WorldWideScience

Sample records for alpha tc3 cells

  1. Assessing activation of hepatic stellate cells by 99mTc-3PRGD2 scintigraphy targeting integrin αvβ3: a feasibility study

    International Nuclear Information System (INIS)

    Zhang, Xin; Xin, Jun; Shi, Yu; Xu, Weina; Yu, Shupeng; Yang, Zhiguang; Liu, Changping; Cao, Li; Guo, Qiyong

    2015-01-01

    Objective: Hepatic stellate cell (HSC) activation, which is accompanied by increased expression of integrin αvβ3, is an important factor in liver fibrogenesis. Molecular imaging targeting the integrin αvβ3 could provide a non-invasive method for evaluating the expression and the function of the integrin αvβ3 on the activated HSCs (aHSCs) in the injured liver, and then provide important prognostic information. 99m Tc-3PRGD2 is such a radiotracer specific for integrin αvβ3. In this study, we aimed to compare the differences in liver uptake and retention of the 99m Tc-3PRGD2 between normal liver and injured liver to evaluate the feasibility of 99m Tc-3PRGD2 scintigraphy for this purpose. Methods: We used planar scintigraphy to assess changes in integrin αvβ3 binding of intravenously-administered 99m Tc-3PRGD2 in the livers of rats with thioacetamide (TAA)-induced liver fibrosis compared with the controls. We co-injected cold c(RGDyK) with 99m Tc-3PRGD2 to assess the specific binding of the radiotracer. We performed Sirius red staining to assess liver fibrosis, immunofluorescent colocalization to identify the location of integrin αvβ3 expressed in the fibrotic liver, and we measured protein and messenger RNA expression of integrin αvβ3 and alpha smooth muscle actin (α-SMA) in the control and fibrotic livers. Results: The fibrotic livers showed enhanced 99m Tc-3PRGD2 uptake and retention. The radiotracer was demonstrated to bind specifically with the integrin αvβ3 mainly expressed on the aHSCs. The liver-to-heart ratio at 30 min post-injection was higher in the fibrotic livers than in the control livers (TAA, 1.98 ± 0.08 vs. control, 1.50 ± 0.12, p < 0.01). The liver t 1/2 was longer than in the controls (TAA, 27.07 ± 10.69 min vs. control, 12.67 ± 4.10 min, p < 0.01). The difference of heart t 1/2 between the two groups was not statistically significant (TAA, 3.13 ± 0.63 min vs. control, 3.41 ± 0.77 min, p = 0.94). Conclusions: 99m Tc-3PRGD2

  2. In vitro phenotypic, genomic and proteomic characterization of a cytokine-resistant murine β-TC3 cell line.

    Directory of Open Access Journals (Sweden)

    Antonina Coppola

    Full Text Available Type 1 diabetes mellitus (T1DM is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival.The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling.In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to

  3. Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in beta TC3 cells and rat pancreatic beta cells

    DEFF Research Database (Denmark)

    Gromada, J; Dissing, S; Kofod, Hans

    1995-01-01

    The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse beta TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open pro...

  4. (99m)Tc-3PRGD 2 SPECT/CT predicts the outcome of advanced nonsquamous non-small cell lung cancer receiving chemoradiotherapy plus bevacizumab.

    Science.gov (United States)

    Ma, Qingjie; Min, Kaiyin; Wang, Ting; Chen, Bin; Wen, Qiang; Wang, Fan; Ji, Tiefeng; Gao, Shi

    2015-07-01

    Functional imaging can help clinicians assess the individual response of advanced nonsquamous non-small cell lung cancer (NSCLC) to chemoradiation therapy plus bevacizumab. Our purpose is to investigate the ability of (99m)Tc-3PRGD2 single photon emission computed tomography/computed tomography (SPECT/CT) in predicting the early response to treatment. Patients with advanced nonsquamous NSCLC diagnosed by histological or cytological examination were imaged with (99m)Tc-3PRGD2 SPECT/CT at 3 time points: 1-3 days before the start of treatment (SPECT1), 40 Gy radiotherapy with 2 cycles of chemotherapy plus bevacizumab (SPECT2) and 4 weeks after chemoradiotherapy plus bevacizumab (SPECT3). The images were evaluated semiquantitatively by measuring the tumor to non-tumor ratio (T/N) and calculating the percentage change in T/N ratio. Short-term outcome was assessed by the treatment response evaluation according to the Response Evaluation Criteria in Solid Tumors criteria as: complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). Patients were divided two groups: responders (CR and PR) and nonresponders (SD and PD). To determine a threshold for percent reduction in T/N ratios, receiver-operating characteristic (ROC) curve analysis was used. Patients were grouped again based on the threshold of P1 (the change percentage from SPECT1 to SPECT2) and P2 (the change percentage from SPECT1 to SPECT3): P1 responders and P1 nonresponders; P2 responders and P2 nonresponders. Patients were followed up starting 4 weeks after completion of therapy and then every 3 months for the first 2 years and every 6 months after 2 years. OS of P1 responders, P1 nonresponders, P2 responders and P2 nonresponders was estimated and graphically illustrated using the Kaplan-Meier method and the log-rank test was used to test the null hypotheses of equal OS in subgroups of patients. A total of 28 patients completed all imaging and treatment. All primary

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor

    OpenAIRE

    1990-01-01

    T cell-specific expression of the human T cell receptor alpha (TCR- alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR- alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP ...

  8. Effects of TNF-alpha on Endothelial Cell Collective Migration

    Science.gov (United States)

    Chen, Desu; Wu, Di; Helim Aranda-Espinoza, Jose; Losert, Wolfgang

    2013-03-01

    Tumor necrosis factor (TNF-alpha) is a small cell-signaling protein usually released by monocytes and macrophages during an inflammatory response. Previous work had shown the effects of TNF-alpha on single cell morphology, migration, and biomechanical properties. However, the effect on collective migrations remains unexplored. In this work, we have created scratches on monolayers of human umbilical endothelial cells (HUVECs) treated with 25ng/mL TNF-alpha on glass substrates. The wound healing like processes were imaged with phase contrast microscopy. Quantitative analysis of the collective migration of cells treated with TNF-alpha indicates that these cells maintain their persistent motion and alignment better than untreated cells. In addition, the collective migration was characterized by measuring the amount of non-affine deformations of the wound healing monolayer. We found a lower mean non-affinity and narrower distribution of non-affinities upon TNF-alpha stimulation. These results suggest that TNF-alpha introduces a higher degree of organized cell collective migration.

  9. Conception of dismantling cell for glove box with alpha contamination

    International Nuclear Information System (INIS)

    Mangin, D.

    1987-01-01

    The new dismantling cell of Valduc treats particularly alpha glove boxes. This cell is conceived to reduce the intervention inside for man with ventilated clothes and to reduce the volume of alpha wastes by utilization of manipulators and appropriate tools. The respect of low level norms (0.1 Ci/ton) for storage of alpha wastes conductes us to make a first decontamination, to ameliorate the detection in quantity of plutonium in the wastes and for wastes with a level upper the norm to make studies on decontamination by Freon 113 [fr

  10. 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)

  11. CIRM Alpha Stem Cell Clinics: Collaboratively Addressing Regenerative Medicine Challenges.

    Science.gov (United States)

    Jamieson, Catriona H M; Millan, Maria T; Creasey, Abla A; Lomax, Geoff; Donohoe, Mary E; Walters, Mark C; Abedi, Mehrdad; Bota, Daniela A; Zaia, John A; Adams, John S

    2018-06-01

    The California Institute for Regenerative Medicine (CIRM) Alpha Stem Cell Clinic (ASCC) Network was launched in 2015 to address a compelling unmet medical need for rigorous, FDA-regulated, stem cell-related clinical trials for patients with challenging, incurable diseases. Here, we describe our multi-center experiences addressing current and future challenges. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    African Journals Online (AJOL)

    Keywords: Alpha thalassemia, sickle cell anaemia patients, Kampala, Uganda. DOI: http://dx.doi.org/10.4314/ahs.v15i2.48. Introduction. In the early 1960's many adults with sickle cell anaemia. (SCA) as well as those with mild disease were reported in Jamaica1. Various factors, both genetic and environmental, are.

  13. 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....

  14. 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.

  15. Human fat cell alpha-2 adrenoceptors. I. Functional exploration and pharmacological definition with selected alpha-2 agonists and antagonists

    International Nuclear Information System (INIS)

    Galitzky, J.; Mauriege, P.; Berlan, M.; Lafontan, M.

    1989-01-01

    This study was undertaken to investigate more fully the pharmacological characteristics of the human fat cell alpha-2 adrenoceptor. Biological assays were performed on intact isolated fat cells while radioligand binding studies were carried out with [ 3 H]yohimbine in membranes. These pharmacological studies brought: (1) a critical definition of the limits of the experimental conditions required for the exploration of alpha-2 adrenergic responsiveness on human fat cells and membranes; (2) an improvement in the pharmacological definition of the human fat cell postsynaptic alpha-2 adrenoceptor. Among alpha-2 agonists, UK-14,304 was the most potent and the relative order of potency was: UK-14,304 greater than p-aminoclonidine greater than clonidine = B-HT 920 greater than rilmenidine. For alpha-2 antagonists, the potency order was: yohimbine greater than idazoxan greater than SK ampersand F-86,466 much greater than benextramine; (3) a description of the impact of benextramine (irreversible alpha-1/alpha-2 antagonist) on human fat cell alpha-2 adrenergic receptors and on human fat cell function; the drug inactivates the alpha-2 adrenergic receptors with a minor impact on beta adrenergic receptors and without noticeable alterations of fat cell function as assessed by preservation of beta adrenergic and Al-adenosine receptor-mediated lipolytic responses; and (4) a definition of the relationship existing between alpha-2 adrenergic receptor occupancy, inhibition of adenylate cyclase activity and antilipolysis with full and partial agonists. The existence of a receptor reserve must be taken into account when evaluating alpha-2 adrenergic receptor distribution and regulation of human fat cells

  16. Asteroid 2008 TC3 Breakup and Meteorite Fractions

    Science.gov (United States)

    Goodrich, C.; Jenniskens, P.; Shaddad, M. H.; Zolensky, M. E.; Fioretti, A. M.

    2017-01-01

    The recovery of meteorites from the impact of asteroid 2008 TC3 in the Nubian Desert of Sudan on October 7, 2008, marked the first time meteorites were collected from an asteroid observed in space by astronomical techniques before impacting. Search teams from the University of Khartoum traced the location of the strewn field and collected about 660 meteorites in four expeditions to the fall region, all of which have known fall coordinates. Upon further study, the Almahata Sitta meteorites proved to be a mixed bag of mostly ureilites (course grained, fine grained, and sulfide-metal assemblages), enstatite chondrites (EL3-6, EH3, EH5, breccias) and ordinary chondrites (H5-6, L4-5). One bencubbinite-like carbonaceous chondrite was identified, as well as one unique Rumuruti-like chondrite and an Enstatite achondrite. New analysis: The analysed meteorites so far suggest a high 30-40 percent fraction of non-ureilites among the recovered samples, but that high fraction does not appear to be in agreement with the meteorites in the University of Khartoum (UoK) collection. Ureilites dominate the meteorites that were recovered by the Sudanese teams. To better understand the fraction of recovered materials that fell to Earth, a program has been initiated to type the meteorites in the UoK collection in defined search areas. At this meeting, we will present some preliminary results from that investigation.

  17. 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...

  18. Functional activities of receptors for tumor necrosis factor-alpha on human vascular endothelial cells.

    NARCIS (Netherlands)

    Paleolog, E.M.; Delasalle, S.A.; Buurman, W.A.; Feldmann, M.

    1994-01-01

    Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the control of endothelial cell function and hence in regulating traffic of circulating cells into tissues in vivo. Stimulation of endothelial cells in vitro by TNF-alpha increases the surface expression of leukocyte adhesion

  19. 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...

  20. 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.

  1. 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

  2. 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

    Mutations in the genes encoding hepatocyte nuclear factor 4alpha (HNF-4alpha) and HNF-1alpha impair insulin secretion and cause maturity onset diabetes of the young (MODY). HNF-4alpha is known to be an essential positive regulator of HNF-1alpha. More recent data demonstrates that HNF-4alpha...... 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...

  3. Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

    Science.gov (United States)

    Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro

    2004-11-01

    GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

  4. Mapping of HNF4alpha target genes in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Boyd, Mette; Bressendorff, Simon; Moller, Jette

    2009-01-01

    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....... The HNF4alpha ChIP-chip data was matched with gene expression and histone H3 acetylation status of the promoters in order to identify HNF4alpha binding to actively transcribed genes with an open chromatin structure. RESULTS: 1,541 genes were identified as potential HNF4alpha targets, many of which have...

  5. 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

  6. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    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

  7. 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

  8. 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.

  9. Monte Carlo Simulations of Necrotic Cell Targeted Alpha Therapy

    International Nuclear Information System (INIS)

    Penfold, S.N.; Brown, M.P.; Bezak, E.

    2011-01-01

    Full text: Hypoxic tumour cells are radioresistant and are significant contributors to the locoregional recurrences and distant metastases that mark treatment failure. Due to restricted circulatory supply, hypoxic tumor cells frequently become necrotic and thus necrotic areas often lie near hypoxic tumour areas. In this study we investigate the feasibility of binding an alpha-emitting conjugate to necrotic cells located in the proximity of hypoxic, viable tumour cells. Monte Carlo radiation transport simulations were performed to investigate the dose distribution resulting from the thorium 227 (Th227) decay chain in a representative tumour geometry. The Geant4 software toolkit was used to simulate the decay and interactions of the Th227 decay chain. The distribution of Th227 was based on a study by Thomlinson and Gray of human lung cancer histological samples (Thomlinson RH, Gray LH. Br J Cancer 1955; 9:539). The normalized dose distribution obtained with Geant4 from a cylindrical Th227 source in water is illustrated in Fig. I. The relative contribution of the different decay channels is displayed, together with a profile through the centre of the accumulated dose map. The results support the hypothesis that significant α-particle doses will be deposited in the hypoxic tumor tissue immediately surrounding the necrotic core (where the majority of Th227 will be located). As an internal a-particle generator, the Th227-radioimmunoconjugate shows potential as an efficient hypoxic tumour sterilizer.

  10. 99mTc-3PRGD2 scintigraphy to stage liver fibrosis and evaluate reversal after fibrotic stimulus withdrawn

    International Nuclear Information System (INIS)

    Zhang, Xin; Guo, Qiyong; Shi, Yu; Xu, Weina; Yu, Shupeng; Yang, Zhiguang; Cao, Li; Liu, Changping; Zhao, Zhoushe; Xin, Jun

    2017-01-01

    Objective: Scintigraphy using 99mTc-3PRGD2 targeting integrin αvβ3 could assess activation of hepatic stellate cells (HSCs). Liver fibrogenesis is intimately associated with activation of HSCs, and the fibrolytic process is accompanied by the reduction of the activated HSCs. In this study, we aimed to evaluate the feasibility of this method to assess the severity of liver fibrosis and the reversal after the fibrotic stimulus withdrawal. Methods: Liver fibrosis of different stages was induced by thioacetamide (TAA) injection for 2, 4 and 6 weeks (n = 6 for each time point). Another 6 rats with 8-week TAA administration (the 8-week group) and 6 rats which were injected with TAA for 6 weeks, and then withdrawn of TAA for 2 weeks (spontaneous recovery rats, SRR) were designed. The ratios of radioactivity detected in the liver vs. the heart at 30 min post-injection of 99m Tc-3PRGD2 (L/H30 min ), the collagen proportionate area (CPA), the protein and mRNA levels of integrin α v , integrin β 3 were analyzed and compared among groups. Results: The Ishak stage scores of the livers in the control and 2, 4, 6-week groups increased when the TAA administration period was extended. L/H30 min increased with the upgrading of liver fibrosis and the differences between each pair of groups were statistically significant (p 30 min in the 8-week group was similar to that in the 6-week group (p > 0.05), but was significantly higher than that in the SRR group (p = 0.005). Conclusions: Scintigraphy using 99m Tc-3PRGD2 may provide a non-invasive method for grading liver fibrosis and assessing liver fibrosis reversal.

  11. 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

  12. 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.

  13. 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

    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......., 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...

  14. 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

  15. Effect of alpha-tocopherol and alpha-tocopheryl quinone on the radiosensitivity of thiol-depleted mammalian cells

    International Nuclear Information System (INIS)

    Hodgkiss, R.J.; Stratford, M.R.; Watfa, R.R.

    1989-01-01

    The effect of hypoxic cell radiosensitizers is increased when mammalian cells are depleted of endogenous glutathione by buthionine sulphoximine pre-treatment in vitro; a similar gain has not been observed in tumors in vivo despite evidence of glutathione depletion in vivo following buthionine sulphoximine treatment. However, concentrations of biological reducing agents other than glutathione were not measured in the in vivo experiments. Other reducing agents found in tumors include alpha-tocopherol, which reduces the sensitizing efficiency of nitro-aromatic sensitizers in thiol-depleted mammalian cells. These data suggest that the failure to observe large gains in misonidazole sensitizing efficiency in thiol-depleted tumors in vivo may be due, in part, to the presence of biological reducing agents such as alpha-tocopherol

  16. 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.

  17. 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.

