISA and co-immunoprecipitation experiments confirmed the A2M-HSV-1 interaction in vitro. Indirect immunofluorescence shows that A2M exacerbated the cytopathic effect induced after HSV-1 infection. However, A2M-pretreated SKNMC cells notably produced fewer HSV-1 particles than did the untreated cells, suggesting that A2M could induce a restrictive infection. Furthermore, high levels of HSV-1 and A2M induced the production of nitric oxide (NO) in SKNMC. Preliminary results suggest that A2M might induce apoptosis in HSV-1-infected cells. These findings affirm the conclusion that A2M may interact directly with HSV-1 and modulate the course of the infection in SKNMC human neuroblastoma cells.

  2. Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells

    NARCIS (Netherlands)

    J. Arts (Janine); J.M.M.F. Janssen (Josine); J.A. Gustafsson (Jan-Ake); C.W.G.M. Löwik (Clemens); H.A.P. Pols (Huib); J.P.T.M. van Leeuwen (Hans); G.G.J.M. Kuiper (George)

    1997-01-01

    textabstractEstrogens have been shown to be essential for maintaining a sufficiently high bone mineral density and ER alpha expression has been demonstrated in bone cells. Recently, a novel estrogen receptor, estrogen receptor beta (ERbeta) has been identified. Here

  3. Relationship between vagal tone, cortisol, TNF-alpha, epinephrine and negative affects in Crohn's disease and irritable bowel syndrome.

    Directory of Open Access Journals (Sweden)

    Sonia Pellissier

    Full Text Available Crohn's disease (CD and irritable bowel syndrome (IBS involve brain-gut dysfunctions where vagus nerve is an important component. The aim of this work was to study the association between vagal tone and markers of stress and inflammation in patients with CD or IBS compared to healthy subjects (controls. The study was performed in 73 subjects (26 controls, 21 CD in remission and 26 IBS patients. The day prior to the experiment, salivary cortisol was measured at 8:00 AM and 10:00 PM. The day of the experiment, subjects completed questionnaires for anxiety (STAI and depressive symptoms (CES-D. After 30 min of rest, ECG was recorded for heart rate variability (HRV analysis. Plasma cortisol, epinephrine, norepinephrine, TNF-alpha and IL-6 were measured in blood samples taken at the end of ECG recording. Compared with controls, CD and IBS patients had higher scores of state-anxiety and depressive symptomatology. A subgroup classification based on HRV-normalized high frequency band (HFnu as a marker of vagal tone, showed that control subjects with high vagal tone had significantly lower evening salivary cortisol levels than subjects with low vagal tone. Such an effect was not observed in CD and IBS patients. Moreover, an inverse association (r =  -0.48; p<0.05 was observed between the vagal tone and TNF-alpha level in CD patients exclusively. In contrast, in IBS patients, vagal tone was inversely correlated with plasma epinephrine (r =  -0.39; p<0.05. No relationship was observed between vagal tone and IL-6, norepinephrine or negative affects (anxiety and depressive symptomatology in any group. In conclusion, these data argue for an imbalance between the hypothalamus-pituitary-adrenal axis and the vagal tone in CD and IBS patients. Furthermore, they highlight the specific homeostatic link between vagal tone and TNF-alpha in CD and epinephrine in IBS and argue for the relevance of vagus nerve reinforcement interventions in those diseases.

  4. Paraoxonase 1 polymorphism Q192R affects the pro-inflammatory cytokine TNF-alpha in healthy males

    Directory of Open Access Journals (Sweden)

    Rimbach Gerald

    2011-05-01

    Full Text Available Abstract Background Human paraoxonase 1 (PON1 is an HDL-associated enzyme with anti-oxidant/anti-inflammatory properties that has been suggested to play an important protective role against coronary heart diseases and underlying atherogenesis. The common PON1 Q192R polymorphism (rs662, A>G, a glutamine to arginine substitution at amino acid residue 192, has been analyzed in numerous association studies as a genetic marker for coronary heart diseases, however, with controversial results. Findings To get a better understanding about the pathophysiological function of PON1, we analyzed the relationships between the Q192R polymorphism, serum paraoxonase activity and serum biomarkers important for atherogenesis. Genotyping a cohort of 49 healthy German males for the Q192R polymorphism revealed an allele distribution of 0.74 and 0.26 for the Q and R allele, respectively, typical for Caucasian populations. Presence of the R192 allele was found to be associated with a significantly increased paraoxonase enzyme activity of 187.8 ± 11.4 U/l in comparison to the QQ192 genotype with 60.5 ± 4.9 U/l. No significant differences among the genotypes were found for blood pressure, asymmetric dimethylarginine, LDL, HDL, triglycerides, and cholesterol. As expected, MIP-2 alpha a cytokine rather not related to atherosclerosis is not affected by the PON1 polymorphism. In contrast to that, the pro-inflammatory cytokine TNF-alpha is enhanced in R192 carriers (163.8 ± 24.7 pg/ml vs 94.7 ± 3.2 pg/ml in QQ192 carriers. Conclusions Our findings support the hypothesis that the common PON1 R192 allele may be a genetic risk factor for atherogenesis by inducing chronic low-grade inflammation.

  5. Alpha-smooth muscle actin in pathological human disc nucleus pulposus cells in vivo and in vitro.

    Science.gov (United States)

    Hastreiter, Dawn; Chao, Jeannie; Wang, Qi; Ozuna, Richard M; Spector, Myron

    2004-01-01

    That a contractile actin isoform has been found in cells of other cartilage tissues in healing and disease states prompted this investigation of the presence of alpha-smooth muscle actin (alpha-SMA) in pathological human intervertebral disc tissue. The presence of this isoform has been reported in human intervertebral disc specimens obtained at autopsy from subjects for whom there were no reported symptoms. An objective of this study was to evaluate the cell density and percentage of alpha-SMA-containing cells in pathological nucleus pulposus tissue obtained from lumbar disc surgery from 17 patients. Additionally, explants of nucleus pulposus material were cultured to determine how alpha-SMA expression changed with time in vitro. Seventy-six 5-mm diameter explants (approximately 2 mm thick) pooled from six lumbar surgeries were cultured for 1, 2, 4, or 6 weeks. Microtomed sections of paraffin-embedded specimens were stained with hematoxylin and eosin or a monoclonal antibody to alpha-SMA. Histologically, cells were categorized as to alpha-SMA phenotype (positive or negative), and the areal cell density was determined. The evaluation of the cultured nucleus pulposus explants also included documentation of the percentage of cells that were round or elongated and the percentage of the cells that were part of a group (group: >/= 2 cells). Every nucleus pulposus section exhibited the presence of alpha-SMA-containing cells, which accounted for approximately 24 percent of the cells in vivo. In vivo, the cell density was significantly higher in older individuals (p = 0.02). The average time for cell outgrowth from the explants was 8.6 days. Approximately 10-15 percent of the cells in the explants stained positive for alpha-SMA. The time in culture had no significant effect on any of the outcome measures except the percentage of alpha-SMA-containing cells that were round (p = 0.008), with values decreasing through 4 weeks and then slightly rising at 6 weeks. The role of

  6. [The dose of alpha interferon in induction and maintainance therapy of hairy cell leukemia].

    Science.gov (United States)

    Kloke, O; May, D; Wandl, U; Niederle, N

    1988-01-01

    The efficacy of different doses of interferon (IFN) alpha in the induction therapy of hairy cell leukemia was studied. Recombinant IFN alpha-2b was administered subcutaneously every second day at an initial dose of 4 x 10(6) U/m2 (7 patients), 2 x 10(6) U/m2 (14 patients) and 1 x 10(6) U/m2 (13 patients). Each dose was effective to induce a normalization of blood cell counts and a reduction of bone marrow infiltration in the majority of patients treated. A delayed increase in granulocyte counts, however, was noted in patients treated with 1 x 10(6) U/m2, and 2 of them only achieved a partial hematological remission after 1 year of treatment. After the induction of stable remission, patients were randomized into 2 different schedules of maintenance therapy. Five of 7 patients receiving IFN alpha-2b at a total dose of 1 x 10(6) U twice a week, and 6/8 patients treated with 2 x 10(6) U once weekly, respectively, received this therapy for 1 year and remained in stable remission.

  7. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-02-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  8. Toxic effects of zearalenone and alpha-zearalenol on the regulation of steroidogenesis and testosterone production in mouse Leydig cells.

    Science.gov (United States)

    Yang, Jianying; Zhang, Yongfa; Wang, Yongqiang; Cui, Sheng

    2007-06-01

    Zearalenone (ZEA) and its derivative alpha-zearalenol (alpha-ZOL) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to animal fertility disturbances and other reproductive pathologies. The previous study demonstrated the toxic effects of ZEA and alpha-ZOL through disturbances in male fertility and other reproductive pathologies in mice. In this study, we further examined the direct biological effects of ZEA and alpha-ZOL on steroidogenesis production, primarily in Leydig cells of mice. Mature mouse Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining. To examine ZEA and alpha-ZOL-induced biological consequences, we measured testosterone secretion and transcription level of 3 key steroidogenic enzymes including 3beta-HSD-1, P450scc and StAR, in ZEA and alpha-ZOL/human chorionicgonadotropin (hCG) co-treated cells. Our results showed that ZEA and alpha-ZOL (10(-4) M, 10(-6) M and 10(-8) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The suppressive effect is correlated with a decrease in the level of transcription of 3beta-HSD-1, P450scc, and StAR (P<0.05).

  9. Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins

    DEFF Research Database (Denmark)

    Rosengren, K Johan; Daly, Norelle L; Fornander, Liselotte M

    2006-01-01

    variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar......alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D......)-Crp4 peptide, in which a conserved Glu(15) residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide...

  10. Pregnancy persistently affects memory T cell populations

    NARCIS (Netherlands)

    Kieffer, Tom E. C.; Faas, Marijke M.; Scherjon, Sicco A.; Prins, Jelmer R.

    Pregnancy is an immune challenge to the maternal immune system. The effects of pregnancy on maternal immunity and particularly on memory T cells during and after pregnancy are not fully known. This observational study aims to show the short term and the long term effects of pregnancy on the

  11. Does Royal jelly affect tumor cells?

    Directory of Open Access Journals (Sweden)

    Shirzad Maryam

    2013-04-01

    Full Text Available Introduction: Royal jelly is a substance that appears to be effective on immune system and it appears to be effective on both prevention and growth of cancer cells. In this study, we aimed to carry out a research to investigate the effect of royal jelly on the growth of WEHI-164 fibrosarcoma cell in syngenic Balb/c mice. Methods: In an experimental study, 28 male Balb/c mice were designated into four equal groups. The mice were subcutaneously injected with 5x105 WEHI-164 tumor cells on the day zero in the chest area of the animal. Animals in groups 1 to 4 were orally given 100, 200, 300 mg/kg of royal jelly or vehicle, respectively. In every individual mouse, the tumour size was measured every 2 days from day 5 (days 5, 7, 9, 11, 13, 15 and 17. Data were statistically analyzed using Kruskal-Wallis and Mann Whitney-U tests. Result: Our results showed that the mean size of tumor in case group was significantly smaller than the control group in days 11, 13, 15 and 17 (P<0.05. No metastasis was seen in test and control groups. Conclusion: With emphasize on antitumor effect of royal jelly, it seems that royal jelly has important role in control and regression of fibrosarcoma cells. Since royal jelly showed a delayed effect in control of fibrosarcoma, we suggest that royal jelly be used at least 10 days before tumor inoculation.

  12. Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans.

    Science.gov (United States)

    Lockhart, Shawn R; Wu, Wei; Radke, Joshua B; Zhao, Rui; Soll, David R

    2005-04-01

    The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature.

  13. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    Science.gov (United States)

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. PMID:26357513

  14. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Prachya Janhom

    2015-01-01

    Full Text Available In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn. act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson’s disease (PD, has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation.

  15. Neuroprotective Effects of Alpha-Mangostin on MPP(+)-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP(+)-induced apoptosis. The effects of alpha-mangostin on MPP(+)-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP(+) on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP(+). Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP(+). The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP(+) treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP(+)-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation.

  16. Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells.

    Science.gov (United States)

    Lipscomb, Elizabeth A; Dugan, Aisling S; Rabinovitz, Isaac; Mercurio, Arthur M

    2003-01-01

    The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the alpha6beta4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the alpha6beta4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced alpha6beta4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of beta4 expression in these cells augmented the formation of alpha6beta1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the alpha6beta4 integrin in invasion and migration that has been demonstrated previously by expression of the beta4 subunit in beta4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the alpha6beta4 integrin may be a useful approach to prevent carcinoma cell progression.

  17. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    Science.gov (United States)

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  18. TISSUE INHIBITOR OF METALLOPROTEINASE 1, MATRIX METALLOPROTEINASE 9, ALPHA-1 ANTITRYPSIN, METALLOTHIONEIN AND UROKINASE TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN SKIN BIOPSIES FROM PATIENTS AFFECTED BY AUTOIMMUNE BLISTERING DISEASES

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2013-07-01

    Full Text Available Introduction: Proteinases and proteinase inhibitors have been described to play a role in autoimmune skin blistering diseases. We studied skin lesional biopsies from patients affected by several autoimmune skin blistering diseases for proteinases and proteinase inhibitors. Methods: We utilized immunohistochemistry to evaluate biopsies for alpha-1-antitrypsin, human matrix metalloproteinase 9 (MMP9, human tissue inhibitor of metalloproteinases 1 (TIMP-1, metallothionein and urokinase type plasminogen activator receptor (uPAR. We tested 30 patients affected by endemic pemphigus, 30 controls from the endemic area, and 15 normal controls. We also tested 30 biopsies from patients with bullous pemphigoid (BP, 20 with pemphigus vulgaris (PV, 8 with pemphigus foliaceus, and 14 with dermatitis herpetiformis (DH. Results: Contrary to findings in the current literature, most autoimmune skin blistering disease biopsies were negative for uPAR and MMP9. Only some chronic patients with El Bagre-EPF were positive to MMP9 in the dermis, in proximity to telocytes. TIMP-1 and metallothionein were positive in half of the biopsies from BP patients at the basement membrane of the skin, within several skin appendices, in areas of dermal blood vessel inflammation and within dermal mesenchymal-epithelial cell junctions.

  19. Analysis of T cell receptor alpha beta variability in lymphocytes infiltrating melanoma primary tumours and metastatic lesions

    DEFF Research Database (Denmark)

    Schøller, J; thor Straten, P; Jakobsen, Annette Birck

    1994-01-01

    The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumour-infiltrating lymphocytes (TIL) in four different primary human malignant melanomas and their corresponding metastatic lesions was characterized using a recently developed method based on the reverse-transcription-couple......The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumour-infiltrating lymphocytes (TIL) in four different primary human malignant melanomas and their corresponding metastatic lesions was characterized using a recently developed method based on the reverse...... usage of the TCR V gene families V alpha 4, V alpha 5, V alpha 22 and V beta 8, whereas the V beta 3 gene family appeared to be expressed together with HLA-A1. Other highly expressed V gene families, apparently not restricted to either HLA-A1 or -A2, were V alpha 1 (expressed in three of four primary...... tumours) and V alpha 21 (expressed in two of four tumours). We found no evidence suggesting any correlations between the haplotypes HLA-A1 and -A2 and preferential V gene family expression in the metastatic lesions, and the only common feature was V alpha 8, which was found to be highly expressed in two...

  20. Prostaglandin F2 alpha stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and mobilizes intracellular Ca2+ in bovine luteal cells

    International Nuclear Information System (INIS)

    Davis, J.S.; Weakland, L.L.; Weiland, D.A.; Farese, R.V.; West, L.A.

    1987-01-01

    The present studies were conducted to determine whether prostaglandin F2 alpha (PGF2 alpha) stimulates the production of ''second messengers'' derived from inositol phospholipid hydrolysis and increases intracellular free Ca2+ ([Ca2+]i) in isolated bovine luteal cells. PGF2 alpha provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (InsP, InsP2, and InsP3, respectively). InsP3 was formed more rapidly than InsP2 or InsP after PGF2 alpha treatment. In addition, PGF2 alpha increased inositol phospholipid turnover, as evidenced by increased 32 PO 4 incorporation into phosphatidic acid and phosphatidylinositol. LiCl (1-20 mM) enhanced inositol phosphate accumulation in response to PGF2 alpha. Maximal increases in InsP3 occurred at 1 microM PGF2 alpha, with half-maximal stimulation occurring at 36 nM. The acute effects of PGF2 alpha on InsP3 levels were independent of reductions in extracellular calcium. Prostaglandins E1 and E2 also stimulated increases in inositol phosphate levels, albeit to a lesser extent. PGF2 alpha also induced rapid and concentration-dependent increases in [Ca2+]i as measured by quin-2 fluorescence. The PGF2 alpha-induced increases in [Ca2+]i were maximal within 30 sec (approximately 2- to 3-fold), and [Ca2+]i remained elevated for 8-10 min. The PGF2 alpha-induced increases in [Ca2+]i were also independent of extracellular calcium. These findings demonstrate that the action of PGF2 alpha is coupled to the phospholipase C-InsP3 and diacylglycerol second messenger system in the corpus luteum

  1. Prostaglandin F2 alpha stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and mobilizes intracellular Ca2+ in bovine luteal cells

    Energy Technology Data Exchange (ETDEWEB)

    Davis, J.S.; Weakland, L.L.; Weiland, D.A.; Farese, R.V.; West, L.A.

    1987-06-01

    The present studies were conducted to determine whether prostaglandin F2 alpha (PGF2 alpha) stimulates the production of ''second messengers'' derived from inositol phospholipid hydrolysis and increases intracellular free Ca2+ ((Ca2+)i) in isolated bovine luteal cells. PGF2 alpha provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (InsP, InsP2, and InsP3, respectively). InsP3 was formed more rapidly than InsP2 or InsP after PGF2 alpha treatment. In addition, PGF2 alpha increased inositol phospholipid turnover, as evidenced by increased /sup 32/PO/sub 4/ incorporation into phosphatidic acid and phosphatidylinositol. LiCl (1-20 mM) enhanced inositol phosphate accumulation in response to PGF2 alpha. Maximal increases in InsP3 occurred at 1 microM PGF2 alpha, with half-maximal stimulation occurring at 36 nM. The acute effects of PGF2 alpha on InsP3 levels were independent of reductions in extracellular calcium. Prostaglandins E1 and E2 also stimulated increases in inositol phosphate levels, albeit to a lesser extent. PGF2 alpha also induced rapid and concentration-dependent increases in (Ca2+)i as measured by quin-2 fluorescence. The PGF2 alpha-induced increases in (Ca2+)i were maximal within 30 sec (approximately 2- to 3-fold), and (Ca2+)i remained elevated for 8-10 min. The PGF2 alpha-induced increases in (Ca2+)i were also independent of extracellular calcium. These findings demonstrate that the action of PGF2 alpha is coupled to the phospholipase C-InsP3 and diacylglycerol second messenger system in the corpus luteum.

  2. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    OpenAIRE

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson’s disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MP...

  3. All-trans retinoic acid combined with 5-Aza-2 Prime -deoxycitidine induces C/EBP{alpha} expression and growth inhibition in MLL-AF9-positive leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Fujiki, Atsushi [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Imamura, Toshihiko, E-mail: imamura@koto.kpu-m.ac.jp [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sakamoto, Kenichi; Kawashima, Sachiko; Yoshida, Hideki; Hirashima, Yoshifumi; Miyachi, Mitsuru; Yagyu, Shigeki; Nakatani, Takuya [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sugita, Kanji [Department of Pediatrics, University of Yamanashi, Yamanashi (Japan); Hosoi, Hajime [Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer We tested whether ATRA and 5-Aza affect AML cell differentiation and growth. Black-Right-Pointing-Pointer Cell differentiation and growth arrest were induced in MLL-AF9-expressing cells. Black-Right-Pointing-Pointer Increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1 were also observed. Black-Right-Pointing-Pointer MLL-AF4/AF5q31-expressing cells are less sensitive to ATRA and 5-Aza. Black-Right-Pointing-Pointer Different MLL fusion has distinct epigenetic properties related to RA pathway. -- Abstract: The present study tested whether all-trans retinoic acid (ATRA) and 5-Aza-2 Prime -deoxycitidine (5-Aza) affect AML cell differentiation and growth in vitro by acting on the CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) and c-Myc axis. After exposure to a combination of these agents, cell differentiation and growth arrest were significantly higher in human and murine MLL-AF9-expressing cells than in MLL-AF4/AF5q31-expressing cells, which were partly associated with increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1, and decreased expression of c-Myc. These findings indicate that MLL-AF9-expressing cells are more sensitive to ATRA and 5-Aza, indicating that different MLL fusion proteins possess different epigenetic properties associated with retinoic acid pathway inactivation.

  4. Co-receptor choice by V alpha14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection.

    Science.gov (United States)

    Engel, Isaac; Hammond, Kirsten; Sullivan, Barbara A; He, Xi; Taniuchi, Ichiro; Kappes, Dietmar; Kronenberg, Mitchell

    2010-05-10

    Mouse natural killer T (NKT) cells with an invariant V alpha14-J alpha18 rearrangement (V alpha14 invariant [V alpha14i] NKT cells) are either CD4(+)CD8(-) or CD4(-)CD8(-). Because transgenic mice with forced CD8 expression in all T cells exhibited a profound NKT cell deficit, the absence of CD8 has been attributed to negative selection. We now present evidence that CD8 does not serve as a coreceptor for CD1d recognition and that the defect in development in CD8 transgene homozygous mice is the result of a reduction in secondary T cell receptor alpha rearrangements. Thymocytes from mice hemizygous for the CD8 transgene have a less severe rearrangement defect and have functional CD8(+) V alpha14i NKT cells. Furthermore, we demonstrate that the transcription factor Th, Poxviruses and Zinc finger, and Krüppel family (Th-POK) is expressed by V alpha14i NKT cells throughout their differentiation and is necessary both to silence CD8 expression and for the functional maturity of V alpha14i NKT cells. We therefore suggest that Th-POK expression is required for the normal development of V alpha14i NKT cells and that the absence of CD8 expression by these cells is a by-product of such expression, as opposed to the result of negative selection of CD8-expressing V alpha14i NKT cells.

  5. Influence of βS-globin haplotypes and hydroxyurea on tumor necrosis factor-alpha levels in sickle cell anemia.

    Science.gov (United States)

    Laurentino, Marília Rocha; Maia, Pedro Aurio; Barbosa, Maritza Cavalcante; Bandeira, Izabel Cristina Justino; Rocha, Lilianne Brito da Silva; Gonçalves, Romelia Pinheiro

    2014-03-01

    Sickle cell anemia is a chronic inflammatory disease characterized by an increased production of proinflammatory cytokines including tumor necrosis factor-alpha. Hydroxyurea, by decreasing the polymerization of hemoglobin, reduces inflammatory states. The effect of the genetic polymorphisms of sickle cell patients on tumor necrosis factor-alpha levels remains unknown. The aim of this study was to investigate the association of tumor necrosis factor-alpha levels with β-globin haplotypes and the use of hydroxyurea. A cross-sectional study was performed of 67 patients with sickle cell anemia diagnosed at steady-state in a referral hospital in Fortaleza, Ceará, Brazil. A group of 26 healthy individuals was used as control. βS-haplotype analysis was performed by restriction fragment length polymorphism-polymerase chain reaction. The tumor necrosis factor-alpha levels were measured by the enzyme-linked immunosorbent assay test. Laboratory data (complete blood count and fetal hemoglobin) and information regarding the use of hydroxyurea were obtained from medical records. Statistical analysis was performed using R software with the Kruskal-Wallis and Mann-Whitney tests. Statistical significance was established for p-values sickle cell anemia had significantly higher tumor necrosis factor-alpha levels than controls (p-values sickle cell anemia patients who were receiving hydroxyurea treatment than those who were not (p-value = 0.1249). Sickle cell anemia patients with Bantu/n genotype had significantly higher levels than patients with the Bantu/Benin genotype (p-value = 0.0021). In summary, βS-globin haplotypes, but not hydroxyurea therapy, have a role in modulating tumor necrosis factor-alpha levels in sickle cell anemia adults at steady-state. Many previous studies have investigated prognosis and inflammatory states in sickle cell anemia patients, but the discovery that tumor necrosis factor-alpha levels vary according to the genetic polymorphism of the patient is a

  6. Expression of the alpha 6 beta 4 integrin by squamous cell carcinomas and basal cell carcinomas: possible relation to invasive potential?

    DEFF Research Database (Denmark)

    Rossen, K; Dahlstrøm, K K; Mercurio, A M

    1994-01-01

    We have studied the expression of alpha 6 beta 4 integrin, a carcinoma laminin receptor in ten squamous cell carcinomas (SCCs) and ten basal cell carcinomas (BCCs) of the skin in order to examine whether changes in alpha 6 beta 4 integrin expression may be related to invasive and metastatic...... potential. Monoclonal antibodies specific for each subunit were applied on cryosections, using a three step indirect peroxidase technique. In normal epidermis the basal cells expressed both the alpha 6 and the beta 4 subunits, and the expression was polarized against the basement membrane. In SCCs...

  7. Phenotypic and functional markers for 1alpha,25-dihydroxyvitamin D(3)-modified regulatory dendritic cells

    DEFF Research Database (Denmark)

    Pedersen, A W; Holmstrøm, K; Jensen, S S

    2009-01-01

    be measured with simple methods to identify regulatory DCs that are applicable for future clinical studies. Human DCs were generated from peripheral blood monocytes in the presence of 1alpha,25-dihydroxyvitamin D(3) (VD3), which gave rise to a phenotype that resembles immature DCs, with the exception of high......The clinical use of dendritic cells (DCs) to induce antigen-specific immune tolerance has been hampered by the lack of a widely acknowledged method for generating human regulatory DCs but even more so by the non-existence of reliable markers. Thus, we set out to find reliable markers that can...

  8. The alpha-cell as target for type 2 diabetes therapy

    DEFF Research Database (Denmark)

    Christensen, Mikkel; Bagger, Jonatan I; Vilsboll, Tina

    2011-01-01

    -coupled receptors in the hepatocytes. Type 2 diabetic patients are characterized by elevated glucagon levels contributing decisively to hyperglycemia in these patients. Accumulating evidence demonstrates that targeting the pancreatic alpha-cell and its main secretory product glucagon is a possible treatment....... Furthermore, potential advantages and limitations of antagonizing the glucagon receptor or suppressing glucagon secretion in the treatment of type 2 diabetes are discussed with a focus on already marketed drugs and drugs in clinical development. It is concluded that the development of novel glucagon receptor...

  9. Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule.

    Science.gov (United States)

    Yu, Dehai; Du, Zhonghua; Li, Wei; Chen, Huaqiu; Ye, Songgen; Hoffman, Andrew R; Cui, Jiuwei; Hu, Ji-Fan

    2017-01-01

    Adult T-cell leukemia/lymphoma (ATL) is a very aggressive T cell malignancy that carries a poor prognosis, primarily due to its resistance to chemotherapy and to life-threatening infectious complications. Interferon-alpha (IFNα) has been used in combination with the anti-retroviral drug zidovudine to treat patients with ATL. However, the efficacy of long-term therapy is significantly limited due to the systemic toxicity of IFNα. We utilized phage display library screening to identify short peptides that specifically bind to Jurkat T lymphocyte leukemia cells. By fusing the Jurkat-binding peptide to the C-terminus of IFNα, we constructed an engineered chimeric IFNα molecule (IFNP) for the treatment of ATL. We found that IFNP exhibited significantly higher activity than wild type IFNα in inhibiting the growth of leukemia cells and inducing cell blockage at the G0/G1 phase. The synthetic IFNP molecule exerted its antitumor activity by upregulating the downstream genes involved in the STAT1 pathway and in apoptosis. Using a cell receptor binding assay, we showed that this Jurkat-binding peptide facilitated the binding affinity of IFNα to the cell surface type I IFN receptor. The isolated Jurkat-binding peptide significantly potentiates the therapeutic activity of IFNα in T lymphocyte leukemia cells. The engineered IFNP molecule may prove to a novel antitumor approach in the treatment of patients with ATL. © 2017 The Author(s). Published by S. Karger AG, Basel.

  10. Protective effects of alpha lipoic acid on high glucose-induced neurotoxicity in PC12 cells.

    Science.gov (United States)

    Najafi, Rezvan; Sharifi, Ali Mohammad; Hosseini, Asieh

    2015-06-01

    Hyperglycemia plays an important role in the development of diabetic neuropathy. In this study, we investigated the protective effects of alpha lipoic acid (ALA) against high glucose-induced neurotoxicity in PC12 cells as a suitable in vitro model for studying neuronal functions. PC12 cells were treated with high glucose (25 mg/ml for 24 h) in the absence and presence of ALA (100 μM for 24 h). The viability of PC12 cells was estimated by using MTT assay. The expression of pro- apoptotic Bax, anti- apoptotic Bcl-2 and caspase 3 protein were evaluated by western blotting. The reactive oxygen species (ROS) levels were determined with 2,7-dichlorodihydro- fluorescein diacetate (H2DCFDA). Biochemical markers of oxidative stress were assessed by using the total antioxidant power (TAP), lipid peroxidation (LPO), ADP/ATP ratio, activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Pretreatment of PC12 cells with ALA, significantly improved high glucose-induced toxicity by increasing activity of antioxidant enzymes CAT and SOD in the PC12 cell. It also increased the concentrations of TAP. An elevated level of cell death and ROS in high glucose conditions, diminished with ALA treatment. Over expression of Bax and caspase 3 protein, elevation of ADP/ATP ratio and LPO level in high glucose- treated PC12 cells, were significantly reduced by ALA. It was concluded that ALA attenuates neurotoxicity induced by high glucose in PC12 cells.

  11. Catechol-o-methyltransferase expression and 2-methoxyestradiol affect microtubule dynamics and modify steroid receptor signaling in leiomyoma cells.

    Directory of Open Access Journals (Sweden)

    Salama A Salama

    Full Text Available CONTEXT: Development of optimal medicinal treatments of uterine leiomyomas represents a significant challenge. 2-Methoxyestradiol (2ME is an endogenous estrogen metabolite formed by sequential action of CYP450s and catechol-O-methyltransferase (COMT. Our previous study demonstrated that 2ME is a potent antiproliferative, proapoptotic, antiangiogenic, and collagen synthesis inhibitor in human leiomyomas cells (huLM. OBJECTIVES: Our objectives were to investigate whether COMT expression, by the virtue of 2ME formation, affects the growth of huLM, and to explore the cellular and molecular mechanisms whereby COMT expression or treatment with 2ME affect these cells. RESULTS: Our data demonstrated that E(2-induced proliferation was less pronounced in cells over-expressing COMT or treated with 2ME (500 nM. This effect on cell proliferation was associated with microtubules stabilization and diminution of estrogen receptor alpha (ERalpha and progesterone receptor (PR transcriptional activities, due to shifts in their subcellular localization and sequestration in the cytoplasm. In addition, COMT over expression or treatment with 2ME reduced the expression of hypoxia-inducible factor -1alpha (HIF-1 alpha and the basal level as well as TNF-alpha-induced aromatase (CYP19 expression. CONCLUSIONS: COMT over expression or treatment with 2ME stabilize microtubules, ameliorates E(2-induced proliferation, inhibits ERalpha and PR signaling, and reduces HIF-1 alpha and CYP19 expression in human uterine leiomyoma cells. Thus, microtubules are a candidate target for treatment of uterine leiomyomas. In addition, the naturally occurring microtubule-targeting agent 2ME represents a potential new therapeutic for uterine leiomyomas.

  12. Interactive effects of fumonisin B1 and alpha-zearalenol on proliferation and cytokine expression in Jurkat T cells.

    Science.gov (United States)

    Luongo, D; Severino, L; Bergamo, P; De Luna, R; Lucisano, A; Rossi, M

    2006-12-01

    Mycotoxins are secondary metabolites of fungi that grow on various food and feed. These compounds elicit a wide spectrum of toxicological effects, including the capacity to alter normal immune function. Feed commodities are usually contaminated with more than one mycotoxin; however, extensive information on the interaction between concomitantly occurring mycotoxins and the consequence for their toxicity is lacking. In the present study, we examined the effects in vitro of fumonisin B1 (FB1) and alpha-zearalenol (alpha-ZEA), alone or in combination, on the immune function in the human lymphoblastoid Jurkat T cell line. Treatment of cells with increasing concentrations of FB1 resulted in a dose-dependent induction of proliferation. In contrast, alpha-ZEA showed a marked inhibitory effect on cell proliferation, even at very low doses, essentially mediated by apoptosis. In stimulated cells pre-incubated with FB1, the levels of IL-2 and IFN gamma mRNAs were similar to control whereas a reduction of cytokine transcripts was reported following alpha-ZEA treatment. Interestingly, co-administration of mycotoxins resulted in further inhibition of both proliferation and IFN gamma mRNA expression when compared with alpha-ZEA alone. In conclusion, FB1 and alpha-ZEA showed different immunomodulation abilities when individually administered. Combination of mycotoxins resulted instead in interactive effects.