  18. Prostaglandin production by melanocytic cells and the effect of alpha-melanocyte stimulating hormone.

    Science.gov (United States)

    Nicolaou, Anna; Estdale, Sian E; Tsatmali, Marina; Herrero, Daniel Pascual; Thody, Anthony J

    2004-07-16

    Prostaglandins are potent mediators of the inflammatory response and are also involved in cancer development. In this study, we show that human melanocytes and FM55 melanoma cells express cyclooxygenase-1 and -2 (COX-1 and -2) and thus have the capability to produce prostaglandins. The FM55 cells produced predominantly PGE2 and PGF2alpha, whereas the HaCaT keratinocyte cell line produced mainly PGE2. The anti-inflammatory peptide, alpha-melanocyte stimulating hormone (alpha-MSH), reduced prostaglandin production in FM55 and HaCaT cells and reversed the effect of the pro-inflammatory cytokine TNF-alpha in the former. These results indicate that melanocytes produce prostaglandins and that alpha-MSH, by inhibiting this response, may play an important role in regulating inflammatory responses in the skin.

  19. 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.

  20. Relative biological effectiveness (RBE) of alpha radiation in cultured porcine aortic endothelial cells.

    Science.gov (United States)

    Thomas, Patricia; Tracy, Bliss; Ping, Tilly; Baweja, Anar; Wickstrom, Mark; Sidhu, Narinder; Hiebert, Linda

    2007-03-01

    Northern peoples can receive elevated radiation doses (1- 10 mSv/y) from transfer of polonium-210 (210Po) through the lichen-caribou-human food chain. Ingested 210Po is primarily blood-borne and thus many of its short range alpha particles irradiate the endothelial cells lining the blood vessels. The relative biological effectiveness (RBE) of alpha particles vs. x-rays was examined in porcine aortic endothelial cells as a surrogate for understanding what might happen to human endothelial cells in northern populations consuming traditional foods. Cultured porcine aortic endothelial cells were exposed to x-ray and 210Po alpha particle radiation. Alpha irradiation was applied to the cell cultures internally via the culture medium and externally, using thin-bottomed culture dishes. The results given here are based on the external irradiation method, which was found to be more reliable. Dose-response curves were compared for four lethal endpoints (cell viability, live cell fraction, release of lactate dehydrogenase [LDH] and clonogenic survival) to determine the relative biological effectiveness (RBE) of alpha radiation. The alpha RBE for porcine cells varied from 1.6-21, depending on the endpoint: 21.2+/-4.5 for cell viability, 12.9+/-2.7 for decrease in live cell number, 5.3+/-0.4 for LDH release to the medium but only 1.6 +/-0.1 for clonogenic survival. The low RBE of 1.6 was due to x-ray hypersensitivity of endothelial cells at low doses.

  1. 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.

  2. Alpha-, gamma- and delta-tocopherols reduce inflammatory angiogenesis in human microvascular endothelial cells.

    Science.gov (United States)

    Wells, Shannon R; Jennings, Merilyn H; Rome, Courtney; Hadjivassiliou, Vicky; Papas, Konstantinos A; Alexander, Jonathon S

    2010-07-01

    Vitamin E, a micronutrient (comprising alpha-, beta-, gamma- and delta-tocopherols, alpha-, beta-, gamma- and delta-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of alpha-, gamma- and delta-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-alpha-induced VCAM-1 as in vitro models of inflammatory angiogenesis. alpha-, gamma- and delta-tocopherols were not toxic to either cell type up to 40 microM. In BEC, confluent cell density was decreased by all concentrations of delta- and gamma-tocopherol (10-40 microM) but not by alpha-tocopherol. LEC showed no change in cell density in response to tocopherols. delta-Tocopherol (40 microM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of gamma-tocopherol, as well as the highest dose of alpha-tocopherol (40 microM), decreased cell invasiveness. delta-Tocopherol had no effect on LEC invasiveness at any molarity. delta-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; alpha- and gamma-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 microM) concentrations of alpha-, gamma- and delta-tocopherol, but showed no effects with smaller doses (10-20 microM) in BEC. gamma-Tocopherol (10-20 microM) and alpha-tocopherol (10 microM), but not delta-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, alpha-, gamma- and delta-tocopherol each dose-dependently reduced TNF-alpha-induced expression of VCAM-1. In LEC, there was no significant change to TNF-alpha-induced VCAM-1 expression with any concentration of alpha-, gamma- or delta-tocopherol. These data demonstrate that physiological levels (0-40 microM) of alpha-, gamma- and delta-tocopherols are nontoxic and dietary tocopherols, especially delta

  3. T-cell receptor V sub. alpha. and C sub. alpha. alleles associated with multiple sclerosis and myasthenia gravis

    Energy Technology Data Exchange (ETDEWEB)

    Oksenberg, J.R.; Cavalli-Sforza, L.L.; Steinman, L. (Stanford Univ., CA (USA)); Sherritt, M.; Bernard, C.C. (LaTrobe Univ., Victoria (Australia)); Begovich, A.B.; Erlich, H.A. (Cetus Corporation, Emeryville, CA (USA))

    1989-02-01

    Polymorphic markers in genes encoding the {alpha} chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, the authors amplified selected sequences derived from the full-length TcR {alpha} cDNA probe. These PcR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, the authors have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V{sub {alpha}} and C{sub {alpha}} markers were identified between patients and healthy individuals.

  4. 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

    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)-C...

  5. 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

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

    DEFF Research Database (Denmark)

    Hong, Jing; Chen, Jianguo; Jeppesen, Per Bendix

    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 (Pacids 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......-cell hypersecretion caused by palmitate and enhanced the expression of genes involved in fatty acid metabolism. This indicates that stevioside may be a promising antidiabetic agent in treatment of type 2 diabetes....

  7. 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...

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

    DEFF Research Database (Denmark)

    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...

  9. Transformation of mouse embryo (C3H 10T1/2) cells by alpha particles

    International Nuclear Information System (INIS)

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

    1977-01-01

    Mammalian cells in culture (C3H mouse 10T1/2 cells) have been shown here for the first time to be transformed by alpha irradiation when cells were irradiated with 5.6 MeV alpha particles from a Tandem Van de Graaff machine. Malignant tumors were induced following inoculation of the transformed cells into syngeneic hosts. Unirradiated control cells injected at the same concentration have, so far, failed to produce tumors. The morphology of the transformed foci was remarkably similar to that obtained by x rays and chemicals but different from virally transformed cells. When the cells were seeded at low density in the exponential growth phase, the transformation frequency per surviving cell increased approximately as the cube of the dose and peaked at an alpha particle fluence between 1.5 and 2.5 x 10 7 alpha particles per cm 2 (205 to 342 rads). The frequency of the transformation was found to be greatly dependent on the number of cells per dish irradiated. Irradiation of larger numbers resulted in much lower frequencies of transformation. The maximum transformation frequency observed in nine separate experiments was 4 percent of the surviving cells. At doses greater than 200 rads the transformation frequency per surviving cell remained constant. The present results permit us to conclude that alpha irradiation may, indeed, be able to exert a direct effect on the genome of the cell to produce malignancy without any external immunological or hormonal influences

  10. 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

  11. alpha-Lactalbumin species variation, HAMLET formation, and tumor cell death.

    Science.gov (United States)

    Pettersson, Jenny; Mossberg, Ann-Kristin; Svanborg, Catharina

    2006-06-23

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.

  12. FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

    Science.gov (United States)

    Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

    2009-12-01

    FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

  13. In vitro cytotoxicity of alpha conjugates for human pancreatic cancer cell lines

    International Nuclear Information System (INIS)

    Qu, C.; Li, Y.; Rizvi, M.A.; Allen, B.; Samra, J.; Smith, R.

    2003-01-01

    Targeted Alpha therapy (TAT) can inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The aim of this study is to demonstrate the cytotoxicity of different alpha conjugates in vitro to human metastatic pancreatic cancer cell lines (CAPAN-1, CFPAN-1 and PANC-1). We are labeling the C595 and J591 (non-specific controls) monoclonal antibodies (Mabs) with 213 Bi were performed according to the standard methods in our laboratory. 213 Bi-C595 is specifically cytotoxic to CAPAN-1, CFPAN-1 and PANC-1cell lines in a concentration-dependent fashion. While non-specific alpha conjugates only killed very small fractions of pancreatic cancer cells. These alpha conjugates might be useful agents for the treatment of micro-metastases in pancreatic cancer patients with over-expression of the targeted receptors

  14. 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.

  15. 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.

  16. The Alpha Stem Cell Clinic: a model for evaluating and delivering stem cell-based therapies.

    Science.gov (United States)

    Trounson, Alan; DeWitt, Natalie D; Feigal, Ellen G

    2012-01-01

    Cellular therapies require the careful preparation, expansion, characterization, and delivery of cells in a clinical environment. There are major challenges associated with the delivery of cell therapies and high costs that will limit the companies available to fully evaluate their merit in clinical trials, and will handicap their application at the present financial environment. Cells will be manufactured in good manufacturing practice or near-equivalent facilities with prerequisite safety practices in place, and cell delivery systems will be specialized and require well-trained medical and nursing staff, technicians or nurses trained to handle cells once delivered, patient counselors, as well as statisticians and database managers who will oversee the monitoring of patients in relatively long-term follow-up studies. The model proposed for Alpha Stem Cell Clinics will initially use the capacities and infrastructure that exist in the most advanced tertiary medical clinics for delivery of established bone marrow stem cell therapies. As the research evolves, they will incorporate improved procedures and cell preparations. This model enables commercialization of medical devices, reagents, and other products required for cell therapies. A carefully constructed cell therapy clinical infrastructure with the requisite scientific, technical, and medical expertise and operational efficiencies will have the capabilities to address three fundamental and critical functions: 1) fostering clinical trials; 2) evaluating and establishing safe and effective therapies, and 3) developing and maintaining the delivery of therapies approved by the Food and Drug Administration, or other regulatory agencies.

  17. Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

    Science.gov (United States)

    Durante, M.; Grossi, G. F.; Gialanella, G.; Pugliese, M.; Nappo, M.; Yang, T. C.

    1995-01-01

    We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS).

  18. Prediction of lung cells oncogenic transformation for induced radon progeny alpha particles using sugarscape cellular automata.

    Science.gov (United States)

    Baradaran, Samaneh; Maleknasr, Niaz; Setayeshi, Saeed; Akbari, Mohammad Esmaeil

    2014-01-01

    Alpha particle irradiation from radon progeny is one of the major natural sources of effective dose in the public population. Oncogenic transformation is a biological effectiveness of radon progeny alpha particle hits. The biological effects which has caused by exposure to radon, were the main result of a complex series of physical, chemical, biological and physiological interactions. The cellular and molecular mechanisms for radon-induced carcinogenesis have not been clear yet. Various biological models, including cultured cells and animals, have been found useful for studying the carcinogenesis effects of radon progeny alpha particles. In this paper, sugars cape cellular automata have been presented for computational study of complex biological effect of radon progeny alpha particles in lung bronchial airways. The model has included mechanism of DNA damage, which has been induced alpha particles hits, and then formation of transformation in the lung cells. Biomarkers were an objective measure or evaluation of normal or abnormal biological processes. In the model, the metabolism rate of infected cell has been induced alpha particles traversals, as a biomarker, has been followed to reach oncogenic transformation. The model results have successfully validated in comparison with "in vitro oncogenic transformation data" for C3H 10T1/2 cells. This model has provided an opportunity to study the cellular and molecular changes, at the various stages in radiation carcinogenesis, involving human cells. It has become well known that simulation could be used to investigate complex biomedical systems, in situations where traditional methodologies were difficult or too costly to employ.

  19. 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

  20. A new gene deletion in the alpha-like globin gene cluster as the molecular basis for the rare alpha-thalassemia-1(--/alpha alpha) in blacks: HbH disease in sickle cell trait.

    Science.gov (United States)

    Steinberg, M H; Coleman, M B; Adams, J G; Hartmann, R C; Saba, H; Anagnou, N P

    1986-02-01

    A novel deletion of at least 26 kilobase of DNA, including both alpha-globin genes, the psi alpha- and psi zeta-globin genes, but sparing the functional zeta-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two alpha-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional zeta-globin genes.

  1. The average number of alpha-particle hits to the cell nucleus required to eradicate a tumour cell population

    International Nuclear Information System (INIS)

    Roeske, John C; Stinchcomb, Thomas G

    2006-01-01

    Alpha-particle emitters are currently being considered for the treatment of micrometastatic disease. Based on in vitro studies, it has been speculated that only a few alpha-particle hits to the cell nucleus are considered lethal. However, such estimates do not consider the stochastic variations in the number of alpha-particle hits, energy deposited, or in the cell survival process itself. Using a tumour control probability (TCP) model for alpha-particle emitters, we derive an estimate of the average number of hits to the cell nucleus required to provide a high probability of eradicating a tumour cell population. In simulation studies, our results demonstrate that the average number of hits required to achieve a 90% TCP for 10 4 clonogenic cells ranges from 18 to 108. Those cells that have large cell nuclei, high radiosensitivities and alpha-particle emissions occurring primarily in the nuclei tended to require more hits. As the clinical implementation of alpha-particle emitters is considered, this type of analysis may be useful in interpreting clinical results and in designing treatment strategies to achieve a favourable therapeutic outcome. (note)

  2. 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

    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......, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials...

  3. 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.

  4. Characterization of estrogen receptors alpha and beta in uterine leiomyoma cells.

    Science.gov (United States)

    Valladares, Francisco; Frías, Ignacio; Báez, Delia; García, Candelaria; López, Francisco J; Fraser, James D; Rodríguez, Yurena; Reyes, Ricardo; Díaz-Flores, Lucio; Bello, Aixa R

    2006-12-01

    Cellular and subcellular localization of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in uterine leiomyomas. Retrospective study. University of La Laguna (ULL) and Canary University Hospital (HUC). Premenopausal and postmenopausal women with uterine leiomyomas. Hysterectomy and myomectomy. Estrogen receptor alpha was only present in smooth muscle cells with variation in the subcellular location in different leiomyomas. Estrogen receptor beta was widely distributed in smooth muscle, endothelial, and connective tissue cells with nuclear location in all cases studied; variations were only found in the muscle cells for this receptor. Estrogens operate in leiomyoma smooth muscle cells through different receptors, alpha and beta. However they only act through the ERbeta in endothelial and connective cells.

  5. 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

  6. Role of the synthase domain of Ags1p in cell wall alpha-glucan biosynthesis in fission yeast

    NARCIS (Netherlands)

    Vos, Alina; Dekker, Nick; Distel, Ben; Leunissen, Jack A. M.; Hochstenbach, Frans

    2007-01-01

    The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides beta-glucan and chitin, alpha-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall alpha-glucan is an essential

  7. HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

    Science.gov (United States)

    Aits, Sonja; Gustafsson, Lotta; Hallgren, Oskar; Brest, Patrick; Gustafsson, Mattias; Trulsson, Maria; Mossberg, Ann-Kristin; Simon, Hans-Uwe; Mograbi, Baharia; Svanborg, Catharina

    2009-03-01

    HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

  8. 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.

  9. Pharmacokinetics and radiation dosimetry of 99mTc-3PRGD2 in healthy individuals: A pilot study

    International Nuclear Information System (INIS)

    Cheng Guanghui; Gao Shi; JI Tiefeng; Ma Qingjie; Wang Qing; Jia Bing; Chen Zuowei

    2012-01-01

    99m Tc-3PRGD 2 is a new SPECT radiotracer for several tumor imaging with high uptake where integrin α v β 3 is highly expressed. This pilot study was to assess the safety, biodistribution and radiation dosimetry of 99m Tc-3PRGD 2 in healthy volunteers. The 10 healthy male volunteers were injected with 99m Tc-3PRGD 2 (786.7±55.8 MBq, 19.1-24.2 mCi). Baseline measurements of vital signs, laboratory safety tests and 12-lead electrocardiogram were recorded before and after injection. Blood and urine samples were collected and radiation counts were obtained at various time points. Whole-body scans and ROIs of identified organs were used for visual analysis and estimating the radiation dosimetry. No adverse reactions were found during the study. 99m Tc-3PRGD 2 exhibited a rapid clearance from the blood with less than 45% of the initial dosage at 10 min after injection and gradual increasing radioactivity in urine with (52.9±6)% of original dose at 1440 min. The whole-body imaging showed high radioactive accumulation in bladder. And the highest 99m Tc-3PRGD 2 uptake was found in the kidneys (3.50×10 -2 mSv/MBq). The 99m Tc-3PRGD 2 exhibited good pharmacokinetic properties and little radiation burden. This study showed that 99m Tc-3PRGD 2 would be a safe and attractive SPECT agent in clinic applications. (authors)

  10. 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....

  11. Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    Science.gov (United States)

    Svensson, Malin; Fast, Jonas; Mossberg, Ann-Kristin; Düringer, Caroline; Gustafsson, Lotta; Hallgren, Oskar; Brooks, Charles L; Berliner, Lawrence; Linse, Sara; Svanborg, Catharina

    2003-12-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.

  12. 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

  13. 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

  14. The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiaomin [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Yongzhang, E-mail: yluo@tsinghua.edu.cn [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China)

    2010-07-16

    Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.

  15. 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...

  16. 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

  17. 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

  18. 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.