  13. Activated endothelial cells elicit paracrine induction of epithelial chloride secretion. 6-Keto-PGF1alpha is an epithelial secretagogue.

    Science.gov (United States)

    Blume, E D; Taylor, C T; Lennon, P F; Stahl, G L; Colgan, S P

    1998-09-15

    Endothelial cells play a central role in the coordination of the inflammatory response. In mucosal tissue, such as the lung and intestine, endothelia are anatomically positioned in close proximity to epithelia, providing the potential for cell-cell crosstalk. Thus, in this study endothelial-epithelial biochemical crosstalk pathways were studied using a human intestinal crypt cell line (T84) grown in noncontact coculture with human umbilical vein endothelia. Exposure of such cocultures to endothelial-specific agonists (LPS) resulted in activation of epithelial electrogenic Cl- secretion and vectorial fluid transport. Subsequent experiments revealed that in response to diverse stimuli (LPS, IL-1alpha, TNF-alpha, hypoxia), endothelia produce and secrete a small, stable epithelial secretagogue into conditioned media supernatants. Further experiments identified this secretagogue as 6-keto-PGF1alpha, a stable hydrolysis product of prostacyclin (PGI2). Results obtained with synthetic prostanoids indicated that 6-keto-PGF1alpha (EC50 = 80 nM) and PGI2 stable analogues (EC50 = 280 nM) activate the same basolaterally polarized, Ca2+-coupled epithelial receptor. In summary, these findings reveal a previously unappreciated 6-keto-PGF1alpha receptor on intestinal epithelia, the ligation of which results in activation of electrogenic Cl- secretion. In addition, these data reveal a novel action for the prostacyclin hydrolysis product 6-keto-PGF1alpha and provide a potential endothelial- epithelial crosstalk pathway in mucosal tissue.

  14. Biodentine Reduces Tumor Necrosis Factor Alpha-induced TRPA1 Expression in Odontoblastlike Cells.

    Science.gov (United States)

    El Karim, Ikhlas A; McCrudden, Maelíosa T C; McGahon, Mary K; Curtis, Tim M; Jeanneau, Charlotte; Giraud, Thomas; Irwin, Chris R; Linden, Gerard J; Lundy, Fionnuala T; About, Imad

    2016-04-01

    The transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression. Immunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies. Immunofluorescent staining revealed TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha-induced TRPA1 responses after Biodentine treatment. In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. Does potentized HgCl2 (Mercurius corrosivus) affect the activity of diastase and alpha-amylase?

    Science.gov (United States)

    Witt, Claudia M; Bluth, Michael; Hinderlich, Stephan; Albrecht, Henning; Lüdtke, Rainer; Weisshuhn, Thorolf E R; Willich, Stefan N

    2006-05-01

    Homeopathic drugs even with dilutions beyond 10(23) (high potencies) are frequently used, although their working mechanism is still unknown. Curative information preserved in solvent structure is postulated to exert biologic effects. The objective was to test for a stimulating or inhibiting effect of high potencies of the homeopathic remedy HgCl2 (Mercurius corrosivus) on two sugar hydrolases. High potencies were produced using stepwise dilution plus shaking. Controls included potentized solvent (aqua bidestillata), equimolar dilutions without shaking, and enzyme-free references. Tested were potencies with dilution factors 1:200 (CC) on diastase extract from winter barley, and 1:100 (C) on alpha-amylase from hog pancreas. Enzyme activity was colorimetrically determined by Lugol's iodine-starch reaction. An inhibiting effect of HgCl2 on enzyme activities was observed only in low potencies and dilutions. Statistically significant differences between potencies and controls were not found in randomized and blinded experiments. This experimental design provided independent reproducible results of cell-free in vitro assays. However, it did not indicate an effect of potentized HgCl2 on hydrolases. Demonstrating potency effects may require additional experimental features.

  16. Cervical squamous carcinoma cells are resistant to the combined action of tumor necrosis factor-alpha and histamine whereas normal keratinocytes undergo cytolysis.

    Science.gov (United States)

    Diaconu, Nicolae-Costin; Rummukainen, Jaana; Mättö, Mikko; Naukkarinen, Anita; Harvima, Rauno J; Pelkonen, Jukka; Harvima, Ilkka T

    2008-02-07

    Previous reports showed that mast cells can typically be found in the peritumoral stroma of cervix carcinomas as well as in many other cancers. Both histamine and TNF-alpha are potent preformed mast cell mediators and they can act simultaneously after release from mast cells. Thus, the effect of TNF-alpha and histamine on cervical carcinoma cell lines was studied. TNF-alpha alone induced slight growth inhibition and cell cycle arrest at G0/G1 phase in SiHa cells, but increased their migration. Histamine alone had no effect on cells. In addition, TNF-alpha and histamine in combination showed no additional effect over that by TNF-alpha alone, although SiHa cells were even pretreated with a protein synthesis inhibitor. Furthermore, TNF-alpha-sensitive ME-180 carcinoma cells were also resistant to the combination effect of TNF-alpha and histamine. In comparison, TNF-alpha or histamine alone induced growth inhibition in a non-cytolytic manner in normal keratinocytes, an effect that was further enhanced to cell cytolysis when both mediators acted in combination. Keratinocytes displayed strong TNF receptor (TNFR) I and II immunoreactivity, whereas SiHa and ME-180 cells did not. Furthermore, cervix carcinoma specimens revealed TNF-alpha immunoreactivity in peritumoral cells and carcinoma cells. However, the immunoreactivity of both TNFRs was less intense in carcinoma cells than that in epithelial cells in cervical specimens with non-specific inflammatory changes. SiHa and ME-180 cells are resistant to the cytolytic effect of TNF-alpha and histamine whereas normal keratinocytes undergo cytolysis, possibly due to the smaller amount of TNFRs in SiHa and ME-180 cells. In the cervix carcinoma, the malignant cells may resist this endogenous cytolytic action and TNF-alpha could even enhance carcinoma cell migration.

  17. The Role of Plasmacytoid Dendritic Cells in Innate and Adaptive Immune Responses against Alpha Herpes Virus Infections

    Directory of Open Access Journals (Sweden)

    Philipp Schuster

    2011-01-01

    Full Text Available In 1999, two independent groups identified plasmacytoid dendritic cells (PDC as major type I interferon- (IFN- producing cells in the blood. Since then, evidence is accumulating that PDC are a multifunctional cell population effectively coordinating innate and adaptive immune responses. This paper focuses on the role of different immune cells and their interactions in the surveillance of alpha herpes virus infections, summarizes current knowledge on PDC surface receptors and their role in direct cell-cell contacts, and develops a risk factor model for the clinical implications of herpes simplex and varicella zoster virus reactivation. Data from studies involving knockout mice and cell-depletion experiments as well as human studies converge into a “spider web”, in which the direct and indirect crosstalk between many cell populations tightly controls acute, latent, and recurrent alpha herpes virus infections. Notably, cells involved in innate immune regulations appear to shape adaptive immune responses more extensively than previously thought.

  18. The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha(1A1)-adrenoceptors

    DEFF Research Database (Denmark)

    Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork

    2008-01-01

    Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice variant...... a cell line stably expressing a functional form of this splice variant. The expression of recombinant alpha(1A1)-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca(2+)](i)) when...... human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole...

  19. Characterization of the binding of radioiodinated hybrid recombinant IFN-alpha A/D to murine and human lymphoid cell lines

    International Nuclear Information System (INIS)

    Faltynek, C.R.; Princler, G.L.; Schwabe, M.; Shata, M.T.; Lewis, G.K.; Kamin-Lewis, R.M.

    1990-01-01

    The hybrid recombinant human interferon (IFN) rIFN-alpha A/D was radioiodinated. Specific binding of [125I]rIFN-alpha A/D was observed with both human and murine cell lines. The binding of [125I]rIFN-alpha A/D to human Daudi cells had similar characteristics to the previously described binding of [125I]rIFN-alpha A or -alpha 2. The following lines of evidence demonstrated that [125I]rIFN-alpha A/D bound with high affinity to the same receptor on murine cells as murine IFN-alpha and -beta: (i) the binding of [125I]rIFN-alpha A/D to murine LBRM cells was inhibited to a similar extent by natural murine IFN-alpha, natural murine IFN-beta, and rIFN-A/D; (ii) the Kd (approximately 2 X 10(-10) M) obtained from both competition experiments and saturation binding experiments with [125I]rIFN-alpha A/D was comparable to the previously reported Kd for the binding of natural murine IFN-alpha and -beta to other murine cell lines; (iii) the size of the cross-linked [125I]rIFN-alpha A/D receptor complex formed on murine LBRM cells was similar to the previously reported cross-linked complex formed after binding radioiodinated natural murine IFN-beta to other murine cell lines. Due to the current lack of readily available recombinant murine IFN-alpha or -beta for radiolabeling and the previously demonstrated biological activity of rIFN-alpha A/D on murine cells, [125I]rIFN-alpha A/D should prove to be a useful reagent for further studies of murine IFN receptors

  20. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

  1. Metabolite Depletion Affects Flux Profiling of Cell Lines

    DEFF Research Database (Denmark)

    Nilsson, A.; Haanstra, J. R.; Teusink, B.

    2018-01-01

    Quantifying the rate of consumption and release of metabolites (i.e., flux profiling) has become integral to the study of cancer. The fluxes as well as the growth of the cells may be affected by metabolite depletion during cultivation.......Quantifying the rate of consumption and release of metabolites (i.e., flux profiling) has become integral to the study of cancer. The fluxes as well as the growth of the cells may be affected by metabolite depletion during cultivation....

  2. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.

    Directory of Open Access Journals (Sweden)

    Teruhisa Takagishi

    Full Text Available Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB. DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1, a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step and activation of endogenous PLCγ-1 (second step leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.

  3. Interferon-alpha-induced changes in surface antigens in a hairy-cell leukemia (JOK-1), and a Burkitt's lymphoma cell line (Daudi) during in vitro culture

    DEFF Research Database (Denmark)

    Nielsen, B; Madsen, P S; Jensen, A W

    1992-01-01

    In further studying the mechanism of action of IFN-alpha in HCL, we cultured the HCL cell line JOK-1 and the IFN-sensitive Burkitt cell line Daudi with and without IFN-alpha and investigated the changes in density of a number of surface antigens by use of mAb and flow cytometry analyses. During...... culture with IFN-alpha, reproducible changes were induced in both cell lines, which were qualitatively similar but differed quantitatively with small and transient changes in JOK-1. Significant decreases in surface antigen expression were observed for CD 19, 23, 37, and for IgM on both cell lines...... of IFN-alpha in HCL was not paralleled by a specific direct effect on JOK-1 in vitro. Our findings therefore do not support the theory that IFN's mechanism of action in vivo is a direct effect on HC, but suggest that indirect effects are involved. Udgivelsesdato: 1992-Mar...

  4. The role of estrogen receptor alpha in mediating chemoresistance in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Jiang Zhinong

    2012-05-01

    Full Text Available Abstract Introduction Previous studies suggested that estrogen receptor alpha (ERα plays an important role in the chemoresistance of breast cancers. However, large random trials failed to demonstrate any benefit of the concurrent estrogen antagonist tamoxifen on the chemotherapy efficacy. Thus, in the present study, the importance of the role of ERα in the chemoresistance of breast cancer cells was investigated. Methods The ERα-transfected Bcap37 cells and natural ERα-positive T47D breast cancer cells were treated using chemotherapeutic agents with or without 17-beta estradiol (E2 pretreatment. Their viabilities were assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. The dead cell rates were determined using propidium iodide dye exclusion tests, and the expression levels of Bcl-2 and Bax were detected through Western blot analysis. The effects of E2 on the growth of breast cancer cells were also determined via cell growth curve and cell cycle analysis. Results ERα activation by E2 increased the sensitivity of natural ERα-positive T47D breast cancer cells to chemotherapeutic agents. However, the increase in ERα expression in ERα-negative Bcap37 breast cancer cells also significantly increased their resistance. These phenomena cannot be explained by asserting that ERα mediated the chemoresistance of breast cancer cells by regulating the expression of Bcl-2 and Bax. Our findings show that ERα activation upregulated the expression of Bcl-2 in natural ERα-positive T47D breast cancer cells, whereas ERα activation by E2 downregulated and upregulated the Bcl-2 and Bax expression levels, respectively, in ERα-transfected Bcap37 cells. This phenomenon was due to the influence of ERα on the growth of breast cancer cells. Specifically, ERα activation enhanced the growth of natural ERα-positive breast cancer cells and thus increased their sensitivity to chemotherapeutic agents. However, ERα activation also

  5. Genetically-induced Estrogen Receptor Alpha mRNA (Esr1) Overexpression Does Not Adversely Affect Fertility or Penile Development in Male Mice

    Science.gov (United States)

    Heath, John; Abdelmageed, Yazeed; Braden, Tim D.; Williams, Carol S.; Williams, John W.; Paulose, Tessie; Hernandez-Ochoa, Isabel; Gupta, Rupesh; Flaws, Jodi A.; Goyal, Hari O.

    2011-01-01

    Previously, we reported that estrogen receptor alpha mRNA (Esr1) or protein (ESR1) overexpression resulting from neonatal exposure to estrogens in rats was associated with infertility and mal-developed penis characterized by reduced length and weight and abnormal accumulation of fat cells. The objective of this study was to determine if mutant male mice overexpressing Esr1 are naturally infertile or have reduced fertility and/or develop abnormal penis. The fertility parameters, including fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per female, were not altered from controls, as a result of Esr1 overexpression. Likewise, penile morphology, including the length, weight, and diameter and os penis development, was not altered from controls. Conversely, weights of the seminal vesicles and bulbospongiosus and levator ani (BS/LA) muscles were significantly (P testosterone concentration were not different from controls. Hence, the genetically-induced Esr1 overexpression alone, without an exogenous estrogen exposure during the neonatal period, is unable to adversely affect the development of the penis as well as other male reproductive organs, except limited, but significant, reductions in weights of the seminal vesicles and BS/LA muscles. PMID:20930192

  6. Characterization of monocyte-derived dendritic cells maturated with IFN-alpha

    DEFF Research Database (Denmark)

    Svane, I M; Nikolajsen, K; Walter, M R

    2006-01-01

    , but the most potent reagents such as LPS or polyriboinosinic polyribocytidylic acid (Poly I:C) are not approved for clinical use. We tested the ability of type I interferon (IFN) to induce such maturation. We found that 24-h IFN-alpha co-culture of day 7 monocyte-derived DC generated with GM-CSF and IL-4......Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required...... induces increased numbers of DC positive for CD54 and CD40 together with the co-stimulatory molecule CD80 but not the activation marker CD83. Also, IFN-alpha maturation leads to an increase in IP-10 and MCP-1 chemokine secretion, but only a minor increase in IL-12p40 secretion. In line with this...

  7. Regression of hepatocarcinoma cells using RNA aptamer specific to alpha-fetoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Ju [Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Yongin 448-701 (Korea, Republic of); Lee, Seong-Wook, E-mail: SWL0208@dankook.ac.kr [Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Yongin 448-701 (Korea, Republic of)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Identification of RNA aptamer specific to AFP with high affinity. Black-Right-Pointing-Pointer Specific induction of HCC proliferation by AFP. Black-Right-Pointing-Pointer Efficient increase in oncogene expression by AFP. Black-Right-Pointing-Pointer Efficient inhibition of AFP-mediated HCC proliferation by the aptamer. Black-Right-Pointing-Pointer Efficient suppression of AFP-induced oncogene expression of by the aptamer. -- Abstract: Alpha-fetoprotein (AFP) is a cancer-associated fetal protein and has long been utilized as a serum fetal defect/tumor marker to monitor distress/disease progression. In addition, AFP is closely associated with the proliferation of hepatocellular carcinoma. Thus, direct targeting of AFP has been recommended for a therapeutic strategy against hepatocellular carcinoma. In this study, we developed and characterized an RNA aptamer that specifically bound to the alpha-fetoprotein using SELEX technology. The aptamer interacted with the AFP with a K{sub D} of {approx}33 nM. Importantly, the identified aptamer specifically and efficiently inhibited the AFP-mediated proliferation of hepatocarcinoma cells in a dose dependent manner. Moreover, the aptamer efficiently down-regulated AFP-induced expression of oncogenes in the cells. These results indicate that an AFP-specific RNA aptamer could be a useful therapeutic and diagnostic agent against AFP-related hepatocellular carcinoma.

  8. A dynamic ratio of the alpha+ and alpha- isoforms of the tight junction protein ZO-1 is characteristic of Caco-2 cells and correlates with their degree of differentiation.

    Science.gov (United States)

    Ciana, Annarita; Meier, Katharina; Daum, Nicole; Gerbes, Stefan; Veith, Michael; Lehr, Claus-Michael; Minetti, Giampaolo

    2010-05-07

    ZO-1 is a peripheral protein that plays a central role in the macromolecular assembly of tight junctions by interacting with integral proteins (occludin, claudins, JAMs) of the membrane of adjoining cells, with the actin cytoskeleton, and with nuclear factors. Human ZO-1 is expressed in all epithelia and some specialized endothelia as variable amounts of two related isoforms, which originate from the alternatively spliced mRNA transcripts alpha(+) and alpha(-) and whose specific differential role is still unknown. Moreover, little is known about the timing of expression of ZO-1 isoforms at the protein and mRNA level. This study shows that during growth of freshly plated Caco-2 cells, the alpha(+)/alpha(-) ratio increased as a result of simultaneous increase of alpha(+) and decrease of alpha(-). Differences in the isoform ratio also correlated with differences in epithelium differentiation. This was determined by aminopeptidase N measurements of cells grown on conventional substrates and on modified, micro/nano-patterned surfaces. A comparable shift of ZO-1 isoforms was not observed in other tumour cell lines of non-intestinal origin (A549, Calu-3). Pancreatic stem cells, propagated without exogenous differentiation stimuli, displayed a slight, stable prevalence of the alpha(-) isoform. Of the intestinal cell lines examined (Caco-2 and T84), only Caco-2 cells displayed a dramatic shift in isoform expression. This suggests that this tumour cell line retains to a higher degree a developmental programme related to the dynamic of enterocytic differentiation in vivo.

  9. Alpha-mangostin induces Ca2+-ATPase-dependent apoptosis via mitochondrial pathway in PC12 cells.

    Science.gov (United States)

    Sato, Ayumi; Fujiwara, Hironori; Oku, Hisae; Ishiguro, Kyoko; Ohizumi, Yasushi

    2004-05-01

    We investigated the cell death effects of eight xanthones on PC12 rat pheochromocytoma cells. Among these compounds, alpha-mangostin, from the fruit hull of Garcinia mangostana L., had the most potent effect with the EC(50) value of 4 microM. Alpha-mangostin-treated PC12 cells demonstrated typical apoptotic DNA fragmentation and caspase-3 cleavage (equivalent to activation). The flow cytometric analysis indicated that this compound induced apoptosis in time-and concentration-dependent manners. Alpha-mangostin showed the features of the mitochondrial apoptotic pathway such as mitochondrial membrane depolarization and cytochrome c release. Furthermore, alpha-mangostin inhibited the sarco(endo)plasmic reticulum Ca(2+)-ATPase markedly. There was a correlation between the Ca(2+)-ATPase inhibitory effects and the apoptotic effects of the xanthone derivatives. On the other hand, c-Jun NH(2)-terminal kinase (JNK/SAPK), one of the signaling molecules of endoplasmic reticulum (ER) stress, was activated with alpha-mangostin treatment. These results suggest that alpha-mangostin inhibits Ca(2+)-ATPase to cause apoptosis through the mitochondrial pathway.

  10. The integrin alpha 6 beta 1 promotes the survival of metastatic human breast carcinoma cells in mice

    DEFF Research Database (Denmark)

    Wewer, U M; Shaw, L M; Albrechtsen, R

    1997-01-01

    The role of the integrin alpha 6 beta 1 in breast carcinoma progression was studied by targeted elimination of this integrin in MDA-MB-435 cells, a human breast carcinoma cell line that is highly metastatic in athymic mice. The strategy used is based on the finding that expression of a cytoplasmi...... in the liver after intrahepatic injection because of extensive apoptosis in the beta 4-delta CYT transfectants. These data suggest that a major function of the alpha 6 beta 1 integrin in breast carcinoma is to facilitate tumorigenesis and promote tumor cell survival in distant organs....

  11. Plasmacytoid dendritic cells accumulate and secrete interferon alpha in lymph nodes of HIV-1 patients.

    Directory of Open Access Journals (Sweden)

    Clara Lehmann

    2010-06-01

    Full Text Available Circulating plasmacytoid dendritic cells (pDC decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNalpha, which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNalpha compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNalpha before undergoing cell death. Since IFNalpha inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4(+ T cells, thus playing into the mechanisms of AIDS immunopathogenesis.

  12. Modelling the electrical activity of pancreatic alpha-cells based on experimental data from intact mouse islets

    DEFF Research Database (Denmark)

    Diderichsen, Paul Matthias; Gopel, S.O.

    2006-01-01

    + current, a KATP conductance, and an unspecific leak current. Tolbutamide closes KATP channels in the alpha-cell, leading to a reduction of the resting conductance from 1.1 nS to 0.4 nS. This causes the alpha-cell to depolarise from -76 mV to 33 mV. When the basal membrane potential passes the range...... between -60 and -35 mV, the alpha-cell generates action potentials. At higher voltages, the alpha-cell enters a stable depolarised state and the electrical activity ceases. The effects of tolbutamide are simulated by gradually reducing the KATP conductance (g (K,ATP) ) from 500 pS to 0 pS. When g (K......,ATP) is between 72 nS and 303 nS, the model generates action potentials in the same voltage range as the alpha-cell. When g (K,ATP) is lower than 72 nS, the model enters a stable depolarised state, and firing of action potentials is inhibited due to voltage-dependent inactivation of the Na+ and T-type Ca2...

  13. Cell volume changes affect gluconeogenesis in the perfused liver of ...

    Indian Academy of Sciences (India)

    Unknown

    In conclusion, gluconeogenesis appears to play a vital role in C. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress. [Goswami C, Datta S, Biswas K and Saha N 2004 Cell volume changes affect gluconeogenesis in the perfused ...

  14. Obesity reversibly depletes the basal cell population and enhances mammary epithelial cell estrogen receptor alpha expression and progenitor activity.

    Science.gov (United States)

    Chamberlin, Tamara; D'Amato, Joseph V; Arendt, Lisa M

    2017-11-29

    Obesity is correlated with an increased risk for developing postmenopausal breast cancer. Since obesity rates continue to rise worldwide, it is important to understand how the obese microenvironment influences normal mammary tissue to increase breast cancer risk. We hypothesized that obesity increases the proportion of luminal progenitor cells, which are thought to be the cells of origin for the most common types of breast cancer, potentially leading to an increased risk for breast cancer. To study the obese microenvironment within the mammary gland, we used a high-fat diet mouse model of obesity and human breast tissue from reduction mammoplasty surgery. We identified changes in breast epithelial cell populations using flow cytometry for cell surface markers, in vitro functional assays and expression of markers on breast tissue sections. In both obese female mice and women, mammary epithelial cell populations demonstrated significant decreases in basal/myoepithelial cells, using either flow cytometry or cell-type-specific markers (SMA and p63). Estrogen receptor alpha (ERα) expression was significantly increased in luminal cells in obese mammary tissue, compared with control mice or breast tissue from lean women. Functional assays demonstrated significantly enhanced mammary epithelial progenitor activity in obese mammary epithelial cells and elevated numbers of ERα-positive epithelial cells that were co-labeled with markers of proliferation. Weight loss in a group of obese mice reversed increases in progenitor activity and ERα expression observed in obese mammary tissue. Obesity enhances ERα-positive epithelial cells, reduces the number of basal/myoepithelial cells, and increases stem/progenitor activity within normal mammary tissue in both women and female mice. These changes in epithelial cell populations induced by obesity are reversible with weight loss. Our findings support further studies to examine how obesity-induced changes in stem/progenitor cells

  15. Isorhamnetin Attenuates Staphylococcus aureus-Induced Lung Cell Injury by Inhibiting Alpha-Hemolysin Expression.

    Science.gov (United States)

    Jiang, Lanxiang; Li, Hongen; Wang, Laiying; Song, Zexin; Shi, Lei; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

    2016-03-01

    Staphylococcus aureus, like other gram-positive pathogens, has evolved a large repertoire of virulence factors as a powerful weapon to subvert the host immune system, among which alpha-hemolysin (Hla), a secreted pore-forming cytotoxin, plays a preeminent role. We observed a concentration-dependent reduction in Hla production by S. aureus in the presence of sub-inhibitory concentrations of isorhamnetin, a flavonoid from the fruits of Hippophae rhamnoides L., which has little antibacterial activity. We further evaluate the effect of isorhamnetin on the transcription of the Hla-encoding gene hla and RNAIII, an effector molecule in the agr system. Isorhamnetin significantly down-regulated RNAIII expression and subsequently inhibited hla transcription. In a co-culture of S. aureus and lung cells, topical isorhamnetin treatment protected against S. aureus-induced cell injury. Isorhamnetin may represent a leading compound for the development of anti-virulence drugs against S. aureus infections.

  16. alpha-1-Antitrypsin (AAT)-modified donor cells suppress GVHD but enhance the GVL effect: a role for mitochondrial bioenergetics

    NARCIS (Netherlands)

    Marcondes, A.M.; Karoopongse, E.; Lesnikova, M.; Margineantu, D.; Welte, T.; Dinarello, C.A.; Hockenbery, D.; Janciauskiene, S.; Deeg, H.J.

    2014-01-01

    Hematopoietic cell transplantation is curative in many patients. However, graft-versus-host disease (GVHD), triggered by alloreactive donor cells, has remained a major complication. Here, we show an inverse correlation between plasma alpha-1-antitrypsin (AAT) levels in human donors and the

  17. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    Science.gov (United States)

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Interferon-alpha-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets.

    Science.gov (United States)

    Riffault, S; Carrat, C; van Reeth, K; Pensaert, M; Charley, B

    2001-01-01

    Transmissible gastroenteritis virus (TGEV) infection of piglets results in a very rapid and massive release of IFN-alpha in serum and secretions. The objective of this work was to characterize the IFN-alpha-producing cells (IPC) in tissues of TGEV-infected piglets. Caesarean-derived colostrum-deprived piglets were infected orally with the TGEV virulent Miller strain and IPC were characterized in situ by immunohistochemistry, using a rabbit anti-pig IFN-alpha antiserum. IPC were almost exclusively detected in intestinal tissues and mesenteric lymph nodes (MLN), as early as 6 h post inoculation (p.i.), with a peak at 12-18 h. They disappeared by 24 h. IPC were localized between enterocytes in the small intestine epithelial layer, in the lamina propria, around the Peyer's patches and, at highest frequency, in MLN. Very few IPC were present in the spleen and popliteal lymph nodes of infected piglets. Double immunohistochemical staining for IFN-alpha and leukocyte markers on MLN cryosections showed that IPC were mainly Swine Leukocyte Antigen (SLA) class II positive, and were not stained by an anti-macrophage (SWC3a) MAb. In addition, double staining with anti-TGEV and anti-IFN-alpha MAbs showed that viral antigens were present in MLN, close to IPC. These results show for the first time the presence of IPC in gut mucosa and gut-associated lymphoid tissues in response to an enteropathogenic virus. Moreover, this work shows that IFN-alpha released in serum is likely to originate almost exclusively from gut IPC triggered locally by viral antigens to produce IFN-alpha, since there were very few IPC in spleen or peripheral lymph nodes. MHC class II molecule expression by gut-associated IPC suggests that these cells may be the in vivo mucosal counterparts of the dendritic cells recently shown to produce IFN-alpha after in vitro viral induction.

  19. Functional expression of four PDH-E(1)alpha recombinant histidine mutants in a human fibroblast cell line with zero endogenous PDH complex activity.

    Science.gov (United States)

    Seyda, A; Robinson, B H

    2000-04-21

    Conserved histidine residues have been implicated in the geometry and catalytic mechanism of the E(1)alpha proteins of the PDH complex. We constructed and expressed a series of PDH-E(1)alpha histidine mutants (H63, H84, H92, and H263) in a cell line with zero PDH complex activity due to a null E(1)alpha allele. Based on immunoblot and enzyme activity analyses, all introduced histidine mutations, with the exception of H92, affected the specific activity of the PDH complex. We showed that H63 and H263 are essential for the activity since mutations introduced at those sites produced a PDH complex with near-zero activity. Mutations introduced at H84 only partially reduced activity, implying that H84 may play a less critical role in the PDH complex. Mutations introduced at H92 caused the absence of immunoreactive material for both the E(1)alpha and E(1)beta subunits and may have impaired import or assembly of precursor peptides into the mature PDH complex. Copyright 2000 Academic Press.

  20. Immunohistochemical expression of alpha-smooth muscle actin and glucocorticoid and calcitonin receptors in central giant-cell lesions.

    Science.gov (United States)

    Maiz, Nancy Noya; de la Rosa-García, Estela; Camacho, María Esther Irigoyen

    2016-04-01

    Central giant-cell lesions (CGCLs) are reactive lesions that consist histologically of spindle-shaped stromal cells, (fibroblasts and myofibroblasts) loosely arranged in a fibrous stroma, multinucleated giant cells and mononuclear cells with haemorrhagic areas. This study identified the immunoexpression of alpha-smooth muscle actin in spindle-shaped stromal cells, and glucocorticoid and calcitonin receptors in multinucleated giant cells and mononuclear cells. Their association with the clinical and radiographic characteristics of these lesions was identified. Thirty-five cases of CGCLs were studied. Expression of alpha-smooth muscle actin, glucocorticoid and calcitonin was evaluated by immunohistochemistry. The labelling index was 100 times the quotient of the number of positive cells divided by the total number of cells of each type. Logistic regression analysis was applied. Alpha-smooth muscle actin was positive (54%) for spindle stromal cells (myofibroblasts). A significant association was observed with root resorption (P = 0.004) and cortical bone destruction (P = 0.024). Glucocorticoid immunoexpression was positive for 99% of the giant cells and 86.7% of the mononuclear cells. Glucocorticoid immunoexpression in the mononuclear cells was associated with root resorption (P = 0.031). A longer evolution time was associated with lower immunoexpression of glucocorticoid (OR 12.4: P = 0.047). Calcitonin immunoexpression was positive in 86% of the giant cells. Immunoexpression of calcitonin was associated with age (P = 0.040). Myofibroblasts are important components of CGCLs, stromal cells and alpha-smooth muscle. Actin immunoexpression was associated with root and cortical bone resorption. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. DOPAL is transmissible to and oligomerizes alpha-synuclein in human glial cells.

    Science.gov (United States)

    Jinsmaa, Yunden; Sullivan, Patricia; Sharabi, Yehonatan; Goldstein, David S

    2016-01-01

    Glial cytoplasmic inclusions (GCIs) containing alpha-synuclein (AS) are a neuropathologic hallmark of multiple system atrophy (MSA). Oligomerized AS is thought to be the pathogenic form of the protein. Glial cells normally express little AS, but they can take up AS from the extracellular fluid. 3,4-Dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in the intra-neuronal metabolism of dopamine (DA), potently oligomerizes AS. In this study we tested whether DOPAL is taken up by human glial cells and augments intracellular oligomerization of AS. DOPAL (exogenous or endogenous from co-incubation with PC12 cells) and AS (native or A53T mutant form) were added to the incubation medium of glial cells (glioblastoma or MO3.13 oligodendrocytes). Glial cellular contents of DOPAL and its intracellular metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at up to 180 min of incubation. Glial cellular AS oligomers were quantified by Western blotting. Neither glioblastoma nor MO3.13 cells contained endogenous catecholamines or AS. Co-incubation of the cells with DA-producing PC12 cells produced time-related increases in DOPAL and DOPAC contents. Similarly, glial cellular DOPAL and DOPAC contents increased rapidly after addition of DOPAL to the medium. After addition of native or A53T-AS, intracellular AS also increased. Incubation of glial cells with both DOPAL and AS enhanced the intracellular oligomerization of native and A53T-AS. DOPAL is transmissible to glial cells and enhances intracellular oligomerization of AS. An interaction of DOPAL with AS might help explain the formation of CGIs in MSA. Published by Elsevier B.V.

  2. Long-Term GABA Administration Induces Alpha Cell-Mediated Beta-like Cell Neogenesis

    DEFF Research Database (Denmark)

    Ben-Othman, Nouha; Vieira, Andhira; Courtney, Monica

    2017-01-01

    , these neo-generated β-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in β-like cell counts, suggestive of α-to-β-like cell conversion processes...

  3. Expression of the Alpha8 Integrin Chain Facilitates Phagocytosis by Renal Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Ines Marek

    2018-03-01

    Full Text Available Background/Aims: Healing of mesangioproliferative glomerulonephritis involves degradation of excess extracellular matrix, resolution of hypercellularity by apoptosis and phagocytosis of apoptotic cells. Integrin receptors participate in the regulation of phagocytosis. In mice deficient for alpha8 integrin (Itga8-/- healing of glomerulonephritis is delayed. As Itga8 is abundant in mesangial cells (MC which are non-professional phagocytes, we hypothesized that Itga8 facilitates phagocytosis of apoptotic cells and matrix components by MC. Methods: MC were isolated from wild type (WT and Itga8-/- mice. Latex beads were coated with matrix components. Apoptosis was induced by cisplatin in macrophages and in DiI-stained MC. After coincubation of latex beads or apoptotic cells with MC, the phagocytosis rate was detected in WT and Itga8-/- MC via fluorescence microscopy and FACS analysis. Results: Itga8-/- MC showed reduced phagocytosis of matrix-coated beads and apoptotic cells compared to WT MC. Reduction of stress fibers was observed in Itga8-/- compared to WT MC. Inhibition of cytoskeletal reorganization by inhibition of Rac1 or ROCK during phagocytosis significantly decreased the rate of phagocytosis by WT MC but not by Itga8-/- MC. Conclusion: The expression of Itga8 facilitates phagocytosis in MC, likely mediated by Itga8-cytoskeleton interactions. An impairment of MC phagocytosis might thus contribute to a delayed glomerular regeneration in Itga8-/- mice.

  4. Tumor necrosis factor-alpha during neonatal brain development affects anxiety- and depression-related behaviors in adult male and female mice.

    Science.gov (United States)

    Babri, Shirin; Doosti, Mohammad-Hossein; Salari, Ali-Akbar

    2014-03-15

    A nascent literature suggests that neonatal infection is a risk factor for the development of brain, behavior and hypothalamic-pituitary-adrenal axis which can affect anxiety- and depression-related behaviors in later life. It has been documented that neonatal infection raises the concentrations of tumor necrosis factor-alpha (TNF-α) in neonate rodents and such infections may result in neonatal brain injury, at least in part, through pro-inflammatory cytokines. In addition, previous studies have shown that TNF-α is involved in cellular differentiation, neurogenesis and programmed cell death during the development of the central nervous system. We investigated for the first time whether neonatal exposure to TNF-α can affect body weight, stress-induced corticosterone (COR), anxiety- and depression-related behaviors in adult mice. In the present study, neonatal mice were treated to recombinant mouse TNF-α (0.2, 0.4, 0.7 and 1 μg/kg) or saline on postnatal days 3 and 5, then adult male and female mice were exposed to different behavioral tests. The results indicated that neonatal TNF-α treatment reduced body weight in neonatal period in both sexes. In addition, this study presents findings indicating that high doses of TNF- increase stress-induced COR levels, anxiety- and depression-related behaviors in adult males, but increase levels of anxiety without significantly influencing depression in adult female mice [corrected]. Our findings suggest that TNF-α exposure during neonatal period can alter brain and behavior development in a dose and sex-dependent manner in mice. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

    International Nuclear Information System (INIS)

    Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y.

    1989-01-01

    The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [ 3 H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 ± 0.017% of hemoglobin in blood of normal adult, 0.11 ± 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [ 3 H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups

  6. Biosynthesis and transport of lysosomal alpha-glucosidase in the human colon carcinoma cell-line Caco-2: secretion from the apical surface

    NARCIS (Netherlands)

    Klumperman, J.; Fransen, J.A.; Boekestijn, J.C.; Oude Elferink, R.P.; Matter, K.; Hauri, H.P.; Tager, J.M.; Ginsel, L.A.

    1991-01-01

    The human adenocarcinoma cell line Caco-2 was used for studies on the biosynthesis and transport of lysosomal acid alpha-glucosidase in polarized epithelial cells. Metabolic labelling revealed that in Caco-2 cells alpha-glucosidase is synthesized as a precursor form of 110 x 10(3) Mr. This form is

  7. Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Miettinen, Johanna A., E-mail: johanna.miettinen@oulu.fi [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Pietilae, Mika [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Salonen, Riikka J. [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Ohlmeier, Steffen [Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 Oulu (Finland); Ylitalo, Kari; Huikuri, Heikki V. [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Lehenkari, Petri [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland)

    2011-04-01

    Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-{alpha}) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-{alpha} exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-{alpha} exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-{alpha} exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-{alpha} exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-{alpha} exposure, which might influence MSC differentiation stage and capacity.

  8. Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

    International Nuclear Information System (INIS)

    Thompson, Pamela D.; Sakwe, Amos; Koumangoye, Rainelli; Yarbrough, Wendell G.; Ochieng, Josiah; Marshall, Dana R.

    2014-01-01

    This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head

  9. Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Pamela D.; Sakwe, Amos [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Koumangoye, Rainelli [Division of Surgical Oncology and Endocrine Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Yarbrough, Wendell G. [Division of Otolaryngology, Departments of Surgery and Pathology and Yale Cancer Center, Yale University, New Haven, CT 06520 (United States); Ochieng, Josiah [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Marshall, Dana R., E-mail: dmarshall@mmc.edu [Department of Pathology, Anatomy and Cell Biology, Meharry Medical College, Nashville, TN 37208 (United States)

    2014-02-15

    This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head

  10. Gut-homing CD4+ T cell receptor alpha beta+ T cells in the pathogenesis of murine inflammatory bowel disease

    DEFF Research Database (Denmark)

    Rudolphi, A; Boll, G; Poulsen, S S

    1994-01-01

    We studied which T cell subsets from the gut-associated lymphoid tissue (GALT) can migrate out of the gut mucosa and repopulate GALT compartments of an immunodeficient (semi)syngeneic host. Many distinct lymphocyte subsets were found in GALT of immunocompetent H-2d (BALB/c, BALB/cdm2, C.B-17......+/+) mice. No antigen receptor-expressing lymphoid cells were found in GALT of congenic C.B-17 scid/scid (scid) mice. The heterotopic transplantation of a full-thickness gut wall graft from the ileum or colon of immunocompetent (C.B-17+/+, BALB/cdm2) donor mice onto immunodeficient scid mice selectively...... reconstituted a CD3+ T cell receptor alpha beta+ CD4+ T cell subset. CD4+ cells of this subset expressed the surface phenotype of mucosa-seeking, memory T cells. In the immunodeficient scid host, this gut-derived CD4+ T cell subset was found in spleen, peritoneal cavity, mesenteric lymph nodes (LN), epithelial...

  11. Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Maneckjee, R.; Minna, J.D. (National Cancer Institute-Navy Medical Oncology Branch, Bethesda, MD (USA) Uniformed Services Univ. of the Health Sciences, Bethesda, MD (USA))

    1990-05-01

    Using specific radioactively-labeled ligands, the authors find that lung cancer cell lines of diverse histologic types express multiple, high-affinity membrane receptors for {mu}, {delta}, and {kappa} opioid agonists and for nicotine and {alpha}-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas {mu}, {delta}, and {kappa} opioid agonists at low concentrations inhibited lung cancer growth in vitro. They also found that lung cancer cells expressed various combinations of immunoreactive opioid peptides ({beta}-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, they tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a tumor suppressor system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer.

  12. Phenotypical and functional characterization of double-negative (CD4-CD8-) alpha beta T-cell receptor positive cells from an immunodeficient patient

    DEFF Research Database (Denmark)

    Illum, N; Ralfkiaer, E; Pallesen, G

    1991-01-01

    We have characterized CD4-CD8- double-negative (DN) alpha beta TCR+ T cells from a patient with immunodeficiency, lymphocytosis, lymphadenopathy, and hepatosplenomegaly. The majority of peripheral blood lymphocytes were DN alpha beta TCR+ T cells as evaluated by FACS and biochemical analysis...... (MoAbs) indicated a polyclonal T-cell expansion. Thymic biopsy showed normal histology, whereas lymph node biopsy samples showed altered histological and immunohistological patterns with markedly expanded paracortical areas containing the DN T cells of the same phenotype as found in peripheral blood T...

  13. Influence of ?S-globin haplotypes and hydroxyurea on tumor necrosis factor-alpha levels in sickle cell anemia

    Directory of Open Access Journals (Sweden)

    Marília Rocha Laurentino

    2014-04-01

    Full Text Available Background: Sickle cell anemia is a chronic inflammatory disease characterized by an increased production of proinflammatory cytokines including tumor necrosis factor-alpha. Hydroxyurea, by decreasing the polymerization of hemoglobin, reduces inflammatory states. The effect of the genetic polymorphisms of sickle cell patients on tumor necrosis factor-alpha levels remains unknown. Objective: The aim of this study was to investigate the association of tumor necrosis factor-alpha levels with β-globin haplotypes and the use of hydroxyurea. Methods: A cross-sectional study was performed of 67 patients with sickle cell anemia diagnosed at steady-state in a referral hospital in Fortaleza, Ceará, Brazil. A group of 26 healthy individuals was used as control. βS-haplotype analysis was performed by restriction fragment length polymorphism-polymerase chain reaction. The tumor necrosis factor-alpha levels were measured by the enzyme-linked immunosorbent assay test. Laboratory data (complete blood count and fetal hemoglobin and information regarding the use of hydroxyurea were obtained from medical records. Statistical analysis was performed using R software with the Kruskal-Wallis and Mann-Whitney tests. Statistical significance was established for p-values < 0.05 for all analyses. Results: The mean age of the participants was 35.48 years. Patients with sickle cell anemia had significantly higher tumor necrosis factor-alpha levels than controls (p-values < 0.0001. Tumor necrosis factor-alpha levels were lower in sickle cell anemia patients who were receiving hydroxyurea treatment than those who were not (p-value = 0.1249. Sickle cell anemia patients with Bantu/n genotype had significantly higher levels than patients with the Bantu/Benin genotype (p-value = 0.0021. Conclusion: In summary, βS-globin haplotypes, but not hydroxyurea therapy, have a role in modulating tumor necrosis factor-alpha levels in sickle cell anemia adults at steady-state. Many

  14. Effect of alpha-interferon alone and combined with other antineoplastic agents on renal cell carcinoma determined by the tetrazolium microculture assay.

    Science.gov (United States)

    Homma, Y; Aso, Y

    1994-01-01

    The antiproliferative effect of various alpha-interferons (alpha-IFNs), alone or combined with other agents, on a renal cell carcinoma cell line was evaluated by the tetrazolium microculture assay to examine the rationale for combination therapies. Cells incubated in 96-week microculture plates at 5 x 10(3)/well were exposed to various agents for 3 days. There were no obvious differences in the growth inhibition caused by the 5 kinds of alpha-IFN examined as single agents. The combination of alpha-IFN with the following agents was also assessed: 5-fluorouracil (5FU), methotrexate (MTX), mitomycin C, bleomycin, cis-diaminedichloroplatinum (CDDP), vinblastine, etoposide (ETOP), alpha-IFN, tumor necrosis factor-alpha (TNF), and alpha-difluoromethylornithine. Synergism was observed for the combination of alpha-IFN+TNF, while the other combinations had additive or subadditive effects. No interference or antagonism was found. Trimodal combinations of alpha-IFN+MTX with either 5FU, ETOP, or CDDP all showed subadditive effects. These results indicated that an increased antiproliferative effect, although not necessarily synergistic, was obtained by the combination of alpha-IFN with a variety of antineoplastic agents, providing a rationale to seek for combination therapies including alpha-IFN for treating renal cell carcinoma.

  15. Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line

    International Nuclear Information System (INIS)

    Choi, Hyun-Jeung; Kim, Tae Yong; Ruiz-Llorente, Sergio; Jeon, Min Ji; Han, Ji Min; Kim, Won Gu; Shong, Young Kee; Kim, Won Bae

    2012-01-01

    Introduction: Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells. Methods: Human thyroid cancer-derived cell lines, TPC-1, were treated with ALA, and changes in NIS mRNA and protein expression were measured. ALA's effect on NIS gene promoter was evaluated, and functional NIS expression was assessed by iodide uptake assay. Results: Treatment with ALA increased NIS mRNA expression up to ten folds of control dose-dependently after 24 h of exposure. ALA increased NIS promoter activity, and increased iodide uptake by 1.6 fold. ALA induced expression of NIS protein, but had no significant effect on the plasma membrane trafficking. ALA increased phosphorylation of CREB and nuclear translocation of pCREB, and co-treatment of ALA and trichostatin A increased iodide uptake by three folds in TPC-1 cells. Conclusions: ALA is a potential agent to increase NIS transcription in TPC-1. It could be used as an adjunctive agent to increase efficacy of radioiodine therapy if combined with a strategy to increase NIS protein trafficking to cell membrane.

  16. Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells.

    Science.gov (United States)

    Zeng, Ling; Zhou, Hai-Yun; Tang, Na-Na; Zhang, Wei-Feng; He, Gui-Jun; Hao, Bo; Feng, Ya-Dong; Zhu, Hong

    2016-05-28

    To investigate the influence of phosphatidylinositol-3-kinase protein kinase B (PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, mRNA and activity levels of hypoxia inducible factor-1 alpha (HIF-1α), glucose transporter 1, hexokinase-II, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting siRNA to assess impact of the high expression of HIF-1α on glycolysis. HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymes and the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions. The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells.

  17. MicroRNA expression in alpha and beta cells of human pancreatic islets.

    Directory of Open Access Journals (Sweden)

    Dagmar Klein

    Full Text Available microRNAs (miRNAs play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98% subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs and 134 were expressed more in β-cells (β-miRNAs. Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different

  18. The alpha-cell as target for type 2 diabetes therapy

    DEFF Research Database (Denmark)

    Christensen, Mikkel; Bagger, Jonatan I; Vilsboll, Tina

    2011-01-01

    -coupled receptors in the hepatocytes. Type 2 diabetic patients are characterized by elevated glucagon levels contributing decisively to hyperglycemia in these patients. Accumulating evidence demonstrates that targeting the pancreatic alpha-cell and its main secretory product glucagon is a possible treatment...... for type 2 diabetes. Several lines of preclinical evidence have paved the way for the development of drugs, which suppress glucagon secretion or antagonize the glucagon receptor. In this review, the physiological actions of glucagon and the role of glucagon in type 2 diabetic pathophysiology are outlined....... Furthermore, potential advantages and limitations of antagonizing the glucagon receptor or suppressing glucagon secretion in the treatment of type 2 diabetes are discussed with a focus on already marketed drugs and drugs in clinical development. It is concluded that the development of novel glucagon receptor...

  19. [Cellular adhesion signal transduction network of tumor necrosis factor-alpha induced hepatocellular carcinoma cells].

    Science.gov (United States)

    Zheng, Yongchang; Du, Shunda; Xu, Haifeng; Xu, Yiyao; Zhao, Haitao; Chi, Tianyi; Lu, Xin; Sang, Xinting; Mao, Yilei

    2014-11-18

    To systemically explore the cellular adhesion signal transduction network of tumor necrosis factor-alpha (TNF-α)-induced hepatocellular carcinoma cells with bioinformatics tools. Published microarray dataset of TNF-α-induced HepG2, human transcription factor database HTRI and human protein-protein interaction database HPRD were used to construct and analyze the signal transduction network. In the signal transduction network, MYC and SP1 were the key nodes of signaling transduction. Several genes from the network were closely related with cellular adhesion.Epidermal growth factor receptor (EGFR) is a possible key gene of effectively regulating cellular adhesion during the induction of TNF-α. EGFR is a possible key gene for TNF-α-induced metastasis of hepatocellular carcinoma.

  20. Direct and remote modulation of L-channels in chromaffin cells: distinct actions on alpha1C and alpha1D subunits?

    Science.gov (United States)

    Baldelli, Pietro; Hernández-Guijo, Jesus Miguel; Carabelli, Valentina; Novara, Monica; Cesetti, Tiziana; Andrés-Mateos, Eva; Montiel, Carmen; Carbone, Emilio

    2004-02-01

    Understanding precisely the functioning of voltage-gated Ca2+ channels and their modulation by signaling molecules will help clarifying the Ca(2+)-dependent mechanisms controlling exocytosis in chromaffin cells. In recent years, we have learned more about the various pathways through which Ca2+ channels can be up- or down-modulated by hormones and neurotransmitters and how these changes may condition chromaffin cell activity and catecolamine release. Recently, the attention has been focused on the modulation of L-channels (CaV 1), which represent the major Ca2+ current component in rat and human chromaffin cells. L-channels are effectively inhibited by the released content of secretory granules or by applying mixtures of exogenous ATP, opioids, and adrenaline through the activation of receptor-coupled G proteins. This unusual inhibition persists in a wide range of potentials and results from a direct (membrane-delimited) interaction of G protein subunits with the L-channels co-localized in membrane microareas. Inhibition of L-channels can be reversed when the cAMP/PKA pathway is activated by membrane permeable cAMP analog or when cells are exposed to isoprenaline (remote action), suggesting the existence of parallel and opposite effects on L-channel gating by distinctly activated membrane autoreceptors. Here, the authors review the molecular components underlying these two opposing signaling pathways and present new evidence supporting the presence of two L-channel types in rat chromaffin cells (alpha1C and alpha1D), which open new interesting issues concerning Ca(2+)-channel modulation. In light of recent findings on the regulation of exocytosis by Ca(2+)-channel modulation, the authors explore the possible role of L-channels in the autocontrol of catecholamine release.

  1. Alpha Particles and X Rays Interact in Inducing DNA Damage in U2OS Cells.

    Science.gov (United States)

    Sollazzo, Alice; Brzozowska, Beata; Cheng, Lei; Lundholm, Lovisa; Haghdoost, Siamak; Scherthan, Harry; Wojcik, Andrzej

    2017-10-01

    Survivors of the atomic bombings of Hiroshima and Nagasaki are monitored for health effects within the Life Span Study (LSS). The LSS results represent the most important source of data about cancer effects from ionizing radiation exposure, which forms the foundation for the radiation protection system. One uncertainty connected to deriving universal risk factors from these results is related to the problem of mixed radiation qualities. The A-bomb explosions generated a mixed beam of the sparsely ionizing gamma radiation and densely ionizing neutrons. However, until now the possible interaction of the two radiation types of inducing biological effects has not been taken into consideration. The existence of such interaction would suggest that the application of risk factors derived from the LSS to predict cancer effects after pure gamma-ray irradiation (such as in the Fukushima prefecture) leads to an overestimation of risk. To analyze the possible interaction of radiation types, a mixed-beam exposure facility was constructed where cells can be exposed to sparsely ionizing X rays and densely ionizing alpha particles. U2OS cells were used, which are stably transfected with a plasmid coding for the DNA repair gene 53BP1 coupled to a gene coding for the green fluorescent protein (GFP). The induction and repair of DNA damage, which are known to be related to cancer induction, were analyzed. The results suggest that alpha particles and X rays interact, leading to cellular and possibly cancer effects, which cannot be accurately predicted based on assuming simple additivity of the individual mixed-beam components.

  2. Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

    Science.gov (United States)

    Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

    2015-04-29

    Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

  3. Study of Alpha Tocopherol, Celecoxib Induced Apoptosis in Human Colorectal Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    Solgui R

    2010-03-01

    Full Text Available Background and Objectives: Chronic, unbridled oxidative damages have been known as the culprits behind many chronic diseases, including cancers, atherosclerosis, diabetes and Alzheimer’s. Cyclooxygenase-2 (COX-2-the main enzyme involved in inducing these processes- plays an important role in tumor development and progression. COX-2 inhibitors should be used at high doses for a long time in order to bring about chemoprevention and induction of anti-tumor effects. For example, celecoxib prevents colorectal tumor growth and induces apoptosis in both in vitro and in vivo models. Disregulation of COX-2 expression coincides with the development of gastrointestinal malignancy in humans and in animal models of colorectal cancer. Increased COX-2 expression in human colorectal adeno-carcinomas has been elucidated when compared with normal adjacent colonic mucosa. The capacity of vitamin E, particularly in α form, to quench free radical damage, induces apoptosis and impact expression of oncogenes makes it an appropriate choice for chemotherapeutic strategies. Studies have shown that carcinogenesis and DNA damage due to UV are inhibited by vitamin E. The goal of this study was to investigate alpha tocopherol and celecoxib induced apoptosis in human colorectal carcinoma cell line.Methods: In this study, HT29 cells were exposed to different concentrations of tocopherol (5, 10, and 20µM and celecoxib (25, 50, 75, 100µM followed by DNA extraction and fragmentation for demonstrating cell death process. Results: The results indicated that celecoxib at lower doses (25, and 50µM could not induce cell death, but at higher doses (75, and 100 µM, DNA fragmentation results typically resembled programmed cell death.Conclusion: ocopherol (5, 10, and 20µM in combination with celecoxib improved the impact of celecoxib on cell death induction and made it the rational notion to be combined with vitamin E in clinical practice.

  4. [Effect of truncated PDGF-alpha receptor on proliferation and expression of c-sis mRNA of vascular smooth muscular cells of pulmonary artery].

    Science.gov (United States)

    Wang, Xiao-Qin; Su, Xu; Liu, Han-Min; Gao, Ju; Li, Feng-Yi; Dong, Li-Qun; Luo, Chun-Hua

    2005-11-01

    To investigate the effect of truncated PDGF-alpha receptor on proliferation expression of c-sis mRNA of pulmonary artery vascular smooth muscular cells(VSMC). Tissue mass culture was done to get vascular smooth muscular cells of pulmonary artery. Different concentrations of truncated PDGF-alpha receptor and adenoviral recombined body (Ad5CMV-PalphaRtr, ACP) were added into the cultures. VSMC proliferation curves were obtained using MTT test. The expression of c-sis mRNA was detected by PCR. ACP at 5 ml/L and 10 ml/L concentrations restrained the proliferation of VSMC apparently with the peak of cell growth being attenuated or eliminated. The curve presented ascending tendency only after 7 days. Affected by 5 ml/L ACP, PDGF-BB exerted no significant accelerating effect on the proliferation of VSMC. The expression of c-sis mRNA was up-regulated under the effect of ACP. Affected by 5 ml/L ACP and PDGF-BB, the expression of c-sis mRNA was down-regulated. ACP at 5 ml/L and 10 ml/L concentration powerful inhibitor of cellular proliferation for pulmonary artery VSMC. It can increase c-sis mRNA expression significantly and the increase seems to be ACP dosage dependent.

  5. The contribution of VHL substrate binding and HIF1-alpha to the phenotype of VHL loss in renal cell carcinoma.

    Science.gov (United States)

    Maranchie, Jodi K; Vasselli, James R; Riss, Joseph; Bonifacino, Juan S; Linehan, W Marston; Klausner, Richard D

    2002-04-01

    Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.

  6. Increased replication of T-cell-tropic HIV strains and CXC-chemokine receptor-4 induction in T cells treated with macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES beta-chemokines.

    Science.gov (United States)

    Dolei, A; Biolchini, A; Serra, C; Curreli, S; Gomes, E; Dianzani, F

    1998-01-22

    To study, in T-lymphoid cells, the effects of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES beta-chemokines on the replication of T-cell-tropic HIV-1 strains, since it has been reported that beta-chemokines interfere with the replication of macrophage-tropic HIV-1 strains, but not T-cell-tropic strains. Freshly phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL) and cultured PHA-activated T cells from healthy volunteers, as well as the C8166 T-cell line, were treated overnight with beta-chemokines before infection with T-cell-tropic HIV-1 isolates, or human T-lymphotropic virus type IIIB. HIV replication was followed by detecting the production of infectious particles, p24 antigen, and viral sequences. CXC-chemokine receptor (CXCR)-4 expression was followed by detection and quantification of specific transcripts. Pretreatment of T cells with MIP-1alpha, MIP-1beta and RANTES affected T-cell-tropic strains, increased the replication of HIV-1beta and HIV-1RPdT strains dose-dependently, as well as virus absorption and provirus DNA accumulation. These findings were associated with increased accumulation of CXCR-4 transcripts, and mediated by the protein tyrosine kinase signalling. Moreover, beta-chemokines stimulated PBL proliferation. Beta-chemokines increase the adsorption and replication of at least some T-cell-tropic HIV-1 strains, and this is related to stimulated expression of the CXCR-4 coreceptor.

  7. Expression of estrogen receptor beta increases integrin alpha1 and integrin beta1 levels and enhances adhesion of breast cancer cells.

    Science.gov (United States)

    Lindberg, Karolina; Ström, Anders; Lock, John G; Gustafsson, Jan-Ake; Haldosén, Lars-Arne; Helguero, Luisa A

    2010-01-01

    Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERalpha and ERbeta). Estrogen-bound ERalpha induces proliferation of mammary epithelial and cancer cells, while ERbeta is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERbeta levels compared to the early stage breast cancers, suggesting that loss of ERbeta could be important in cancer development. Analysis of ERbeta-/- mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERbeta is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERalpha and ERbeta. As ERbeta is widely found in breast cancer but not in cell lines, we used ERalpha positive T47-D and MCF-7 human breast cancer cells to generate cells with inducible ERbeta expression. Furthermore, the colon cancer cell lines SW480 and HT-29 were also used. Integrin alpha1 mRNA and protein levels increased following ERbeta expression. Integrin beta1-the unique partner for integrin alpha1-increased only at the protein level. ERbeta expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERbeta increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERbeta expression was associated to less cell migration. These results indicate that ERbeta affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells.

  8. Prothymosin alpha interacts with free core histones in the nucleus of dividing cells.

    Science.gov (United States)

    Covelo, Guillermo; Sarandeses, Concepción S; Díaz-Jullien, Cristina; Freire, Manuel

    2006-11-01

    The acidic protein prothymosin alpha (ProTalpha), with a broad presence in mammalian cells, has been widely considered to have a role in cell division, through an unrevealed mechanism in which histones may be involved in view of their ability to interact with ProTalpha in vitro. Results of co-immunoprecipitation experiments presented here demonstrate that ProTalpha interacts in vivo with core histones in proliferating B-lymphocytes (NC-37 cells). This interaction occurs with histones H3, H2A, H2B and H4 located free in the nucleoplasm, whereas no interaction was detected with histone H1, mono-nucleosome particles or chromatin. Moreover, the core histones form part of a nuclear multiprotein complex of about 700 kDa separated by ProTalpha-Sepharose affinity, with components including H3 and H4 acetyltranferases, H3 methyltransferases, hnRNP isotypes A3, A2/B1 and R, ATP-dependent and independent DNA helicases II, beta-actin and vimentin, all co-purifying by gel filtration. This indicates that the interaction of ProTalpha with core histones in the nucleus may be related to the structural modification of histones H3 and H4, and hence to chromatin activity, raising the possibility that the other proteins in the nuclear complex may play a role in this process.

  9. Induction of apoptosis in murine clonal osteoblasts expressed by human T-cell leukemia virus type I tax by NF-kappa B and TNF-alpha.

    Science.gov (United States)

    Kitajima, I; Nakajima, T; Imamura, T; Takasaki, I; Kawahara, K; Okano, T; Tokioka, T; Soejima, Y; Abeyama, K; Maruyama, I

    1996-02-01

    We investigated the effects of various cytokines in the presence of human T-cell leukemia virus type I (HTLV-I) tax protein in murine clonal osteoblasts, MC3T3-E1 cells. Skeletal remodeling by osteoclasts and osteoblasts is coordinated by cytokines, which are activated by HTLV-I tax protein via nuclear factor-kappa B (NF-kappa B). MC3T3-E1 cells were cocultured with an irradiated HTLV-I-producing lymphocyte cell line, MT-2. After coculture, the tumor necrosis factor-alpha (TNF-alpha) level in the medium was markedly elevated during the 7 days of culture, and MC3T3-E1 cells underwent apoptotic cell death. Marked apoptosis was also observed in MC3T3-E1 cells treated with MT-2 culture medium and in HTLV-I tax-expressing MC3T3-E1 clones, which both expressed high levels of TNF-alpha. This apoptosis was prevented by treatment with neutralizing anti-TNF-alpha antibody (alpha TNF). HTLV-I tax protein and TNF-alpha induced activation of NF-kappa B in apoptotic MC3T3-E1 cells. Decreased NF-kappa B activation was observed in HTLV-I tax-expressing MC3T3-E1 cells treated with alpha TNF. Our results suggest that HTLV-I tax activated NF-kappa B and subsequently TNF-alpha, leading to apoptosis of osteoblasts.

  10. The role of alpha-synuclein in melanin synthesis in melanoma and dopaminergic neuronal cells.

    Directory of Open Access Journals (Sweden)

    Tianhong Pan

    Full Text Available The relatively high co-occurrence of Parkinson's disease (PD and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM, the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR and inhibit tyrosine hydroxylase (TH, both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA, led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in

  11. Features of the alpha-cell population organization in pancreas of spontaneously hypertensive rats (SHR

    Directory of Open Access Journals (Sweden)

    T. V. Abramova

    2017-08-01

    Full Text Available Alpha cells of pancreatic islets constitute the second largest population of pancreatic endocrinocytes, and the glucagon synthesized in them plays an important role in the regulation of glucose homeostasis. At the same time, glucagon-suppressive therapy is defined as the strategically main source of success of therapy for patients with type 2 diabetes mellitus. In clinical practice, diabetes is often associated with hypertension in the metabolic syndrome, which causes interest in the pathophysiology of α-endocrinocytes. The aim of the study was to investigate the distribution parameters of α-cells in the pancreatic islets of SHR and to characterize the morphological and functional state of glucagon-synthesizing endocrinocytes Materials and methods. In the histological sections of the pancreas, glucagon was detected by the immunofluorescence method; the area of pancreatic islets, as well as the number of α-cells in them, the concentration of immunoreactive glucagon in these cells, the specific indices of the distribution of islets, α-cells and glucagon per unit area of the gland. The results were processed with a package of statistical programs, to assess the reliability of the differences in the groups, the Student’s t-test and the Wilcoxon W-test were used. Results. In normoglycemic SHR, the number of islets containing glucagon-synthesizing α-cells was 10% more (p <0.05 than in normotensive Wistar rats. In the pancreatic tissue, single α-endocrine cells were found in rats of both lines, not forming separate islets. In giant islets of SHR there were 72% more α-endocrine cells than in Wistar rats, and in large islets this difference was twofold. Accordingly, α-cells of these islets contributed significantly to higher glucagon levels in the pancreas of hypertensive rats: in giant islets, the amount of the hormone was 80%, and in large islets – 3.6 times higher than in normotensive rats. In this paper we discuss the mechanisms that lead

  12. Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

    Science.gov (United States)

    Poli, Alessandro; Fiume, Roberta; Baldanzi, Gianluca; Capello, Daniela; Ratti, Stefano; Gesi, Marco; Manzoli, Lucia; Graziani, Andrea; Suh, Pann-Ghill; Cocco, Lucio; Follo, Matilde Y

    2017-09-01

    Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Malignant T cells express lymphotoxin alpha and drive endothelial activation in cutaneous T cell lymphoma

    DEFF Research Database (Denmark)

    Lauenborg, Britt; Christensen, Louise; Ralfkiaer, Ulrik

    2015-01-01

    Lymphotoxin α (LTα) plays a key role in the formation of lymphatic vasculature and secondary lymphoid structures. Cutaneous T cell lymphoma (CTCL) is the most common primary lymphoma of the skin and in advanced stages, malignant T cells spreads through the lymphatic to regional lymph nodes...... to internal organs and blood. Yet, little is known about the mechanism of the CTCL dissemination. Here, we show that CTCL cells express LTα in situ and that LTα expression is driven by aberrantly activated JAK3/STAT5 pathway. Importantly, via TNF receptor 2, LTα functions as an autocrine factor by stimulating...... expression of IL-6 in the malignant cells. LTα and IL-6, together with VEGF promote angiogenesis by inducing endothelial cell sprouting and tube formation. Thus, we propose that LTα plays a role in malignant angiogenesis and disease progression in CTCL and may serve as a therapeutic target in this disease....

  14. Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo.

    Directory of Open Access Journals (Sweden)

    Elodie Angot

    Full Text Available Several people with Parkinson's disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10-22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.