  19. Alpha Particles Induce Autophagy in Multiple Myeloma Cells.

    Science.gov (United States)

    Gorin, Jean-Baptiste; Gouard, Sébastien; Ménager, Jérémie; Morgenstern, Alfred; Bruchertseifer, Frank; Faivre-Chauvet, Alain; Guilloux, Yannick; Chérel, Michel; Davodeau, François; Gaschet, Joëlle

    2015-01-01

    Radiation emitted by the radionuclides in radioimmunotherapy (RIT) approaches induce direct killing of the targeted cells as well as indirect killing through the bystander effect. Our research group is dedicated to the development of α-RIT, i.e., RIT using α-particles especially for the treatment of multiple myeloma (MM). γ-irradiation and β-irradiation have been shown to trigger apoptosis in tumor cells. Cell death mode induced by (213)Bi α-irradiation appears more controversial. We therefore decided to investigate the effects of (213)Bi on MM cell radiobiology, notably cell death mechanisms as well as tumor cell immunogenicity after irradiation. Murine 5T33 and human LP-1 MM cell lines were used to study the effects of such α-particles. We first examined the effects of (213)Bi on proliferation rate, double-strand DNA breaks, cell cycle, and cell death. Then, we investigated autophagy after (213)Bi irradiation. Finally, a coculture of dendritic cells (DCs) with irradiated tumor cells or their culture media was performed to test whether it would induce DC activation. We showed that (213)Bi induces DNA double-strand breaks, cell cycle arrest, and autophagy in both cell lines, but we detected only slight levels of early apoptosis within the 120 h following irradiation in 5T33 and LP-1. Inhibition of autophagy prevented (213)Bi-induced inhibition of proliferation in LP-1 suggesting that this mechanism is involved in cell death after irradiation. We then assessed the immunogenicity of irradiated cells and found that irradiated LP-1 can activate DC through the secretion of soluble factor(s); however, no increase in membrane or extracellular expression of danger-associated molecular patterns was observed after irradiation. This study demonstrates that (213)Bi induces mainly necrosis in MM cells, low levels of apoptosis, and autophagy that might be involved in tumor cell death.

  20. Alpha-particles induce autophagy in multiple myeloma cells

    Directory of Open Access Journals (Sweden)

    Joelle Marcelle Gaschet

    2015-10-01

    Full Text Available Objectives: Radiations emitted by the radionuclides in radioimmunotherapy (RIT approaches induce direct killing of the targeted cells as well as indirect killing through bystander effect. Our research group is dedicated to the development of α-RIT, i.e RIT using α-particles especially for the treatment of multiple myeloma (MM. γ-irradiation and β-irradiation have been shown to trigger apoptosis in tumor cells. Cell death mode induced by 213Bi α-irradiation appears more controversial. We therefore decided to investigate the effects of 213Bi on MM cell radiobiology, notably cell death mechanisms as well as tumor cell immunogenicity after irradiation.Methods: Murine 5T33 and human LP-1 multiple myeloma (MM cell lines were used to study the effects of such α-particles. We first examined the effects of 213Bi on proliferation rate, double strand DNA breaks, cell cycle and cell death. Then, we investigated autophagy after 213Bi irradiation. Finally, a co-culture of dendritic cells (DC with irradiated tumour cells or their culture media was performed to test whether it would induce DC activation.Results: We showed that 213Bi induces DNA double strand breaks, cell cycle arrest and autophagy in both cell lines but we detected only slight levels of early apoptosis within the 120 hours following irradiation in 5T33 and LP-1. Inhibition of autophagy prevented 213Bi induced inhibition of proliferation in LP-1 suggesting that this mechanism is involved in cell death after irradiation. We then assessed the immunogenicity of irradiated cells and found that irradiated LP-1 can activate DC through the secretion of soluble factor(s, however no increase in membrane or extracellular expression of danger associated molecular patterns (DAMPs was observed after irradiation.Conclusion: This study demonstrates that 213Bi induces mainly necrosis in MM cells, low levels of apoptosis and also autophagy that might be involved in tumor cell death.

  1. 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......-labelled riboprobes were used. The prominent myeloid cell infiltrate in diseased tissues comprised F4/80+, Mac-l+ macrophages, neutrophils, dendritic cells and activated macrophages. TNF-alpha transcription and translation were associated with activated macrophages in the lamina propria. Activated macrophages...

  2. 99mTc-3PRGD2 Scintimammography in Palpable and Nonpalpable Breast Lesions

    Directory of Open Access Journals (Sweden)

    Lin Liu

    2014-07-01

    Full Text Available The aim of this study was to explore the diagnostic performance of 99mTc-3(poly-(ethylene glycol,PEG4-RGD2 (99mTc-3PRGD2 scintimammography (SMM in patients with either palpable or nonpalpable breast lesions and compare SMM to mammography to assess the possible incremental value of SMM in breast cancer detection. We also investigated the αvβ3 expression in malignant and benign breast lesions. Ninety-four patients with 110 lesions were included in this study. Mammograms were evaluated according to the Breast Imaging Reporting and Data System (BI-RADS by a specialized imaging radiologist. Prone SMM was performed 1 hour after injection of 99mTc-3PRGD2. Scintigraphic images were interpreted independently by two experienced nuclear medicine physicians using a three-point system, and the kappa value was calculated to determine the interreader agreement. The McNemar test was used to compare SMM and mammography with respect to sensitivity, specificity, and accuracy. Diagnostic values for breast cancer detection were evaluated for each lesion. Immunohistochemistry was performed to evaluate integrin αvβ3 expression. Histopathology revealed 46 malignant lesions and 64 benign lesions. The overall sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of SMM were 83%, 73%, 77%, 69%, and 85%, respectively. The kappa value between the two reviewers was 0.63. The diagnostic values of SMM were higher than those of mammography in evaluating overall breast lesions. A sensitivity of 91% was achieved when SMM and mammography results were combined with 60% of all false-negative mammography findings classified as true-positive results by SMM. Integrin αvβ3 expression was positively identified using SMM imaging. SMM is a promising tool to avoid unnecessary biopsies when used in addition to mammography and can be used to image αvβ3 expression in breast cancer with good image quality.

  3. Paracrine GABA and insulin regulate pancreatic alpha cell proliferation in a mouse model of type 1 diabetes.

    Science.gov (United States)

    Feng, Allen L; Xiang, Yun-Yan; Gui, Le; Kaltsidis, Gesthika; Feng, Qingping; Lu, Wei-Yang

    2017-06-01

    This study aimed to elucidate the mechanism of increased proliferation of alpha cells in recent-onset type 1 diabetes. Pancreatic beta cells express GAD and produce γ-aminobutyric acid (GABA), which inhibits alpha cell secretion of glucagon. We explored the roles of GABA in alpha cell proliferation in conditions corresponding to type 1 diabetes in a mouse model and in vitro. Type 1 diabetes was induced by injecting the mice with streptozotocin (STZ). Some of the STZ-injected mice were treated with GABA (10 mg/kg daily) for 12 days. Isolated pancreatic islets were treated with STZ or STZ together with GABA for 2 days. The effects of GABA treatment on STZ-induced alpha cell proliferation in vivo and in vitro were assessed. The effect of muscimol, a GABA receptor agonist, on αTC1-6 cell proliferation was also examined. STZ injection substantially decreased levels of GAD, GABA and insulin in pancreatic beta cells 12 h after injection; this was followed by an upsurge of phosphorylated mechanistic target of rapamycin (p-mTOR) in the alpha cells at day 1, and a significant increase in alpha cell mass at day 3. Treating STZ-injected mice with GABA largely restored the immunodetectable levels of insulin and GAD in the beta cells and significantly decreased the number of aldehyde dehydrogenase 1 family, member A3 (ALDH1a3)-positive cells, alpha cell mass and hyperglucagonaemia. STZ treatment also increased alpha cell proliferation in isolated islets, which was reversed by co-treatment with GABA. Muscimol, together with insulin, significantly lowered the level of cytosolic Ca 2+ and p-mTOR, and decreased the proliferation rate of αTC1-6 cells. GABA signalling critically controls the alpha cell population in pancreatic islets. Low intraislet GABA may contribute to alpha cell hyperplasia in early type 1 diabetes.

  4. Chemical effect on Tc 3d3/2 core hole lifetime

    International Nuclear Information System (INIS)

    Koever, L.; Toth, J.; Cserny, I.

    1986-01-01

    The Tc 3d lines are often used for identification of various forms of technetium. The accuracy of the chemical analysis of Tc compounds is influenced by the Coster-Kronig broadening and by its dependence on the chemical state of Tc. The experiment reported used X-ray photoelectron spectroscopy to investigate the 3d core line widths obtained from spectra of Tc metal and of technetium-dioxide samples. A deconvolution algorithm was used for data evaluation. The results were compared with other experimental data. (D.Gy.)

  5. The insulinotropic effect of endothelin-1 is mediated by glucagon release from the islet alpha cells

    DEFF Research Database (Denmark)

    Brock, B; Gregersen, S; Kristensen, K

    1999-01-01

    AIMS/HYPOTHESIS: The circulating concentrations of endothelin-1 (ET-1), a peptide derived from endothelium, are increased in hypertension and diabetes. Endothelin-1 has recently been shown to be an insulinotropic agent. The mechanism of action of endothelin-1 on the endocrine pancreas has not yet...... been clarified. METHODS: We investigated the action of endothelin-1 on the insulin secretion, the binding of (125)I-ET-1 to beta cells as well as its effects on purified beta and non-beta cells from normal rats. The expression of endothelin receptors in alpha- and beta-cell lines and in normal rat...... from purified beta cells. Endothelin-1-(100 nmol/l) increased, however, both insulin and glucagon secretion from a mixture of purified beta and non-beta cells indicating that alpha cells seem to have a key role for the action of ET-1 on insulin secretion. CONCLUSION/INTERPRETATION: The insulinotropic...

  6. 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.

  7. Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Song, Kai; Zhu, Fei; Zhang, Han-zhong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); First Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan (China)

    2012-08-15

    Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black

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

    Science.gov (United States)

    Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

    2009-08-07

    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 mass and curing diabetes in animals that have been chemically depleted of beta cells.

  9. Omentin inhibits TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via ERK/NF-{kappa}B pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Xia, E-mail: zhongxia1977@126.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Li, Xiaonan; Liu, Fuli; Tan, Hui [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Shang, Deya, E-mail: wenhuashenghuo1@163.com [Department of Emergency, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Black-Right-Pointing-Pointer Omentin reduces expression of ICAM-1 and VCAM-1 induced by TNF-{alpha} in HUVECs. Black-Right-Pointing-Pointer Omentin inhibits TNF-{alpha}-induced ERK and NF-{kappa}B activation in HUVECs. Black-Right-Pointing-Pointer Omentin supreeses TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 via ERK/NF-{kappa}B pathway. -- Abstract: In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-{alpha} (TNF-{alpha}) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-{alpha}-activated signal pathway of nuclear factor-{kappa}B (NF-{kappa}B) by preventing NF-{kappa}B inhibitory protein (I{kappa}B{alpha}) degradation and NF-{kappa}B/DNA binding activity. Omentin pretreatment significantly inhibited TNF-{alpha}-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-{alpha}-induced NF-{kappa}B activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-{alpha}. These results suggest that omentin may inhibit TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-{kappa}B pathway.

  10. 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...... 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...

  11. 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...

  12. Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

    Science.gov (United States)

    Ribeiro, Maisa; Teixeira, Sarah R; Azevedo, Monarko N; Fraga, Ailton C; Gontijo, Antônio Pm; Vêncio, Eneida F

    2017-04-01

    To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

  13. Tumor necrosis factor alpha production in irradiated cells in vitro

    International Nuclear Information System (INIS)

    Koeteles, G.J.; Bognar, G.; Kubasova, T.

    1994-01-01

    Normal and tumor cell lines were used to investigate tumor necrosis factor (TNFα) production and its radiation sensitivity. The cells were irradiated with gamma rays using different doses from 0.25 Gy up to 5 Gy. The number of plated cells, changes of proliferation and TNFα production were determined during the following four post-irradiation days. For TNFα quantity measurement immuno-radiometric assay (IRMA) and enzyme amplified sensitivity assay (EASIA) was used. The results suggest that though gamma irradiation decreased cell proliferation in a dose dependent manner, the quantity produced in the post-irradiation period increased considerably in each irradiated sample. (N.T.) 3 refs.; 2 figs.; 1 tab

  14. 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.

  15. 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...

  16. Alpha-defensins 1-3 release by dendritic cells is reduced by estrogen

    Directory of Open Access Journals (Sweden)

    Sperling Rhoda

    2011-08-01

    Full Text Available Abstract Background During pregnancy the immune system of the mother must protect any activation that may negatively affect the fetus. Changes in susceptibility to infection as well as resolution of some autoimmune disorders represent empirical evidence for pregnancy related alterations in immunity. Sex hormones reach extremely high levels during pregnancy and have been shown to have direct effects on many immune functions including the antiviral response of dendritic cells. Among the immunologically active proteins secreted by monocyte derived DCs (MDDC are the alpha-defensins 1-3. This family of cationic antimicrobial peptides has a broad spectrum of microbicidal activity and has also been shown to link innate to adaptive immunity by attracting T cells and immature DCs, which are essential for initiating and polarizing the immune response. Methods We compare culture-generated monocyte derived DCs (MDDCs with directly isolated myeloid dendritic cells (mDCs and plasmacytoid dendritic cells (pDCs and measure their alpha-defensins 1-3 secretion by ELISA both, in basal situations and after hormone (E2 or PG treatments. Moreover, using a cohort of pregnant women we isolated mDCs from blood and also measure the levels of these anti-microbial peptides along pregnancy. Results We show that mDCs and pDCs constitutively produce alpha-defensins 1-3 and at much higher levels than MDDCs. Alpha-defensins 1-3 production from mDCs and MDDCs but not pDCs is inhibited by E2. PG does not affect alpha-defensins 1-3 in any of the populations. Moreover, alpha-defensins 1-3 production by mDCs was reduced in the later stages of pregnancy in 40% of the patients. Conclusions Here, we demonstrate that mDCs and pDCs secrete alpha-defensins 1-3 and present a novel effect of E2 on the secretion of alpha-defensins 1-3 by dendritic cells.

  17. Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

    Science.gov (United States)

    Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

    1999-03-15

    We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

  18. TNF alpha induces ABCA1 through NF-kappa B in macrophages and in phagocytes ingesting apoptotic cells

    NARCIS (Netherlands)

    Gerbod-Giannone, Marie-Christine; Li, Yankun; Holleboom, Adriaan; Han, Seongah; Hsu, Li-Chung; Tabas, Ira; Tall, Alan R.

    2006-01-01

    Recent evidence suggests that tumor necrosis factor alpha (TNF alpha) signaling in vascular cells can have antiatherogenic consequences, but the mechanisms are poorly understood. TNFa is released by free cholesterol loaded apoptotic macrophages, and the clearance of these cells by phagocytic

  19. 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

  20. Alpha chain determinants on the membrane of immunoglobulin synthesizing cells

    NARCIS (Netherlands)

    Hijmans, W.; Schuit, H.R.E.; Radl, J.; Vossen, J.M.J.J.

    1974-01-01

    In a study of surface immunoglobulins (Ig) on lymphocytes from patients with paraproteinemia (1), we observed that a variable number of plasma cells not only contained intracellular Ig, but also had Ig on their surface, as shown in the vital technique of immunofluorescence. Moreover, in the bone

  1. Antibodies against alpha-synuclein reduce oligomerization in living cells.

    Directory of Open Access Journals (Sweden)

    Thomas Näsström

    Full Text Available Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.

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

    Science.gov (United States)

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

    2002-05-01

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

  3. The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells.

    Science.gov (United States)

    Bolduc, Gilles R; Madoff, Lawrence C

    2007-12-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.

  4. 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)

  5. 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.

  6. Radiobiological Effects of Alpha-Particles from Astatine-211: From DNA Damage to Cell Death

    Energy Technology Data Exchange (ETDEWEB)

    Claesson, Kristina

    2011-05-15

    In recent years, the use of high linear energy transfer (LET) radiation for radiotherapeutic applications has gained increased interest. Astatine-211 (211At) is an alpha-particle emitting radionuclide, promising for targeted radioimmunotherapy of isolated tumor cells and microscopic clusters. To improve development of safe radiotherapy using 211At it is important to increase our knowledge of the radiobiological effects in cells. During radiotherapy, both tumors and adjacent normal tissue will be irradiated and therefore, it is of importance to understand differences in the radio response between proliferating and resting cells. The aim of this thesis was to investigate effects in fibroblasts with different proliferation status after irradiation with alpha-particles from 211At or X-rays, from inflicted DNA damage, to cellular responses and biological consequences. Throughout this work, irradiation was performed with alpha-particles from 211A or X-rays. The induction and repair of double-strand breaks (DSBs) in human normal fibroblasts were investigated using pulsed-field gel electrophoresis and fragment analysis. The relative biological effectiveness (RBE) of 211At for DSB induction varied between 1.4 and 3.1. A small increase of DSBs was observed in cycling cells compared to stationary cells. The repair kinetics was slower after 211At and more residual damage was found after 24 h. Comparison between cells with different proliferation status showed that the repair was inefficient in cycling cells with more residual damage, regardless of radiation quality. Activation of cell cycle arrests was investigated using immunofluorescent labeling of the checkpoint kinase Chk2 and by measuring cell cycle distributions with flow cytometry analysis. After alpha-particle irradiation, the average number of Chk2-foci was larger and the cells had a more affected cell cycle progression for several weeks compared with X-irradiated cells, indicating a more powerful arrest after 211At

  7. Molecular pathways in the bystander response of cells exposed to very low fluences of alpha particles

    International Nuclear Information System (INIS)

    Little, J.B.