  15. Ethanol increases tumor necrosis factor-alpha receptor-1 (TNF-R1) levels in hepatic, intestinal, and cardiac cells.

    Science.gov (United States)

    Rodriguez, Diego A; Moncada, Claudio; Núñez, Marco T; Lavandero, Sergio; Ponnappa, Biddanda C; Israel, Yedy

    2004-05-01

    Chronic ethanol consumption leads to cell injury in virtually every tissue. Tumor necrosis factor-alpha (TNF-alpha) constitutes a major factor in the development of alcohol-induced liver injury. In alcohol-dependent subjects, elevated levels of plasma TNF-alpha are strongly predictive of mortality. Binding of TNF-alpha to TNF-alpha receptor-1 (TNF-R1) activates death domain pathways, leading to necrosis and apoptosis in most tissues, and it also increases the expression of intercellular adhesion molecules (i.e., ICAM-1), which promote inflammation. We determined whether ethanol exposure leads to increases in cellular TNF-R1. We incubated HepG2 human hepatoma cells and H4-II-E-C3 rat hepatoma cells with 25, 50, and 100 mM ethanol for various intervals of time up to 48 h. Human colonic adenocarcinoma cells (Caco-2 cells) and neonatal rat primary cardiomyocytes were also incubated with different concentrations of ethanol. Levels of TNF-R1 were measured either by a sandwich enzyme-linked immunosorbent assay (ELISA) method or by determining the extracellular transmembrane domain of TNF-R1 by an intact-cell ELISA method. Ethanol exposure for 48 h increased TNF-R1 levels in human hepatoma cells in a dose-dependent manner. Levels increased significantly by 164% at 50 mM and by 240% at 100 mM ethanol. Effects were time dependent and did not reach a plateau at 48 h. Similar increases in TNF-R1 were also observed in rat hepatoma cells (90% at 50 mM and 230% at 100 mM ethanol). Under similar conditions, Caco-2 cells showed a significant 80% increase in TNF-R1 levels at 200 mM ethanol, a concentration found in intestine. Neonatal rat primary cardiomyocytes showed TNF-R1 increases of 36% at 50 mM and 44% at 100 mM ethanol. These results indicate that exposure of different cell types to pharmacologic concentrations of ethanol increases TNF-R1 levels and may augment TNF-alpha-mediated cell injury in different tissues.

  16. Nitric oxide mediated DNA double strand breaks induced in proliferating bystander cells after {alpha}-particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Han Wei [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Chen Shaopeng [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, 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, Tat Chee Avenue, Kowloon Tong (Hong Kong); Wu Lijun [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China)

    2010-02-03

    Low-dose {alpha}-particle exposures comprise 55% of the environmental dose to the human population and have been shown to induce bystander responses. Previous studies showed that bystander effect could induce stimulated cell growth or genotoxicity, such as excessive DNA double strand breaks (DSBs), micronuclei (MN), mutation and decreased cell viability, in the bystander cell population. In the present study, the stimulated cell growth, detected with flow cytometry (FCM), and the increased MN and DSB, detected with p53 binding protein 1 (53BP1) immunofluorescence, were observed simultaneously in the bystander cell population, which were co-cultured with cells irradiated by low-dose {alpha}-particles (1-10 cGy) in a mixed system. Further studies indicated that nitric oxide (NO) and transforming growth factor {beta}1 (TGF-{beta}1) played very important roles in mediating cell proliferation and inducing MN and DSB in the bystander population through treatments with NO scavenger and TGF-{beta}1 antibody. Low-concentrations of NO, generated by spermidine, were proved to induce cell proliferation, DSB and MN simultaneously. The proliferation or shortened cell cycle in bystander cells gave them insufficient time to repair DSBs. The increased cell division might increase the probability of carcinogenesis in bystander cells since cell proliferation increased the probability of mutation from the mis-repaired or un-repaired DSBs.

  17. Structural and Functional Studies of Ig[alpha beta] and Its Assembly with the B Cell Antigen Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Radaev, Sergei; Zou, Zhongcheng; Tolar, Pavel; Nguyen, Khanh; Nguyen, AnhThao; Krueger, Peter D.; Stutzman, Nicole; Pierce, Susan; Sun, Peter D. (NIH)

    2010-11-09

    The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Ig{beta} form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Ig{alpha} displays a similar fold as Ig{beta}. An Ig{alpha}{beta} heterodimer model was generated based on the unique disulfide-bonded Ig{beta} dimer. Solution binding studies showed that the extracellular domains of Ig{alpha}{beta} preferentially recognize the constant region of BCR with chain specificity, suggesting a role for Ig{alpha}{beta} to enhance BCR chain signaling. Cluster mutations on Ig{alpha}, Ig{beta}, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the C4 domain of mIgM. These studies provide the first structural model for understanding BCR function.

  18. Radiation and biophysical studies on cells and viruses. Progress report, April 1, 1976--June 30, 1977. [Gamma radiation, alpha particles

    Energy Technology Data Exchange (ETDEWEB)

    Cole, A.

    1977-01-01

    Progress is reported on the following research projects: genetic structure of DNA, chromosomes, and nucleoproteins; particle beam studies of radiosensitive sites; division delay in CHO cells induced by partly penetrating alpha particles; location of cellular sites for mutation induction; sites for radioinduced cell transformation using partly penetrating particle beams; gamma-ray and particle irradiation of nucleoproteins and other model systems; quantitation of surface antigens on normal and neoplastic cells by x-ray fluorescence; hyperthermic effects on cell survival and DNA repair mechanisms; and studies on radioinduced cell transformation. (HLW)

  19. Prolonged storage of red blood cells affects aminophospholipid translocase activity

    NARCIS (Netherlands)

    Verhoeven, A. J.; Hilarius, P. M.; Dekkers, D. W. C.; Lagerberg, J. W. M.; de Korte, D.

    2006-01-01

    BACKGROUND AND OBJECTIVES: Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting

  20. TNF-alpha stimulation increases dental pulp stem cell migration in vitro through integrin alpha-6 subunit upregulation.

    Science.gov (United States)

    Shi, Lei; Fu, Shanqi; Fahim, Sidra; Pan, Shuang; Lina, He; Mu, Xiaodan; Niu, Yumei

    2017-03-01

    The dissemination of stem cells into tissues requiring inflammatory and reparative response is fundamentally dependent upon their chemotactic migration. Expression of TNF-α is up regulated in inflamed pulps. Dental pulp cells are also known to express integrin α6 subunit. Expression of integrin subunit α6 has been linked to the acquisition of migratory potential in a wide variety of cell types in both pathological and physiological capacities. Therefore, in this study we examined the effects of a pleiotropic cytokine TNF-α on the migration of hDPSCs and investigated its relationship with expression of integrin α6 in hDPSCs during chemotactic migration. hDPSC cultures were established. Protein expression profile of α6 integrin subunit was determined. Effect of exogenous TNF-α (50ng/mL) on hDPSCs' migration potential was evaluated by transwell inserts and in vitro scratch assay. Upregulation/downregulation of TNF-α mediated migration was assayed in presence/absence of integrin α6 respectively. To suppress integrin α6 expression, cells were transfected with integrin α6 siRNA and then cell migration and cytoskeletal changes were evaluated. Our results showed significant increase of hDPSCs' migration after stimulation with TNF-α. By knockdown of integrin α6, which is upregulated by TNF-α, we observed a decrease in the TNF-α directed chemotaxis of hDPSCs. In this study, we show that activation of integrin α6 brought about by TNF-α led to an increase in migratory activity in DPSCs in vitro thus describing a novel association between a cytokine TNF-α and α6 chain of an adhesion receptor integrin in regulating migration of hDPSCs. Copyright © 2016. Published by Elsevier Ltd.

  1. Crossreactive T Cells Spotlight the Germline Rules for [alpha beta] T Cell-Receptor Interactions with MHC Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Shaodong; Huseby, Eric S.; Rubtsova, Kira; Scott-Browne, James; Crawford, Frances; Macdonald, Whitney A.; Marrack, Philippa; Kappler, John W. (HHMI); (NJMRC)

    2008-10-31

    To test whether highly crossreactive {alpha}{beta} T cell receptors (TCRs) produced during limited negative selection best illustrate evolutionarily conserved interactions between TCR and major histocompatibility complex (MHC) molecules, we solved the structures of three TCRs bound to the same MHC II peptide (IA{sup b}-3K). The TCRs had similar affinities for IA{sup b}-3K but varied from noncrossreactive to extremely crossreactive with other peptides and MHCs. Crossreactivity correlated with a shrinking, increasingly hydrophobic TCR-ligand interface, involving fewer TCR amino acids. A few CDR1 and CDR2 amino acids dominated the most crossreactive TCR interface with MHC, including V{beta}8 48Y and 54E and V{alpha}4 29Y, arranged to impose the familiar diagonal orientation of TCR on MHC. These interactions contribute to MHC binding by other TCRs using related V regions, but not usually so dominantly. These data show that crossreactive TCRs can spotlight the evolutionarily conserved features of TCR-MHC interactions and that these interactions impose the diagonal docking of TCRs on MHC.

  2. Resistin-like molecule alpha1 (Fizz1 recruits lung dendritic cells without causing pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Madala Satish K

    2012-06-01

    Full Text Available Abstract Background Resistin-like molecule alpha or found in inflammatory zone protein (Fizz1 is increased in pulmonary epithelial cells and also in limited amounts by other lung cells during various lung injuries and fibrosis. However, the direct role of Fizz1 produced in the pulmonary epithelium has not been determined. Methods Fizz1 Transgenic mice (CCSP/Fizz1 were generated that overexpress Fizz1 in the lung epithelium under the control of a doxycycline (Dox inducible lung epithelial cell specific promoter Scgb1a1 (Clara cell secretory protein, CCSP. Histology and FACS analysis of lung cells were used to identify the direct effects of Fizz1 in the transgenic mice (Dox treated when compared with control (CCSP/- mice. Intratracheal bleomycin sulfate or silica in saline and saline alone were used to study the role of Fizz1 during bleomycin- and silica-induced pulmonary fibrosis in CCSP/Fizz1 and CCSP/- mice. Weight change, pulmonary inflammation, and fibrosis were assessed 10 days post bleomycin or 28 days post silica challenge. Results When CCSP/Fizz1 mice were fed Dox food, elevated Fizz1 protein was detected in lung homogenates by western blot. Lungs of mice in which Fizz1 was induced in the epithelium contained increased lung cells staining for CD11c and F4/80 by FACS analysis consistent with increased dendritic cells however, no changes were observed in the percentage of interstitial macrophages compared to CCSP/- controls. No significant changes were found in the lung histology of CCSP/Fizz1 mice after up to 8 weeks of overexpression compared to CCSP/- controls. Overexpression of Fizz1 prior to challenge or following challenge with bleomycin or silica did not significantly alter airway inflammation or fibrosis compared to control mice. Conclusions The current study demonstrates that epithelial cell derived Fizz1 is sufficient to increase the bone-marrow derived dendritic cells in the lungs, but it is not sufficient to cause lung

  3. Effect of fluoxetine and adenosine receptor NECA agonist on G alpha q/11 protein of C6 glioma cells

    Czech Academy of Sciences Publication Activity Database

    Kovářů, H.; Kovářů, F.; Lisá, Věra

    2012-01-01

    Roč. 33, č. 6 (2012), s. 614-618 ISSN 0172-780X Institutional support: RVO:67985823 Keywords : C6 glioma cells * SSRI antidepressant * G alpha q/11 signalling * G protein coupled receptor Subject RIV: ED - Physiology Impact factor: 0.932, year: 2012

  4. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    Science.gov (United States)

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. WilsonU.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  5. BINDING OF STEROIDS AND ENVIRONMENTAL CHEMICALS TO THE RAINBOW TROUT ANDROGEN RECEPTOR ALPHA EXPRESSED IN COS CELLS

    Science.gov (United States)

    Binding of Steroids and Environmental Chemicals to the Rainbow Trout Androgen Receptor Alpha Expressed in COS Cells. Mary C. Cardon, L. Earl Gray. Jr., Phillip C. Hartig and Vickie S. Wilson U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology...

  6. Alpha-arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication

    Directory of Open Access Journals (Sweden)

    Anais Castagnola

    2017-10-01

    Full Text Available Insecticidal crystal (Cry proteins produced by the bacterium Bacillus thuringiensis (Bt target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we describe the secreted proteome (secretome of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to control buffer treatment. The Cry1Ac-induced secretome caused higher proliferation and differentiation and an overall reduction in total cell mortality over time in primary H. virescens midgut stem cell cultures when compared to treatment with control buffer secretome. Differential proteomics identified four proteins with significant differences in abundance comparing Cry1Ac-treated and control secretomes. The most significant difference detected in the Cry1Ac secretome was an arylphorin subunit alpha protein not detected in the control secretome. Feeding of purified alpha-arylphorin to H. virescens larvae resulted in midgut hyperplasia and significantly reduced susceptibility to Cry1Ac toxin compared to controls. These data identify alpha-arylphorin as a protein with a new putative role in the midgut regeneration process in response to Cry1Ac intoxication and possibly pathogen/abiotic stress, identifying alpha-arylphorin as a potential gene to target with insecticidal gene silencing for pest control.

  7. Cell surface estrogen receptor alpha is upregulated during subchronic metabolic stress and inhibits neuronal cell degeneration.

    Directory of Open Access Journals (Sweden)

    Cristiana Barbati

    Full Text Available In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP, a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2 was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a

  8. Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond

    Science.gov (United States)

    Liu, Kuang-Kai; Chen, Mei-Fang; Chen, Po-Yi; Lee, Tony J. F.; Cheng, Chia-Liang; Chang, Chia-Ching; Ho, Yen-Peng; Chao, Jui-I.

    2008-05-01

    Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

  9. ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Qing Lu

    Full Text Available Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs, which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen in vascular injury require the estrogen receptor alpha (ERα. ERα transduces the effects of estrogen via a classical DNA binding, "genomic" signaling pathway and via a more recently-described "rapid" signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα that is specifically defective in rapid signaling, but is competent to regulate transcription through the "genomic" pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen.

  10. ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells

    Science.gov (United States)

    Lu, Qing; Schnitzler, Gavin R.; Ueda, Kazutaka; Iyer, Lakshmanan K.; Diomede, Olga I.; Andrade, Tiffany; Karas, Richard H.

    2016-01-01

    Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, “genomic” signaling pathway and via a more recently-described “rapid” signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the “genomic” pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen. PMID:27035664

  11. Genomics of signaling crosstalk of estrogen receptor alpha in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Peter Dudek

    Full Text Available BACKGROUND: The estrogen receptor alpha (ERalpha is a ligand-regulated transcription factor. However, a wide variety of other extracellular signals can activate ERalpha in the absence of estrogen. The impact of these alternate modes of activation on gene expression profiles has not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: We show that estrogen, growth factors and cAMP elicit surprisingly distinct ERalpha-dependent transcriptional responses in human MCF7 breast cancer cells. In response to growth factors and cAMP, ERalpha primarily activates and represses genes, respectively. The combined treatments with the anti-estrogen tamoxifen and cAMP or growth factors regulate yet other sets of genes. In many cases, tamoxifen is perverted to an agonist, potentially mimicking what is happening in certain tamoxifen-resistant breast tumors and emphasizing the importance of the cellular signaling environment. Using a computational analysis, we predicted that a Hox protein might be involved in mediating such combinatorial effects, and then confirmed it experimentally. Although both tamoxifen and cAMP block the proliferation of MCF7 cells, their combined application stimulates it, and this can be blocked with a dominant-negative Hox mutant. CONCLUSIONS/SIGNIFICANCE: The activating signal dictates both target gene selection and regulation by ERalpha, and this has consequences on global gene expression patterns that may be relevant to understanding the progression of ERalpha-dependent carcinomas.

  12. Fusobacterium nucleatum infection of colonic cells stimulates MUC2 mucin and tumor necrosis factor alpha.

    Science.gov (United States)

    Dharmani, Poonam; Strauss, Jaclyn; Ambrose, Christian; Allen-Vercoe, Emma; Chadee, Kris

    2011-07-01

    The etiology of inflammatory bowel disease is not completely known, but it is influenced by the presence of normal gut microflora as well as yet-unrecognized pathogens. The anaerobic, Gram-negative bacterial species Fusobacterium nucleatum is a common resident of the human mouth and gut and varies in its pathogenic potential. In this study, we demonstrate that highly invasive F. nucleatum isolates derived from the inflamed guts of Crohn's disease patients evoked significantly greater MUC2 and tumor necrosis factor alpha (TNF-α) gene expression than minimally invasive strains isolated from the noninflamed gut in human colonic epithelial cells and in a rat ligated colonic loop model of infection. Only live F. nucleatum induced mucin secretion and TNF-α expression in direct contact with and/or during invasion of colonic cells. In rat colons, mucin secretion was augmented in response to a highly invasive F. nucleatum isolate but was unaffected by treatment with a minimally invasive strain. Taken together, these studies reveal that F. nucleatum may represent a challenging pathogen in the etiology of gut inflammatory diseases and highlight the importance of different pathotypes of candidate bacterial species in disease pathogenesis.

  13. Standoff alpha radiation detection for hot cell imaging and crime scene investigation

    Science.gov (United States)

    Kerst, Thomas; Sand, Johan; Ihantola, Sakari; Peräjärvi, Kari; Nicholl, Adrian; Hrnecek, Erich; Toivonen, Harri; Toivonen, Juha

    2018-02-01

    This paper presents the remote detection of alpha contamination in a nuclear facility. Alpha-active material in a shielded nuclear radiation containment chamber has been localized by optical means. Furthermore, sources of radiation danger have been identified in a staged crime scene setting. For this purpose, an electron-multiplying charge-coupled device camera was used to capture photons generated by alpha-induced air scintillation (radioluminescence). The detected radioluminescence was superimposed with a regular photograph to reveal the origin of the light and thereby the alpha radioactive material. The experimental results show that standoff detection of alpha contamination is a viable tool in radiation threat detection. Furthermore, the radioluminescence spectrum in the air is spectrally analyzed. Possibilities of camera-based alpha threat detection under various background lighting conditions are discussed.

  14. The neuropeptide alpha-melanocyte-stimulating hormone is critically involved in the development of cytotoxic CD8+ T cells in mice and humans.

    Directory of Open Access Journals (Sweden)

    Karin Loser

    Full Text Available BACKGROUND: The neuropeptide alpha-melanocyte-stimulating hormone is well known as a mediator of skin pigmentation. More recently, it has been shown that alpha-melanocyte-stimulating hormone also plays pivotal roles in energy homeostasis, sexual function, and inflammation or immunomodulation. Alpha-melanocyte-stimulating hormone exerts its antiinflammatory and immunomodulatory effects by binding to the melanocortin-1 receptor, and since T cells are important effectors during immune responses, we investigated the effects of alpha-melanocyte-stimulating hormone on T cell function. METHODOLOGY/PRINCIPAL FINDINGS: T cells were treated with alpha-melanocyte-stimulating hormone, and subsequently, their phenotype and function was analyzed in a contact allergy as well as a melanoma model. Furthermore, the relevance of alpha-melanocyte-stimulating hormone-mediated signaling for the induction of cytotoxicity was assessed in CD8(+ T cells from melanoma patients with functional and nonfunctional melanocortin-1 receptors. Here we demonstrate that the melanocortin-1 receptor is expressed by murine as well as human CD8(+ T cells, and we furthermore show that alpha-melanocyte-stimulating hormone/melanocortin-1 receptor-mediated signaling is critical for the induction of cytotoxicity in human and murine CD8(+ T cells. Upon adoptive transfer, alpha-melanocyte-stimulating hormone-treated murine CD8(+ T cells significantly reduced contact allergy responses in recipient mice. Additionally, the presented data indicate that alpha-melanocyte-stimulating hormone via signaling through a functional melanocortin-1 receptor augmented antitumoral immunity by up-regulating the expression of cytotoxic genes and enhancing the cytolytic activity in tumor-specific CD8(+ T cells. CONCLUSIONS/SIGNIFICANCE: Together, these results point to an important role of alpha-melanocyte-stimulating hormone in MHC class I-restricted cytotoxicity. Therefore, treatment of contact allergies or

  15. Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.

    OpenAIRE

    Aperia, A; Ibarra, F; Svensson, L B; Klee, C; Greengard, P

    1992-01-01

    The alpha-adrenergic agonist oxymetazoline increased Na+,K(+)-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the alpha 1-adrenergic antagonist prazosin or the alpha 2-adrenergic antagonist yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K(+)-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as well as by FK 506, an immun...

  16. Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Kristina Ramanauskiene

    2016-01-01

    Full Text Available Lemon balm (Melissa officinalis L. has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC50 290.5 μM after the incubation of 24 h and LC50 171.3 μM after 48 h. RA at concentration 80–130 μM suppresses the cell proliferation and has an antioxidant effect. 200 μM and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 μM–200 μM RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies.

  17. Genes affecting β-cell function in type 1 diabetes

    DEFF Research Database (Denmark)

    Fløyel, Tina; Kaur, Simranjeet; Pociot, Flemming

    2015-01-01

    Type 1 diabetes (T1D) is a multifactorial disease resulting from an immune-mediated destruction of the insulin-producing pancreatic β cells. Several environmental and genetic risk factors predispose to the disease. Genome-wide association studies (GWAS) have identified around 50 genetic regions...... that affect the risk of developing T1D, but the disease-causing variants and genes are still largely unknown. In this review, we discuss the current status of T1D susceptibility loci and candidate genes with focus on the β cell. At least 40 % of the genes in the T1D susceptibility loci are expressed in human...... islets and β cells, where they according to recent studies modulate the β-cell response to the immune system. As most of the risk variants map to noncoding regions of the genome, i.e., promoters, enhancers, intergenic regions, and noncoding genes, their possible involvement in T1D pathogenesis as gene...

  18. Nkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells.

    Science.gov (United States)

    Binot, A-C; Manfroid, I; Flasse, L; Winandy, M; Motte, P; Martial, J A; Peers, B; Voz, M L

    2010-04-15

    In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in beta-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of alpha-cells but has no effect on beta-, delta-, or epsilon-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of beta-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both beta- and alpha-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for alpha-cell than for beta-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control alpha- and beta-cell differentiation. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  19. Sertoli cell specific knockdown of RAR-related orphan receptor (ROR) alpha at puberty reduces sperm count in rats.

    Science.gov (United States)

    Mandal, Kamal; Sarkar, Rajesh K; Sen Sharma, Souvik; Jain, Ayushi; Majumdar, Subeer S

    2018-01-30

    Globally, there is an alarming decline in sperm count. Very often hormonal supplementation fails to restore normal sperm count. Sertoli cells (Sc) present within seminiferous tubules provide appropriate niche and factors required for the differentiation of germ cells (Gc) into mature sperm (spermatogenesis). Functionally compromised Sc may be one of the reasons for failure of hormones to facilitate normal spermatogenesis. Although role of secretory proteins and signaling molecules of Sc has been studied well, role of transcription factors regulating sperm count has not been addressed appropriately. Retinoic acid receptor-related orphan receptor (ROR)-alpha is one of such transcription factors reported in testis but its role in testicular function is not yet known. In a separate study, we found abundant ROR-alpha binding sites on promoter regions of several genes upregulated in pubertal rat Sc as compared to infant Sc. Immunostaining studies also revealed presence of ROR alpha in nucleus of pubertal Sc. We generated a transgenic knockdown rat model expressing shRNA targeted to ROR-alpha under Sc specific promoter, which is transcriptionally active only at and after puberty. ROR-alpha knockdown animals were found to have abnormal association of Sc and Gc, including Gc sloughing and restricted release of sperm. The knockdown animals displayed compromised spermatogenesis leading to significant reduction in sperm count. This is the first report describing the Sc specific role of ROR-alpha in maintaining quantitatively normal sperm output. Identification of various such molecules can generate avenues to limit or reverse an alarmingly declining sperm count witnessed globally in men. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. CREM Alpha Enhances IL-21 Production in T Cells In Vivo and In Vitro

    Science.gov (United States)

    Ohl, Kim; Wiener, Anastasia; Lippe, Ralph; Schippers, Angela; Zorn, Carolin; Roth, Johannes; Wagner, Norbert; Tenbrock, Klaus

    2016-01-01

    The cAMP-responsive element modulator alpha (CREMα) plays a role in autoimmunity and, in particular, in systemic lupus erythematosus. CREMα negatively regulates IL-2 transcription and activates IL-17 expression by direct transcriptional mechanisms. To understand the role of CREM in autoimmunity, we recently generated a mouse with a transgenic overexpression of CREMα selectively in T cells. This mouse is characterized by enhanced IL-17 and IL-21 expression. We, herein, dissect the transcriptional mechanisms of enhanced IL-21 transcription in these mice. T cells of CREMα transgenic mice display an enhanced binding of CREMα to the CD3ζ chain promoter resulting in decreased CD3ζ chain expression. This is accompanied by a decreased excitation threshold and enhanced Ca2+ influx, which is known to induce IL-21 expression via NFATc2 activation. However, CREMα directly binds to cAMP-response element (CRE) half-site within the Il-21 promoter, which results in enhanced promoter activity shown by promoter reporter assays. CREMα-induced IL-21 transcription is not abrogated in the presence of cyclosporine A but depends on an intact CRE site within the IL-21 promoter, which suggests that CREM largely enhances IL-21 expression by direct transcriptional regulation. IL-21 transcription is critical for IL-17 generation in these mice, since IL-21 receptor blockade downregulates IL-17 transcription to wild-type levels. Finally, this is of functional relevance since CREMα transgenic mice display enhanced disease activity in dextran sodium sulfate-induced colitis accompanied by higher local IL-21 expression. Thus, we describe two novel mechanisms of CREMα-dependent IL-21 transcription. Since T cells of systemic lupus erythematosus patients are characterized by enhanced IL-21 transcription, this might also be of functional relevance in humans. PMID:28066428

  1. Study of Alpha Tocopherol, Celecoxib Induced Apoptosis in Human Colorectal Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    R Solgui

    2012-05-01

    Full Text Available

    Background and Objectives: Chronic, unbridled oxidative damages have been known as the culprits behind many chronic diseases, including cancers, atherosclerosis, diabetes and Alzheimer’s. Cyclooxygenase-2 (COX-2-the main enzyme involved in inducing these processes- plays an important role in tumor development and progression. COX-2 inhibitors should be used at high doses for a long time in order to bring about chemoprevention and induction of anti-tumor effects. For example, celecoxib prevents colorectal tumor growth and induces apoptosis in both in vitro and in vivo models. Disregulation of COX-2 expression coincides with the development of gastrointestinal malignancy in humans and in animal models of colorectal cancer. Increased COX-2 expression in human colorectal adeno-carcinomas has been elucidated when compared with normal adjacent colonic mucosa. The capacity of vitamin E, particularly in α form, to quench free radical damage, induces apoptosis and impact expression of oncogenes makes it an appropriate choice for chemotherapeutic strategies. Studies have shown that carcinogenesis and DNA damage due to UV are inhibited by vitamin E. The goal of this study was to investigate alpha tocopherol and celecoxib induced apoptosis in human colorectal carcinoma cell line.

    Methods: In this study, HT29 cells were exposed to different concentrations of tocopherol (5, 10, and 20µM and celecoxib (25, 50, 75, 100µM followed by DNA extraction and fragmentation for demonstrating cell death process.

    Results: The results indicated that celecoxib at lower doses (25, and 50µM could not induce cell death, but at higher doses (75, and 100 µM, DNA fragmentation results typically resembled programmed cell death.

  2. alpha-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells

    NARCIS (Netherlands)

    Kazmierczak, Anna; Strosznajder, Joanna B.; Adamczyk, Agata

    2008-01-01

    alpha-Synuclein is the fundamental component of Lewy bodies which occur in the brain of 60% of sporadic and familial Alzheimer's disease patients. Moreover, a proteolytic fragment of alpha-synuclein, the so-called non-amyloid component of Alzheimer's disease amyloid, was found to be an integral part

  3. cPLA2alpha-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic beta-cells

    DEFF Research Database (Denmark)

    Juhl, Kirstine; Høy, Marianne; Olsen, Hervør L

    2003-01-01

    Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha...... from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination...... of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited...

  4. Modulation of cell proliferation and differentiation of human lung carcinoma cells by the interferon-alpha

    Czech Academy of Sciences Publication Activity Database

    Krejčová, Daniela; Procházková, Jiřina; Kubala, Lukáš; Pacherník, J.

    2009-01-01

    Roč. 28, č. 3 (2009), s. 294-301 ISSN 0231-5882 R&D Projects: GA ČR(CZ) GA524/06/1197; GA ČR(CZ) GP524/06/P345 Grant - others:GA ČR(CZ) GA301/08/0717 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : ABC transporter proteins * cell cycle * interferons Subject RIV: BO - Biophysics Impact factor: 0.741, year: 2009

  5. Alpha 1 Antitrypsin Inhibits Dendritic Cell Activation and Attenuates Nephritis in a Mouse Model of Lupus.

    Directory of Open Access Journals (Sweden)

    Ahmed S Elshikha

    Full Text Available Systemic lupus erythematosus (SLE is an autoimmune disorder with a worldwide distribution and considerable mortality and morbidity. Although the pathogenesis of this disease remains elusive, over-reactive dendritic cells (DCs play a critical role in the disease development. It has been shown that human alpha-1 antitrypsin (hAAT has protective effects in type 1 diabetes and rheumatoid arthritis mouse models. In the present study, we tested the effect of AAT on DC differentiation and functions, as well as its protective effect in a lupus-prone mouse model. We showed that hAAT treatment significantly inhibited LPS (TLR4 agonist and CpG (TLR9 agonist -induced bone-marrow (BM-derived conventional and plasmacytoid DC (cDC and pDC activation and reduced the production of inflammatory cytokines including IFN-I, TNF-α and IL-1β. In MRL/lpr mice, hAAT treatment significantly reduced BM-derived DC differentiation, serum autoantibody levels, and importantly attenuated renal pathology. Our results for the first time demonstrate that hAAT inhibits DC activation and function, and it also attenuates autoimmunity and renal damage in the MRL/lpr lupus model. These results imply that hAAT has a therapeutic potential for the treatment of SLE in humans.

  6. Tumor necrosis factor alpha maintains denervation-induced homeostatic synaptic plasticity of mouse dentate granule cells

    Directory of Open Access Journals (Sweden)

    Denise eBecker

    2013-12-01

    Full Text Available Neurons which lose part of their input respond with a compensatory increase in excitatory synaptic strength. This observation is of particular interest in the context of neurological diseases, which are accompanied by the loss of neurons and subsequent denervation of connected brain regions. However, while the cellular and molecular mechanisms of pharmacologically induced homeostatic synaptic plasticity have been identified to a certain degree, denervation-induced homeostatic synaptic plasticity remains not well understood. Here, we employed the entorhinal denervation in vitro model to study the role of tumor necrosis factor alpha (TNFα on changes in excitatory synaptic strength of mouse dentate granule cells following partial deafferentation. Our experiments disclose that TNFα is required for the maintenance of a compensatory increase in excitatory synaptic strength at 3/4 days postlesion (dpl, but not for the induction of synaptic scaling at 1 - 2 dpl. Furthermore, laser capture microdissection (LMD combined with quantitative PCR (qPCR demonstrates an increase in TNFα-mRNA levels in the denervated zone, which is consistent with our previous finding on a local, i.e., layer-specific increase in excitatory synaptic strength at 3 - 4 dpl. Immunostainings for the glial fibrillary acidic protein (GFAP and TNFα suggest that astrocytes are a source of TNFα in our experimental setting. We conclude that TNFα-signaling is a major regulatory system that aims at maintaining the homeostatic synaptic response of denervated neurons.

  7. Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

    Science.gov (United States)

    Baptista, Melisa J; O'Farrell, Casey; Daya, Sneha; Ahmad, Rili; Miller, David W; Hardy, John; Farrer, Matthew J; Cookson, Mark R

    2003-05-01

    Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

  8. A highly conserved phenylalanine in the alpha, beta-T cell receptor (TCR) constant region determines the integrity of TCR/CD3 complexes

    DEFF Research Database (Denmark)

    Caspar-Bauguil, S; Arnaud, J; Huchenq, A

    1994-01-01

    In the present study, we have investigated the importance of a phenylalanine (phe195) in the Tcr-C alpha region on Tcr-alpha,beta/CD3 membrane expression. An exchange of phe195 with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or ...

  9. Curcumin affects cell survival and cell volume regulation in human renal and intestinal cells

    International Nuclear Information System (INIS)

    Kössler, Sonja; Nofziger, Charity; Jakab, Martin; Dossena, Silvia; Paulmichl, Markus

    2012-01-01

    Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underlying mechanisms of these diverse effects are complex, not fully elucidated and subject of intense scientific debate. Despite increasing evidence indicating that different cation channels can be a molecular target for curcumin, very little is known about the effect of curcumin on chloride channels. Since, (i) the molecular structure of curcumin indicates that the substance could potentially interact with chloride channels, (ii) chloride channels play a role during the apoptotic process and regulation of the cell volume, and (iii) apoptosis is a well known effect of curcumin, we set out to investigate whether or not curcumin could (i) exert a modulatory effect (direct or indirect) on the swelling activated chloride current ICl swell in a human cell system, therefore (ii) affect cell volume regulation and (iii) ultimately modulate cell survival. The ICl swell channels, which are essential for regulating the cell volume after swelling, are also known to be activated under isotonic conditions as an early event in the apoptotic process. Here we show that long-term exposure of a human kidney cell line to extracellular 0.1–10 μM curcumin modulates ICl swell in a dose-dependent manner (0.1 μM curcumin is ineffective, 0.5–5.0 μM curcumin increase, while 10 μM curcumin decrease the current), and short-term exposure to micromolar concentrations of curcumin does not affect ICl swell neither if applied from the extracellular nor from the intracellular side – therefore, a direct effect of curcumin on ICl

  10. Factors affecting daughter cells' arrangement during the early bacterial divisions.

    Directory of Open Access Journals (Sweden)

    Pin-Tzu Su

    Full Text Available On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum.

  11. Does usnic acid affect microtubules in human cancer cells?

    Directory of Open Access Journals (Sweden)

    MA. O'Neill

    Full Text Available Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules or taxol (which stabilizes microtubules. Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

  12. Supramolecular Peptide Nanofiber Morphology Affects Mechanotransduction of Stem Cells.

    Science.gov (United States)

    Arslan, Elif; Hatip Koc, Meryem; Uysal, Ozge; Dikecoglu, Begum; Topal, Ahmet E; Garifullin, Ruslan; Ozkan, Alper D; Dana, Aykutlu; Hermida-Merino, Daniel; Castelletto, Valeria; Edwards-Gayle, Charlotte; Baday, Sefer; Hamley, Ian; Tekinay, Ayse B; Guler, Mustafa O

    2017-10-09

    Chirality and morphology are essential factors for protein function and interactions with other biomacromolecules. Extracellular matrix (ECM) proteins are also similar to other proteins in this sense; however, the complexity of the natural ECM makes it difficult to study these factors at the cellular level. The synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as model systems. In this work, we demonstrate that mechanosensory responses of stem cells are directly regulated by the chirality and morphology of ECM-mimetic peptide nanofibers with strictly controlled characteristics. Structural signals presented on l-amino acid containing cylindrical nanofibers (l-VV) favored the formation of integrin β1-based focal adhesion complexes, which increased the osteogenic potential of stem cells through the activation of nuclear YAP. On the other hand, twisted ribbon-like nanofibers (l-FF and d-FF) guided the cells into round shapes and decreased the formation of focal adhesion complexes, which resulted in the confinement of YAP proteins in the cytosol and a corresponding decrease in osteogenic potential. Interestingly, the d-form of twisted-ribbon like nanofibers (d-FF) increased the chondrogenic potential of stem cells more than their l-form (l-FF). Our results provide new insights into the importance and relevance of morphology and chirality of nanomaterials in their interactions with cells and reveal that precise control over the chemical and physical properties of nanostructures can affect stem cell fate even without the incorporation of specific epitopes.

  13. Stilbene Glucoside, a Putative Sleep Promoting Constituent from Polygonum multiflorum Affects Sleep Homeostasis by Affecting the Activities of Lactate Dehydrogenase and Salivary Alpha Amylase.

    Science.gov (United States)

    Wei, Qian; Ta, Guang; He, Wenjing; Wang, Wei; Wu, Qiucheng

    2017-01-01

    Chinese herbal medicine (CHM) has been used for treating insomnia for centuries. The most used CHM for insomnia was Polygonum multiflorum. However, the molecular mechanism for CHM preventing insomnia is unknown. Stilbene glucoside (THSG), an important active component of P. multiflorum, may play an important role for treating insomnia. To test the hypothesis, Kunming mice were treated with different dosages of THSG. To examine the sleep duration, a computer-controlled sleep-wake detection system was implemented. Electroencephalogram (EEG) and electromyogram (EMG) electrodes were implanted to determine sleep-wake state. RT-PCR and Western blot was used to measure the levels of lactate dehydrogenase (LDH) and saliva alpha amylase. Spearman's rank correlation coefficient was used to identify the strength of correlation between the variables. The results showed that THSG significantly prolonged the sleep time of the mice (palpha amylase (palpha amylase (pamylase were negatively associated with sleep duration (palpha amylase.

  14. Global metabolic profile identifies choline kinase alpha as a key regulator of glutathione-dependent antioxidant cell defense in ovarian carcinoma.

    Science.gov (United States)

    Granata, Anna; Nicoletti, Roberta; Perego, Paola; Iorio, Egidio; Krishnamachary, Balaji; Benigni, Fabio; Ricci, Alessandro; Podo, Franca; Bhujwalla, Zaver M; Canevari, Silvana; Bagnoli, Marina; Mezzanzanica, Delia

    2015-05-10

    Epithelial Ovarian Cancer (EOC) "cholinic phenotype", characterized by increased intracellular phosphocholine content sustained by over-expression/activity of choline kinase-alpha (ChoKα/CHKA), is a metabolic cellular reprogramming involved in chemoresistance with still unknown mechanisms.By stable CHKA silencing and global metabolic profiling here we demonstrate that CHKA knockdown hampers growth capability of EOC cell lines both in vitro and in xenotransplant in vivo models. It also affected antioxidant cellular defenses, decreasing glutathione and cysteine content while increasing intracellular levels of reactive oxygen species, overall sensitizing EOC cells to current chemotherapeutic regimens. Natural recovering of ChoKα expression after its transient silencing rescued the wild-type phenotype, restoring intracellular glutathione content and drug resistance. Rescue and phenocopy of siCHKA-related effects were also obtained by artificial modulation of glutathione levels. The direct relationship among CHKA expression, glutathione intracellular content and drug sensitivity was overall demonstrated in six different EOC cell lines but notably, siCHKA did not affect growth capability, glutathione metabolism and/or drug sensitivity of non-tumoral immortalized ovarian cells. The "cholinic phenotype", by recapitulating EOC addiction to glutathione content for the maintenance of the antioxidant defense, can be therefore considered a unique feature of cancer cells and a suitable target to improve chemotherapeutics efficacy.

  15. CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells in the tumor capsule of skin sweat gland neoplasms.

    Science.gov (United States)

    Nakayama, Hirofumi; Enzan, Hideaki; Miyazaki, Eriko; Moriki, Toshiaki; Toi, Makoto; Zhang, Yanhu

    2002-01-01

    To elucidate the roles of CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells at the tumor border of skin sweat gland neoplasms, we examined expression of stromal cell markers in the tumor capsule of 19 skin sweat gland neoplasms (16 mixed tumors of the skin and three nodular hidradenomas) using monoclonal antibodies to CD34, CD31, cytokeratin 14 (CK14), alpha-smooth muscle actin (ASMA) and high molecular weight caldesmon (HCD). We regarded CD34-positive, CD31-, CK14-, ASMA- and HCD-negative stromal cells to be CD34-positive stromal cells, and ASMA-positive, HCD-, CK14-, CD34- and CD31-negative stromal cells to be ASMA-positive stromal cells. CD34-positive stromal cells were detected in the tumor capsule of all 19 of the tumors examined. In nine of the 16 mixed tumors (56%) and all of the three nodular hidradenomas, ASMA-positive stromal cells were detected at the immediate inner side of the CD34-positive stromal cell layers. These results indicate that cellular components in the tumor capsules of mixed tumors of the skin and nodular hidradenomas are CD34-positive stromal cells and ASMA-positive stromal cells, and suggest that stromal cells of these two cell types are associated with tumor capsule formation of skin sweat gland neoplasms.

  16. Effects of Alpha Particle and Proton Beam Irradiation as Putative Cross-Talk between A549 Cancer Cells and the Endothelial Cells in a Co-Culture System

    International Nuclear Information System (INIS)

    Riquier, Hélène; Abel, Denis; Wera, Anne-Catherine; Heuskin, Anne-Catherine; Genard, Géraldine; Lucas, Stéphane; Michiels, Carine

    2015-01-01

    Background: High-LET ion irradiation is being more and more often used to control tumors in patients. Given that tumors are now considered as complex organs composed of multiple cell types that can influence radiosensitivity, we investigated the effects of proton and alpha particle irradiation on the possible radioprotective cross-talk between cancer and endothelial cells. Materials and Methods: We designed new irradiation chambers that allow co-culture study of cells irradiated with a particle beam. A549 lung carcinoma cells and endothelial cells (EC) were exposed to 1.5 Gy of proton beam or 1 and 2 Gy of alpha particles. Cell responses were studied by clonogenic assays and cell cycle was analyzed by flow cytometry. Gene expression studies were performed using Taqman low density array and by RT-qPCR. Results: A549 cells and EC displayed similar survival fraction and they had similar cell cycle distribution when irradiated alone or in co-culture. Both types of irradiation induced the overexpression of genes involved in cell growth, inflammation and angiogenesis. Conclusions: We set up new irradiation chamber in which two cell types were irradiated together with a particle beam. We could not show that tumor cells and endothelial cells were able to protect each other from particle irradiation. Gene expression changes were observed after particle irradiation that could suggest a possible radioprotective inter-cellular communication between the two cell types but further investigations are needed to confirm these results

  17. Effects of Alpha Particle and Proton Beam Irradiation as Putative Cross-Talk between A549 Cancer Cells and the Endothelial Cells in a Co-Culture System

    Energy Technology Data Exchange (ETDEWEB)

    Riquier, Hélène; Abel, Denis [URBC-NARILIS, University of Namur, 61 rue de Bruxelles, Namur 5000 (Belgium); Wera, Anne-Catherine; Heuskin, Anne-Catherine [LARN-PMR, NARILIS, University of Namur, Namur 5000 (Belgium); Genard, Géraldine [URBC-NARILIS, University of Namur, 61 rue de Bruxelles, Namur 5000 (Belgium); Lucas, Stéphane [LARN-PMR, NARILIS, University of Namur, Namur 5000 (Belgium); Michiels, Carine, E-mail: carine.michiels@unamur.be [URBC-NARILIS, University of Namur, 61 rue de Bruxelles, Namur 5000 (Belgium)

    2015-03-18

    Background: High-LET ion irradiation is being more and more often used to control tumors in patients. Given that tumors are now considered as complex organs composed of multiple cell types that can influence radiosensitivity, we investigated the effects of proton and alpha particle irradiation on the possible radioprotective cross-talk between cancer and endothelial cells. Materials and Methods: We designed new irradiation chambers that allow co-culture study of cells irradiated with a particle beam. A549 lung carcinoma cells and endothelial cells (EC) were exposed to 1.5 Gy of proton beam or 1 and 2 Gy of alpha particles. Cell responses were studied by clonogenic assays and cell cycle was analyzed by flow cytometry. Gene expression studies were performed using Taqman low density array and by RT-qPCR. Results: A549 cells and EC displayed similar survival fraction and they had similar cell cycle distribution when irradiated alone or in co-culture. Both types of irradiation induced the overexpression of genes involved in cell growth, inflammation and angiogenesis. Conclusions: We set up new irradiation chamber in which two cell types were irradiated together with a particle beam. We could not show that tumor cells and endothelial cells were able to protect each other from particle irradiation. Gene expression changes were observed after particle irradiation that could suggest a possible radioprotective inter-cellular communication between the two cell types but further investigations are needed to confirm these results.

  18. Quantitation of alpha-linolenic acid elongation to eicosapentaenoic and docosahexaenoic acid as affected by the ratio of n6/n3 fatty acids

    Directory of Open Access Journals (Sweden)

    Somoza Veronika

    2009-02-01

    Full Text Available Abstract Background Conversion of linoleic acid (LA and alpha-linolenic acid (ALA to their higher chain homologues in humans depends on the ratio of ingested n6 and n3 fatty acids. Design and methods In order to determine the most effective ratio with regard to the conversion of ALA to eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA, human hepatoma cells were incubated with varying ratios of [13C] labeled linoleic acid ([13C]LA- and alpha-linolenic acid ([13C]ALA-methylesters. Regulative cellular signal transduction pathways involved were studied by determinations of transcript levels of the genes encoding delta-5 desaturase (D5D and delta-6 desaturase (D6D, peroxisome proliferator-activated receptor alpha (PPARα and sterol regulatory element binding protein 1c (SREBP-1c. Mitogen-activated protein kinase kinase 1 (MEK1 and mitogen-activated protein kinase kinase kinase 1 (MEKK1 were also examined. Results Maximum conversion was observed in cells incubated with the mixture of [13C]LA/[13C]ALA at a ratio of 1:1, where 0.7% and 17% of the recovered [13C]ALA was converted to DHA and EPA, respectively. Furthermore, differential regulation of enzymes involved in the conversion at the transcript level, dependent on the ratio of administered n6 to n3 fatty acids in human hepatocytes was demonstrated. Conclusion Formation of EPA and DHA was highest at an administered LA/ALA ratio of 1:1, although gene expression of PPARα, SREBP-1c and D5D involved in ALA elongation were higher in the presence of ALA solely. Also, our findings suggest that a diet-induced enhancement of the cell membrane content of highly unsaturated fatty acids is only possible up to a certain level.

  19. B cell progenitors are arrested in maturation but have intact VDJ recombination in the absence of Ig-alpha and Ig-beta.

    Science.gov (United States)

    Pelanda, Roberta; Braun, Uschi; Hobeika, Elias; Nussenzweig, Michel C; Reth, Michael

    2002-07-15

    Ig-alpha and Ig-beta mediate surface expression and signaling of diverse B cell receptor complexes on precursor, immature, and mature B cells. Their expression begins before that of the Ig chains in early progenitor B cells. In this study, we describe the generation of Ig-alpha-deficient mice and their comparative analysis to mice deficient for Ig-beta, the membrane-IgM, and recombination-activating gene 2 to determine the requirement of Ig-alpha and Ig-beta in survival and differentiation of pro-B cells. We find that in the absence of Ig-alpha, B cell development does not progress beyond the progenitor stage, similar to what is observed in humans lacking this molecule. However, neither in Ig-alpha- nor in Ig-beta-deficient mice are pro-B cells impaired in V(D)J recombination, in the expression of intracellular Ig micro-chains, or in surviving in the bone marrow microenvironment. Finally, Ig-alpha and Ig-beta are not redundant in their putative function, as pro-B cells from Ig-alpha and Ig-beta double-deficient mice are similar to those from single-deficient animals in every aspect analyzed.

  20. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  1. PAR1 activation affects the neurotrophic properties of Schwann cells.

    Science.gov (United States)

    Pompili, Elena; Fabrizi, Cinzia; Somma, Francesca; Correani, Virginia; Maras, Bruno; Schininà, Maria Eugenia; Ciraci, Viviana; Artico, Marco; Fornai, Francesco; Fumagalli, Lorenzo

    2017-03-01

    Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Association of apoptosis with the inhibition of extracellular signal-regulated protein kinase activity in the tumor necrosis factor alpha-resistant ovarian carcinoma cell line UCI 101.

    Science.gov (United States)

    Yazlovitskaya, E M; Pelling, J C; Persons, D L

    1999-05-01

    Tumor necrosis factor-alpha (TNF alpha) can function as both an autocrine and a paracrine growth factor and may therefore play a role in ovarian tumor progression. TNF alpha initiates multiple cellular responses, many of which are mediated through the mitogen-activated protein kinase pathways, which transduce signals from the TNF alpha receptors through the cytoplasm to the nucleus, resulting in regulation of gene expression. We examined the role of c-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinase (ERK) 1 and 2 in the cellular growth response to TNF alpha in the ovarian carcinoma cell line UCI 101. JNK1 activity was increased to a maximum level ninefold above the basal level after 10-20 min of treatment with 10 ng/mL TNF alpha. A maximum threefold induction of ERK1/2 activity was observed after 1 min of treatment. At concentrations up to 100 ng/mL, TNF alpha had neither a stimulatory nor an inhibitory effect on growth of UCI 101 cells. However, inhibition of TNF alpha-induced ERK1/2 activity by the MAP/ERK kinase 1 inhibitor PD 98059 resulted in 60% inhibition of cell growth in TNF alpha-treated UCI 101 cells. This decrease in cell growth was accompanied by apoptosis, as demonstrated by the presence of a 180-bp DNA ladder. Thus, the inhibition of TNF alpha-induced ERK1/2 activity was associated with induction of apoptosis in the TNF alpha-resistant cell line UCI 101. Inhibition of TNF alpha-induced ERK1/2 activity was accompanied by a subsequent transient increase in TNF alpha-induced JNK1 activity. The significance of this increase with respect to apoptosis induction remains to be determined. These findings demonstrated that ERK1/2 activity can modulate cellular sensitivity to TNF alpha and suggested that the balance between the levels of ERK1/2 and JNK1 activation may be critical in the cellular growth response to TNF alpha.

  3. Enhancement of the adhesive and spreading potentials of ovarian carcinoma RMG-1 cells due to increased expression of integrin alpha5beta1 with the Lewis Y-structure on transfection of the alpha1,2-fucosyltransferase gene.

    Science.gov (United States)

    Yan, Li-Mei; Lin, Bei; Zhu, Lian-Cheng; Hao, Ying-Ying; Qi, Yue; Wang, Chang-Zhi; Gao, Song; Liu, Shui-Ce; Zhang, Shu-Lan; Iwamori, Masao

    2010-07-01

    Le(Y) antigen is known to be associated with malignant properties including metastasis and a poor prognosis of ovarian carcinomas. To clarify the mechanisms underling these properties, we established ovarian carcinoma-derived cells exhibiting enhanced expression of Le(Y) by transfection with alpha1,2-fucosyltransferase and compared their cellular properties with those of the original cells. So the human alpha1,2-fucosyltransferase gene was transfected into ovarian carcinoma-derived RMG-1 cells, which are known to contain Le(X), a precursor of Le(Y), and RMG-1-hFUT cells exhibiting enhanced expression of Le(Y) were established by selection with anti-Le(Y) antibodies, and their adhesive and spreading potentials on fibronectin-coated plates were compared with those of RMG-1 cells. Results showed that the relative expression of Le(Y) in RMG-1-hFUT cells was about 20-fold that in RMG-1 cells, and that of integrin alpha5beta1 and an integrin-mediated signal transduction molecule, focal adhesion kinase, was also increased in RMG-1-hFUT cells. Interestingly, anti-Le(Y) antibodies were revealed to immunoprecipitate integrin alpha5beta1, indicating that its oligosaccharides are composed of Le(Y), the amounts of which was substantially elevated in RMG-1-hFUT cells. The adhesion and spreading potentials on fibronectin-coated plates of RMG-1-hFUT cells were significantly enhanced in comparison to those of RMG-1 cells, and were greatly suppressed by anti-Le(Y) antibodies, indicating that Le(Y) is involved in the integrin-fibronectin interaction. These results suggested that transfection of the alpha1,2-fucosyltransferase gene into ovarian carcinoma-derived cells brought about elevated expression of integrin alpha5beta1 with Le(Y), resulting in enhancement of the adhesion and spreading potentials of cells through the integrin-fibronection interaction, which was inhibited by anti-Le(Y) antibodies. Thus, Le(Y) in integrin alpha5beta1 was thought to be involved in the enhanced cell

  4. SUPPLEMENTATION OF PATIENTS WITH HOMOZYGOUS SICKLE-CELL DISEASE WITH ZINC, ALPHA-TOCOPHEROL, VITAMIN-C, SOYBEAN OIL, AND FISH OIL

    NARCIS (Netherlands)

    MUSKIET, FAJ; MUSKIET, FD; MEIBORG, G; SCHERMER, JG

    1991-01-01

    Thirteen patients (aged 0.7-17.9 y) with homozygous sickle cell disease were supplemented with alpha-tocopherol, vitamin C, zinc, and soybean oil (suppl 1; for 8 mo) and alpha-tocopherol, vitamin C, and fish oil (suppl 2; for 7 mo). Urinary zinc (suppl 1), plasma vitamin C, plasma cholesterol ester

  5. Hypoxia inducible factor 1-alpha (HIF-1 alpha is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

    Directory of Open Access Journals (Sweden)

    Elisa Conde

    Full Text Available Acute tubular necrosis (ATN caused by ischemia/reperfusion (I/R during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α, using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

  6. Depletion of nuclear import protein karyopherin alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells.

    Science.gov (United States)

    Vuorinen, Elisa M; Rajala, Nina K; Ihalainen, Teemu O; Kallioniemi, Anne

    2018-03-27

    Nucleocytoplasmic transport is a tightly regulated process carried out by specific transport machinery, the defects of which may lead to a number of diseases including cancer. Karyopherin alpha 7 (KPNA7), the newest member of the karyopherin alpha nuclear importer family, is expressed at a high level during embryogenesis, reduced to very low or absent levels in most adult tissues but re-expressed in cancer cells. We used siRNA-based knock-down of KPNA7 in cancer cell lines, followed by functional assays (proliferation and cell cycle) and immunofluorescent stainings to determine the role of KPNA7 in regulation of cancer cell growth, proper mitosis and nuclear morphology. In the present study, we show that the silencing of KPNA7 results in a dramatic reduction in pancreatic and breast cancer cell growth, irrespective of the endogenous KPNA7 expression level. This growth inhibition is accompanied by a decrease in the fraction of S-phase cells as well as aberrant number of centrosomes and severe distortion of the mitotic spindles. In addition, KPNA7 depletion leads to reorganization of lamin A/C and B1, the main nuclear lamina proteins, and drastic alterations in nuclear morphology with lobulated and elongated nuclei. Taken together, our data provide new important evidence on the contribution of KPNA7 to the regulation of cancer cell growth and the maintenance of nuclear envelope environment, and thus deepens our understanding on the impact of nuclear transfer proteins in cancer pathogenesis.

  7. Expression of the alpha subunit of human chorionic gonadotropin is specifically correlated with tumorigenic expression in human cell hybrids.

    OpenAIRE

    Stanbridge, E J; Rosen, S W; Sussman, H H

    1982-01-01

    The expression of HeLa parent phenotype protein markers, the alpha subunit of human chorionic gonadotropin and placental alkaline phosphatase isoenzymes, has been evaluated in paired tumorigenic and nontumorigenic HeLa-fibroblast human cell hybrids. Both of these proteins have been used clinically as markers of malignancy. The results showed that both are expressed in the hybrids. Expression of the gonadotropin subunit in the hybrids is specifically correlated with tumorigenicity; the placent...

  8. Cell-specific immuno-localization of progesterone receptor alpha in the rabbit ovary during pregnancy and after parturition.

    Science.gov (United States)

    Abd-Elkareem, Mahmoud

    2017-05-01

    Progesterone receptor alpha (PRA) has a central coordinator role in the ovarian functions in mammals. The aim of this study was to investigate the immunolocalization of PRA in the rabbit ovary during pregnancy and after parturition. The rabbit ovary during pregnancy and after parturition had moderate cytoplasmic and moderate to intense nuclear PRA immunostaining in the ovarian surface epithelial cells, stromal cells and interstitial gland cells. The PRA was also present in granulosa cells and theca interna cells of the growing, small antral and mature Graafian follicles. Theca interna cells of the atretic antral follicle in addition to endothelial and fibroblast cells had PRA immunoreactivity. The PRA were also observed in the theca externa smooth muscle-like cells of the growing and antral follicles and in the telocytes. In the present study, the corpora haemorrhagica and early developing corpora lutea had, slight cytoplasmic and nuclear PRA immunostaining in the large lutein and small lutein cells. The endothelial cells of the corpora haemorrhagica and corpora lutea had an intense nuclear PRA immune signal. The corpora lutea at an advanced stage of development had moderate cytoplasmic and nuclear PRA immunostaining in the large lutein cells and intense nuclear PRA immunostaining in the small lutein cells. The regressed corpora lutea did not have PRA immunostaining in the apoptotic large lutein cells and moderate cytoplasmic and intense nuclear PRA immunostaining in the small lutein cells. Copyright © 2017. Published by Elsevier B.V.

  9. Ubiquitous hazardous metal lead induces TNF-{alpha} in human phagocytic THP-1 cells: Primary role of ERK 1/2

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Mohd Imran [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Islam, Najmul [Department of Biochemistry, J.N Medical College, Aligarh Muslim University, Aligarh (India); Sahasrabuddhe, Amogh A. [Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow (India); Mahdi, Abbas Ali [Department of Biochemistry, C.S.M. Medical University, Lucknow (India); Siddiqui, Huma; Ashquin, Mohd [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Ahmad, Iqbal, E-mail: ahmadi@sify.com [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India)

    2011-05-15

    Induction of tumor necrosis factor-{alpha} (TNF-{alpha}) in response to lead (Pb) exposure has been implicated in its immunotoxicity. However, the molecular mechanism by which Pb upregulates the level of TNF-{alpha} is wagely known. An attempt was therefore made to elucidate the mechanistic aspect of TNF-{alpha} induction, mainly focusing transcriptional and post transcriptional regulation via mitogen activated protein kinases (MAPKs) activation. We observed that exposure of Pb to human monocytic THP-1 cells resulted in significant enhanced production of TNF-{alpha} m-RNA and protein secretion. Moreover, the stability of TNF-{alpha} m-RNA was also increased as indicated by its half life. Notably, activation of ERK 1/2, p38 and JNK in Pb exposed THP-1 was also evident. Specific inhibitor of ERK1/2, PD 98059 caused significant inhibition in production and stability of TNF-{alpha} m-RNA. However, SB 203580 partially inhibited production and stability of TNF-{alpha} m-RNA. Interestingly, a combined exposure of these two inhibitors completely blocked modulation of TNF-{alpha} m-RNA. Data tends to suggest that expression and stability of TNF-{alpha} induction due to Pb exposure is mainly regulated through ERK. Briefly, these observations are useful in understanding some mechanistic aspects of proinflammatory and immunotoxicity of Pb, a globally acknowledged key environmental contaminant.

  10. Activity of interferon alpha, interleukin 6 and insulin in the regulation of differentiation in A549 alveolar carcinoma cells.

    Science.gov (United States)

    McCormick, C; Freshney, R I; Speirs, V

    1995-02-01

    The differentiation of A549, a human tumour cell line from type II pneumocytes, can be induced by a crude fibroblast-derived factor (FDF) isolated from the conditioned medium of glucocorticoid-treated lung fibroblasts. In the present report, we have used alkaline phosphatase as a differentiation marker to investigate the activity of a number of growth factors as potential candidates for this paracrine activity. This showed that insulin, interleukin 6 (IL-6), and interferon alpha (IFN-alpha) could simulate the activity of conditioned medium. Their effects were dexamethasone (DX) dependent, additive and reversible with a half-life of 1 week. Transforming growth factor alpha and beta, IL-1 alpha and epidermal growth factor, were all inhibitory, and inhibition was opposed, partially or completely, by DX. The most potent inducer was IL-6, but as DX was shown to decrease the concentration of IL-6 in lung fibroblast-conditioned medium it seems an unlikely candidate for FDF. Unlike FDF, all of the positive-acting factors were shown to induce plasminogen activator. FDF has also been shown to be active in the absence of DX. This suggests that differentiation-inducing activity may be present in several paracrine factors, but that so far a candidate for FDF has not been identified.

  11. Do cell phones affect establishing electronic working length?

    Science.gov (United States)

    Hurstel, Justine; Guivarc'h, Maud; Pommel, Ludovic; Camps, Jean; Tassery, Hervé; Cohen, Stephen; Bukiet, Frédéric

    2015-06-01

    affected the measurements (not significant). The electronic working length measurements gave the same results as the visual examination, and this length was not influenced by direct contact with a cell phone (not significant). It was also possible to determine the electronic working length under all the experimental conditions. Within the limitations of the present study, it can be concluded that patients can keep their cell phones on during root canal therapy without any adverse effect on electronic working length determination. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  12. Amiodarone affects Ebola virus binding and entry into target cells.

    Science.gov (United States)

    Salata, Cristiano; Munegato, Denis; Martelli, Francesco; Parolin, Cristina; Calistri, Arianna; Baritussio, Aldo; Palù, Giorgio

    2018-03-02

    Ebola Virus Disease is one of the most lethal transmissible infections characterized by a high fatality rate. Several research studies have aimed to identify effective antiviral agents. Amiodarone, a drug used for the treatment of arrhythmias, has been shown to inhibit filovirus infection in vitro by acting at the early step of the viral replication cycle. Here we demonstrate that amiodarone reduces virus binding to target cells and slows down the progression of the viral particles along the endocytic pathway. Overall our data support the notion that amiodarone interferes with Ebola virus infection by affecting cellular pathways/targets involved in the viral entry process.

  13. Peripheral blood IFN-gamma-secreting V alpha 24(+)V beta 11(+) NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load

    NARCIS (Netherlands)

    Molling, JW; Kolgen, W; van der Vliet, HJJ; Boomsma, MF; Kruizenga, H; Smorenburg, CH; Molenkamp, BG; Langendijk, JA; Leemans, CR; von Blomberg, BME; Scheper, RJ; van den Eertwegh, AJM

    2005-01-01

    Natural killer T (NKT) cells are CDld-restricted lvmphoid cells and are characterized by an invariant T-cell receptor, which in humans consists of a V alpha 24 chain paired with a V beta 11 chain. These cells are known for their rapid production of large amounts of cytokines (e.g., IFN-gamma and

  14. Genome rearrangement affects RNA virus adaptability on prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Kendra ePesko

    2015-04-01

    Full Text Available Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wildtype gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense nonsegmented RNA viruses (order Mononegavirales have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV variants with the nucleocapsid (N gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N-gene translocation towards the 5’ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function, especially on PC3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective

  15. Mechanisms of Alpha-Synuclein Action on Neurotransmission: Cell-Autonomous and Non-Cell Autonomous Role

    Directory of Open Access Journals (Sweden)

    Marco Emanuele

    2015-05-01

    Full Text Available Mutations and duplication/triplication of the alpha-synuclein (αSyn-coding gene have been found to cause familial Parkinson’s disease (PD, while genetic polymorphisms in the region controlling the expression level and stability of αSyn have been identified as risk factors for idiopathic PD, pointing to the importance of wild-type (wt αSyn dosage in the disease. Evidence that αSyn is present in the cerebrospinal fluid and interstitial brain tissue and that healthy neuronal grafts transplanted into PD patients often degenerate suggests that extracellularly-released αSyn plays a role in triggering the neurodegenerative process. αSyn’s role in neurotransmission has been shown in various cell culture models in which the protein was upregulated or deleted and in knock out and transgenic animal, with different results on αSyn’s effect on synaptic vesicle pool size and mobilization, αSyn being proposed as a negative or positive regulator of neurotransmitter release. In this review, we discuss the effect of αSyn on pre- and post-synaptic compartments in terms of synaptic vesicle trafficking, calcium entry and channel activity, and we focus on the process of exocytosis and internalization of αSyn and on the spreading of αSyn-driven effects due to the presence of the protein in the extracellular milieu.

  16. Alpha-Asarone Protects Endothelial Cells from Injury by Angiotensin II

    Directory of Open Access Journals (Sweden)

    Hai-Xia Shi

    2014-01-01

    Full Text Available α-Asarone is the major therapeutical constituent of Acorus tatarinowii Schott. In this study, the potential protective effects of α-asarone against endothelial cell injury induced by angiotensin II were investigated in vitro. The EA.hy926 cell line derived from human umbilical vein endothelial cells was pretreated with α-asarone (10, 50, 100 µmol/L for 1 h, followed by coincubation with Ang II (0.1 µmol/L for 24 h. Intracellular nitric oxide (NO and reactive oxygen species (ROS were detected by fluorescent dyes, and phosphorylation of endothelial nitric oxide synthase (eNOS at Ser1177 was determined by Western blotting. α-Asarone dose-dependently mitigated the Ang II-induced intracellular NO reduction (P0.05 was not affected after 24 h of incubation with α-asarone at 1–100 µmol/L. Therefore, α-asarone protects against Ang II-mediated damage of endothelial cells and may be developed to prevent injury to cardiovascular tissues.

  17. Use of antibodies against the variable regions of the T-cell receptor alpha/beta heterodimer for the study of cutaneous T-cell lymphomas.