    2000-01-01

    Full text: We have examined biological effects in cell populations exposed to very low mean doses of alpha radiation by which only a small fraction of the cells are actually traversed by an alpha particle. We showed earlier that an enhanced frequency of sister chromatid exchanges and HPRT mutations occur in the non-irradiated, 'bystander' cells. The frequency of mutations induced by a single alpha particle traversing the nucleus of a cell was increased nearly fivefold at the lowest fluence studied, a result of mutations occurring in bystander cells. This was associated with a similar increase in the induction of micronuclei, indicating the induction of DNA damage in bystander cells. In order to gain information concerning molecular pathways, we studied changes in gene expression in bystander cells in confluent cultures of human diploid fibroblasts or mouse embryo-derived fibroblasts (MEFs) by western analysis and in-situ immunofluorescence. The expression levels of p53, p21 Waf1 and p34 cdc2 were significantly modulated in bystander cells. The upregulation of p53 and p21 Waf1 did not occur in cultures irradiated at low density, and was markedly reduced in the presence of the gap junction inhibitor lindane. The importance of gap-junction mediated intercellular communication was confirmed in connexin-43 knockout MEFs. Western blot analyses and electrophoretic mobility shift assays indicate that the bystander response is suppressed by incubation with superoxide dismutase as well as an inhibitor of NADPH oxidase, and is associated with the induction of NFKB, suggesting the effect is mediated by oxidative stress. The stress-activated protein kinase p38 and its downstream effector ATF2 are also induced in bystander cells independent of oxidative stress. These results will be discussed in terms of whether activation of the p53 damage response pathway is the direct result of signaling from irradiated cells, or rather is a consequence of DNA induced damage in the bystander

  8. Traversal of cells by radiation and absorbed fraction estimates for electrons and alpha particles

    International Nuclear Information System (INIS)

    Eckerman, K.F.; Ryman, J.C.; Taner, A.C.; Kerr, G.D.

    1986-01-01

    Consideration of the pathlength which radiation traverses in a cell is central to algorithms for estimating energy deposition on a cellular level. Distinct pathlength distributions occur for radionuclides: (1) uniformly distributed in space about the cell (referred to as μ-randomness); (2) uniformly distributed on the surface of the cell (S-randomness); and (3) uniformly distributed within the cell volume (I-randomness). For a spherical cell of diameter d, the mean pathlengths are 2/3d, and 3/4d, respectively, for these distributions. Algorithms for simulating the path of radiation through a cell are presented and the absorbed fraction in the cell and its nucleus are tabulated for low energy electrons and alpha particles emitted on the surface of spherical cells. The algorithms and absorbed fraction data should be of interest to those concerned with the dosimetry of radionuclide-labeled monoclonal antibodies. 8 references, 3 figures, 2 tables

  9. Traversal of cells by radiation and absorbed fraction estimates for electrons and alpha particles

    International Nuclear Information System (INIS)

    Eckerman, K.F.; Ryman, J.C.; Taner, A.C.; Kerr, G.D.

    1985-01-01

    Consideration of the pathlength which radiation traverses in a cell is central to algorithms for estimating energy deposition on a cellular level. Distinct pathlength distributions occur for radionuclides: (1) uniformly distributed in space about the cell (referred to as μ-randomness); (2) uniformly distributed on the surface of the cell (S-randomness); and (3) uniformly distributed within the cell volume (I-randomness). For a spherical cell of diameter d, the mean pathlengths are 2/3d, 1/2d, and 3/4d, respectively, for these distributions. Algorithms for simulating the path of radiation through a cell are presented and the absorbed fraction in the cell and its nucleus are tabulated for low energy electrons and alpha particles emitted on the surface of spherical cells. The algorithms and absorbed fraction data should be of interest to those concerned with the dosimetry of radionuclide-labeled monoclonal antibodies. 8 refs., 3 figs., 2 tabs

  10. 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

  11. 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.

  12. alpha(4)beta(7) independent pathway for CD8(+) T cell-mediated intestinal immunity to rotavirus.

    Science.gov (United States)

    Kuklin, N A; Rott, L; Darling, J; Campbell, J J; Franco, M; Feng, N; Müller, W; Wagner, N; Altman, J; Butcher, E C; Greenberg, H B

    2000-12-01

    Rotavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin alpha(4)ss(7) is not essential for CD8(+) T cells to migrate to the intestine or provide immunity to RV. Mice deficient in ss7 expression (ss7(-/-)) and unable to express alpha(4)ss(7) integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8(+) T cells in ss7(-/-) animals prolonged viral shedding, and transfer of immune ss7(-/-) CD8(+) T cells into chronically infected Rag-2-deficient mice resolved RV infection as efficiently as wt CD8(+) T cells. Paradoxically, alpha(4)ss(7)(hi) memory CD8(+) T cells purified from wt mice that had been orally immunized cleared RV more efficiently than alpha(4)ss(7)(low) CD8(+) T cells. We explained this apparent contradiction by demonstrating that expression of alpha(4)ss(7) on effector CD8(+) T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8(+) T cells primarily of an alpha(4)ss(7)(hi) phenotype, but subcutaneous immunization yields both alpha(4)ss(7)(hi) and alpha(4)ss(7)(low) immune CD8(+) T cells with anti-RV effector capabilities. Thus, alpha(4)ss(7) facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8(+) T cell immunity.

  13. Monte Carlo modelling of damage to haemopoietic stem cells from internally deposited alpha-emitters

    International Nuclear Information System (INIS)

    Utteridge, T.D.; University of South Australia, Pooraka, SA; Charlton, D.E.; Turner, M.S.; Beddoe, A.H.; Leong, A. S-Y.; Milios, J.; Fazzalari, N.; To, L.B.

    1996-01-01

    Full text: Monte Carlo modelling of alpha particle radiation dose to haemopoietic stem cells from radon decay in human marrow fat cells was undertaken following Richardson et al's (Brit J Radiol, 64, 608-624, 1991) proposition that such exposure could induce leukaemia, and epidemiological observations that uranium miners have not developed an excess of leukaemia (Tomasek L. et al, Lancet, 341, 919-923, 1993). The dose to haemopoietic stem cells from alpha emitting radiopharmaceuticals proposed for radiotherapy is also important in risk assessment. Haemopoietic stem cells (presumed to be the targets for leukaemia) were identified as CD34+CD38- mononuclear cells (Terstappen LWMM et al, Blood, 77, 1218-1227, 1991) and their diameters measured using image analysis. The distribution of stem cell distances from fat cells was also measured. The model was used with Monte Carlo treatment of the alpha particle flux from radon and its short lived decay products to estimate (a) the dose and LET distributions for the three stem cell diameters; (b) the number of passages per hit; and (c) stem cell survival. The stem cell population exhibited a trimodal distribution, with mean diameters of 5.7, 11.6 and 14.8 μm; a trimodal distribution has previously been identified in mice (Visser J et al, Exper Hematol Today, 21-27, 1977). At 40% fat in a human lumbar vertebra 3 section, approximately half the stem cells were located on, or very close to the edge, of fat cells in marrow sections. This agrees with the predicted distribution of distances between fat and stem cells obtained using a 3-D model with randomly distributed stem cells. At an air activity of 20 Bq m -3 (ie the UK average indoor radon concentration used by Richardson et al mentioned above) about 0.1 stem cells per person-year were hit and survived; at 100 Bq m -3 about 1 stem cell per person-year was hit and survived. Across the range of radon concentrations encountered in residential and underground miner exposures

  14. 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.

  15. Variability and repertoire size of T-cell receptor V alpha gene segments.

    Science.gov (United States)

    Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

    The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

  16. Coronin-1A links cytoskeleton dynamics to TCR alpha beta-induced cell signaling.

    Directory of Open Access Journals (Sweden)

    Bénédicte Mugnier

    Full Text Available Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR-induced immunological synapse (IS formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of alpha beta T cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-kappaB (I kappa B. Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts alpha beta T cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.

  17. Controlled alpha-sexithiophene nanostructure formation in standard and inverted configuration organic solar cells

    DEFF Research Database (Denmark)

    Radziwon, Michal Jędrzej; Goszczak, Arkadiusz Jaroslaw; Fernandes Cauduro, André Luis

    -type domains in the active organic layer. The molecular packing in these is of same importance, as it strongly affects the carrier transport in the cells. In this work, we present the study of alpha-sexithiophene (α 6T) temperature dependent growth for standard, on gold anodes, and inverted, on electron...... accepting C60 layers, solar cell configurations. Furthermore, a comparative study of the correlation between the α-6T morphology and device performance parameters for standard and inverted solar cell configurations is presented. The morphology of the α 6T layer is controlled by means of the substrate...

  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

    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...... antagonists are confronted with several safety issues. At present, available pharmacological agents based on the glucose-dependent glucagonostatic effects of GLP-1 represent the most favorable way to apply constraints to the alpha-cell in type 2 diabetes.......-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...

  19. Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    Science.gov (United States)

    Hallgren, Oskar; Aits, Sonja; Brest, Patrick; Gustafsson, Lotta; Mossberg, Ann-Kristin; Wullt, Björn; Svanborg, Catharina

    2008-01-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer. The mechanisms of tumor cell death remain unclear, however. Immediately after the encounter with tumor cells, HAMLET invades the cells and causes mitochondrial membrane depolarization, cytochrome c release, phosphatidyl serine exposure, and a low caspase response. A fraction of the cells undergoes morphological changes characteristic of apoptosis, but caspase inhibition does not rescue the cells and Bcl-2 overexpression or altered p53 status does not influence the sensitivity of tumor cells to HAMLET. HAMLET also creates a state of unfolded protein overload and activates 20S proteasomes, which contributes to cell death. In parallel, HAMLET translocates to tumor cell nuclei, where high-affinity interactions with histones cause chromatin disruption, loss of transcription, and nuclear condensation. The dying cells also show morphological changes compatible with macroautophagy, and recent studies indicate that macroautophagy is involved in the cell death response to HAMLET. The results suggest that HAMLET, like a hydra with many heads, may interact with several crucial cellular organelles, thereby activating several forms of cell death, in parallel. This complexity might underlie the rapid death response of tumor cells and the broad antitumor activity of HAMLET.

  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. 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

    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...... compartments with CD4+ T cells from normal GALT plays an essential role in the pathogenesis of IBD in an immunodeficient host.......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...

  2. Cell motility in chronic lymphocytic leukemia: defective Rap1 and alphaLbeta2 activation by chemokine.

    Science.gov (United States)

    Till, Kathleen J; Harris, Robert J; Linford, Andrea; Spiller, David G; Zuzel, Mirko; Cawley, John C

    2008-10-15

    Chemokine-induced activation of alpha4beta1 and alphaLbeta2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of alphaLbeta2; engagement of alpha4beta1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in alphaLbeta2 activation and determine how it is corrected. We show here that the alphaLbeta2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both alpha4beta1 and alphaLbeta2 is defective, autocrine VEGF and chemokine are necessary to activate alpha4beta1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for alphaLbeta2 activation for motility and TEM. The present study not only clarifies the nature of the alphaLbeta2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL.

  3. Purification and characterization of a bioactive alpha-fetoprotein produced by HEK-293 cells.

    Science.gov (United States)

    Lin, Bo; Peng, Guoqing; Feng, Haipeng; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Wang, Qiaoyun; Xie, Xieju; Zhu, Mingyue; Li, Mengsen

    2017-08-01

    Alpha-fetoprotein (AFP) is a biomarker that is used to diagnose hepatocellular carcinoma (HCC) and can promote malignancy in HCC. AFP is an important target in the treatment of liver cancer. To obtain enough AFP to screen for AFP inhibitors, we expressed and purified AFP in HEK-293 cells. In the present study, we produced AFP in the cells and harvested highly pure rAFP (or recombinant expression AFP in HEK-293 cells). We also analysed the bioactivity of rAFP and found that rAFP promoted growth of the human HCC cells, antagonize paclitaxel inhibition of HCC cell proliferation, suppress expression of active caspase-3, and promote expression of Ras and survivin. This study provides a method to produce significant amounts of AFP for use in biochemical assays and functional studies and to screen AFP inhibitors for use in HCC therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Irradiation of bone lining cells from bone-seeking alpha-emitters

    International Nuclear Information System (INIS)

    Kruglikov, I.; Polig, E.

    1993-01-01

    The influence of bone remodeling and the non-uniform distribution of alpha-emitters on the hit statistics is discussed. It is shown that for the first generation of bone lining cells, bone remodeling decreases the probability of no hits to the nuclei of these cells whereas the randomness of the spatial distribution of nuclide increases this probability. For the subsequent generations bone remodeling as well as spatial distribution of nuclide increase the probability of no hits. The most conservative estimations for the variance of hits and probability of no hits, which are defined by the minimums of these values, are obtained. (orig.)

  5. alpha-Tocopheryl succinate promotes selective cell death induced by vitamin K3 in combination with ascorbate.

    Science.gov (United States)

    Tomasetti, M; Strafella, E; Staffolani, S; Santarelli, L; Neuzil, J; Guerrieri, R

    2010-04-13

    A strategy to reduce the secondary effects of anti-cancer agents is to potentiate the therapeutic effect by their combination. A combination of vitamin K3 (VK3) and ascorbic acid (AA) exhibited an anti-cancer synergistic effect, associated with extracellular production of H(2)O(2) that promoted cell death. The redox-silent vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) was used in combination with VK3 and AA to evaluate their effect on prostate cancer cells. Prostate cancer cells were sensitive to alpha-TOS and VK3 treatment, but resistant to AA upto 3.2 mM. When combined, a synergistic effect was found for VK3-AA, whereas alpha-TOS-VK3 and alpha-TOS-AA combination showed an antagonist and additive effect, respectively. However, sub-lethal doses of AA-VK3 combination combined with a sub-toxic dose of alpha-TOS showed to induce efficient cell death that resembles autoschizis. Associated with this cell demise, lipid peroxidation, DNA damage, cytoskeleton alteration, lysosomal-mitochondrial perturbation, and release of cytochrome c without caspase activation were observed. Inhibition of lysosomal proteases did not attenuate cell death induced by the combined agents. Furthermore, cell deaths by apoptosis and autoschizis were detected. These finding support the emerging idea that synergistic combinations of some agents can overcome toxicity and other side-effects associated with high doses of single drugs creating the opportunity for therapeutically relevant selectivity.

  6. Spatial Characterization of Polycyclic Aromatic Hydrocarbons in 2008 TC3 Samples

    Science.gov (United States)

    Sabbah, Hassan; Morrow, A.; Zare, R. N.; Jenniskens, P.

    2009-09-01

    Hassan Sabbah1, Amy L. Morrow1, Richard N. Zare1 and Petrus Jenniskens2 1Stanford University, Stanford, California 94305, 2 SETI Institute, Carl Sagan Center, 515 North Whisman Road, Mountain View, California 94043, USA. In October 2006 a small asteroid (2-3 meters) was observed in outer space. On October 7, 2008, it entered the Earth's atmosphere creating a fireball over Northern Sudan. Some 280 meteorites were collected by the University of Khartoum. In order to explore the existence of organic materials, specifically polycyclic aromatic hydrocarbons (PAHs), we applied two-step laser desorption laser ionization mass spectrometry (L2MS) to some selected fragments. This technique consists of desorbing with a pulsed infrared laser beam the solid materials into a gaseous phase with no fragmentation followed by resonance enhanced multiphoton ionization to analyze the PAH content. L2MS was already applied to an array of extraterrestrial objects including interplanetary dust particles IDPs, carbonaceous chondrites and comet coma particles. Moreover, spatial resolution of PAHs in 2008 TC3 samples was achieved to explore the heterogeneity within individual fragments. The results of these studies and their contribution to understanding the formation of this asteroid will be discussed.

  7. LONG-LIVED BONE MARROW PLASMA CELLS DURING IMMUNE RESPONSE TO ALPHA (1→3 DEXTRAN

    Directory of Open Access Journals (Sweden)

    I. N. Chernyshova

    2015-01-01

    Full Text Available Production kinetics and some functional properties of long-lived marrow plasma cells were studied in mice immunized with T-independent type 2 antigens. Alpha (1→3 dextran was used as an antigen for immunization. The mice were immunized by dextran, and the numbers of IgM antibody producing cells were determined by ELISPOT method. The cell phenotype was determined by cytofluorimetric technique. In the area of normal bone marrow lymphocytes ~4% of T and ~85% of B cells were detected. About 35% of the cells expressed a plasmocyte marker (CD138; 3% were CD138+IgM+, and about 6% of the lymphocytes were double-positive for CD138+IgA+. Among spleen lymphocytes, 50% of T and 47% of B cells were detected. About 1.5% lymphocytes were CD138+, and 0.5% were positive for CD138 and IgM. Time kinetics of antibody-producing cells in bone marrow and spleen was different. In spleen populations, the peak amounts of antibody-secreting cells have been shown on the day 4; the process abated by the day 28. Vice versa, the numbers of the antibody-producing cells in bone marrow started to increase on the day 4. The process reached its maximum on day 14, and after 28th day became stationary. The in vitro experiments have shown that supplementation of bone marrow cells from immune mice with dextran did not influence their functional activity. It was previously shown for cells responding to T-dependent antigens only. A specific marker for the long-lived plasma cells is still unknown. However, these cells possess a common CD138 marker specific for all plasma cells. A method for isolation of bone marrow CD138+ cells was developed. The CD138+ cells were of 87-97% purity, being enriched in long-lived bone marrow cells, and produced monospecific antibodies.

  8. Dexamethasone protection from TNF-alpha-induced cell death in MCF-7 cells requires NF-kappaB and is independent from AKT

    Directory of Open Access Journals (Sweden)

    Mejía Salvador

    2006-02-01

    Full Text Available Abstract Background The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death. Results Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected. Conclusion NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1.

  9. Dexamethasone protection from TNF-alpha-induced cell death in MCF-7 cells requires NF-kappaB and is independent from AKT.

    Science.gov (United States)

    Machuca, Catalina; Mendoza-Milla, Criselda; Córdova, Emilio; Mejía, Salvador; Covarrubias, Luis; Ventura, José; Zentella, Alejandro

    2006-02-21

    The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death. Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected. NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1.

  10. Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

    Science.gov (United States)

    van der Kaaij, R M; Yuan, X-L; Franken, A; Ram, A F J; Punt, P J; van der Maarel, M J E C; Dijkhuizen, L

    2007-07-01

    In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.

  11. 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...