    Science.gov (United States)

    Ralfkiaer, E; Wollf-Sneedorff, A; Vejlsgaard, G L

    1991-11-01

    Recent studies have suggested that antibodies against the variable (V) regions of the T-cell antigen receptor (TCR) may be used as markers for clonality and malignancy in T-cell infiltrates. We have investigated this by examining biopsy samples from 45 patients with cutaneous T-cell lymphomas (CTCL) for reactivity with seven antibodies against different V-gene families on the TCR alpha/beta heterodimer, i.e. ICI (V beta 5a), W112 (V beta 5b), OT145 (V beta 6a), 16G8 (V beta 8a), S511 (V beta 12a), F1 (V alpha 2a) and LC4 (alpha beta Va). Serial biopsies were available in 13 patients and a total of 62 samples were studied. The neoplastic cells in five cases were positive for either V beta 5 (one case), V beta 6 (one case), V beta 8 (two cases) or V beta 12 (one case). In the remaining 40 cases, no staining was seen of the neoplastic cells. These findings indicate that while antibodies against the TCR V-regions may be used as clonotypic markers for certain T-cell neoplasms, there is as yet not a sufficient number of anti-TCR V-region antibodies available for the routine diagnosis of these conditions.

  18. Food does not affect the pharmacokinetics of tesaglitazar, a novel dual peroxisome proliferator-activated receptor alpha/gamma agonist.

    Science.gov (United States)

    Samuelsson, S; Johansson, S; Halldórsdóttir, S; Stenhoff, H; Ohman, K P

    2006-09-01

    Tesaglitazar is a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist in development to treat lipid and glucose abnormalities associated with type 2 diabetes. This study evaluated the effects of food on tesaglitazar pharmacokinetics. In an open, randomized, 2-way crossover study, 20 healthy men received tesaglitazar 1 mg during fasting and after a high-fat, high-calorie breakfast. Blood samples were taken to assess pharmacokinetic variables. Systemic exposure to tesaglitazar was unaffected by food intake. Estimated ratios were 0.99 (90% confidence interval [CI], 0.94-1.04) for fed/fasted area under plasma concentration-time curve and 0.82 (90% CI, 0.78-0.86) for fed/fasted maximum plasma concentration (C(max)). Mean C(max) was approximately 18% lower (0.41 [95% CI, 0.38-0.43] versus 0.50 [95% CI, 0.47-0.53] mumol/L), and median time to C(max) was increased (2.00 vs 0.75 h) in fed versus fasted state. The median difference of t(max) was 1.25 h (P = .0001, signed-rank test). Tesaglitazar was well tolerated. Tesaglitazar pharmacokinetics is unaffected by food intake, allowing once-daily administration of tesaglitazar with or without food in clinical practice.

  19. Hypoxia and the Presence of Human Vascular Endothelial Cells Affect Prostate Cancer Cell Invasion and Metabolism

    Directory of Open Access Journals (Sweden)

    Ellen Ackerstaff

    2007-12-01

    Full Text Available Tumor progression and metastasis are influenced by hypoxia, as well as by interactions between cancer cells and components of the stroma, such as endothelial cells. Here, we have used a magnetic resonance (MRcompatible invasion assay to further understand the effects of hypoxia on human prostate cancer cell invasion and metabolism in the presence and absence of human umbilical vein endothelial cells (HUVECs. Additionally, we compared endogenous activities of selected proteases related to invasion in PC-3 cells and HUVECs, profiled gene expression of PC-3 cells by microarray, evaluated cell proliferation of PC-3 cells and HUVECs by flow cytometry, under hypoxic and oxygenated conditions. The invasion of less-invasive DU-145 cells was not affected by either hypoxia or the presence of HUVECs. However, hypoxia significantly decreased the invasion of PC-3 cells. This hypoxia-induced decrease was attenuated by the presence of HUVECs, whereas under oxygenated conditions, HUVECs did not alter the invasion of PC-3 cells. Cell metabolism changed distinctly with hypoxia and invasion. The endogenous activity of selected extracellular proteases, although altered by hypoxia, did not fully explain the hypoxia-induced changes in invasion. Gene expression profiling indicated that hypoxia affects multiple cellular functions and pathways.

  20. Comparison of alpha-Type-1 polarizing and standard dendritic cell cytokine cocktail for maturation of therapeutic monocyte-derived dendritic cell preparations from cancer patients

    DEFF Research Database (Denmark)

    Trepiakas, Redas; Pedersen, Anders Elm; Met, Ozcan

    2008-01-01

    The current "gold standard" for generation of dendritic cell (DC) used in DC-based cancer vaccine studies is maturation of monocyte-derived DCs with tumor necrosis factor-alpha (TNF-alpha)/IL-1beta/IL-6 and prostaglandin E(2) (PGE(2)). Recently, a protocol for producing so-called alpha-Type-1...... generation and to be superior to the standard DC (sDC) cocktail as it induced fully mature DCs with potent IL-12p70 secretion together with CCR7 expression which is necessary for priming of a TH1 response and for migration to the draining lymph node, respectively. In this study, we tested the adaptation...

  1. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  2. [1 alpha(OH) D3 (Alfarol) is effective for the treatment of chronic B cell leukemia: a case report].

    Science.gov (United States)

    Hashimoto, E; Takeuchi, H; Saitou, M; Hirashima, K

    1989-10-01

    We reported a case of chronic B-cell leukemia reacted to the administration of 1 alpha (OH)D3 (Alfarol-CHUGAI Pharm. Co.), The patient showed pancytopenia with IgM-kappa type monoclonal protein in the serum. The bone marrow aspiration was failed due to a dry tap, but the biopsied specimen showed a marked infiltration of small sized lymphoid cells with wide cytoplasm. The leukemic cells from peripheral blood showed a morphology of atypical hairy cells, Surface markers of the leukemic cells were IgM, D(kappa)+, CD 19+, CD 20+, CD 21- and HLADR+, The leukemic cells showed no L-tartrate resistant acid phosphatase sensitivity. This case was diagnosed as a chronic B-cell leukemia closely related to a hairy cell leukemia. The treatment with estrogen-chlorambucil compound (Bestrabucil-KUREHA Chem, Co.) or splenic irradiation was not effective. However, after two months' administration of Alfarol the regular blood transfusion was not needed because of increment of the Hb concentration. After eight months of its administration, the bone marrow aspirate showed a marked decrease in the number of the leukemic cells and a restoration of normal hematopoietic cells. This experience suggested that Alfarol in usefull for the treatment of chronic B cell leukemia including hairy cell leukemia and chronic lymphocytic leukemia.

  3. Ultraviolet B irradiation of skin induces mast cell degranulation and release of tumour necrosis factor-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, L.J. [University of Queensland, Brisbane, QLD (Australia). Dept. of Dentistry, Immunopathology Unit

    1995-06-01

    In the `sunburn` response in skin, dermal blood vessels are activated and traffic of dendritic Langerhans` cells altered. While these changes have been attributed to the cytokine TNF-{alpha}, the source of this acutely released TNF has not been identified. This report demonstrates that the `sunburn` response, both in vivo and in vitro, is accompanied by rapid degranulation of cutaneous mast cells, with consequential release of intracellular stores of TNF. Epidermal keratinocytes were only minor contributors to local TNF production. Expression of the TNF-inducible CD62E (E-selectin/ELAM-1) and CD54 adhesion molecules on cutaneous endothelium occurred 2 hours following mast cell degranulation, and this event was sensitive to blockade of mast cells with disodium cromoglycate. These results indicate that TNF release in skin in the acute sunburn response can largely be attributed to mast cells. 47 refs., 5 tabs., 2 figs.

  4. Essential role for retinoic acid in the promotion of CD4+ T cell effector responses via retinoic acid receptor alpha

    Science.gov (United States)

    Hall, J.A.; Cannons, J.L.; Grainger, J.R.; Santos, L.M. Dos; Hand, T.W.; Naik, S.; Wohlfert, E.A.; Chou, D.B.; Oldenhove, G.; Robinson, M.; Grigg, M.E.; Kastenmayer, R.; Schwartzberg, P.L.; Belkaid, Y.

    2012-01-01

    SUMMARY Vitamin A and its metabolite, retinoic acid (RA), have recently been implicated in the regulation of immune homeostasis via the peripheral induction of regulatory T cells. Here we show that RA is also required to elicit proinflammatory CD4+ helper T cell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) is the critical mediator of these effects. Strikingly, antagonism of RAR signaling and deficiency in RARα(Rara−/−) results in a cell autonomous CD4+ T cell activation defect. Altogether, these findings reveal a fundamental role for the RA/RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive T cell responses. PMID:21419664

  5. Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets

    DEFF Research Database (Denmark)

    Adriaenssens, Alice E; Svendsen, Berit; Lam, Brian Y H

    2016-01-01

    expressed in alpha, beta and delta cells. The gene encoding the ghrelin receptor, Ghsr, was highlighted as being highly expressed and enriched in delta cells. Activation of the ghrelin receptor raised cytosolic calcium levels in primary pancreatic delta cells and enhanced somatostatin secretion in perfused...... pancreases, correlating with a decrease in insulin and glucagon release. The inhibition of insulin secretion by ghrelin was prevented by somatostatin receptor antagonism. CONCLUSIONS/INTERPRETATION: Our transcriptomic database of genes expressed in the principal islet cell populations will facilitate...

  6. Alpha-mangostin promotes myoblast differentiation by modulating the gene-expression profile in C2C12 cells.

    Science.gov (United States)

    Horiba, Taro; Katsukawa, Masahiro; Abe, Keiko; Nakai, Yuji

    2014-01-01

    Alpha-mangostin, a xanthone contained mostly in mangosteen pericarp, has been reported to exert various biological functions. However, little is known about involvement of this xanthone in the muscle differentiation process. Here, we report the effect of α-mangostin on murine skeletal muscle-derived C2C12 myoblasts. α-mangostin stimulated myoblast differentiation leading to myotube formation. DNA microarray analysis revealed that genes associated with myoblast differentiation and muscle cell component formation were up-regulated in α-mangostin-treated cells. These results indicate that α-mangostin promotes myoblast differentiation through modulating the gene-expression profile in myoblasts.

  7. Collagen gel contraction serves to rapidly distinguish epithelial- and mesenchymal-derived cells irrespective of alpha-smooth muscle actin expression

    DEFF Research Database (Denmark)

    Nielsen, Helga Lind; Gudjonsson, Thorarinn; Villadsen, René

    2004-01-01

    contractility (10.0% reduction in gel height) than their true mesenchymal counterparts (53.1% reduction in gel height). To test whether at all force generation could be induced in the nonmesenchymal cells by alpha-sm actin, transductions were performed to obtain a tetracycline-dependent expression. Expression...... compartment and the stromal compartment express alpha smooth muscle actin (alpha-sm actin) as part of a myoepithelial or a myofibroblastic differentiation program, respectively. Moreover, because both epithelial- and mesenchymal-derived cells are nontumorigenic, other means of discrimination are warranted....... Here, we describe the contraction of hydrated collagen gels as a rapid functional assay for the distinction between epithelial- and mesenchymal-derived stromal-like cells irrespective of the status of alpha-sm actin expression. Three epithelial-derived cell lines and three genuine mesenchymal...

  8. Tributyltin (TBT) and Dibutyltin (DBT) Alter Secretion of Tumor Necrosis Factor Alpha (TNFα) from Human Natural Killer (NK) Cells and a Mixture of T cells and NK Cells

    Science.gov (United States)

    Hurt, Kelsi; Hurd-Brown, Tasia; Whalen, Margaret

    2012-01-01

    Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de-worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha (α). TNFα is an important regulator of adaptive and innate immune responses. TNFα promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24 h, 48 h, and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 µM) on TNFα secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200 - 2.5 nM) decreased TNFα secretion from NK cells. In the T/NK cells 200 nM TBT decreased secretion while 100-5 nM TBT increased secretion of TNFα. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNFα secretion while lower concentrations showed increased secretion. The effects of BTs on TNFα secretion are seen at concentrations present in human blood. PMID:23047847

  9. Evidence for mutagenic repair in V79 cell mutant with aphidicolin-resistant DNA polymerce-alpha

    International Nuclear Information System (INIS)

    Liu, P.K.; Chang, C.C.; Trosko, J.E.

    1984-01-01

    An aphidicolin-resistant (aphr) mutant of Chinese hamster V79 cells, aphr -4-2, is shown to be slow-growing, sensitive to ultraviolet (UV) irradiation, hypermutable for spontaneous and UV-induced mutations, and known to contain an aphr mutant DNA polymerase-alpha, with a 10-fold reduction in the Km for dCTP but not for dATP. We show here that the mutant had a normal repair replication measured by unscheduled DNA synthesis assay. The mutant was specifically sensitive and hypermutable to UV and N-methyl-N'-nitro-N-nitrosoguanidine, but it had normal sensitivity to ionizing radiation and dimethyl sulfate. Unlike the V79 (wt) cells, the mutant exhibited further enhancement in the already elevated mutability following UV and conditioned medium treatment. The mutant characteristic is explained by the presence of an error-prone long-patch excision repair synthesis. The association in the mutant properties--an aphr DNA polymerase-alpha, UV sensitivity, and hypermutability to UV-induced mutation--provides the genetic evidence that DNA polymerase-alpha is likely to be involved in UV-induced DNA repair synthesis

  10. Transcriptional Response of Human Cells to Microbeam Irradiation with 2.1 MeV Alpha Particles

    Science.gov (United States)

    Hellweg, C. E.; Bogner, S.; Spitta, L.; Arenz, A.; Baumstark-Khan, C.; Greif, K. D.; Giesen, U.

    Within the next decades an increasing number of human beings in space will be simultaneously exposed to different stimuli especially microgravity and radiation To assess the risks for humans during long-duration space missions the complex interplay of these parameters at the cellular level must be understood Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors Activation of the Nuclear Factor kappa B NF- kappa B pathway as a possible anti-apoptotic route represents such an important cellular stress response A screening assay for detection of NF- kappa B-dependent gene activation using the destabilized variant of Enhanced Green Fluorescent Protein d2EGFP as reporter protein had been developed It consists of Human Embryonic Kidney HEK 293 Cells stably transfected with a receptor-reporter-construct carrying d2EGFP under the control of a NF- kappa B response element Clones positive for Tumor Necrosis Factor alpha TNF- alpha inducible d2EGFP expression were selected as cellular reporters Irradiation was performed either with X-rays 150 kV 19 mA at DLR Cologne or with 2 1 MeV alpha particles LET sim 160 keV mu m at PTB Braunschweig After irradiation the following biological endpoints were determined i cell survival via the colony forming ability test ii time-dependent activation of NF- kappa B dependent d2EGFP gene expression using flow cytometry iii quantitative RT-PCR

  11. Fabrication of substrates with curvature for cell cultivation by alpha-particle irradiation and chemical etching of PADC films

    Science.gov (United States)

    Ng, C. K. M.; Tjhin, V. T.; Lin, A. C. C.; Cheng, J. P.; Cheng, S. H.; Yu, K. N.

    2012-05-01

    In the present paper, we developed a microfabrication technology to generate cell-culture substrates with identical chemistry and well-defined curvature. Micrometer-sized pits with curved surfaces were created on a two-dimensional surface of a polymer known as polyallyldiglycol carbonate (PADC). A PADC film was first irradiated by alpha particles and then chemically etched under specific conditions to generate pits with well-defined curvature at the incident positions of the alpha particles. The surface with these pits was employed as a model system for studying the effects of substrate curvature on cell behavior. As an application, the present work studied mechanosensing of substrate curvature by epithelial cells (HeLa cells) through regulation of microtubule (MT) dynamics. We used end-binding protein 3-green fluorescent protein (EB3-GFP) as a marker of MT growth to show that epithelial cells having migrated into the pits with curved surfaces had significantly smaller MT growth speeds than those having stayed on flat surfaces without the pits.

  12. Alpha-mangostin induces apoptosis through activation of reactive oxygen species and ASK1/p38 signaling pathway in cervical cancer cells

    OpenAIRE

    Lee, Chien-Hsing; Ying, Tsung-Ho; Chiou, Hui-Ling; Hsieh, Shu-Ching; Wen, Shiua-Hua; Chou, Ruey-Hwang; Hsieh, Yi-Hsien

    2017-01-01

    Alpha-mangostin, a natural xanthonoid, has been reported to possess the anti-cancer property in various types of human cancer. However, its effects and mechanism of ?-mangostin in cervical cancer remain unclear. We found that ?-mangostin effectively inhibited cell viability, resulted in loss of mitochondrial membrane potential (MMP), release of cytochrome C, increase of Bax, decrease of Bcl-2, and activation of caspase-9/caspase-3 cascade in cervical cancer cells. Alpha-mangostin elevated the...

  13. An altered gp100 peptide ligand with decreased binding by TCR and CD8alpha dissects T cell cytotoxicity from production of cytokines and activation of NFAT

    Directory of Open Access Journals (Sweden)

    Niels eSchaft

    2013-09-01

    Full Text Available Altered peptide ligands (APLs provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100(280-288 APLs with respect to T cell cytotoxicity, production of cytokines and activation of Nuclear Factor of Activated T cells (NFAT by human T cells gene-engineered with a gp100-HLA-A2-specific TCRalpha/beta. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3, which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6 elicited T cell cytotoxicity and production of IFNgamma, and to a lesser extent TNFalpha, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, TCR-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wt peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8alpha. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8alpha. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications.

  14. Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

    Science.gov (United States)

    Squillaro, Tiziana; Antonucci, Ivana; Alessio, Nicola; Esposito, Anna; Cipollaro, Marilena; Melone, Mariarosa Anna Beatrice; Peluso, Gianfranco; Stuppia, Liborio; Galderisi, Umberto

    2017-12-01

    Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity. © 2017 Wiley Periodicals, Inc.

  15. Alpha mangostin Inhibits Hepatic Stellate Cells Activation Through TGF-β/Smad and Akt Signaling Pathways: An in vitro Study in LX2.

    Science.gov (United States)

    Rahmaniah, Rahmaniah; Yuyuntia, Yuyuntia; Soetikno, Vivian; Arozal, Wawaimuli; Antarianto, Radiana Dhewayani; Louisa, Melva

    2018-03-01

    Alpha mangostin has been reported to have activity for the treatment of liver fibrosis in the rats. However, the mechanisms of action are poorly understood. This study was aimed to investigate the effect of alpha mangostin on hepatic stellate cells (HSC) activation and proliferation through TGF-β/Smad and Akt signaling pathways. Immortalized HSC, LX2 cells, were incubated with TGF-β with or without alpha mangostin (5 or 10 μM). Sorafenib 10 µM was used as positive control. LX2 viability was counted using trypan blue exclusion method. The effect of alpha mangostin on TGF-β concentrations, and the expressions of proliferation and fibrogenic markers were evaluated. Alpha mangostin treatment resulted in a reduced proliferation of HSC, decreased Ki-67 and p-Akt expressions. These findings were followed with decreased concentrations of TGF-β in the medium of cells treated with alpha mangostin, decreased expressions of COL1A1, TIMP1, PAI1, α-SMA, and p-Smad3 as fibrogenic markers. These effects were shown to be dose-dependent. Alpha mangostin inhibits hepatic stellate cells proliferation and activation through TGF-β/Smad and Akt signaling pathways in dose dependent manner. © Georg Thieme Verlag KG Stuttgart · New York.

  16. Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.

    Science.gov (United States)

    Aperia, A; Ibarra, F; Svensson, L B; Klee, C; Greengard, P

    1992-01-01

    The alpha-adrenergic agonist oxymetazoline increased Na+,K(+)-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the alpha 1-adrenergic antagonist prazosin or the alpha 2-adrenergic antagonist yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K(+)-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as well as by FK 506, an immunosuppressant agent known to inhibit calcineurin; these results indicate that the action of oxymetazoline is mediated via activation of calcineurin (a calcium/calmodulin-dependent protein phosphatase). Activation of the Na+,K(+)-ATPase by either oxymetazoline or A23187 was associated with a greater than 2-fold increase in its affinity for Na+. The results provide a biochemical mechanism by which norepinephrine, released from renal nerve terminals, stimulates Na+ retention. PMID:1380157

  17. Expression of the GABA(A) receptor alpha6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABA(A) receptors

    DEFF Research Database (Denmark)

    Carlson, B X; Belhage, B; Hansen, Gert Helge

    1997-01-01

    Da (alpha6 subunit) radioactive peaks in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [3H]Ro15-4513 in the 51-kDa peak; however...... that the major effect of THIP was to increase alpha6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in alpha6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest...

  18. Function of the integrin alpha 6 beta 1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor

    DEFF Research Database (Denmark)

    Shaw, L M; Chao, C; Wewer, U M

    1996-01-01

    The involvement of the alpha 6 beta a integrin, a laminin receptor, in breast carcinoma progression needs to be addressed rigorously. We report that a human breast carcinoma cell line, MDA-MB-435, known to be highly invasive and metastatic, expresses three potential integrin laminin receptors......, and their capacity to invade Matrigel was reduced significantly. These findings support the hypothesis that alpha 6 beta 1 is important for breast cancer progression. Moreover, this approach is a powerful method that should be useful in assessing the role of alpha 6 beta 1 in other cells....

  19. Loss of Gadkin Affects Dendritic Cell Migration In Vitro.

    Directory of Open Access Journals (Sweden)

    Hannah Schachtner

    Full Text Available Migration is crucial for the function of dendritic cells (DCs, which act as outposts of the immune system. Upon detection of pathogens, skin- and mucosa-resident DCs migrate to secondary lymphoid organs where they activate T cells. DC motility relies critically on the actin cytoskeleton, which is regulated by the actin-related protein 2/3 (ARP2/3 complex, a nucleator of branched actin networks. Consequently, loss of ARP2/3 stimulators and upstream Rho family GTPases dramatically impairs DC migration. However, nothing is known yet about the relevance of ARP2/3 inhibitors for DC migration. We previously demonstrated that the AP-1-associated adaptor protein Gadkin inhibits ARP2/3 by sequestering it on intracellular vesicles. Consistent with a role of Gadkin in DC physiology, we here report Gadkin expression in bone marrow-derived DCs and show that its protein level and posttranslational modification are regulated upon LPS-induced DC maturation. DCs derived from Gadkin-deficient mice were normal with regards to differentiation and maturation, but displayed increased actin polymerization. While the actin-dependent processes of macropinocytosis and cell spreading were not affected, loss of Gadkin significantly impaired DC migration in vitro, however, in vivo DC migration was unperturbed suggesting the presence of compensatory mechanisms.

  20. Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

    Science.gov (United States)

    Sobkowiak, Robert; Zielezinski, Andrzej; Karlowski, Wojciech M; Lesicki, Andrzej

    2017-10-01

    Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

  1. Glycoconjugate with terminal alpha galactose. A property common to basal cells and a subpopulation of columnar cells of numerous epithelia in mouse and rat.

    Science.gov (United States)

    Flint, F F; Schulte, B A; Spicer, S S

    1986-01-01

    Glycoconjugates associated with the basal cell layer of various types of epithelia in the mouse and rat were examined histochemically with a battery of lectin-horseradish peroxidase (HRP) conjugates of differing sugar binding specificities. Basal cells in paraffin sections of composite tissue blocks stained with an isolectin from Griffonia simplicifolia (GSA I-B4) specific for terminal alpha-galactose residues but failed to react with the other lectins. Basal cells in epithelium lining striated and excretory ducts of salivary and lacrimal glands, tongue, esophagus, trachea, renal calyx, ureter, urinary bladder, urethra, epididymis and vas deferens stained selectively and intensely for content of a glycoconjugate with terminal alpha-galactose. This galacto-conjugate appeared associated with the plasmalemma of basal cells. Basal cells with a galactocalyx formed an intermittent to continuous layer generally increasing in prevalence distally in glandular duct systems. A minor population of pyramido-columnar cells with cytosolic GSA I-B4 reactivity occurred in striated ducts and appeared less numerous in intralobular excretory ducts and more prevalent in extraglandular ducts. In trachea and renal pelvis, the GSA I-B4 positive cell profiles ranged from low cuboidal to tall pyramidal in contour, but the latter appeared not to reach the lumen. In contrast, no GSA I-B4 positive basal cells were seen in any segment of the pancreatic or bile ducts or in the epithelium of the gastrointestinal tract. These findings suggest that the basal cells found in similar sites in different epithelia and possessing in common a unique alpha-galactoconjugate may function in a manner common to all and not simply in providing progenitor cells for epithelial renewal.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Breast cancer cells in three-dimensional culture display an enhanced radioresponse after coordinate targeting of integrin alpha5beta1 and fibronectin.

    Science.gov (United States)

    Nam, Jin-Min; Onodera, Yasuhito; Bissell, Mina J; Park, Catherine C

    2010-07-01

    Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that beta1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific alpha heterodimer of beta1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare alpha-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of alpha5beta1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of alpha5beta1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of alpha5beta1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of alpha5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and alpha5beta1-integrin as targets for breast cancer therapy. Copyright 2010 AACR.

  3. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  4. Alpha-1-Antitrypsin Antagonizes Cisplatin-Induced Cytotoxicity in Prostate Cancer (PC3) and Melanoma Cancer (A375) Cell Lines.

    Science.gov (United States)

    Ljujic, Mila; Mijatovic, Sanja; Bulatovic, Mirna Z; Mojic, Marija; Maksimovic-Ivanic, Danijela; Radojkovic, Dragica; Topic, Aleksandra

    2017-04-01

    Increased circulating alpha-1-antitrypsin (AAT) correlates with cancer stage/aggressiveness, but its role in cancer biology is unclear. We revealed antagonistic effect of AAT to cisplatin-induced cytotoxicity in prostate (PC3) and melanoma (A375) cancer cell lines. Moreover, AAT abrogated cytotoxicity of MEK inhibitor U0126 in PC3 cell line. Weaker antagonistic effect of AAT on cytotoxicity of PI3/Akt and NF-kB inhibitors was also observed. In addition, cisplatin increased AAT gene expression in transfected PC3 cells. However, AAT derived from transfected PC3 cells did not antagonize cisplatin-induced cytotoxicity. In conclusion, these results suggest possible association between high circulating AAT and cisplatin resistance.

  5. Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins.

    Science.gov (United States)

    Vickaryous, Nicola K; Teh, Evelyn M; Stewart, Bruce; Dolphin, Peter J; Too, Catherine K L; McLeod, Roger S

    2003-03-21

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the

  6. Intracellular Modifiers of Integrin Alpha 6p Production in Aggressive Prostate and Breast Cancer Cell Lines

    Science.gov (United States)

    Kacsinta, Apollo D.; Rubenstein, Cynthia S.; Sroka, Isis C.; Pawar, Sangita; Gard, Jaime M.; Nagle, Raymond B.; Cress, Anne E.

    2014-01-01

    Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent “inside-out” signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent “inside-out” signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype. PMID:25450398

  7. T-cell abnormalities after mediastinal irradiation for lung cancer. The in vitro influence of synthetic thymosin alpha-1

    International Nuclear Information System (INIS)

    Schulof, R.S.; Chorba, T.L.; Cleary, P.A.; Palaszynski, S.R.; Alabaster, O.; Goldstein, A.L.

    1985-01-01

    The effects of mediastinal irradiation (RT) on the numbers and functions of purified peripheral blood T-lymphocytes from patients with locally advanced non-small cell lung cancer were evaluated. The patients were candidates for a randomized trial to evaluate the immunorestorative properties of synthetic thymosin alpha-1. Twenty-one patients studied before RT did not exhibit any significant difference in T-cell numbers or function compared to age-matched healthy subjects. However, 41 patients studied within 1 week after completing RT exhibited significant depressions of E-rosette-forming cells at 4 degrees C (E4 degrees-RFC)/mm3, E-rosette-forming cells at 29 degrees C (E29 degrees-RFC)/mm3, OKT3/mm3, OKT4/mm3, and OKT8/mm3 (P . 0.0001); total T-cell percentages (%OKT3, P . 0.01); and T-cell proliferative responses in mixed lymphocyte cultures (MLR) (P . 0.01) and to the mitogen phytohemagglutinin under suboptimal conditions (P less than or equal to 0.03). Nine patients studied before and after RT showed a significant increase in OKT4/OKT8 (P . 0.01) following RT. A short-term in vitro incubation with thymosin alpha-1 could enhance MLR of T-cells in 12 of 27 patients with post-RT abnormalities. In 13 patients who were treated with placebo, the RT-induced depression of T-cell numbers and function persisted for at least 3 to 4 months. In addition, in 12 patients progressive decreases developed in %E4 degrees-RFC, %OKT3, %OKT4, and OKT4/OKT8, which always preceded clinical relapse

  8. Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

    Czech Academy of Sciences Publication Activity Database

    Neužil, Jiří; Dong, L.F.; Wang, X.F.; Zingg, J.M.

    2006-01-01

    Roč. 343, č. 4 (2006), s. 1113-1117 ISSN 0006-291X Institutional research plan: CEZ:AV0Z50520514 Keywords : apoptosis * tocopherol-associated protein * alpha-tocopheryl succinate Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.855, year: 2006

  9. The effect of a metalloproteinase inhibitor (GI5402) on tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha receptors during human endotoxemia

    NARCIS (Netherlands)

    Dekkers, P. E.; Lauw, F. N.; ten Hove, T.; te Velde, A. A.; Lumley, P.; Becherer, D.; van Deventer, S. J.; van der Poll, T.

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is released from the cell surface by cleavage of pro-TNF-alpha by metalloproteinases (MPs). In cell cultures, inhibition of MPs has been found not only to reduce the release of TNF-alpha, but also to enhance the surface expression of TNF-alpha and TNF-alpha

  10. Alpha-tocopherol ameliorates experimental autoimmune encephalomyelitis through the regulation of Th1 cells

    Directory of Open Access Journals (Sweden)

    Haikuo Xue

    2016-05-01

    Full Text Available Objective(s: Multiple sclerosis (MS is a serious neurological autoimmune disease, it commonly affects young adults. Vitamin E (Vit E is an important component of human diet with antioxidant activity, which protects the body’s biological systems. In order to assess the effect of Vit E treatment on this autoimmune disease, we established experimental autoimmune encephalomyelitis (EAE, the animal model of MS, and treated EAE with α-tocopherol (AT which is the main content of Vit E. Materials and Methods:Twenty C57BL/6 adult female mice were used and divided into two groups randomly. EAE was induced with myelin oligodendrocyte glycoprotein (MOG, and one group was treated with AT, at a dose of 100 mg/kg on the 3th day post-immunization with MOG, the other group was treated with 1% alcohol. Mice were euthanized on day 14, post-immunization, spleens were removed for assessing splenocytes proliferation and cytokine profile, and spinal cords were dissected to assess the infiltration of inflammatory cells in spinal cord. Results:AT was able to attenuate the severity of EAE and delay the disease progression. H&E staining and fast blue staining indicated that AT reduced the inflammation and the demyelination reaction in the spinal cord. Treatment with AT significantly decreased the proliferation of splenocytes. AT also inhibited the production of IFN-γ (Th1 cytokine, though the other cytokines were only affected slightly. Conclusion:According to the results, AT ameliorated EAE, through suppressing the proliferation of T cells and the Th1 response. AT may be used as a potential treatment for MS.

  11. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    Energy Technology Data Exchange (ETDEWEB)

    Tandle, Anita T. [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Calvani, Maura; Uranchimeg, Badarch [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Zahavi, David [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Melillo, Giovanni [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Libutti, Steven K., E-mail: slibutti@montefiore.org [Department of Surgery, Montefiore-Einstein Center for Cancer Care, Albert Einstein College of Medicine, Greene Medical Arts Pavilion, 4th Floor 3400, Bainbridge Avenue, Bronx, New York 10467 (United States)

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  12. 17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

    Science.gov (United States)

    Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

    2010-03-01

    The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

  13. Peroxisome Proliferator-Activated Receptor-alpha Gene Level Differently Affects Lipid Metabolism and Inflammation in Apolipoprotein E2 Knock-In Mice

    NARCIS (Netherlands)

    Lalloyer, Fanny; Wouters, Kristiaan; Baron, Morgane; Caron, Sandrine; Vallez, Emmanuelle; Vanhoutte, Jonathan; Bauge, Eric; Shiri-Sverdlov, Ronit; Hofker, Marten; Staels, Bart; Tailleux, Anne

    Objective-Peroxisome proliferator-activated receptor-alpha (PPAR alpha) is a ligand-activated transcription factor that controls lipid metabolism and inflammation. PPAR alpha is activated by fibrates, hypolipidemic drugs used in the treatment of dyslipidemia. Previous studies assessing the influence

  14. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Milatovic, Dejan [Department of Pediatrics/Pediatric Toxicology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Splittgerber, Ryan [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Fan, Guo-Huang [Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37221 (United States); Richmond, Ann, E-mail: ann.richmond@vanderbilt.edu [VA Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP

  15. Maternal obesity accelerates fetal pancreatic beta-cell but not alpha-cell development in sheep: prenatal consequences.