  12. Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

    Science.gov (United States)

    Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

    2008-10-01

    Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

  13. Silencing alpha-fetoprotein inhibits VEGF and MMP-2/9 production in human hepatocellular carcinoma cell.

    Science.gov (United States)

    Meng, Wenbo; Li, Xun; Bai, Zhongtian; Li, Yan; Yuan, Jinqiu; Liu, Tao; Yan, Jun; Zhou, Wence; Zhu, Kexiang; Zhang, Hui; Li, Yumin

    2014-01-01

    Alpha-fetoprotein not only serves as a diagnostic marker for liver cancer, but also posses a variety of biological functions. However, the role of Alpha-fetoprotein on tumor angiogenesis and cell invasion remains incompletely understood. In this study, we aimed to evaluate if Alpha-fetoprotein can regulate the major angiogenic factors and matrix metalloproteinases in human liver cancer cells. Alpha-fetoprotein silencing was achieved by Stealth RNAi. Expression of Alpha-fetoprotein was examined by a full-automatic electrochemistry luminescence immunity analyzer. Expression of VEGF, VEGFR-2, MMP-9, and MMP-2 was examined by Western blot and immunocytochemistry. Apoptosis was detected by TUNEL assay. Angiogenesis was detected by in vitro angiogenesis assay kit. Silencing of Alpha-fetoprotein led to an increased apoptosis, which was associated with a decreased expression of vascular endothelial growth factor, vascular endothelial growth factor receptor 2, matrix metalloproteinases-2/9. These results suggest that Alpha-fetoprotein may play a regulatory role on angiogenesis and cell invasion during liver cancer development.

  14. Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Takeuchi, Kentaro; Inada, Hirohiko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yamasaki, Daisuke [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Doi, Takefumi [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2009-11-20

    Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial {beta}-oxidation. The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a ligand-activated transcription factor that plays an important role in the regulation of {beta}-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPAR{alpha} and found that PPAR{alpha} induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPAR{alpha} regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

  15. 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.

  16. ArF excimer laser modulation of TNF-alpha and gelatinase B in NIH 3T3 cells

    International Nuclear Information System (INIS)

    Naudy-Vives, C.; Courant, D.; Perot, J.C.; Garcia, J.; Fretier, P.; Court, L.; Dormont, D.

    1995-01-01

    The effects on TNF-alpha and gelatinase B activity in mammalian cells induced by 193 nm argon fluoride excimer laser have been investigated. The data show that a secretion of 92 kDa type IV collagenase and TNF-alpha were increased in cell culture supernatants. Moreover, the 193 nm laser radiation produces a decrease of cell proliferation and an increase of cell activation 8 hours after irradiation. The total protein amount increases with the delivered dose. Same, but less effects were obtained after exposure to a conventional UV lamp at 254 nm. (author)

  17. 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...

  18. Effect of cetuximab in combination with alpha-radioimmunotherapy in cultured squamous cell carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Nestor, Marika, E-mail: marika.nestor@bms.uu.s [Unit of Otolaryngology and Head and Neck Surgery, Department of Surgical Sciences, Uppsala University, S-751 85 Uppsala (Sweden); Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Uppsala University, S-751 85 Uppsala (Sweden); Sundstroem, Magnus [Unit of Molecular Pathology, Department of Genetics and Pathology, Uppsala University (Sweden); Anniko, Matti [Unit of Otolaryngology and Head and Neck Surgery, Department of Surgical Sciences, Uppsala University, S-751 85 Uppsala (Sweden); Tolmachev, Vladimir [Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Uppsala University, S-751 85 Uppsala (Sweden)

    2011-01-15

    Aim: The monoclonal antibody cetuximab, targeting the epidermal growth factor receptor (EGFR), is a promising molecular targeting agent to be used in combination with radiation for anticancer therapy. In this study, effects of cetuximab in combination with alpha-emitting radioimmunotherapy (RIT) in a panel of cultured human squamous cell carcinomas (SCCs) were assessed. Methods: SCC cell lines were characterized and treated with cetuximab in combination with anti-CD44v6 RIT using the astatinated chimeric monoclonal antibody U36 ({sup 211}At-cMAb U36). Effects on {sup 211}At-cMAb U36 uptake, internalization and cell proliferation were then assessed in SCC cells. Results: Cetuximab in combination with {sup 211}At-cMAb U36 mediated increased growth inhibition compared to RIT or cetuximab alone in two cell lines. However, cetuximab also mediated radioprotective effects compared to RIT alone in two cell lines. The radioprotective effects occurred in the cell lines in which cetuximab clearly inhibited cell growth during radiation exposure. Cetuximab treatment also influenced {sup 211}At-cMAb-U36 uptake and internalization, suggesting interactions between CD44v6 and EGFR. Conclusions: Results from this study demonstrate the vast importance of further clarifying the mechanisms of cetuximab and radiation response, and the relationship between EGFR and suitable RIT targets. This is important not only in order to avoid potential radioprotective effects, but also in order to find and utilize potential synergistic effects from these combinations.

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

    Directory of Open Access Journals (Sweden)

    Dehai Yu

    2017-06-01

    Full Text Available Background/Aims: 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. Methods: 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. Results: 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. Conclusion: 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.

  20. Effect of cetuximab in combination with alpha-radioimmunotherapy in cultured squamous cell carcinomas

    International Nuclear Information System (INIS)

    Nestor, Marika; Sundstroem, Magnus; Anniko, Matti; Tolmachev, Vladimir

    2011-01-01

    Aim: The monoclonal antibody cetuximab, targeting the epidermal growth factor receptor (EGFR), is a promising molecular targeting agent to be used in combination with radiation for anticancer therapy. In this study, effects of cetuximab in combination with alpha-emitting radioimmunotherapy (RIT) in a panel of cultured human squamous cell carcinomas (SCCs) were assessed. Methods: SCC cell lines were characterized and treated with cetuximab in combination with anti-CD44v6 RIT using the astatinated chimeric monoclonal antibody U36 ( 211 At-cMAb U36). Effects on 211 At-cMAb U36 uptake, internalization and cell proliferation were then assessed in SCC cells. Results: Cetuximab in combination with 211 At-cMAb U36 mediated increased growth inhibition compared to RIT or cetuximab alone in two cell lines. However, cetuximab also mediated radioprotective effects compared to RIT alone in two cell lines. The radioprotective effects occurred in the cell lines in which cetuximab clearly inhibited cell growth during radiation exposure. Cetuximab treatment also influenced 211 At-cMAb-U36 uptake and internalization, suggesting interactions between CD44v6 and EGFR. Conclusions: Results from this study demonstrate the vast importance of further clarifying the mechanisms of cetuximab and radiation response, and the relationship between EGFR and suitable RIT targets. This is important not only in order to avoid potential radioprotective effects, but also in order to find and utilize potential synergistic effects from these combinations.

  1. 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

  2. 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.

  3. 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...

  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. Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

    Science.gov (United States)

    Herr, D; Keck, C; Tempfer, C; Pietrowski, Detlef

    2004-12-01

    The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells. Copyright 2004 Wiley-Liss, Inc.

  6. In vitro study of alpha 2-adrenoceptor turnover and metabolism using the adenocarcinoma cell line HT29

    International Nuclear Information System (INIS)

    Paris, H.; Taouis, M.; Galitzky, J.

    1987-01-01

    The biosynthesis rate of the receptor was studied in postconfluent HT29 cells, when its density expressed as fmol/mg of cell membrane protein is constant, by following the recovery of the receptor binding capacity after blockade with the non-reversible alpha-adrenergic antagonist benextramine. Study of the inhibition of [ 3 H]yohimbine and [ 3 H]UK-14,304 binding showed that benextramine was a more potent antagonist at alpha 2-adrenoceptor than phenoxybenzamine. The incubation of intact HT29 cells for 30 min in the presence of 10(-5) M benextramine irreversibly blocked more than 95% of the alpha 2-adrenoceptors and totally suppressed the inhibitory effect of UK-14,304 on cyclic AMP production. The blockade appeared specific, since benextramine effects were prevented by alpha 2-adrenergic agents. Moreover, neither vasoactive intestinal polypeptide responsiveness nor other tested aspects of the regulation of the adenylate cyclase was altered by the treatment. Study of the time course of receptor recovery after irreversible blockade indicated that alpha 2-adrenoceptors reappeared in the cells with a monoexponential kinetic. The linearization of the repopulation curve obtained with the labeled antagonist [ 3 H]yohimbine allowed the determination of the rate constant for receptor degradation (k = 0.0268 +/- 0.0025 hr-1) and the rate of receptor synthesis (6.91 +/- 0.64 fmol/mg of cell membrane protein/hr) corresponding to the synthesis of about 500 receptors/cell/hr. The alpha 2-adrenoceptor half-life was 26 +/- 3 hr. Measurement of the biological effects associated to the alpha-adrenoceptor stimulation during the course of receptor recovery indicated a relationship between the number of cell receptors and the percentage of inhibition of the cyclic AMP accumulation induced by forskolin

  7. Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor.

    Science.gov (United States)

    Matsuura, Takashi; Uematsu, Takashi; Yamaoka, Minoru; Furusawa, Kiyofumi

    2004-03-01

    The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients.

  8. 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 cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent 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.

  9. Localisation of malignant germ-cell tumours by external scanning after injection of radiolabelled anti-alpha-fetoprotein

    International Nuclear Information System (INIS)

    Halsall, A.K.; Fairweather, D.S.; Bradwell, A.R.; Blackburn, J.C.; Howell, A.; Dykes, P.W.; Reeder, A.; Hine, K.R.

    1981-01-01

    Sheep IgG antibody to alpha-fetoprotein was labelled with 131 I and used to identify human germ-cell tumours by emission scanning. Eleven patients were studied after resection of their primary tumours. Ten had malignant teratoma and one an endodermal sinus tumour. All eight patients with raised serum alpha-fetoprotein concentrations had metastases apparent in the antibody scans. Of the remaining three patients with normal serum alpha-fetoprotein concentrations, two had positive scans. Three of the patients with positive results were scanned twice; the second scans were negative after treatment, when the alpha-fetoprotein concentrations had returned to normal. These results suggest that antibody scans are useful in the clinical management of patients with germ-cell tumours. (author)

  10. 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

  11. Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.

    Science.gov (United States)

    Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang

    2009-07-31

    Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

  12. ADAM12 and alpha9beta1 integrin are instrumental in human myogenic cell differentiation

    DEFF Research Database (Denmark)

    Lafuste, Peggy; Sonnet, Corinne; Chazaud, Bénédicte

    2005-01-01

    of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion...

  13. Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

    Science.gov (United States)

    Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

    1989-06-01

    A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

  14. Enhancement of alpha particles-induced cell transformation by oxygen free radicals and tumor necrosis factor released from phagocytes

    International Nuclear Information System (INIS)

    Gong Yifen; Guo Renfeng; Zhu Maoxiang; Shou Jiang; Ge Guixiu; Yang Zhihua; Hieber, L.; Peters, K.; Schippel, C.

    1997-01-01

    To illustrate the role of several endogenous factors released from phagocytes under chronic inflammation in radiation-induced cancer. C 3 T 10 T 1/2 and SHE cells were used as targets, and 238 Pu alpha source was used in alpha irradiation. The enhancement of TF in alpha particles-induced cell transformation by PMA-stimulated human blood and zymosan-stimulated U-937 cells was studied using formation of transformed foci. Transformation frequency (TF) of C 3 H 10 T 1/2 cells exposed to alpha particles of 0.5 Gy increased 2.1 and 2.8 fold by PMA-and PMA-stimulated neutrophils, respectively. TF of irradiated SHE cells at a dose of 0.5 Gy increased 12 fold by the addition of the supernatant of macrophage-like U-937 cell line. It was shown that TF of irradiated SHE cells at above dose increased 8 fold by the supernatant treated with anti-TNF-α could be subcultured continuously in vitro. The cells at 40 th passage and two lines of monoclone cells have the ability to develop malignant tumors in nude mice. The overdose of free radicals and TNF-α released from neutrophils and macrophages have played an important role in low dose radiation-induced cancer

  15. 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.

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

    International Nuclear Information System (INIS)

    Lee, Young Ju; Lee, Seong-Wook

    2012-01-01

    Highlights: ► Identification of RNA aptamer specific to AFP with high affinity. ► Specific induction of HCC proliferation by AFP. ► Efficient increase in oncogene expression by AFP. ► Efficient inhibition of AFP-mediated HCC proliferation by the aptamer. ► 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 D of ∼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.

  17. 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.

  18. 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.

  19. Choline kinase-alpha by regulating cell aggressiveness and drug sensitivity is a potential druggable target for ovarian cancer.

    Science.gov (United States)

    Granata, A; Nicoletti, R; Tinaglia, V; De Cecco, L; Pisanu, M E; Ricci, A; Podo, F; Canevari, S; Iorio, E; Bagnoli, M; Mezzanzanica, D

    2014-01-21

    Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by (1)H-magnetic spectroscopy analysis; (III) a 35-36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with conventional drugs.

  20. 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

  1. Xanthophylls and alpha-tocopherol decrease UVB-induced lipid peroxidation and stress signaling in human lens epithelial cells.

    Science.gov (United States)

    Chitchumroonchokchai, Chureeporn; Bomser, Joshua A; Glamm, Jayme E; Failla, Mark L

    2004-12-01

    Epidemiological studies suggest that consumption of vegetables rich in the xanthophylls lutein (LUT) and zeaxanthin (ZEA) reduces the risk for developing age-related cataract, a leading cause of vision loss. Although LUT and ZEA are the only dietary carotenoids present in the lens, direct evidence for their photoprotective effect in this organ is not available. The present study examined the effects of xanthophylls and alpha-tocopherol (alpha-TC) on lipid peroxidation and the mitogen-activated stress signaling pathways in human lens epithelial (HLE) cells following ultraviolet B light (UVB) irradiation. When presented with LUT, ZEA, astaxanthin (AST), and alpha-TC as methyl-beta-cyclodextrin complexes, HLE cells accumulated the lipophiles in a concentration- and time-dependent manner with uptake of LUT exceeding that of ZEA and AST. Pretreatment of cultures with either 2 micromol/L xanthophyll or 10 micromol/L alpha-TC for 4 h before exposure to 300 J/m(2) UVB radiation decreased lipid peroxidation by 47-57% compared with UVB-treated control HLE cells. Pretreatment with the xanthophylls and alpha-TC also inhibited UVB-induced activation of c-JUN NH(2)-terminal kinase (JNK) and p38 by 50-60 and 25-32%, respectively. There was substantial inhibition of UVB-induced JNK and p38 activation for cells containing xanthophylls/mg, respectively, whereas >2.3 nmol alpha-TC/mg protein was required to significantly decrease UVB-induced stress signaling. These data suggest that xanthophylls are more potent than alpha-TC for protecting human lens epithelial cells against UVB insult.

  2. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Hui-Hsuan [School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Tsai, Chia-Wen [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Chou, Fen-Pi [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Wang, Chau-Jong [Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Hsuan, Shu-Wen [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China); Wang, Cheng-Kun [E-Chyun Dermatology Clinic, No.70, Sec. 3, Jhonghua E. Rd., East District, Tainan, Taiwan (China); Chen, Jing-Hsien [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China)

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  3. 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...

  4. Survival of alpha particle irradiated cells as a function of the shape and size of the sensitive volume (nucleus)

    International Nuclear Information System (INIS)

    Stinchcomb, T.G.; Roeske, J.C.

    1995-01-01

    Microdosimetry is the study of the stochastic variation of energy deposited within sub-cellular targets. As such, the size and shape of the critical target (i.e. cell nucleus) are essential when considering microdosimetric quantities. In this work, a microdosimetric analysis examines the expected cell survival as a function of the size and shape of the cell nucleus under conditions of irradiation emitting alpha particles. The results indicate that, in general, cell survival is relatively insensitive to changes in the shape of the cell nucleus when the volume is held constant. However, cell survival is a strong function of the variation in the size of the target. These results are useful when analysing the results of cell survival experiments for alpha particle emitters. (Author)

  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. 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...... polarized dendritic cells (alphaDC1) in serum-free medium was published based on maturation of monocyte-derived DCs with TNF-alpha/IL-1-beta/polyinosinic:polycytidylic acid (poly-I:C)/interferon (IFN)-alpha and IFN-gamma. This DC maturation cocktail was described to fulfill the criteria for optimal DC......-regulation of inhibitory molecules such as PD-L1, ILT2, ILT3 as compared to sDC. Although alphaDC1 matured DCs secreted more IL-12p70 and IL-23 these DCs had lower or similar stimulatory capacity compared to sDCs when used as stimulating cells in mixed lymphocyte reaction (MLR) or for induction of autologous influenza...

  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-thalassaemia and response to hydroxyurea in sickle cell anaemia.

    Science.gov (United States)

    Darbari, Deepika S; Nouraie, Mehdi; Taylor, James G; Brugnara, Carlo; Castro, Oswaldo; Ballas, Samir K

    2014-04-01

    Hydroxyurea (HU) reduces vaso-occlusive crises (VOC) and other complications of sickle cell anaemia (SCA). Alpha-thalassaemia is a known modifier of SCA. Studies on the efficacy of HU in SCA patients with α-thalassaemia have yielded varying results. To determine the effect of α-thalassaemia in response to HU therapy in the Multicenter Study of Hydroxyurea (MSH) cohort. We compared the laboratory parameters and VOC incidence in the MSH cohort stratified by the presence or the absence of α-thalassaemia. Hydroxyurea showed significant (P = 0.001 for all baseline vs. follow-up comparisons) treatment effect on red cell indices irrespective of α-globin gene deletion. The magnitude of the HU-related changes was similar for mean corpuscular volume (MCV) (no α-thalassaemia 13 fl and α-thalassaemia 13 fl) and mean corpuscular haemoglobin (MCH) (no α-thalassaemia 4 pg and α-thalassaemia 4 pg) in both groups. Foetal haemoglobin (HbF) and F-cells also increased significantly with HU treatment in both groups. Total haemoglobin increased after HU treatment in both groups, but the increase was smaller and not statistically significant in patients with α-thalassaemia. In contrast, HU-related reduction in VOCs was more pronounced in patients with α-thalassaemia (VOC incidence rate ratio HU/placebo: 0.63 for α-thalassaemia and 0.54 for no α-thalassaemia (P for interaction 0.003). Hydroxyurea decreases VOCs in SCA patients with and without α-thalassaemia, and the degree of VOC reduction was more pronounced in the patients with alpha-thalassaemia. Despite the lower baseline values, changes in standard laboratory parameters such as MCV and HbF percent remain useful in monitoring HU therapy in the presence of α-thalassaemia. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Alpha 1-adrenergic stimulation of phosphatidylinositol turnover and respiration of brown fat cells

    International Nuclear Information System (INIS)

    Mohell, N.; Wallace, M.; Fain, J.N.

    1984-01-01

    The alpha-adrenergic agonist phenylephrine (in the presence of the beta-adrenergic antagonist alprenolol) stimulated respiration and incorporation of [ 3 H]glycerol and [ 32 P] P/sub i/ into phosphatidylinositol of hamster brown fat cells in a concentration-dependent manner. Both responses were preferentially inhibited by prazosin as compared with yohimbine, indicating alpha 1 specificity. Uniquely, prazosin inhibition of phenylephrine-stimulated phosphatidylinositol metabolism had two components, since 30% of the response was inhibited by less than 1 nM prazosin, 10 nM gave no further inhibition, and 100 nM prazosin completely inhibited the response. The phosphatidylinositol response was still present in Ca 2 +-free buffer, although reduced in magnitude. The concentration relationships of the effects of agonists and antagonists were compared with those of previous results of [ 3 H]prazosin binding and with phenylephrine potency to compete for binding. On the basis of these comparisons, it is suggested that the highly prazosin-sensitive part of the phosphatidylinositol response may be closely associated with receptor occupation

  10. 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...

  11. Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells

    International Nuclear Information System (INIS)

    Wada, Hiroshi; Tanemura, Masahiro; Umeshita, Koji; Doki, Yuichiro; Mori, Masaki; Nagano, Hiroaki; Yamamoto, Hirofumi; Noda, Takehiro; Murakami, Masahiro; Kobayashi, Shogo; Marubashi, Shigeru; Eguchi, Hidetoshi; Takeda, Yutaka

    2009-01-01

    The combination therapy of interferon (IFN)-alpha and 5-fluorouracil (5-FU) improved the prognosis of the patients with hepatocellular carcinoma (HCC). To determine the molecular mechanisms of the anti-tumor and anti-angiogenic effects, we examined the direct anti-proliferative effects on human umbilical vein endothelial cells (HUVEC) and indirect effects by regulating secretion of angiogenic factors from HCC cells. The direct effects on HUVEC were examined by TUNEL, Annexin-V assays and cell cycles analysis. For analysis of the indirect effects, the apoptosis induced by the conditioned medium from HCC cell treated by IFN-alpha/5-FU and expression of angiogenic factors was examined. IFN-alpha and 5-FU alone had anti-proliferative properties on HUVEC and their combination significantly inhibited the growth (compared with control, 5-FU or IFN alone). TUNEL and Annexin-V assays showed no apoptosis. Cell cycle analysis revealed that IFN-alpha and 5-FU delayed cell cycle progression in HUVEC with S-phase accumulation. The conditioned medium from HuH-7 cells after treatment with IFN/5-FU significantly inhibited HUVEC growth and induced apoptosis, and contained high levels of angiopoietin (Ang)-1 and low levels of vascular endothelial growth factor (VEGF) and Ang-2. Knockdown of Ang-1 in HuH-7 cells abrogated the anti-proliferative effects on HUVEC while knockdown of Ang-2 partially rescue the cells. These results suggested that IFN-alpha and 5-FU had direct growth inhibitory effects on endothelial cells, as well as anti-angiogenic effects through regulation of angiogenic factors released from HCC cells. Modulation of VEGF and Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC

  12. 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.

  13. 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.

  14. Infection of Human Fallopian Tube Epithelial Cells with Neisseria gonorrhoeae Protects Cells from Tumor Necrosis Factor Alpha-Induced Apoptosis

    Science.gov (United States)

    Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E.; Christodoulides, Myron; Velasquez, Luis

    2006-01-01

    Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-α antibodies; and (iii) the addition of exogenous TNF-α induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-α-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-α-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

  15. [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.

  16. Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

    International Nuclear Information System (INIS)

    Lind, I.; Ahnert-Hilger, G.; Fuchs, G.; Gratzl, M.

    1987-01-01

    Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86 Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules

  17. [beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Brown, C.A.; Mahuran, D.J. (Univ. of Toronto (Canada))

    1993-08-01

    In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

  18. Use of track-end alpha particles from 241Am to study radiosensitive sites in CHO cells

    International Nuclear Information System (INIS)

    Datta, R.; Cole, A.; Robinson, S.

    1976-01-01

    Monolayers of CHO cells placed on membrane filters were irradiated with alpha particles from a 241 Am source. Particle penetration into the cells was controlled by placing the cell sample at various distances from the source. Dosimetric and spectrometric measurements were performed at comparable positions using a parallel plate ionization chamber and a scintillation crystal spectrometer. Cell survival, as measured by conventional cloning techniques, was single hit in form. A pronounced minimum in mean lethal dose of 29 rad was observed for alpha particle beams that penetrated only about 3 μm into the cell. A pronounced maximum in inactivation cross section of 90 μm 2 , equal to about half the projected area of the nucleus, occurred for beams that penetrated only 5 to 7 μm into the cell. Thus, a single alpha particle penetration several micrometers within the cell nucleus was effective in killing the cell, while fully penetrating beams were actually less efficient; the latter beams required multiple particle traversals and about three times the cell dose to achieve the same effect. These results support the proposal that radiosensitive sites are located in a thin peripheral region of the nucleus

  19. 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.

  20. RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

    Science.gov (United States)

    Yu, W; Sanders, B G; Kline, K

    1997-01-01

    RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

  1. 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.

  2. Alpha radiation-induced alterations of the proliferation kinetics, chromatin structure and gene expression in mammalian cells

    International Nuclear Information System (INIS)

    Hieber, L.

    1983-01-01

    Exponentially growing mammalian cells were exposed to 3.4 MeV alpha particles. The chromatin of cells arrested in G2 by alpha irradiation was severely damaged, though all cells were still capable to condensate their chromatin after fusion with mitotic cells. In addition to the common types of aberrations (breaks, gaps, dicentrics and exchanges) cells were found possessing one or more chromosomes with long stretches of undercondensed chromatin. Repair of these lesions was indicated by site specific unscheduled DNA synthesis and by the observation that condensation of these regions improved during G2 arrest. Furthermore, during G2 arrest the synthesis of two cellular proteins was stimulated. This was studied by two-dimensional gel electrophoresis of 35 S-methionine labeled cellular proteins. All these findings provided evidence that radiation-induced G2 arrest is caused by chromatin damage, which prevents regular chromosome condensation for mitosis. (orig./MG) [de

  3. Type-I interferon receptor expression: its circadian rhythm and downregulation after interferon-alpha administration in peripheral blood cells from renal cancer patients.

    Science.gov (United States)

    Shiba, Masahiro; Nonomura, Norio; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Inoue, Hitoshi; Tsujimura, Akira; Nishimura, Kazuo; Okuyama, Akihiko

    2009-04-01

    To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. Our findings might support the establishment of an optimal schedule for IFN-alpha administration.

  4. 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.

  5. 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

  6. 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...

  7. [Experimental study on aging effect of Angelica sinensis polysaccharides combined with cytarabine on human leukemia KG1alpha cell lines].

    Science.gov (United States)

    Xu, Chun-Yan; Geng, Shan; Liu, Jun; Zhu, Jia-Hong; Zhang, Xian-Ping; Jiang, Rong; Wang, Ya-Ping

    2014-04-01

    The latest findings of our laboratory showed that Angelica sinensis polysaccharide (ASP) showed a definite effect in regulating the aging of hematopoietic stem cells. Leukemia is a type of malignant hematopoietic tumor in hematopoietic stem cells. There have been no relevant reports about ASP's effect in regulating the aging of leukemia cells. In this study, human acute myeloid leukemia (AML) KG1alpha cell lines in logarithmic growth phase were taken as the study object, and were divided into the ASP group, the cytarabine (Ara-C) group, the ASP + Ara-C group and the control group. The groups were respectively treated with different concentration of ASP, Ara-C and ASP + Ara-C for different periods, with the aim to study the effect of ASP combined with Ara-C in regulating the aging of human acute myeloid leukemia KG1alpha cell lines and its relevant mechanism. The results showed that ASP, Ara-C and ASP + Ara-C could obviously inhibit KG1alpha cell proliferation in vitro, block the cells in G0/G1 phase. The cells showed the aging morphological feature. The percentage of positive stained aging cells was dramatically increased, and could significantly up-regulate the expression of aging-related proteins P16 and RB, which were more obvious in the ASP + Ara-C group. In conclusion, the aging mechanism of KG1alpha cell induced by ASP and Ara-C may be related to the regulation of the expression of aging-related proteins, suggesting that the combined administration of ASP and anticancer drugs plays a better role in the treatment of leukemia .

  8. 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.

  9. Cell cycle sensitivity of HL-60 cells to the differentiation-inducing effects of 1-alpha,25-dihydroxyvitamin D3

    International Nuclear Information System (INIS)

    Studzinski, G.P.; Bhandal, A.K.; Brelvi, Z.S.

    1985-01-01

    A recently described system for monocyte-like differentiation of HL-60 cells was utilized to determine if the initiation of this pathway can be linked to a set of replicative cellular events. The standard induction system consisted of a 4-h exposure to 100 nM 1-alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] followed by determination of nonspecific esterase and phagocytic activity 24 h later. The cell cycle status was ascertained by the incorporation of [ 3 H]thymidine and autoradiography. Studies in which cell cycle block in the G1/S phase boundary region was produced by a partial inhibition of DNA synthesis with thymidine, or sodium butyrate, showed that the exposure of such semisynchronous cultures to 1,25(OH)2D3 resulted in an increased proportion of differentiated cells. Conversely, blocking the cell cycle with vinblastine (G2/M block) or theobromine (mid-G1 block) inhibited the initiation of differentiation by 1,25(OH)2D3. Experiments in which the differentiated cells were examined for the cell cycle position at the time of the exposure to 1,25(OH)2D3 by [ 3 H]thymidine labeling and autoradiography confirmed that the late G1 and early S phase cells are those which predominate in the differentiated fraction of 1,25(OH)2D3-treated HL-60 cultures. These results link pre- and early replicative cellular events to the induction of monocytic differentiation by 1,25(OH)2D3

  10. 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.

  11. Study of substrate topographical effects on epithelial cell behavior using etched alpha-particle tracks on PADC films

    International Nuclear Information System (INIS)

    Ng, C.K.M.; Poon, W.L.; Li, W.Y.; Cheung, T.; Cheng, S.H.; Yu, K.N.

    2008-01-01

    Micrometer-size pits on the surface of a polymer (polyallyldiglycol carbonate or PADC) substrate created by alpha-particle irradiation and subsequent chemical etching were used to study the topographical effects alone on cell behavior. Vinculin, the cell adhesion and membrane protrusion protein, was used as an indicator of cytoskeletonal reorganization on the substrate and localization of vinculin was used to demonstrate the presence of focal adhesions. In our experiments, vinculin expressed in epithelial HeLa cells cultured on PADC films with track-etch pits, but not in cells cultured on the raw or chemically etched blank films. In other words, vinculin expression was induced by the topography of track-etch pits, while etching of the substrate alone (without alpha-particle irradiation) did not cause up-regulation of vinculin protein expression. HeLa cells cultured on PADC films with track-etch pits also showed changes in cell proliferation, cell area and cell circularity, and were largely contained by the pits. In other words, the cell membrane edges tended to be in contact with the pits. By comparing the correlation between the positions of HeLa cells and the pits, and that between the positions of cells and computer-simulated pits, the tendency for membrane edges of HeLa cells to be in contact with the pits was recognized. This could be explained by inhibition of membrane protrusion at the pits. In conclusion, substrate track-etch pits were an important determinant of epithelial cell behaviors

  12. 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)

  13. 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

  14. Interleukin-4 inhibits both paracrine and autocrine tumor necrosis factor-alpha-induced proliferation of B chronic lymphocytic leukemia cells

    NARCIS (Netherlands)

    van Kooten, C.; Rensink, I.; Aarden, L.; van Oers, R.

    1992-01-01

    The proliferative response of purified malignant B cells from 26 patients with chronic lymphocytic leukemia (CLL) was investigated in vitro. In the majority of these patients, a proliferative response could be induced by the combination of tumor necrosis factor (TNF)-alpha and PMA. The concentration

  15. Intermitted pharmacologic pretreatment by xenon, isoflurane, nitrous oxide, and the opioid morphine prevents tumor necrosis factor alpha-induced adhesion molecule expression in human umbilical vein endothelial cells

    NARCIS (Netherlands)

    Weber, Nina C.; Kandler, Jennis; Schlack, Wolfgang; Grueber, Yvonne; Frädorf, Jan; Preckel, Benedikt

    2008-01-01

    BACKGROUND: The barrier properties of the endothelium are of critical importance during pathophysiologic processes. These barrier properties depend on an intact cytoskeleton and are regulated by cell adhesion molecules. Tumor necrosis factor alpha (TNF-alpha) is known to induce cell adhesion

  16. 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

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

    International Nuclear Information System (INIS)

    Liu, K-K; Chen, P-Y; Lee, Tony J F; Chao, J-I; Chen, M-F; Cheng, C-L; Chang, C-C; Ho, Y-P

    2008-01-01

    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

  18. 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.

  19. 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.

  20. Structural basis for alpha fetoprotein-mediated inhibition of caspase-3 activity in hepatocellular carcinoma cells.

    Science.gov (United States)

    Lin, Bo; Zhu, Mingyue; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Li, Mengsen

    2017-10-01

    Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients. © 2017 UICC.

  1. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

    Science.gov (United States)

    Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

    2006-10-01

    Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

  2. 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.

  3. In vivo administration of interferon alpha and interleukin 2 induces proliferation of lymphoid cells in the organs of mice

    International Nuclear Information System (INIS)

    Puri, R.K.; Travis, W.D.; Rosenberg, S.A.

    1990-01-01

    We have previously shown that interleukin 2 (IL-2) synergizes with interferon alpha (IFN-alpha) in mediating the regression of established pulmonary and hepatic metastases and the reduction of intradermal tumor in various murine tumor models. To understand the mechanism of synergy, we have examined lymphoid cell proliferation in various organs of mice in response to IL-2 and IFN-alpha administration. We have utilized a technique for labeling newly synthesized DNA in vivo with 5-[125I]iodo-2'-deoxyuridine to examine proliferation of endogenous cells in response to IL-2 and IL-2 plus IFN-alpha. A proliferation index was calculated by dividing cpm in the tissues treated with cytokines by cpm obtained in corresponding tissues of control mice. After 4 days of IL-2 administration, a significant uptake of 5-[125I]iodo-2'-deoxyuridine was observed in the lungs, liver, kidneys, and spleen (proliferation index of 13, 10.3, 3.6, and 3.2, respectively). IFN-alpha alone mediated very little incorporation of radiolabel but when administered in combination with IL-2 a reduction of IL-2-induced proliferation was seen on day 4. For example 19,272 +/- 4,556 cpm (mean +/- SE) were obtained in the liver of IL-2-treated mice, compared to 8,103 +/- 2,111 cpm in livers of IL-2 plus IFN-alpha-treated mice (P less than 0.05). Similar inhibition of IL-2-induced proliferation was observed in the lungs, kidneys, and spleen. In contrast, on days 7 or 8, higher uptake of radiolabel was obtained in IFN-alpha plus IL-2-treated lungs, liver, and kidneys, compared to organs of mice treated with IL-2 alone or IFN-alpha alone. A proliferation index of 30.5, 9.8, and 10 was obtained in the lungs, liver, and kidneys of IL-2- plus IFN-alpha-treated animals, compared to 9.6, 3.6, and 5.5 in the corresponding organs of IL-2-treated mice

  4. G2M arrest and apoptosis in murine T lymphoma cells following exposure to 212Bi alpha particle irradiation

    International Nuclear Information System (INIS)

    Palayoor, S.T.; Humm, J.L.; Macklis, R.M.

    1993-01-01

    Asynchronous exponentially growing EL4 murine T lymphoma cells were exposed either to high LET α-radiation from 212 Bi-DTPA or to γ-radiation from a 137 Cs source. Radiation-induced cell cycle perturbation was studied by flow cytometry. Alpha irradiation, like γ, transiently arrested cells in the G2M phase in a dose-dependent manner. The maximum percentages of cells accumulated in G2M 18 h after α- and γ-irradiation were comparable, though the dose-response relationships differed. The ''RBE'' value for G2M block for α- versus γ-radiation was approx. 4. (author)

  5. 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

    challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors...... 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...... for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol....

  6. 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.)

  7. 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.

  8. Efficacy of dendritic cells matured early with OK-432 (Picibanil), prostaglandin E2, and interferon-alpha as a vaccine for a hormone refractory prostate cancer cell line.