    Science.gov (United States)

    Ford, Stephen P; Zhang, Liren; Zhu, Meijun; Miller, Myrna M; Smith, Derek T; Hess, Bret W; Moss, Gary E; Nathanielsz, Peter W; Nijland, Mark J

    2009-09-01

    Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring.

  16. Immunoreactivity for alpha-smooth muscle actin characterizes a potentially aggressive subgroup of little basal cell carcinomas

    Directory of Open Access Journals (Sweden)

    L Pilloni

    2009-06-01

    Full Text Available Basal cell carcinoma (BCC is a very common malignant skin tumor that rarely metastatizes, but is often locally aggressive. Several factors, like large size (more than 3 cm, exposure to ultraviolet rays, histological variants, level of infiltration and perineural or perivascular invasion, are associated with a more aggressive clinical course. These morphological features seem to be more determinant in mideface localized BCC, which frequently show a significantly higher recurrence rate. An immunohistochemical profile, characterized by reactivity of tumor cells for p53, Ki67 and alpha-SMA has been associated with a more aggressive behaviour in large BCCs. The aim of this study was to verify if also little (less than 3 cm basal cell carcinomas can express immunohistochemical markers typical for an aggressive behaviour.

  17. Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage

    DEFF Research Database (Denmark)

    Porse, Bo T; Bryder, David; Theilgaard-Mönch, Kim

    2005-01-01

    dissociate the ability of C/EBP alpha to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid...... progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation...

  18. Comparison of the cytotoxic effect of lapachol, alpha-lapachone and pentacyclic 1,4-naphthoquinones on human leukemic cells.

    Science.gov (United States)

    Salustiano, Eduardo J S; Netto, Chaquip D; Fernandes, Renata F; da Silva, Alcides J M; Bacelar, Thiago S; Castro, Carolina P; Buarque, Camilla D; Maia, Raquel C; Rumjanek, Vivian M; Costa, Paulo R R

    2010-04-01

    The pentacyclic 1,4-naphthoquinones 1a-d were cytotoxic (IC(50) approximately 2-7 microM) to human leukemic cell lines K562 (oxidative stress-resistant), Lucena-1 (MDR phenotype) and Daudi. Fresh leukemic cells obtained from patients, some with the MDR phenotype, were also sensitive to these compounds. The pentacyclic 1,4-naphthoquinones 1a and 1c induced apoptotic cell death in cells from leukemic patients as determined by flow cytometry. Conversely, the cell lines were highly insensitive to lapachol (2) and alpha-lapachone (3). Mitomycin-C inhibited cell proliferation at concentrations as low as 0.5 microM. The low toxicity against lymphocytes activated by phytohemagglutinin shows that these compounds are selective for the cancer cells studied. Previous data suggest that these compounds (1a-d) can be bioactivated in situ by reduction followed by rearrangement leading to enones, which are powerful alkylating agents. In contrast, lapachol (2) and beta-lapachone (3), which cannot be bioactivated by reduction, showed little activity against the same cell lines.

  19. Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

    Directory of Open Access Journals (Sweden)

    Park Raekil

    2006-01-01

    Full Text Available Abstract Background We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi and that interleukin 1 alpha (IL-1 alpha up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. Methods The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM. Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. Results Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. Conclusion We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.

  20. Hydroxysteroid dehydrogenases in bovine and porcine granulosa cells convert zearalenone into its hydroxylated metabolites alpha-zearalenol and beta-zearalenol.

    Science.gov (United States)

    Malekinejad, H; Colenbrander, B; Fink-Gremmels, J

    2006-05-01

    The enzymes 3alpha- and 3beta-hydroxysteroid dehydrogenase (3alpha- and 3beta-HSD) play a pivotal role in synthesis of various steroid hormones including oestradiol and testosterone. The structure of the mycotoxin zearalenone resembles many characteristics of steroids and binds to oestrogen receptors as an agonist. Consequently, it is suggested that zearalenone is also a substrate for 3alpha-HSD and 3beta-HSD. 3alpha-HSD and 3beta-HSD isoforms are expressed in the liver and kidney but also in many steroidogenic tissues. It was the aim of the present study to demonstrate the presence of these enzymes in granulosa cells, which were obtained from bovine and porcine ovaries, and to investigate whether zearalenone is a substrate for these enzymes. The results show a species-specific expression pattern in the granulosa cells of both species. Moreover, it was demonstrated that zearalenone when added to the culture medium, is converted into alpha-zearalenol and beta-zearalenol. Corresponding to the apparent expression profile, in porcine granulosa cells predominantly alpha-zearalenol was formed, whereas bovine granulosa cells preferentially converted zearalenone into beta-zearalenol. This is the first report demonstrating the extrahepatic biotransformation of zearalenone in target tissues.

  1. Azithromycin reduces tumor necrosis factor-alpha production in lipopolysaccharide-stimulated THP-1 monocytic cells by modification of stress response and p38 MAPK pathway.

    Science.gov (United States)

    Ikegaya, S; Inai, K; Iwasaki, H; Naiki, H; Ueda, T

    2009-08-01

    Macrolide antibiotics are known to have a variety of immunomodulatory effects in addition to antimicrobial activity, but the mechanisms of immunomodulation are still unclear. We investigated in vitro the effect of azithromycin on tumor necrosis factor alpha (TNF-alpha) production in lipopolysaccharide (LPS)-stimulated THP-1 cells, a human monocytic cell line, and compared the results with those for other macrolides, minocycline and ofloxacin. In the presence of LPS, treatment with azithromycin (AZM) resulted in a significant decrease in LPS-induced TNF-alpha production compared to that with other antimicrobial agents. the results of phosphorylation of three MAPKs, ERK, JNK and p38, indicated that the phospho-p38 level was reduced by AZM. Ikappab-alpha, an inhibitor of NFkappab, was not disrupted by the antibiotics. LPS-induced TNF-alpha release from THP-1 cells was inhibited in the presence of KNK437, a potent 70-kDa heat shock protein (HSP-70) inhibitor. Interestingly, the induction of HSP-70 by LPS was attenuated with the concurrent addition of AZM in the cells. AZM was found to restrain TNF-alpha production by monocytes at least in part by modifying the HSp-70 and p38 related signaling pathways to LPS stimulation.

  2. HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Kudová, Jana; Procházková, J.; Vašíček, Ondřej; Perečko, Tomáš; Sedláčková, M.; Pešl, M.; Pachernik, J.; Kubala, Lukáš

    2016-01-01

    Roč. 11, č. 6 (2016) E-ISSN 1932-6203 R&D Projects: GA ČR GA13-29358S Grant - others:Grantová agentura ČR - GA ČR(CZ) GJ15-13443Y Institutional support: RVO:68081707 Keywords : INDUCIBLE FACTOR 1-ALPHA * GENE-EXPRESSION * CARDIAC DIFFERENTIATION Subject RIV: BO - Biophysics Impact factor: 2.806, year: 2016

  3. A multi-omics approach identifies key hubs associated with cell type-specific responses of airway epithelial cells to staphylococcal alpha-toxin.

    Directory of Open Access Journals (Sweden)

    Erik Richter

    Full Text Available Responsiveness of cells to alpha-toxin (Hla from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

  4. Vitamin E analogue, D-alpha tocopherol succinate, enhances x-ray induced growth delay of human adenocarcinoma cancer cell line

    International Nuclear Information System (INIS)

    Jaworska, A.; Ottesen, T.E.

    2003-01-01

    The purpose of this study was to assess the effects of d-alpha Tocopherol succinate (alpha-TS) in modifying radiation-induced viability reduction and apoptosis occurrence in the model for normal and cancer cells. Our hypothesis was that alpha-TS enhances the growth-inhibitory effect of x-irradiation in cancer cells and that the effect is more pronounced in these cells than in normal cells. Murine NIH 3T3 Swiss albino embryonic cells and HT29 human Caucasian colon adenocarcinoma cells were used in the experiments. Alpha-TS was added to the cultures 1 h prior to irradiation with doses of 2 or 5Gy of x-ray. After irradiation cells were incubated for 73 h. Trypan blue exclusion viability test and estimation of apoptosis and necrosis were made. Apoptotic and necrotic cells were counted in fluorescence microscope using fluorescence dyes: propidium iodide and Hoechst 33342. For experiments with the dose of 5 Gy at least five series of experiments were performed. At lower doses (up to approximately 25μM/ml) treatment with alpha-TS alone enhanced growth of both cell lines. At higher doses treatment with alpha-TS alone delayed the growth of the cell cultures, accompanied by 20-25% necrosis. At the concentrations higher than 25μM/mL alpha-TS alone caused growth delay of both cell cultures, being much more pronounced for the cancer cell line HT29. At the concentrations of 50 μM/mL, responsible for about 30-60% of growth delay, there was observed a synergy effect for x-rays and alpha-TS for both cell lines. The effect was more pronounced for HT29 cells (DMF=0.48 for HT29 versus DMF=0.73 for NIH 3T3). These results may confirm the views of the literature reports suggesting that use of vitamin E together with radiation could be favorable for colon cancer treatment; however, more experiments using more advanced techniques are needed

  5. The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of alpha 1 type-XI and alpha 2 type-V collagen chains.

    Science.gov (United States)

    Kleman, J P; Hartmann, D J; Ramirez, F; van der Rest, M

    1992-11-15

    The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.

  6. Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and alpha particles

    Science.gov (United States)

    2012-01-01

    Background Little is known about the cellular effects of exposure to mixed beams of high and low linear energy transfer radiation. So far, the effects of combined exposures have mainly been assessed with clonogenic survival or cytogenetic methods, and the results are contradictory. The gamma-H2AX assay has up to now not been applied in this context, and it is a promising tool for investigating the early cellular response to mixed beam irradiation. Purpose To determine the dose response and repair kinetics of gamma-H2AX ionizing radiation-induced foci in VH10 human fibroblasts exposed to mixed beams of 241Am alpha particles and X-rays. Results VH10 human fibroblasts were irradiated with each radiation type individually or both in combination at 37°C. Foci were scored for repair kinetics 0.5, 1, 3 and 24 h after irradiation (one dose per irradiation type), and for dose response at the 1 h time point. The dose response effect of mixed beam was additive, and the relative biological effectiveness for alpha particles (as compared to X-rays) was of 0.76 ± 0.52 for the total number of foci, and 2.54 ± 1.11 for large foci. The repair kinetics for total number of foci in cells exposed to mixed beam irradiation was intermediate to that of cells exposed to alpha particles and X-rays. However, for mixed beam-irradiated cells the frequency and area of large foci were initially lower than predicted and increased during the first 3 hours of repair (while the predicted number and area did not). Conclusions The repair kinetics of large foci after mixed beam exposure was significantly different from predicted based on the effect of the single dose components. The formation of large foci was delayed and they did not reach their maximum area until 1 h after irradiation. We hypothesize that the presence of low X-ray-induced damage engages the DNA repair machinery leading to a delayed DNA damage response to the more complex DNA damage induced by alpha particles. PMID:23121736

  7. PGF2alpha induced differential expression of genes involved in turnover of extracellular matrix in rat decidual cells

    Directory of Open Access Journals (Sweden)

    Callegari Eduardo A

    2005-01-01

    Full Text Available Abstract In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied. Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM. Genes such as gelatinase A (MMP2, cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2 and 3 (TIMP3, plasminogen activator inhibitor1 (PAI1, tissue type plasminogen activator (tPA, urokinase plasminogen activator (tPA, endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3, plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation.

  8. Decellularization of bovine anterior cruciate ligament tissues minimizes immunogenic reactions to alpha-gal epitopes by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Yoshida, Ryu; Vavken, Patrick; Murray, Martha M

    2012-10-01

    Rupture of ACL is a common injury. While the current surgical treatments are effective, many patients still suffer from precocious osteoarthritis, and there is an increasing interest in bioengineering approaches to improve ACL repair. Bovine collagen is a material currently in use for tissue engineering of ligaments. The alpha-gal epitopes found on bovine cells are a source of immunogenic stimulus for human cells. In this study, we wished to determine if those epitopes could be removed sufficiently to mitigate an immunogenic response using either a decellularization protocol or decellularization followed by alpha-galactosidase treatment. Bovine ACLs were treated with Triton-X, sodium deoxycholate, ribonuclease, and deoxyribonuclease to remove cells. A subset of the decellularized tissues was further treated with alpha-galactosidase. Human peripheral blood mononuclear cells (PBMCs) were exposed to untreated, decellularized, and alpha-galactosidase-treated tissues, and PBMC migration and IL-6 release were measured. PBMCs were significantly more attracted to untreated ACL compared to decellularized or alpha-galactosidase-treated tissue, but no difference was seen between the two treatment groups. PBMCs also released significantly more IL-6 when exposed to untreated tissue compared to decellularized ACL or alpha-galactosidase-treated ACL, but no difference was seen between the two treatment groups. Immunohistochemistry using anti-alpha-gal antibody detected the epitopes throughout the untreated ACL, but similar areas of reaction were not seen on decellularized or alpha-galactosidase-treated ACL. These results suggest that our decellularization protocol minimizes the immunogenic reactions of human PBMCs to bovine ACL tissue. Therefore, decellularized bovine ACL tissue may be a safe, effective biomaterial for ACL injury treatments. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Signal regulatory protein alpha (SIRPalpha cells in the adaptive response to ESAT-6/CFP-10 protein of tuberculous mycobacteria.

    Directory of Open Access Journals (Sweden)

    W Ray Waters

    2009-07-01

    Full Text Available Early secretory antigenic target-6 (ESAT-6 and culture filtrate protein-10 (CFP-10 are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRPalpha (also referred to as macrophage fusion receptor or CD172a is essential for multi-nucleate giant cell formation.In the present study, ESAT-6/CFP-10 complex and SIRPalpha interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a(+ (SIRPalpha-expressing cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail, but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis DeltaRD1. Sorted CD172a(+ cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8alpha. Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c(+, CD172a(+, and CD3(+ cells, including CD172a-expressing multi-nucleated giant cells.These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a(+ cells, bind to CD172a(+ cells, and induce multi-nucleated giant cells expressing CD172a.

  10. Effect of tissue-specific acetylcholinesterase inhibitor C-547 on alpha 3 beta 4 and alpha beta epsilon delta acetylcholine receptors in COS cells

    Czech Academy of Sciences Publication Activity Database

    Lindovský, Jiří; Petrov, K.; Krůšek, Jan; Reznik, V.S.; Nikolsky, E. E.; Vyskočil, František

    2012-01-01

    Roč. 688, 1-3 (2012), s. 22-26 ISSN 0014-2999 R&D Projects: GA MŠk(CZ) LC554; GA ČR(CZ) GA202/09/0806; GA AV ČR(CZ) IAA500110905; GA AV ČR(CZ) IAA100110501; GA AV ČR(CZ) IAA5011411 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : nicotinic ACh receptor * alpha 3 beta 4 * alpha beta epsilon delta * C-547 * anti-cholinesterase Subject RIV: ED - Physiology Impact factor: 2.592, year: 2012

  11. T-cell receptor v-alpha and v-Beta gene usage in interleukin-2-cultured tumor-infiltrating lymphocytes from patients with breast-cancer

    DEFF Research Database (Denmark)

    Andersen, E; Scholler, J; Straten, P

    1994-01-01

    of restricted V gene expression. The mean number of V alpha segments per TIL culture was higher than the number of V beta segments per culture. A significant negative correlation was observed between the number of CD4+ cells and the number of V beta segments per culture, and no other correlations between...... surface through the T cell receptor (TCR) complex. We have studied the phenotype, cytotoxicity, and expression of TCR variable (V) alpha and beta chain on in vitro IL-2-cultured TIL isolated from primary malignant breast tumors from 11 patients. 10/11 cultures were dominated by CD4(+) (T-helper) cells...

  12. Alpha Thalassemia (For Parents)

    Science.gov (United States)

    ... the body has a problem producing alpha globin Beta thalassemia : when the body has a problem producing beta ... Transfusion Blood Test: Hemoglobin Electrophoresis Sickle Cell Disease Beta Thalassemia Blood All About Genetics Prenatal Genetic Counseling Genetic ...

  13. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    Science.gov (United States)

    Lin, Yu-Fen; Nagasawa, Hatsumi; Little, John B; Kato, Takamitsu A; Shih, Hung-Ying; Xie, Xian-Jin; Wilson, Paul F; Brogan, John R; Kurimasa, Akihiro; Chen, David J; Bedford, Joel S; Chen, Benjamin P C

    2014-01-01

    We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  14. Suppression of transformed foci, induced by alpha radiation of C3H 10T1/2 cells, by untransformed cells

    International Nuclear Information System (INIS)

    Lloyd, E.L.; Gemmell, M.A.; Henning, C.B.

    1978-01-01

    The C3H 10T1/2 CL8 cell line obtained from a mouse embryo has been widely used for screening chemical carcinogens. Transformed foci are easily distinguishable in this system as crisscrossed, piled-up cells which stain more deeply than the surrounding untransformed cells. When these foci are ringcloned and subcultured, they have been shown to give rise to malignant tumors in C3H immunodepressed mice. Previous work showed that such malignant transformations, which occurred with a dose dependent frequency, could be induced by alpha particle irradiation. The present study, in turn, demonstrates that the expression of these transformations can be completely suppressed by co-cultivating the transformed cells with a large number of untransformed cells. The precise ratio of the number of untransformed cells to transformed cells to give complete suppression was found to vary in different experiments. Maximum effects were seen when a small number of transformed cells in low passage were used. These experiments may provide at least a partial explanation for the greatly increased frequency of transformations per cell irradiated in vitro, compared with the number of tumors observed after irradiation of the same number of cells in vivo. In addition, if conditions could be optimized whereby transformed foci could reproducibly be eliminated by the use of a known number of untransformed cells, this might have important applications in the prevention and treatment of certain human cancers

  15. Altered cell adhesion activity by pervanadate due to the dissociation of alpha-catenin from the E-cadherin.catenin complex.

    Science.gov (United States)

    Ozawa, M; Kemler, R

    1998-03-13

    Leukemia cells (K562) that grow as non-adhesive single cells and have no endogenous cadherin were transfected with an E-cadherin expression vector, and cell clones stably expressing E-cadherin on their surface were established. The expression of E-cadherin induced the up-regulation of catenins, and E-cadherin became associated with catenins. The transfected cells grew as floating aggregates. Cell aggregation was Ca2+-dependent and was inhibited by E-cadherin antibodies. The aggregates dissociated into single cells on the addition of pervanadate. Pervanadate caused a dramatic augmentation of the phosphorylation of E-cadherin, beta-catenin, and gamma-catenin (plakoglobin), but alpha-catenin was not detectably phosphorylated. After pervanadate treatment, beta-catenin and gamma-catenin migrated more slowly on gel electrophoresis, suggesting changes in their conformations due to eventual changes in their phosphorylation levels. In the treated cells, a significant amount of alpha-catenin was dissociated from the E-cadherin.catenin complex. Aggregates of cells expressing an E-cadherin chimeric molecule covalently linked with alpha-catenin were not dissociated on pervanadate treatment, supporting the idea that the dissociation of alpha-catenin from the complex underlies the observed E-cadherin dysfunction.

  16. Whole blood assay for NK activity in splenectomized and non-splenectomized hairy cell leukemia patients during IFN-alpha-2b treatment

    DEFF Research Database (Denmark)

    Nielsen, B; Hokland, P; Ellegaard, J

    1989-01-01

    Natural killer cell (NK) activity in peripheral blood (PB) was followed longitudinally for up to 2 yr after initiation of low-dose IFN-alpha-2b therapy in nine hairy cell leukemia (HCL) patients. A whole blood NK (WB-NK) assay was employed in order to measure the NK activity per unit blood. The p...

  17. ALPHA-ADRENOCEPTOR REGULATION OF INOSITOL PHOSPHATES, INTERNAL CALCIUM AND MEMBRANE CURRENT IN DDT1 MF-2 SMOOTH-MUSCLE CELLS

    NARCIS (Netherlands)

    NELEMANS, A; HOITING, B; MOLLEMAN, A; DUIN, M; DENHERTOG, A

    1990-01-01

    The effect of alpha-1-adrenoceptor stimulation on inositol phosphates (InsPs), intracellular Ca2+ and membrane current was measured in vas deferens DDT1 MF-2 cells. The InsPs were analyzed after labelling the cells with [H-3]myo-inositol using high performance liquid chromatography and the internal

  18. Bone marrow stromal cell defects and 1 alpha,25-dihydroxyvitamin D3 deficiency underlying human myeloid leukemias.

    Science.gov (United States)

    Blazsek, I; Farabos, C; Quittet, P; Labat, M L; Bringuier, A F; Triana, B K; Machover, D; Reynes, M; Misset, J L

    1996-01-01

    Primary myelodysplasia (MDP) and acute and chronic myelogenous leukemias (AML, CML) are considered disorders of clonal stem cell division. Several constitutive gene defects that contribute to the development of abnormal cell behavior have been identified in the hematopoietic cells. The role of bone marrow stroma cells in leukemogenesis, however, has not been established. We studied the organization of the bone marrow (BM) microenvironment to see if it was impaired during the initiation and progression of these malignancies. The buffy coat, hematon, and plasma fractions were separated from BM aspirates taken from healthy donors and diseased subjects at distinct clinical stages. The structural integrity of the BM microenvironment was evaluated analyzing the morphogenetic unit, the hematon. The hematon is a multicellular complex that includes fibroblasts, adipocytes, endothelial cells, resident macrophages, hematopoietic cobblestone area-forming cells (CAFC), high-proliferative potential colony-forming cells (HPP-CFC), granulocyte-macrophage colony-forming unit (GM-CFU), burst-forming unit erythroid (BFU-E), and terminally differentiated cells in normal BM. Hematon complexes were present in most BM aspirates from healthy donors (46H+/55). But they were absent from most of the patients with MDP (21H+/62) and AML (5H+/24) in the first perceptible phase, and from those with CML throughout the disease (5H+/55). Hematon complexes were present in the BM aspirate in 22/36 AML patients at clinical remission after chemotherapy or differentiation therapy. The hematon fraction isolated from normal BM, contained 25 times more 25-hydroxyvitamin D3 and about 500-fold more 1alpha,25-dihydroxyvitamin D3 than the BM plasma. The concentration of 1alpha,25-dihydroxyvitamin D3 was low or undetectable in the BM plasma of some, but not all, patients with MDP (18/35) or AML (9/24). Thus, in the BM microenvironment, the metabolism of low-density lipids and lipophylic hormones are severely

  19. Increased frequency of CD4{sup -}8{sup -}T cells bearing T-cell receptor {alpha}{beta} chains in peripheral blood of atomic bomb survivors exposed to high doses

    Energy Technology Data Exchange (ETDEWEB)

    Yoichiro Kusunoki; Seishi Kyoizumi; Yuko Hirai; Shoichiro Fujita; Mitoshi Akiyama [Radiation Effects Research Foundation, Hiroshima (Japan)

    1994-07-01

    A rare T-cell subpopulation, CD4{sup -z}8{sup -}{alpha}{beta} cells, may be differentiated through a pathway (or pathways) different from the pathway(s) of conventional CD4+ or CD8+ cells. In the present study, the frequencies of CD4{sup -}8{sup -} T cells in peripheral-blood {alpha}{beta} T cells in 409 atomic bomb survivors were determined to investigate late effects of radiation on the composition of human T-cell subpopulations. The frequency of CD4{sup -}8{sup -}{alpha}{beta} T-cell decreased significantly with the subject`s age and was higher in females than males. A significant increase in the frequency was found in the survivors exposed to more than 1.5Gy, suggesting that the previous radiation exposure altered differentiation and development of T cells. 25 refs., 4 figs., 3 tabs.

  20. Prostaglandin F2alpha differentially affects mRNA expression relating to angiogenesis, vasoactivation and prostaglandins in the early and mid corpus luteum in the cow.

    Science.gov (United States)

    Shirasuna, Koumei; Sasahara, Kiemi; Matsui, Motozumi; Shimizu, Takashi; Miyamoto, Akio

    2010-08-01

    Administration of prostaglandin (PG) F(2alpha) in cattle during the mid-luteal phase (Days 8-12 of the estrous cycle) drastically reduces the plasma progesterone concentrations and the volume of the corpus luteum (CL). However, PGF(2alpha) does not induce luteolysis during the early luteal phase (up to Day 5 of the estrous cycle). To characterize the possible distinct difference in acute response to a luteolytic dose of PGF(2alpha) administration, we determined various mRNA expressions in the early and mid CL relating to angiogenesis, vasoactivation and PG-related factors at 30 min after PGF(2alpha) injection in cyclic cows. The experiments were conducted on Day 4 (early CL) and Days 10-12 (mid CL). Cows were either injected with 500 microg PGF(2alpha) analogue or saline as the control (early CL control, n=5; early CL PGF(2alpha) treated, n=5; mid CL control, n=5; mid CL PGF(2alpha) treated, n=7). Thirty min after injection of PGF(2alpha) or saline, the cows were ovariectomized transvaginally, and the CL tissues were collected from regions designated as the periphery and center of the CL. Administration of PGF(2alpha) up-regulated the mRNA expressions of angiogenic-related factors such as vascular endothelial growth factors, vasohibin, fibroblast growth factor 2 and insulin-like growth factor-II in the early CL, whereas PGF(2alpha) down-regulated these mRNA expressions in the mid CL. In the vasoactive factors, PGF(2alpha) stimulated the mRNA expressions of endothelin-1, angiotensin converting enzyme, endothelial nitric oxide synthase (NOS) and inducible NOS in the periphery area of the mid CL, but not in the early CL. However, PGF(2alpha) drastically down-regulated PGF(2alpha) receptor mRNA expression in both regions of the early and mid CL. The results indicated a clear difference in the acute action of PGF(2alpha) depending not only on the luteal phase (immature vs. mature) but also the region (periphery vs. center) within the CL at 30 min after PGF(2alpha

  1. Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line.

    Science.gov (United States)

    Baker, Olga J; Camden, Jean M; Redman, Robert S; Jones, Jonathan E; Seye, Cheikh I; Erb, Laurie; Weisman, Gary A

    2008-11-01

    Sjögren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

  2. Human immunodeficiency syndromes affecting human natural killer cell cytolytic activity

    Directory of Open Access Journals (Sweden)

    Daniel Denis Billadeau

    2014-01-01

    Full Text Available NK cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T-cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synapse. Concurrently, lytic granules undergo minus-end directed movement and accumulate at the microtubule-organizing center (MTOC through the interaction with microtubule motor proteins, followed by polarization of the lethal cargo toward the target cell. Ultimately, myosin-dependent movement of the lytic granules toward the NK cell plasma membrane through F-actin channels, along with SNARE-dependent fusion promotes lytic granule release into the cleft between the NK cell and target cell resulting in target cell killing. Herein, we will discuss several disease-causing mutations in primary immunodeficiency syndromes and how they impact NK cell-mediated killing by disrupting distinct steps of this tightly regulated process.

  3. Modulation of GLO1 Expression Affects Malignant Properties of Cells

    Directory of Open Access Journals (Sweden)

    Antje Hutschenreuther

    2016-12-01

    Full Text Available The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO. Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1 that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

  4. A Comparitive Assessement of Cytokine Expression in Human-Derived Cell Lines Exposed to Alpha Particles and X-Rays

    Directory of Open Access Journals (Sweden)

    Vinita Chauhan

    2012-01-01

    Full Text Available Alpha- (α- particle radiation exposure has been linked to the development of lung cancer and has been identified as a radiation type likely to be employed in radiological dispersal devices. Currently, there exists a knowledge gap concerning cytokine modulations associated with exposure to α-particles. Bio-plex technology was employed to investigate changes in proinflammatory cytokines in two human-derived cell lines. Cells were irradiated at a dose of 1.5 Gy to either α-particles or X-rays at equivalent dose rates. The two cell lines exhibited a unique pattern of cytokine expression and the response varied with radiation type. Of the 27 cytokines assessed, only vascular endothelin growth factor (VEGF was observed to be modulated in both cell lines solely after α-particle exposure, and the expression of VEGF was shown to be dose responsive. These results suggest that certain proinflammatory cytokines may be involved in the biological effects related to α- particle exposure and the responses are cell type and radiation type specific.

  5. SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction.

    Science.gov (United States)

    Pasquier, Jennifer; Abu-Kaoud, Nadine; Abdesselem, Houari; Madani, Aisha; Hoarau-Véchot, Jessica; Thawadi, Hamda Al; Vidal, Fabien; Couderc, Bettina; Favre, Gilles; Rafii, Arash

    2015-08-01

    The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites. Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases. We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion. We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases.

  6. SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction

    International Nuclear Information System (INIS)

    Pasquier, Jennifer; Abu-Kaoud, Nadine; Abdesselem, Houari; Madani, Aisha; Hoarau-Véchot, Jessica; Thawadi, Hamda Al.; Vidal, Fabien; Couderc, Bettina; Favre, Gilles; Rafii, Arash

    2015-01-01

    The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites. Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases. We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion. We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases. The online version of this article (doi:10.1186/s12885-015-1556-7) contains supplementary material, which is available to authorized users

  7. Induction of promotive rather than suppressive immune responses from a novel NKT cell repertoire Valpha19 NKT cell with alpha-mannosyl ceramide analogues consisting of the immunosuppressant ISP-I as the sphingosine unit.

    Science.gov (United States)

    Shimamura, Michio; Okamoto, Naoki; Huang, Yi-Ying; Yasuoka, Jouji; Morita, Kenji; Nishiyama, Akira; Amano, Yuusuke; Mishina, Tadashi

    2006-05-01

    A 2-substituted 2-aminopropane-1,3-diol or 2-aminoethanol is the minimum structure required for the immunosuppressive activity of ISP-I, an antibiotic isolated from the culture broth of Isaria sinclairil. A series of alpha-mannosyl ceramide (alpha-ManCer) analogues was derived from 2-substituted 2-aminopropane-1,3-diols or 2-aminoethanols in place of sphingosine. The newly synthesized glycosides were evaluated for their effects on immune responses. In contrast to the immunosuppressive activity of the precursors, the alpha-ManCer analogues induced immunopromotive responses from invariant Valpha19-Jalpha26 transgenic mouse lymphocytes more effectively than the original alpha-ManCer. Collectively, it is strongly suggested that the 2-substituted 2-aminopropane-1,3-diols and 2-aminoethanols mimic sphingosine in the alpha-ManCer analogues so that they potentially acquire specific antigenicity toward Valpha19 NKT cell, a novel NKT cell subset.

  8. Piper and Vismia Species from Colombian Amazonia Differentially Affect Cell Proliferation of Hepatocarcinoma Cells

    Directory of Open Access Journals (Sweden)

    Leandro J. Lizcano

    2014-12-01

    Full Text Available There is an increasing interest to identify plant-derived natural products with antitumor activities. In this work, we have studied the effects of aqueous leaf extracts from Amazonian Vismia and Piper species on human hepatocarcinoma cell toxicity. Results showed that, depending on the cell type, the plants displayed differential effects; thus, Vismia baccifera induced the selective killing of HepG2, while increasing cell growth of PLC-PRF and SK-HEP-1. In contrast, these two last cell lines were sensitive to the toxicity by Piper krukoffii and Piper putumayoense, while the Piperaceae did not affect HepG2 growth. All the extracts induced cytotoxicity to rat hepatoma McA-RH7777, but were innocuous (V. baccifera at concentrations < 75 µg/mL or even protected cells from basal death (P. putumayoense in primary cultures of rat hepatocytes. In every case, cytotoxicity was accompanied by an intracellular accumulation of reactive oxygen species (ROS. These results provide evidence for the anticancer activities of the studied plants on specific cell lines and suggest that cell killing could be mediated by ROS, thus involving mechanisms independent of the plants free radical scavenging activities. Results also support the use of these extracts of the Vismia and Piper genera with opposite effects as a model system to study the mechanisms of the antitumoral activity against different types of hepatocarcinoma.

  9. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    International Nuclear Information System (INIS)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B.

    1991-01-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism

  10. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. (Univ. of Missouri-Columbia (USA))

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  11. Function of the integrin alpha 6 beta 1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor

    DEFF Research Database (Denmark)

    Shaw, L M; Chao, C; Wewer, U M

    1996-01-01

    The involvement of the alpha 6 beta a integrin, a laminin receptor, in breast carcinoma progression needs to be addressed rigorously. We report that a human breast carcinoma cell line, MDA-MB-435, known to be highly invasive and metastatic, expresses three potential integrin laminin receptors...... function that involved expression of a cytoplasmic domain deletion mutant of the beta 4 integrin subunit by cDNA transfection. Stable transfectants of MDA-MB-435 cells that expressed this mutant beta 4 subunit were inhibited dramatically in their ability to adhere and migrate on laminin matrices......, and their capacity to invade Matrigel was reduced significantly. These findings support the hypothesis that alpha 6 beta 1 is important for breast cancer progression. Moreover, this approach is a powerful method that should be useful in assessing the role of alpha 6 beta 1 in other cells....