    Science.gov (United States)

    Yoo, Changhee; Do, Hyun-Ah; Jeong, In Gab; Park, Hongzoo; Hwang, Jung-Jin; Hong, Jun Hyuk; Cho, Jin Seon; Choo, Myong-Soo; Ahn, Hanjong; Kim, Choung-Soo

    2010-09-01

    Dendritic cells (DCs) are potent antigen-presenting cells. OK432 (Picibanil) was introduced as a potent stimulator of DC maturation in combination with prostaglandin-E(2) and interferon-alpha. We compared the efficacy of a DC-prostate cancer vaccine using early-mature DCs stimulated with OK432, PGE2 and INF-alpha (OPA) with that of vaccines using other methods. On days 3 or 7 of DC culture, TNF-alpha (T), TNF-alpha and LPS (TL) or OPA were employed as maturation stimulators. DU145 cells subjected to heat stress were hybridized with mature DCs using polyethyleneglycol. T cells were sensitized by the hybrids, and their proliferative and cytokine secretion activities and cytotoxicity were measured. The yields of early-mature DCs were higher, compared to yields at the conventional maturation time (P<0.05). In the early maturation setting, the mean fusion ratios, calculated from the fraction of dual-positive cells, were 13.3%, 18.6%, and 39.9%, respectively (P=0.051) in the T only, TL, and OPA-treated groups. The function of cytotoxic T cells, which were sensitized with the hybrids containing DCs matured early with OPA, was superior to that using other methods. The antitumor effects of DC-DU145 hybrids generated with DCs subjected to early maturation with the OPA may be superior to that of the hybrids using conventional maturation methods.

  9. 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...

  10. 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 facto...

  11. 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

  12. Cell death triggered by alpha-emitting {sup 213}Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death

    Energy Technology Data Exchange (ETDEWEB)

    Seidl, Christof; Schroeck, Hedwig; Seidenschwang, Sabine; Beck, Roswitha; Schwaiger, Markus; Senekowitsch-Schmidtke, Reingard [Technische Universitaet Muenchen, Department of Nuclear Medicine, Munich (Germany); Schmid, Ernst [National Research Center for Environment and Health, Institute of Radiation Biology, GSF, Neuherberg (Germany); Abend, Michael [German Armed Forces, Institute of Radiobiology, Munich (Germany); Becker, Karl-Friedrich [Technische Universitaet Muenchen, Institute of Pathology, Munich (Germany); National Research Center for Environment and Health, Institute of Pathology, GSF, Neuherberg (Germany); National Research Center for Environment and Health, Institute of Molecular Immunology, GSF, Munich (Germany); Apostolidis, Christos; Nikula, Tuomo K. [European Commission, Institute for Transuranium Elements, Karlsruhe (Germany); Kremmer, Elisabeth [National Research Center for Environment and Health, Institute of Molecular Immunology, GSF, Munich (Germany)

    2005-03-01

    Radioimmunotherapy with {alpha}-particle-emitting nuclides, such as{sup 213}Bi, is a promising concept for the elimination of small tumour nodules or single disseminated tumour cells. The aim of this study was to investigate cellular damage and the mode of cell death triggered by {sup 213}Bi-immunoconjugates. Human gastric cancer cells (HSC45-M2) expressing d9-E-cadherin were incubated with different levels of activity of {sup 213}Bi-d9MAb targeting d9-E-cadherin and {sup 213}Bi-d8MAb, which does not bind to d9-E-cadherin. Micronucleated (M) cells, abnormal (A) cells and apoptotic (A) [(MAA)] cells were scored microscopically in the MAA assay following fluorescent staining of nuclei and cytoplasm. Chromosomal aberrations were analysed microscopically following Giemsa staining. The effect of z-VAD-fmk, known to inhibit apoptosis, on the prevention of cell death was investigated following treatment of HSC45-M2 cells with sorbitol as well as {sup 213}Bi-d9MAb. Activation of caspase 3 after incubation of HSC45-M2 cells with both sorbitol and {sup 213}Bi-d9MAb was analysed via Western blotting. Following incubation of HSC45-M2 human gastric cancer cells expressing d9-E-cadherin with {sup 213}Bi-d9MAb the number of cells killed increased proportional to the applied activity concentration. Microscopically visible effects of {alpha}-irradiation of HSC45-M2 cells were formation of micronuclei and severe chromosomal aberrations. Preferential induction of these lesions with specific {sup 213}Bi-d9MAb compared with unspecific {sup 213}Bi-d8MAb (not targeting d9-E-cadherin) was not observed if the number of floating, i.e. unbound {sup 213}Bi-immunoconjugates per cell exceeded 2 x 10{sup 4}, most likely due to intense crossfire. In contrast to sorbitol-induced cell death, cell death triggered by {sup 213}Bi-immunoconjugates was independent of caspase 3 activation and could not be inhibited by z-VAD-fmk, known to suppress the apoptotic pathway. {sup 213}Bi-immunoconjugates seem

  13. Clinical value of imaging using antibody to alpha fetoprotein in germ cell tumours

    International Nuclear Information System (INIS)

    Hitchins, R.N.; Begent, R.H.J.; Green, A.J.; Searle, F.; Bagshawe, K.D.; Charing Cross Group of Hospitals, London; Van Heyningen, V.

    1989-01-01

    Germ cell tumours (GCT) producing alpha fetoprotein (aFP) can be imaged by external scintigraphy after intraveneous administration of radiolabelled antibody directed against aFP. Antibody imaging (AI) by this method was used in an attempt to guide surgical resection of deposits of drug-resistant or recurrent GCT. 30 patients with GCT and raised aFP in whom site of tumour was not known were investigated by AI and conventional imaging methods. All but one were heavily pretreated. Where tumour appeared localised, resection was attempted. Tumour was found in all sites positive by both AI and conventional imaging. AI produced false-positive results in one of 30 patients and false-negative results in 9 patients. Computerised tomography was false-positive in one case and false-negative in three. In these patients, AI gave true-negative and true-positive results, respectively. Of 11 patients with positive AI in whom resection was attempted, 6 achieved sustained complete response with up to 5 years follow-up. We conclude AI and conventional imaging methods to be complementary in selection for surgery of patients with drug-resistant or recurrent GCT. (orig.) [de

  14. 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.

  15. In vitro cytotoxicity of human recombinant tumor necrosis factor alpha in association with radiotherapy in a human ovarian carcinoma cell line

    International Nuclear Information System (INIS)

    Manetta, A.; Lucci, J.; Soopikian, J.; Granger, G.; Berman, M.L.; DiSaia, P.J.

    1990-01-01

    It has been speculated that tumor necrosis factor alpha (TNF-alpha) may decrease the cytotoxicity of radiotherapy by increasing the scavenging of toxic superoxide radicals. Because of the possible clinical implications, the cytotoxicity of TNF-alpha in combination with radiotherapy (RT) was compared with that of RT alone in a human ovarian cancer cell line. NIH:OVCAR-3 cells were incubated with TNF-alpha at 10.0, 1.0, 0.1, and 0.01 microgram/ml. Plates were divided into two groups; one received 150 cGy of radiotherapy and the other received no further therapy. Seventy-two hours later, supernatants were aspirated and viable cells were stained with a 1% solution of crystal violet. Survival of cells treated with RT plus TNF-alpha was expressed as a percentage of surviving irradiated controls. Analysis of results revealed minimal additive cell killing effect between TNF-alpha and radiotherapy at all concentrations of tumor necrosis factor, with the greatest difference noted in the group treated with 10 micrograms/ml TNF-alpha. A continued radiotherapy dose-response study with TNF-alpha showed a similar additive, not radioprotective, effect. This may have implication as a potentiator of RT in some human tumors

  16. MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

    Science.gov (United States)

    Tran, An Hue Vy; Han, Se Hee; Kim, Joon; Grasso, Francesca; Kim, In San; Han, Ye Sun

    2017-07-01

    Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. {sup 99m}Tc-3PRGD{sub 2} SPECT to monitor early response to neoadjuvant chemotherapy in stage II and III breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Bin; Chen, Bin; Wang, Ting; Chen, Minglong; Ji, Tiefeng; Gao, Shi; Ma, Qingjie [China-Japan Union Hospital of Jilin University, Department of Nuclear Medicine, Changchun (China); Song, Yan [China-Japan Union Hospital of Jilin University, Department of Breast Surgery, Changchun (China); Wang, Xueju [China-Japan Union Hospital of Jilin University, Department of Pathology, Changchun (China)

    2015-08-15

    Monitoring of response to neoadjuvant chemotherapy (NCT) is important for optimal management of patients with breast cancer. {sup 99m}Tc-3PRGD{sub 2} SPECT is a newly developed imaging modality for evaluating tumor vascular status. In this study, we investigated the application of {sup 99m}Tc-3PRGD{sub 2} SPECT in evaluating therapy response to NCT in patients with stage II or III breast cancer. Thirty-three patients were scheduled to undergo {sup 99m}Tc-3PRGD{sub 2} SPECT at baseline, after the first and second cycle of NCT. Four patients had extremely low {sup 99m}Tc-3PRGD{sub 2} uptake at baseline, and were not included in the subsequent studies. Changes in tumor to nontumor (T/N) ratio were compared with pathological tumor responses classified using the residual cancer burden system. Receiver operator characteristic analysis was used to compare the power to identify responders between the end of the first and the end of the second cycle of NCT. The impact of breast cancer subtype on {sup 99m}Tc-3PRGD{sub 2} uptake was evaluated. The correlation between {sup 99m}Tc-3PRGD{sub 2} uptake and pathological tumor response was also evaluated in each breast cancer subtype. Surgery was performed after four cycles of NCT and pathological analysis revealed 18 responders and 15 nonresponders. In patients with clearly visible {sup 99m}Tc-3PRGD{sub 2} uptake at baseline, the sensitivity, specificity, and negative predictive value of {sup 99m}Tc-3PRGD{sub 2} SPECT were 86.7 %, 85.7 % and 86.7 % after the first cycle of NCT, and 92.9 %, 93.3 % and 93.3 % after the second cycle, respectively. Among these patients, the HER-2-positive group demonstrated both higher T/N ratios and a greater change in T/N ratio than patients with other breast cancer subtypes (P < 0.05). A strong correlation was found between changes in T/N ratio and pathological tumor response in the HER-2-positive group (P < 0.03). {sup 99m}Tc-3PRGD{sub 2} SPECT seems to be useful for determining the pathological

  18. Proliferative and antiproliferative effects of interferon-gamma and tumor necrosis factor-alpha on cell lines derived from cervical and ovarian malignancies

    International Nuclear Information System (INIS)

    Mutch, D.G.; Massad, L.S.; Kao, M.S.; Collins, J.L.

    1990-01-01

    Four human cell lines derived from cervical carcinomas (ME-180, SiHa, HT-3, and MS751) and three human cell lines derived from ovarian carcinomas (SK-OV-3, Caov-3, and NIH:OVCAR-3) were analyzed in vitro to determine the effect of recombinant interferon-gamma and recombinant human tumor necrosis factor-alpha on cell growth and survival. The effects of interferon-gamma, tumor necrosis factor-alpha, and both interferon-gamma and tumor necrosis factor-alpha on cell growth were measured after 24 and 72 hours of incubation by the incorporation of chromium 51. The results of this analysis showed that all seven cell lines were resistant to the antiproliferative action of tumor necrosis factor-alpha, that the growth of most cell lines was inhibited by interferon-gamma by 72 hours of incubation, and that after 72 hours of incubation all cell lines demonstrated a synergistic antiproliferative response to the combination of interferon-gamma and tumor necrosis factor-alpha. However, the effects of these cytokines on cell growth were found to differ among cell lines and varied with the concentration and the duration of incubation. The growth of one cell line (Caov-3) was stimulated by both tumor necrosis factor-alpha and interferon-gamma. These results suggest that the clinical effects of these cytokines on the growth of gynecologic cancers may be more complex than previously supposed

  19. EMMPRIN promotes melanoma cells malignant properties through a HIF-2alpha mediated up-regulation of VEGF-receptor-2.

    Directory of Open Access Journals (Sweden)

    Faten Bougatef

    Full Text Available EMMPRIN's expression in melanoma tissue was reported to be predictive of poor prognosis. Here we demonstrate that EMMPRIN up-regulated VEGF receptor-2 (VEGFR-2 in two different primary melanoma cell lines and consequently increased migration and proliferation of these cells while inhibiting their apoptosis. SiRNA inhibition of VEGFR-2 expression abrogated these EMMPRIN effects. EMMPRIN regulation of VEGFR-2 was mediated through the over-expression of HIF-2alpha and its translocation to the nucleus where it forms heterodimers with HIF-1beta. These results were supported by an in vivo correlation between the expression of EMMPRIN with that of VEGFR-2 in human melanoma tissues as well as with the extent of HIF-2alpha localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2alpha/VEGFR-2 mediated mechanism, with an autocrine role in melanoma cell malignancy. The inhibition of EMMPRIN in cancer may thus simultaneously target both the VEGFR-2/VEGF system and the matrix degrading proteases to block tumor cell growth and invasion.

  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. Interleukin-1 alpha modulates collagen gene expression in cultured synovial cells.

    Science.gov (United States)

    Mauviel, A; Teyton, L; Bhatnagar, R; Penfornis, H; Laurent, M; Hartmann, D; Bonaventure, J; Loyau, G; Saklatvala, J; Pujol, J P

    1988-01-01

    The effects of porcine interleukin-1 (IL-1) alpha on collagen production were studied in cultured human rheumatoid synovial cells. Addition of 0.05-5 ng of IL-1/ml into the cultures resulted in a dose-dependent decreased rate of collagen released into the medium over 24 h. To determine whether this inhibition was due to secondary action of prostaglandin E2 (PGE2) secreted in response to IL-1, cultures were incubated in presence of various inhibitors of arachidonate metabolism. Depending on the cell strains, these inhibitors were able to suppress or diminish the effect of IL-1, suggesting that PGE2 is involved in the mechanism. Depression of collagen production caused by IL-1 mainly affected type I collagen and therefore led to a change in the type I/type III collagen ratio in the extracellular medium. Steady-state levels of mRNA for types I and III procollagens were estimated by dot-blot hybridization and compared with the amounts of respective collagens produced in the same cultures. IL-1 generally increased procollagen type I mRNA, but to a variable extent, as did indomethacin (Indo). Depending on the cell strain, the combination of indo and IL-1 could elevate the mRNA level of type I procollagen compared with Indo alone. These results did not correlate with the production rate of collagen in the medium, which was diminished by exposure to IL-1. The level of mRNA for collagen type III was not greatly changed by incubation with IL-1, and a better correlation was generally observed with the amount of type III collagen found in the medium. These data suggest that an additional control mechanism at translational or post-translational level must exist, counterbalancing the stimulatory effect of IL-1 on collagen mRNA transcription. It is likely that IL-1 could modulate the production of collagen in synovial cells by an interplay of different mechanisms, some of them limiting the effect of primary elevation of the steady-state mRNA level. Images Fig. 3. Fig. 4. Fig. 5

  2. Inhibition of polymerases-alpha and -beta completely blocks DNA repair induced by UV irradiation in cultured mouse neuronal cells

    International Nuclear Information System (INIS)

    Licastro, F.; Sarafian, T.; Verity, A.M.; Walford, R.L.

    1985-01-01

    The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain

  3. Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4

    NARCIS (Netherlands)

    Gascan, H.; Aversa, G. G.; Gauchat, J. F.; van Vlasselaer, P.; Roncarolo, M. G.; Yssel, H.; Kehry, M.; Spits, H.; de Vries, J. E.

    1992-01-01

    In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell

  4. In vivo assessment of the antiproliferative properties of interferon-alpha during immunotherapy: Ki-67 (MIB-1) in patients with metastatic renal cell carcinoma

    DEFF Research Database (Denmark)

    Donskov, F; Marcussen, N; Hokland, M

    2004-01-01

    The aim of the present study was to investigate the in vivo antiproliferative effect of interferon alpha (IFN-alpha) in patients with metastatic renal cell carcinoma (mRCC). Core needle biopsies of metastatic and/or the primary kidney cancer were obtained before interleukin-2 (IL-2)- and IFN...

  5. 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

  6. The fibroblast growth factor receptor (FGFR) agonist FGF1 and the neural cell adhesion molecule-derived peptide FGL activate FGFR substrate 2alpha differently

    DEFF Research Database (Denmark)

    Chen, Yongshuo; Li, Shizhong; Berezin, Vladimir

    2010-01-01

    Activation of fibroblast growth factor (FGF) receptors (FGFRs) both by FGFs and by the neural cell adhesion molecule (NCAM) is crucial in the development and function of the nervous system. We found that FGFR substrate 2alpha (FRS2alpha), Src homologous and collagen A (ShcA), and phospholipase-Cg...

  7. 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.

  8. Analysis and Quantitation of NF-[kappa]B Nuclear Translocation in Tumor Necrosis Factor Alpha (TNF-[alpha]) Activated Vascular Endothelial Cells

    Science.gov (United States)

    Fuseler, John W.; Merrill, Dana M.; Rogers, Jennifer A.; Grisham, Matthew B.; Wolf, Robert E.

    2006-07-01

    Nuclear factor kappa B (NF-[kappa]B) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-[kappa]Bs, which prevent entry into the nucleus. Following cellular stimulation, the I-[kappa]Bs are rapidly degraded, activating NF-[kappa]B. The active form of NF-[kappa]B rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-[kappa]B is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-[kappa]B (p65 subunit, p65-NF-[kappa]B) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-[kappa]B protein in the nucleus determined biochemically. The appearance of p65-NF-[kappa]B in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-[kappa]B can be reliably determined from IOD measurements of the immunofluorescent labeled protein.

  9. 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.

  10. Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.