  12. Growth arrest and decrease of alpha-SMA and type I collagen expression by palmitic acid in the rat hepatic stellate cell line PAV-1.

    Science.gov (United States)

    Abergel, Armand; Sapin, Vincent; Dif, Nicolas; Chassard, Christophe; Darcha, Claude; Marcand-Sauvant, Julie; Gaillard-Martinie, Brigitte; Rock, Edmond; Dechelotte, Pierre; Sauvant, Patrick

    2006-05-01

    Liver fibrosis is characterized by an activation of hepatic stellate cells (HSC). During primary culture HSC evolve from a quiescent into an activated phenotype which is characterized by alpha-smooth muscle actin (alpha-SMA) up-regulation, increase in cell growth, and extracellular matrix secretion. HSC culture with trans-resveratrol can lead to deactivation of myofibroblast-like HSC. We used an HSC line, PAV-1, to check the role of retinol and palmitic acid in the deactivation process of HSC. Using mass and metabolic-based methods, Western blot and immunocytochemistry assays, we demonstrated that treatment with palmitic acid (75 muM) alone or in combination with retinol (2 muM) significantly decreased cell proliferation and alpha-SMA expression. We also established that the association of both compounds strongly decreased collagen type I expression. Our results suggest the potential use of palmitic acid alone or in combination with retinol to induce HSC deactivation.

  13. Critical factors affecting cell encapsulation in superporous hydrogels

    International Nuclear Information System (INIS)

    Desai, Esha S; Tang, Mary Y; Gemeinhart, Richard A; Ross, Amy E

    2012-01-01

    We recently showed that superporous hydrogel (SPH) scaffolds promote long-term stem cell viability and cell driven mineralization when cells were seeded within the pores of pre-fabricated SPH scaffolds. The possibility of cell encapsulation within the SPH matrix during its fabrication was further explored in this study. The impact of each chemical component used in SPH fabrication and each step of the fabrication process on cell viability was systematically examined. Ammonium persulfate, an initiator, and sodium bicarbonate, the gas-generating compound, were the two components having significant toxicity toward encapsulated cells at the concentrations necessary for SPH fabrication. Cell survival rates were 55.7% ± 19.3% and 88.8% ± 9.4% after 10 min exposure to ammonium persulfate and sodium bicarbonate solutions, respectively. In addition, solution pH change via the addition of sodium bicarbonate had significant toxicity toward encapsulated cells with cell survival of only 50.3% ± 2.5%. Despite toxicity of chemical components and the SPH fabrication method, cells still exhibited significant overall survival rates within SPHs of 81.2% ± 6.8% and 67.0% ± 0.9%, respectively, 48 and 72 h after encapsulation. This method of cell encapsulation holds promise for use in vitro and in vivo as a scaffold material for both hydrogel matrix encapsulation and cell seeding within the pores. (paper)

  14. Aggregate formation affects ultrasonic disruption of microalgal cells.

    Science.gov (United States)

    Wang, Wei; Lee, Duu-Jong; Lai, Juin-Yih

    2015-12-01

    Ultrasonication is a cell disruption process of low energy efficiency. This study dosed K(+), Ca(2+) and Al(3+) to Chlorella vulgaris cultured in Bold's Basal Medium at 25°C and measured the degree of cell disruption under ultrasonication. Adding these metal ions yielded less negatively charged surfaces of cells, while with the latter two ions large and compact cell aggregates were formed. The degree of cell disruption followed: control=K(+)>Ca(2+)>Al(3+) samples. Surface charges of cells and microbubbles have minimal effects on the microbubble number in the proximity of the microalgal cells. Conversely, cell aggregates with large size and compact interior resist cell disruption under ultrasonication. Staining tests revealed high diffusional resistance of stains over the aggregate interior. Microbubbles may not be effective generated and collapsed inside the compact aggregates, hence leading to low cell disruption efficiencies. Effective coagulation/flocculation in cell harvesting may lead to adverse effect on subsequent cell disruption efficiency. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Mechanisms of cell death induced by the neutrophil antimicrobial peptides alpha-defensins and LL-37.

    NARCIS (Netherlands)

    Aarbiou, J.; Tjabringa, G.S.; Verhoosel, R.M.; Ninaber, D.K.; White, S.R.; Peltenburg, L.T.; Rabe, K.F.; Hiemstra, P.S.

    2006-01-01

    OBJECTIVE: The aim of this study was to investigate the mechanisms of cell death mediated by the antimicrobial peptides neutrophil defensins (human neutrophil peptides 1-3 [HNP1-3]) and LL-37. MATERIALS AND METHODS: HNP1-3- and LL-37-mediated cell death was assessed in human lung epithelial cells

  16. Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree.

    Directory of Open Access Journals (Sweden)

    Tomoki Yagai

    Full Text Available Cholangiocarcinoma (CC is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC at the peribiliary gland (PBG of large bile ducts and liver stem/progenitor cell (LPC at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5 is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. In vitro experiments using siRNA for SAMD5 revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines.SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.

  17. Plasma cell deficiency in human subjects with heterozygous mutations in Sec61 translocon alpha 1 subunit (SEC61A1).

    Science.gov (United States)

    Schubert, Desirée; Klein, Marie-Christine; Hassdenteufel, Sarah; Caballero-Oteyza, Andrés; Yang, Linlin; Proietti, Michele; Bulashevska, Alla; Kemming, Janine; Kühn, Johannes; Winzer, Sandra; Rusch, Stephan; Fliegauf, Manfred; Schäffer, Alejandro A; Pfeffer, Stefan; Geiger, Roger; Cavalié, Adolfo; Cao, Hongzhi; Yang, Fang; Li, Yong; Rizzi, Marta; Eibel, Hermann; Kobbe, Robin; Marks, Amy L; Peppers, Brian P; Hostoffer, Robert W; Puck, Jennifer M; Zimmermann, Richard; Grimbacher, Bodo

    2017-08-04

    Primary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation. We identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs. Whole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out ex vivo in primary cells of patients and in vitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival. We investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On in vitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines. We describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding our knowledge about the pathogenesis of antibody deficiencies. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. All rights reserved.

  18. Tumor necrosis factor alpha inhibits the suppressive effect of regulatory T cells on the hepatitis B virus-specific immune response.

    Science.gov (United States)

    Stoop, Jeroen N; Woltman, Andrea M; Biesta, Paula J; Kusters, Johannes G; Kuipers, Ernst J; Janssen, Harry L A; van der Molen, Renate G

    2007-09-01

    Chronicity of hepatitis B virus (HBV) infection is characterized by a weak immune response to the virus. CD4+CD25+ regulatory T cells (Treg) are present in increased numbers in the peripheral blood of chronic HBV patients, and these Treg are capable of suppressing the HBV-specific immune response. The aim of this study was to abrogate Treg-mediated suppression of the HBV-specific immune response. Therefore, Treg and a Treg-depleted cell fraction were isolated from peripheral blood of chronic HBV patients. Subsequently, the suppressive effect of Treg on the response to HBV core antigen (HBcAg) and tetanus toxin was compared, and the effect of exogenous tumor necrosis factor alpha (TNF-alpha), interleukin-1-beta (IL-1beta), or neutralizing antibodies against interleukin-10 (IL-10) or transforming growth factor beta (TGF-beta) on Treg-mediated suppression was determined. The results show that Treg of chronic HBV patients had a more potent suppressive effect on the response to HBcAg compared with the response to tetanus toxin. Neutralization of IL-10 and TGF-beta or exogenous IL-1beta had no effect on Treg-mediated suppression of the anti-HBcAg response, whereas exogenous TNF-alpha partially abrogated Treg-mediated suppression. Preincubation of Treg with TNF-alpha demonstrated that TNF-alpha had a direct effect on the Treg. No difference was observed in the type II TNF receptor expression by Treg from chronic HBV patients and healthy controls. Treg-mediated suppression of the anti-HBV response can be reduced by exogenous TNF-alpha. Because chronic HBV patients are known to produce less TNF-alpha, these data implicate an important role for TNF-alpha in the impaired antiviral response in chronic HBV.

  19. First quantitative assay of alpha-Gal in soft tissues: presence and distribution of the epitope before and after cell removal from xenogeneic heart valves.

    Science.gov (United States)

    Naso, F; Gandaglia, A; Iop, L; Spina, M; Gerosa, G

    2011-04-01

    Decellularized xenograft heart valves might be the ideal scaffolds for tissue engineered heart valves as the alternative to the currently used biological and mechanical prostheses. However, removal of the alpha-Gal epitope is a prerequisite to avoid hyperacute rejection of untreated xenograft material. The aim of this study was to develop an ELISA soft-tissue assay for alpha-Gal quantification in xenograft heart valves before and after a detergent-based (TriCol) or equivalent cell removal procedure. Leaflets from porcine valves were enzymatically digested to expose the epitope and reacted with the alpha-Gal monoclonal antibody M86 for its recognition. Rabbit erythrocytes were used as a reference for the quantification of alpha-Gal. Native aortic and pulmonary leaflets exhibited different epitope concentration: 4.33×10(11) vs. 7.12×10(11)/10 mg wet tissue (pvalves revealed a different alpha-Gal distribution within and among different leaflets. The pattern was consistent with immunofluorescence analysis and was unrelated to microvessel density distribution. After TriCol treatment alpha-Gal was no longer detectable in both pulmonary and aortic decellularized valves, confirming the ability of this method to remove both cells and alpha-Gal antigen. These results hold promise for a reliable quantitative evaluation of alpha-Gal in decellularized valves obtained from xenograft material for tissues engineering purposes. Additionally, this method is applicable to further evaluate currently used xenograft bioprostheses. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  20. Alpha-phellandrene-induced apoptosis in mice leukemia WEHI-3 cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Hsu, Shu-Chun; Lu, Kung-Wen; Ma, Yi-Shih; Wu, Chih-Chung; Lu, Hsu-Feng; Chen, Jaw-Chyun; Lin, Jaung-Geng; Wu, Ping-Ping; Chung, Jing-Gung

    2016-11-01

    Although reports have shown that α-phellandrene (α-PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α-PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α-PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α-PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI-3 cells in vitro. Results indicated that α-PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub-G1 phase (apoptosis) in WEHI-3 cells. α-PA increased the productions of reactive oxygen species (ROS) and Ca 2+ and decreased the levels of mitochondrial membrane potential (ΔΨ m ) in dose- and time-dependent manners in WEHI-3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α-PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI-3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α-PA induces cell cycle arrest and apoptosis in WEHI-3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640-1651, 2016. © 2015 Wiley Periodicals, Inc.

  1. Regulation of PGE2 signaling pathways and TNF-alpha signaling pathways on the function of bone marrow-derived dendritic cells and the effects of CP-25.

    Science.gov (United States)

    Li, Ying; Sheng, Kangliang; Chen, Jingyu; Wu, Yujing; Zhang, Feng; Chang, Yan; Wu, Huaxun; Fu, Jingjing; Zhang, Lingling; Wei, Wei

    2015-12-15

    This study was to investigate PGE2 and TNF-alpha signaling pathway involving in the maturation and activation of bone marrow dendritic cells (DCs) and the effect of CP-25. Bone marrow DCs were isolated and stimulated by PGE2 and TNF-alpha respectively. The markers of maturation and activation expressed on DCs, such as CD40, CD80, CD83, CD86, MHC-II, and the ability of antigen uptake of DCs were analyzed by flow cytometry. The proliferation of T cells co-cultured with DCs, the signaling pathways of PGE2-EP4-cAMP and TNF-alpha-TRADD-TRAF2-NF-κB in DCs were analyzed. The results showed that both PGE2 and TNF-alpha up-regulated the expressions of CD40, CD80, CD83, CD86, and MHC-II, decreased the antigen uptake of DCs, and DCs stimulated by PGE2 or TNF-alpha could increase T cell proliferation. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased significantly the expressions of CD40, CD80, CD83, CD86 and MHC-II, increased the antigen uptake of DCs, and suppressed T cell proliferation induced by DCs. PGE2 increased the expressions of EP4, NF-κB and down-regulated cAMP level of DCs. TNF-alpha could also up-regulate TNFR1, TRADD, TRAF2, and NF-κB expression of DCs. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased the expressions of EP4 and NF-κB, increased cAMP level in DCs stimulated by PGE2. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) also could down-regulate significantly TNFR1, TRADD, TRAF2, and NF-κB expression in DCs stimulated by TNF-alpha. These results demonstrate that PGE2 and TNF-alpha could enhance DCs functions by mediating PGE2-EP4-cAMP pathway, TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathway respectively. CP-25 might inhibit the function of DCs through regulating PGE2-EP4-cAMP and TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Decreased Progesterone Receptor B/A Ratio in Endometrial Cells by Tumor Necrosis Factor-Alpha and Peritoneal Fluid from Patients with Endometriosis.

    Science.gov (United States)

    Chae, Uisoo; Min, Jin Young; Kim, Sung Hoon; Ihm, Hyo Jin; Oh, Young Sang; Park, So Yun; Chae, Hee Dong; Kim, Chung Hoon; Kang, Byung Moon

    2016-11-01

    Progesterone resistance is thought to be a major factor that contributes to progression of endometriosis. However, it is not clear what causes progesterone resistance in endometriosis. This study aimed to assess whether cytokines or peritoneal fluid can affect progesterone receptor (PR) expression in endometrial cells and to verify whether PR expression is reduced in endometriosis. The PR-B/A ratio was measured via real-time polymerase chain reaction after in vitro culture, in which endometrial cells were treated with either tumor necrosis factor-alpha (TNF-α), interleukin-1 beta, or peritoneal fluid obtained from women with advanced-stage endometriosis. Immunohistochemistry was performed to compare PR-B expression between eutopic and ectopic endometrial tissues from women with and without advanced-stage endometriosis. The PR-B/A ratio was significantly decreased by treatment with either TNF-α (p=0.011) or peritoneal fluid from women with advanced-stage endometriosis (p=0.027). Immunoreactivity of PR-B expression was significantly lower during the secretory phase than during the proliferative phase in endometrial tissues from control subjects (pendometriosis compared with eutopic endometrium tissues from control subjects. Progesterone resistance in endometriosis may be caused by proinflammatory conditions in the pelvic peritoneal microenvironment.

  3. Stratification of alpha ganglion cells and ON/OFF directionally selective ganglion cells in the rabbit retina

    OpenAIRE

    ZHANG, JIAN; LI, WEI; HOSHI, HIDEO; MILLS, STEPHEN L.; MASSEY, STEPHEN C.

    2005-01-01

    The correlation between cholinergic sensitivity and the level of stratification for ganglion cells was examined in the rabbit retina. As examples, we have used ON or OFF α ganglion cells and ON/OFF directionally selective (DS) ganglion cells. Nicotine, a cholinergic agonist, depolarized ON/OFF DS ganglion cells and greatly enhanced their firing rates but it had modest excitatory effects on ON or OFF α ganglion cells. As previously reported, we conclude that DS ganglion cells are the most sens...

  4. Cyclic nucleotides differentially regulate the synthesis of tumour necrosis factor-alpha and interleukin-1 beta by human mononuclear cells

    NARCIS (Netherlands)

    Endres, S; Fülle, H J; Sinha, B; Stoll, D; Dinarello, C A; Gerzer, R; Weber, P.C.

    Recent reports have shown that phosphodiesterase (PDE) inhibitors suppress production of tumour necrosis factor-alpha (TNF-alpha) in mouse macrophages. In the present study we show that theophylline, pentoxifylline and 3-isobutyl-1-methylxanthine markedly suppress the lipopolysaccharide

  5. Both PAX4 and MAFA are expressed in a substantial proportion of normal human pancreatic alpha cells and deregulated in patients with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Rémy Bonnavion

    Full Text Available Pax4 and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A are two transcription factors crucial for normal functions of islet beta cells in the mouse. Intriguingly, recent studies indicate the existence of notable difference between human and rodent islet in terms of gene expression and functions. To better understand the biological role of human PAX4 and MAFA, we investigated their expression in normal and diseased human islets, using validated antibodies. PAX4 was detected in 43.0±5.0% and 39.1±4.0% of normal human alpha and beta cells respectively. We found that MAFA, detected in 88.3±6.3% insulin(+cells as in the mouse, turned out to be also expressed in 61.2±6.4% of human glucagons(+ cells with less intensity than in insulin(+ cells, whereas MAFB expression was found not only in the majority of glucagon(+ cells (67.2±7.6%, but also in 53.6±10.5% of human insulin(+ cells. Interestingly, MAFA nuclear expression in both alpha and beta cells, and the percentage of alpha cells expressing PAX4 were found altered in a substantial proportion of patients with type 2 diabetes. Both MAFA and PAX4 display, therefore, a distinct expression pattern in human islet cells, suggesting more potential plasticity of human islets as compared with rodent islets.

  6. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    Directory of Open Access Journals (Sweden)

    María Inés Marchesini

    Full Text Available Choloylglycine hydrolase (CGH, E.C. 3.5.1.24 is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  7. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    Science.gov (United States)

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  8. Radiosensitivity of Prostate Cancer Cell Lines for Irradiation from Beta Particle-emitting Radionuclide ¹⁷⁷Lu Compared to Alpha Particles and Gamma Rays.

    Science.gov (United States)

    Elgqvist, Jörgen; Timmermand, Oskar Vilhelmsson; Larsson, Erik; Strand, Sven-Erik

    2016-01-01

    The purpose of the present study was to investigate the radiosensitivity of the prostate cancer cell lines LNCaP, DU145, and PC3 when irradiated with beta particles emitted from (177)Lu, and to compare the effect with irradiation using alpha particles or gamma rays. Cells were irradiated with beta particles emitted from (177)Lu, alpha particles from (241)Am, or gamma rays from (137)Cs. A non-specific polyclonal antibody was labeled with (177)Lu and used to irradiate cells in suspension with beta particles. A previously described in-house developed alpha-particle irradiator based on a (241)Am source was used to irradiate cells with alpha particles. External gamma-ray irradiation was achieved using a standard (137)Cs irradiator. Cells were irradiated to absorbed doses equal to 0, 0.5, 1, 2, 4, 6, 8, or 10 Gy. The absorbed doses were calculated as mean absorbed doses. For evaluation of cell survival, the tetrazolium-based WST-1 assay was used. After irradiation, WST-1 was added to the cell solutions, incubated, and then measured for level of absorbance at 450 nm, indicating the live and viable cells. LNCaP, DU145, and PC3 cell lines all had similar patterns of survival for the different radiation types. No significant difference in surviving fractions were observed between cells treated with beta-particle and gamma-ray irradiation, represented for example by the surviving fraction values (mean±SD) at 2, 6, and 10 Gy (SF2, SF6, and SF10) for DU145 after beta-particle irradiation: 0.700±0.090, 0.186±0.050 and 0.056±0.010, respectively. A strong radiosensitivity to alpha particles was observed, with SF2 values of 0.048±0.008, 0.018±0.006 and 0.015±0.005 for LNCaP, DU145, and PC3, respectively. The surviving fractions after irradiation using beta particles or gamma rays did not differ significantly at the absorbed dose levels and dose rates used. Irradiation using alpha particles led to a high level of cell killing. The results show that the beta-particle emitter

  9. Differential responses of cells from human skin keratinocyte and bovine mammary epithelium to attack by pore-forming Staphylococcus aureus alpha-toxin.

    Science.gov (United States)

    Suriyaphol, Gunnaporn; Sarikaputi, Meena; Suriyaphol, Prapat

    2009-11-01

    Human skin keratinocytes HaCat attacked by Staphylococcus aureus alpha-toxin showed a transient drop of cellular ATP levels whereas in toxin-perforated bovine mammary epithelial cells (BMEC), the ATP levels dropped more slowly. Morphologically, during the ATP level depletion, HaCat cell developed a spacious intracellular vacuole together with the transient influx of trypan blue. WST-1 signal, which tested the function of mitochondrial enzyme in viable cells, also decreased concomitantly. On the other hand, BMEC excluded trypan blue and vacuolation was not observed throughout the experiment. We conclude that mammary epithelial cells resist the toxin better than keratinocytes. This is the first report showing that alpha-toxin enhances transient membrane permeability to large molecules, temporary vacuole formation and the transient defect of mitochondrial enzyme in viable cells without cell lysis.

  10. psychosocial impact of sickle cell disease on mothers of affected

    African Journals Online (AJOL)

    2007-09-09

    Sep 9, 2007 ... Many previous studies (1-3,9,14) have described the negative psychosocial impact of SCD on affected children and their families. This paper tries to examine whether there are any characteristics or peculiar problems (in terms of scope and severity) exerted via SCD by comparing mothers, of affected.

  11. How does metabolism affect cell death in cancer?

    Science.gov (United States)

    Villa, Elodie; Ricci, Jean-Ehrland

    2016-07-01

    In cancer research, identifying a specificity of tumor cells compared with 'normal' proliferating cells for targeted therapy is often considered the Holy Grail for researchers and clinicians. Although diverse in origin, most cancer cells share characteristics including the ability to escape cell death mechanisms and the utilization of different methods of energy production. In the current paradigm, aerobic glycolysis is considered the central metabolic characteristic of cancer cells (Warburg effect). However, recent data indicate that cancer cells also show significant changes in other metabolic pathways. Indeed, it was recently suggested that Kreb's cycle, pentose phosphate pathway intermediates, and essential and nonessential amino acids have key roles. Renewed interest in the fact that cancer cells have to reprogram their metabolism in order to proliferate or resist treatment must take into consideration the ability of tumor cells to adapt their metabolism to the local microenvironment (low oxygen, low nutrients). This variety of metabolic sources might be either a strength, resulting in infinite possibilities for adaptation and increased ability to resist chemotherapy-induced death, or a weakness that could be targeted to kill cancer cells. Here, we discuss recent insights showing how energetic metabolism may regulate cell death and how this might be relevant for cancer treatment. © 2015 FEBS.

  12. STEM CELL ORIGIN DIFFERENTLY AFFECTS BONE TISSUE ENGINEERING STRATEGIES.

    Directory of Open Access Journals (Sweden)

    Monica eMattioli-Belmonte

    2015-09-01

    Full Text Available Bone tissue engineering is a promising research area for the improvement of traditional bone grafting procedure drawbacks. Thanks to the capability of self-renewal and multi-lineage differentiation, stem cells are one of the major actors in tissue engineering approaches, and adult mesenchymal stem cells (MSCs are considered to be appropriate for regenerative medicine strategies. Bone marrow MSCs (BM-MSCs are the earliest- discovered and well-known stem cell population used in bone tissue engineering. However, several factors hamper BM-MSC clinical application and subsequently, new stem cell sources have been investigated for these purposes. The successful identification and combination of tissue engineering, scaffold, progenitor cells, and physiologic signalling molecules enabled the surgeon to design, recreate the missing tissue in its near natural form. On the basis of these considerations, we analysed the capability of two different scaffolds, planned for osteochondral tissue regeneration, to modulate differentiation of adult stem cells of dissimilar local sources (i.e. periodontal ligament, maxillary periosteum as well as adipose-derived stem cells, in view of possible craniofacial tissue engineering strategies. We demonstrated that cells are differently committed toward the osteoblastic phenotype and therefore, considering their peculiar features, they may alternatively represent interesting cell sources in different stem cell-based bone/periodontal tissue regeneration approaches.

  13. Comparative Study of Various Delivery Methods for the Supply of Alpha-Ketoglutarate to the Neural Cells for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Tanushree Vishnoi

    2013-01-01

    Full Text Available Delivery of growth factors or bioactive molecules plays an important role in tissue engineering, as the duration to which these are supplied can modulate the cell fate. Thus, the delivery method plays an important role, and the same is presented in this work wherein the exogenous supply of alpha-ketoglutarate (α-KG gave better results for fast proliferating cells as compared to delivery by microspheres or microspheres incorporated scaffolds which can be used while culturing slow growing cells. All these studies were performed in two dimensional (2D and three dimensional (3D setups in which chitosan-gelatin-polypyrrole has been used as 3-D scaffolds. Chitosan and gelatin microspheres alone as well as incorporated in the cryogels were characterized. MTT assay done using neuro-2a cell line showed approximately 42% and 70% increment in cellular proliferation when gelatin and chitosan microspheres were added in a 3-D setup, respectively, as compared to the control. Biochemical analysis of ammonia showed 6-fold reductions in ammonia level in a 3-D setup compared to the control. We also studied the synthesis of a neurotransmitter-like glutamate and found that its concentration increased up to 0.25 mg/ml when the microspheres were added exogenously in a 3-D system.

  14. TNF-alpha impairs the S-G2/M cell cycle checkpoint and cyclobutane pyrimidine dimer repair in premalignant skin cells: Role of the PI3K-Akt pathway

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.; Calay, D.

    2008-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is induced by UVB radiation and has been implicated in the early stages of skin carcinogenesis. Here, we show that in normal keratinocytes and the transformed keratinocyte cell lines, HaCaT and A431, TNF-alpha stimulates protein kinase B/Akt, which results...... cycling. TNF-alpha enhanced apoptosis less potently and did not increase the level of CPD or stimulate cell cycle progression in normal keratinocytes. Our data suggest that TNF-alpha overrides the G2/M checkpoint in premalignant skin cells and allows for some cells containing unrepaired CPD to enter...... in activation of the survival complex mTORC1 (mammalian target of rapamycin complex 1) and inhibition of the proapoptotic proteins Bad and Fox03a. In UVB-irradiated HaCaT cells (10-20 mJ cm(-2)), TNF-alpha increased the proportion of cycling cells and enhanced the rate of apoptosis. A significantly higher...

  15. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

    Science.gov (United States)

    Marchesini, María I; Morrone Seijo, Susana M; Guaimas, Francisco F; Comerci, Diego J

    2016-01-01

    Brucella abortus , the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus , ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus .

  16. Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

    Science.gov (United States)

    Garcia, J A; Ferreira, H L; Vieira, F V; Gameiro, R; Andrade, A L; Eugênio, F R; Flores, E F; Cardoso, T C

    2017-06-01

    Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy. © 2015 John Wiley & Sons Ltd.

  17. [CD4+ alpha beta T cell and gamma delta T cell responses to BCG in patients with pulmonary tuberculosis--comparison with healthy controls].

    Science.gov (United States)

    Tsukaguchi, K; Okamura, H; Tokuyama, T; Okamoto, Y; Fu, A; Yamamoto, C; Nakaya, M; Kobayashi, A; Yoneda, T; Narita, N

    1997-12-01

    We demonstrated that CD4+ alpha beta (CD4+) and gamma delta T cell subsets from healthy donors had similar effector functions (cytotoxicity and cytokine production) in response to mycobacterial antigens, despite differences in the antigens recognized. To elucidate the pathogenesis of pulmonary tuberculosis, this study was undertaken to compare T cell functions between patients with pulmonary tuberculosis with no complications and healthy controls. Both resting and activated CD4+ and gamma delta T cells from the patient group proliferated in response to live BCG at a significantly lower rate than those from the control group. The cytotoxicity of BCG-pulsed monocytes and IFN-gamma production in both the CD4+ and gamma delta T cells from patients was significantly lower than those of controls. In contrast to IFN-gamma, significantly higher IL-10 production by both CD4+ and gamma delta T cells from patients was detected. The proliferative responses to BCG by CD4+ and gamma delta T cells from patients after antituberculous therapy were partially restored, but remained at lower levels compared with controls. These results suggest that not only a general deterioration in CD4+ and gamma delta T cells effector functions, but also suppressive factors (such as IL-10) might be responsible for the pathogenesis of pulmonary tuberculosis, and that the low response to BCG by both CD4+ and gamma delta T cells in patients with tuberculosis is in part attributable to patient predisposition.

  18. Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

    Directory of Open Access Journals (Sweden)

    Bernhard F Gibbs

    Full Text Available Stem cell factor (SCF is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1 in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

  19. Comparative study of toxic effects of zearalenone and its two major metabolites alpha-zearalenol and beta-zearalenol on cultured human Caco-2 cells.

    Science.gov (United States)

    Abid-Essefi, S; Bouaziz, C; Golli-Bennour, E El; Ouanes, Z; Bacha, Hassen

    2009-01-01

    Zearalenone (ZEN) is a fusarotoxin converted predominantly into alpha-zearalenol (alpha-Zol) and beta-zearalenol (beta-Zol) by hepatic hydroxysteroid dehydrogenases. The feeding of naturally contaminated grains with ZEN was associated with hyperestrogenic and adverse effects on humans and animals. There is a lack of information on the attribution of the toxic effects of these toxins. One wonders if these effects are due to the parent molecule (ZEN) or to its major metabolites (alpha-Zol and beta-Zol). Using human Caco-2 cells, we looked for the molecular mechanisms of toxicity of ZEN, alpha-Zol, and beta-Zol. Toxicity effects were studied by MTT viability assay and oxidative stress induction by measuring malondialdehyde (MDA) generation. To check whether the oxidative stress induction was associated to DNA lesions, we looked for DNA fragmentation by means of the Comet and the diphenylamine assays. To specify cell death pathway, we investigated caspase-3 activation, confirmed by poly(ADP-ribose) polymerase cleavage and by Bcl-2 depletion. Our results clearly demonstrated that ZEN as well as its two metabolites presented variable toxic effects. They induced cell death and an increase in MDA generation. These effects were associated to DNA fragmentation as well as caspase-3 activation. The observed toxic effects seem to be relieved by the metabolism of ZEN into alpha-Zol and beta-Zol. 2009 Wiley Periodicals, Inc.

  20. Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

    Science.gov (United States)

    Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

    1996-01-01

    In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

  1. Increased alpha-defensins 1-3 production by dendritic cells in HIV-infected individuals is associated with slower disease progression.

    Directory of Open Access Journals (Sweden)

    Marta Rodríguez-García

    Full Text Available BACKGROUND: Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC produce alpha-defensins1-3 and that alpha-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of alpha-defensins1-3 production by DC on disease progression. METHODOLOGY/PRINCIPAL FINDINGS: Monocyte-derived DC (MDDC were analyzed comparatively in healthy controls (HC and HIV-infected patients, including untreated "elite" and "viremic" controllers, untreated viremic non-controllers and antiretroviral-treated patients. We found that production of alpha-defensins1-3 was significantly increased in MDDC from HIV-infected patients versus HC, and this increase was mainly due to that observed in controllers, while in non-controllers the increase was not statistically significant (controllers vs. HC, p<0.005; controllers vs. non-controllers p<0.05. Secreted alpha-defensins1-3 by immature MDDC positively correlated with CD4 T cell counts in controllers, but not in non-controllers. Moreover, independently of their clinical classification, HIV-infected patients with higher alpha-defensins1-3 secretion by immature MDDC showed slower disease progression, measured as no decrease in the number of CD4+ T-cells below 350 cell/mm(3, lower increase of plasma viral load and no initiation of treatment over time. Plasma alpha-defensins1-3 levels lacked any relationship with immunologic and virologic parameters. CONCLUSIONS/SIGNIFICANCE: High production of alpha-defensins1-3 by immature DCs appears as a host protective factor against progression of HIV-1 infection, suggesting potential diagnostic, therapeutic and preventive implications. This protective effect may arise from the activity of alpha-defensins1-3 to damage the virions prior and/or after their

  2. Alpha- and beta-cell abnormalities in haemoglobin A1c-defined prediabetes and type 2 diabetes

    DEFF Research Database (Denmark)

    Calanna, Salvatore; Scicali, Roberto; Di Pino, Antonino

    2014-01-01

    separate days after a 75 g-oral glucose tolerance test and an isoglycaemic intravenous glucose infusion, respectively. Blood was analysed for glucose, insulin, C-peptide, glucagon, and incretin hormones. Insulinogenic index, disposition index, glucagon suppression, and incretin effect were evaluated......-diagnosed type 2 diabetic patients. The patients with type 2 diabetes showed lower insulinogenic index (P = 0.0003), disposition index (P effect was significantly (P ... compared to subjects with HbA1c-defined prediabetes and controls. Plasma levels of incretin hormones were similar across the three groups. HbA1c associated negatively with insulinogenic index, disposition index, and incretin effect. Our findings show clear alpha- and beta-cell dysfunction in HbA1c...

  3. Beta-globin gene cluster haplotypes and alpha-thalassemia in sickle cell disease patients from Trinidad.