    Science.gov (United States)

    Lee, Kwang Hoon; Chung, Hae-Shin; Kim, Hyoung Sup; Oh, Sang-Ho; Ha, Moon-Kyung; Baik, Ja-Hyun; Lee, Sungnack; Bang, Dongsik

    2003-07-01

    To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

  11. Cytokines interleukin-1beta and tumor necrosis factor-alpha regulate different transcriptional and alternative splicing networks in primary beta-cells

    DEFF Research Database (Denmark)

    Ortis, Fernanda; Naamane, Najib; Flamez, Daisy

    2010-01-01

    by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells. RESEARCH DESIGN AND METHODS: Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h......-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression...... of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine...

  12. Divergent effects of 17-{beta}-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-{alpha}-induced neointima formation

    Energy Technology Data Exchange (ETDEWEB)

    Nintasen, Rungrat [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom); Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University (Thailand); Riches, Kirsten; Mughal, Romana S. [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom); Viriyavejakul, Parnpen; Chaisri, Urai; Maneerat, Yaowapa [Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University (Thailand); Turner, Neil A. [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom); Porter, Karen E., E-mail: medkep@leeds.ac.uk [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} augments neointimal hyperplasia in human saphenous vein. Black-Right-Pointing-Pointer TNF-{alpha} induces detrimental effects on endothelial and smooth muscle cell function. Black-Right-Pointing-Pointer Estradiol exerts modulatory effects on TNF-induced vascular cell functions. Black-Right-Pointing-Pointer The modulatory effects of estradiol are discriminatory and cell-type specific. -- Abstract: Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-{alpha} on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-{alpha} (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-{alpha}, an effect that was abolished by co-culture with E2. TNF-{alpha} increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-{alpha} alone. SV-EC migration was significantly impaired by TNF-{alpha} (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-{alpha} increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-{alpha} potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there

  13. 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.

  14. Effects of tumor necrosis factor-alpha and interferon-gamma on expressions of matrix metalloproteinase-2 and -9 in human bladder cancer cells.

    Science.gov (United States)

    Shin, K Y; Moon, H S; Park, H Y; Lee, T Y; Woo, Y N; Kim, H J; Lee, S J; Kong, G

    2000-10-31

    We have investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon (INF-gamma), the potent Bacillus Calmette-Guerin (BCG)-induced cytokines on the production of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP in high grade human bladder cancer cell lines, T-24, J-82 and HT-1376 cell lines. MMP-2 expression and activity were decreased in T-24 cells treated with both cytokines in a dose dependent manner. However, J-82 cells treated with TNF-alpha and INF-gamma revealed dose dependent increases of MMP-9 expression and activity with similar baseline expression and activity of MMP-2. HT-1376 cells after exposure to TNF-alpha only enhanced the expression and activity of MMP-9. These results indicate that TNF-alpha and INF-gamma could regulate the production of MMP-2 or MMP-9 on bladder cancer cells and their patterns of regulation are cell specific. Furthermore, this diverse response of bladder cancer cells to TNF-alpha and INF-gamma suggests that BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs.

  15. 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

  16. Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

    Science.gov (United States)

    Sugita, Sunao; Horie, Shintaro; Nakamura, Orie; Maruyama, Kazuichi; Takase, Hiroshi; Usui, Yoshihiko; Takeuchi, Masaru; Ishidoh, Kazumi; Koike, Masato; Uchiyama, Yasuo; Peters, Christoph; Yamamoto, Yoshimi; Mochizuki, Manabu

    2009-10-15

    Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

  17. Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

    Science.gov (United States)

    Bhatti, L; Behle, K; Stevens, R H

    1992-10-01

    Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number

  18. Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    Science.gov (United States)

    Mossberg, Ann-Kristin; Wullt, Björn; Gustafsson, Lotta; Månsson, Wiking; Ljunggren, Eva; Svanborg, Catharina

    2007-09-15

    We studied if bladder cancers respond to HAMLET (human alpha-lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native alpha-lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers. (c) 2007 Wiley-Liss, Inc.

  19. 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.

  20. Characteristics and mechanisms of the bystander response in monolayer cell cultures exposed to very low fluences of alpha particles

    International Nuclear Information System (INIS)

    Little, John B.; Azzam, Edouard I.; Toledo, Sonia M. de; Nagasawa, Hatsumi

    2005-01-01

    When confluent cultures of mammalian cells are irradiated with very low fluences of alpha particles whereby only occasional cells receive any radiation exposure, genetic changes are observed in the non-irradiated ('bystander') cells. Upregulation of the p53 damage-response pathway as well as activation of proteins in the MAPK family occurred in bystander cells; p53 was phosphorylated on the serine 15 residue suggesting that the upregulation of p53 was a consequence of DNA damage. Damage signals were transmitted to bystander cells through gap junctions, as confirmed by the use of genetically manipulated cells including connexin43 knockouts. Expression of connexin43 was markedly enhanced by irradiation. A moderate bystander effect was observed for specific gene mutations and chromosomal aberrations. This effect was markedly enhanced in cells defective in the non-homologous end joining DNA repair pathway. Finally, an upregulation of oxidative metabolism occurred in bystander cells; the increased levels of reactive oxygen species appeared to be derived from flavine-containing oxidase enzymes. We hypothesize that genetic effects observed in non-irradiated bystander cells are a consequence of oxidative base damage; >90% of mutations in bystander cells were point mutations. When bystander cells cannot repair DNA double strand breaks, they become much more sensitive to the induction of chromosomal aberrations and mutations, the latter consisting primarily of deletion mutants. While we propose that the genetic effects occurring in bystander cells are a consequence of oxidative stress, the nature of the signal that initiates this process remains to be determined

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Fabrication of substrates with curvature for cell cultivation by alpha-particle irradiation and chemical etching of PADC films

    International Nuclear Information System (INIS)

    Ng, C.K.M.; Tjhin, V.T.; Lin, A.C.C.; Cheng, J.P.; Cheng, S.H.; Yu, K.N.

    2012-01-01

    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.

  6. Differential modulation of a radiation-induced bystander effect in glioblastoma cells by pifithrin-alpha and wortmannin

    Energy Technology Data Exchange (ETDEWEB)

    Shao Chunlin, E-mail: clshao@shmu.edu.c [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China); Zhang Jianghong [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China); Prise, Kevin M. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Lisburn Road, Belfast BT9 7AB (United Kingdom)

    2010-03-15

    The implication of radiation-induced bystander effect (RIBE) for both radiation protection and radiotherapy has attracted significant attention, but a key question is how to modulate the RIBE. The present study found that, when a fraction of glioblastoma cells in T98G population were individually targeted with precise helium particles through their nucleus, micronucleus (MN) were induced and its yield increased non-linearly with radiation dose. After co-culturing with irradiated cells, additional MN could be induced in the non-irradiated bystander cells and its yield was independent of irradiation dose, giving direct evidence of a RIBE. Further results showed that the RIBE could be eliminated by pifithrin-alpha (p53 inhibitor) but enhanced by wortmannin (PI3K inhibitor). Moreover, it was found that nitric oxide (NO) contributed to this RIBE, and the levels of NO of both irradiated cells and bystander cells could be extensively diminished by pifithrin-alpha but insignificantly reduced by wortmannin. Our results indicate that RIBE can be modulated by p53 and PI3K through a NO-dependent and NO-independent pathway, respectively.

  7. 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...

  8. A case of reversible dilated cardiomyopathy after alpha-interferon therapy in a patient with renal cell carcinoma.

    Science.gov (United States)

    Kuwata, Akiko; Ohashi, Masuo; Sugiyama, Masaya; Ueda, Ryuzo; Dohi, Yasuaki

    2002-12-01

    A 47-year-old man with renal cell carcinoma underwent nephrectomy, and postoperative chemotherapy was performed with recombinant alpha-interferon. Five years later, he experienced dyspnea during physical exertion. An echocardiogram revealed dilatation and systolic dysfunction of the left ventricle, and thallium-201 myocardial scintigraphy showed diffuse heterogeneous perfusion. We diagnosed congestive heart failure because of cardiomyopathy induced by alpha-interferon therapy. Withdrawal of interferon therapy and the combination of an angiotensin-converting enzyme inhibitor, diuretics, and digitalis improved left ventricular systolic function. Furthermore, myocardial scintigraphy using [123I] beta-methyl-p-iodophenylpentadecanoic acid (123I-BMIPP) or [123 I]metaiodobenzylguanidine (123I-MIBG) revealed normal perfusion after the improvement of congestive heart failure. This is a rare case of interferon-induced cardiomyopathy that resulted in normal myocardial images in 123I-BMIPP and 123I-MIBG scintigrams after withdrawal of interferon therapy.

  9. The effect of incorporation of SDF-1alpha into PLGA scaffolds on stem cell recruitment and the inflammatory response.

    Science.gov (United States)

    Thevenot, Paul T; Nair, Ashwin M; Shen, Jinhui; Lotfi, Parisa; Ko, Cheng-Yu; Tang, Liping

    2010-05-01

    Despite significant advances in the understanding of tissue responses to biomaterials, most implants are still plagued by inflammatory responses which can lead to fibrotic encapsulation. This is of dire consequence in tissue engineering, where seeded cells and bioactive components are separated from the native tissue, limiting the regenerative potential of the design. Additionally, these interactions prevent desired tissue integration and angiogenesis, preventing functionality of the design. Recent evidence supports that mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) can have beneficial effects which alter the inflammatory responses and improve healing. The purpose of this study was to examine whether stem cells could be targeted to the site of biomaterial implantation and whether increasing local stem cell responses could improve the tissue response to PLGA scaffold implants. Through incorporation of SDF-1alpha through factor adsorption and mini-osmotic pump delivery, the host-derived stem cell response can be improved resulting in 3X increase in stem cell populations at the interface for up to 2 weeks. These interactions were found to significantly alter the acute mast cell responses, reducing the number of mast cells and degranulated mast cells near the scaffold implants. This led to subsequent downstream reduction in the inflammatory cell responses, and through altered mast cell activation and stem cell participation, increased angiogenesis and decreased fibrotic responses to the scaffold implants. These results support that enhanced recruitment of autologous stem cells can improve the tissue responses to biomaterial implants through modifying/bypassing inflammatory cell responses and jumpstarting stem cell participation in healing at the implant interface. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. 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

  11. 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

    Mesenchymal-like cells in the stroma of breast cancer may arise as a consequence of plasticity within the epithelial compartment, also referred to as epithelial-mesenchymal transition, or by recruitment of genuine mesenchymal cells from the peritumoral stroma. Cells of both the epithelial...... 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...

  12. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    -alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

  13. PKC-alpha modulation by miR-483-3p in platinum-resistant ovarian carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Arrighetti, Noemi, E-mail: Noemi.Arrighetti@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Cossa, Giacomo, E-mail: Gia.Cossa@gmail.com [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); De Cecco, Loris, E-mail: Loris.Dececco@istitutotumori.mi.it [Functional Genomics and Bioinformatics, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Stucchi, Simone, E-mail: Simone.Stucchi@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Carenini, Nives, E-mail: Nives.Carenini@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Corna, Elisabetta, E-mail: Elisabetta.Corna@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Gandellini, Paolo, E-mail: Paolo.Gandellini@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Zaffaroni, Nadia, E-mail: Nadia.Zaffaroni@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Perego, Paola, E-mail: paola.perego@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Gatti, Laura, E-mail: Laura.Gatti@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy)

    2016-11-01

    The occurrence of drug resistance limits the efficacy of platinum compounds in the cure of ovarian carcinoma. Since microRNAs (miRNAs) may contribute to this phenomenon by regulating different aspects of tumor cell response, the aim of this study was to exploit the analysis of expression of miRNAs in platinum sensitive/resistant cells in an attempt to identify potential regulators of drug response. MiR-483-3p, which may participate in apoptosis and cell proliferation regulation, was found up-regulated in 4 platinum resistant variants, particularly in the IGROV-1/Pt1 subline, versus parental cells. Transfection of a synthetic precursor of miR-483-3p in IGROV-1 parental cells elicited a marked up-regulation of the miRNA levels. Growth-inhibition and colony-forming assays indicated that miR-483-3p over-expression reduced cell growth and conferred mild levels of cisplatin resistance in IGROV-1 cells, by interference with their proliferative potential. Predicted targets of miR-483-3p included PRKCA (encoding PKC-alpha), previously reported to be associated to platinum-resistance in ovarian carcinoma. We found that miR-483-3p directly targeted PRKCA in IGROV-1 cells. In keeping with this finding, cisplatin sensitivity of IGROV-1 cells decreased upon molecular/pharmacological inhibition of PKC-alpha. Overall, our results suggest that overexpression of miR-483-3p by ovarian carcinoma platinum-resistant cells may interfere with their proliferation, thus protecting them from DNA damage induced by platinum compounds and ultimately representing a drug-resistance mechanism. The impairment of cell growth may account for low levels of drug resistance that could be relevant in the clinical setting. - Highlights: • miR-483-3p is up-regulated in ovarian carcinoma cells resistant to platinum drugs. • Ectopic expression of miR-483-3p in IGROV-1 confers mild levels of Pt-resistance. • Overexpression of miR-483-3p down-regulates PRKCA levels in ovarian carcinoma cells. • miR 483

  14. 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...

  15. Epigenetic Basis for the Regulation of Estrogen Receptor Alpha Activity in Breast Cancer Cells

    Science.gov (United States)

    2009-04-01

    Contreras, J.I., Prescott , M.S., Dagenais, S.L., Wu, R., Yee, J., Orringer, M.B., Misek, D.E., Hanash, S.M., et al. (2002). The hepatocyte nuclear... Microbiology . All Rights Reserved. Coactivator Function Defines the Active Estrogen Receptor Alpha Cistrome† Mathieu Lupien,1‡ Jérôme Eeckhoute,1

  16. 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

  17. Functional inhibition of NF-kappa B signal transduction in alpha v alpha beta 3 integrin expressing endothelial cells by using RGD-PEG-modified adenovirus with a mutant I kappa B gene

    NARCIS (Netherlands)

    Ogawara, K; Kuldo, JM; Oosterhuis, K; Kroesen, BJ; Rots, MG; Trautwein, C; Kimura, T; Haisma, HJ; Molema, G

    2006-01-01

    In order to selectively block nuclear factor kappa B (NF-kappa B)-dependent signal transduction in angiogenic endothelial cells, we constructed an alpha v beta 3 integrin specific adenovirus encoding dominant negative I kappa B (dnI kappa B) as a therapeutic gene. By virtue of RGD modification of

  18. Preparation of. alpha. -deuterated L-amino acids using E. coli cells containing tryptophanase. Poluchenie. alpha. -dejterirovannykh L-aminokislot s ispol'zovaniem kletok E. coli, soderzhashchikh triptofanazy

    Energy Technology Data Exchange (ETDEWEB)

    Faleev, N G; Ruvinov, S B; Saporovskaya, M B; Belikov, V M; Zakomyrdina, L N; Sakharova, I S; Torchinskij, Yu M [AN SSSR, Moscow (USSR). Inst. Ehlementoorganicheskikh Soedinenij AN SSSR, Moscow (USSR). Inst. Molekulyarnoj Biologii AN SSSR, Moscow (USSR)

    1989-10-01

    Method for preparation of a series of {alpha}-deuterated L-amino acids of high optical purity with quantitative chemica yield, suing stereospecific isotopic exchange in D{sub 2}O under the effect of E.coli cells with high tryptophanase activity was developed.

  19. 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.

  20. 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.

  1. A peptide derived from alpha-fetoprotein inhibits the proliferation induced by estradiol in mammary tumor cells in culture.

    Science.gov (United States)

    Sierralta, Walter D; Epuñan, Maria J; Reyes, José M; Valladares, Luis E; Andersen, Thomas T; Bennett, James A; Jacobson, Herbert I; Pino, Ana M

    2008-01-01

    This study was aimed to obtain additional information on the activity of a cyclized 9-amino acid peptide (cP) containing the active site of alpha fetoprotein, which inhibits the estrogen-stimulated proliferation of tumor cells in culture and of xenografts in immunodeficient mice. Breast cancer cells cultured in the presence of 2 nM estradiol were exposed to cP for different periods and their proliferation, estradiol binding parameters, clustering tendency and expression of E-cadherin and p21Cip1 were analyzed by biochemical and cell biology methods. The proliferation of MCF7 cells was significantly decreased by the addition of 2 microg/ml cP to the medium. cP did not increase cell death rate nor alter the number of binding sites for estradiol nor the endogenous aromatase activity of MCF7 cells. cP also decreased the proliferation of estrogen-dependent ZR75-1 cells but had no effect on estrogen-independent MDA-MB-231 cells. An increased nuclear p21Cip1 expression detected after cP treatment suggests that cP slows MCF7 cell proliferation via this regulator. We propose that cP could represent a novel breast cancer therapeutic agent whose mechanism of action is different from that of tamoxifen or of inhibitors of aromatase.

  2. 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

  3. 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

    cytometry and analysed by RNA sequencing. The role of the ghrelin receptor was validated by imaging delta cell calcium concentrations using islets with delta cell restricted expression of the calcium reporter GCaMP3, and in perfused mouse pancreases. RESULTS: A database was constructed of all genes...... 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...

  4. A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

    Science.gov (United States)

    Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

    2009-06-01

    The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

  5. Distance distribution of bystander effects in alpha-particle irradiated cell populations using a CR-39-based culture dish

    International Nuclear Information System (INIS)

    Gaillard, S.; Pusset, D.; Toledo, S.M. de; Azzam, E.I.; Fromm, M.

    2008-01-01

    Propagation of induced biological effects from irradiated to non-irradiated cells is known to occur in cell cultures exposed to low fluences of charged particles. These bystander effects are currently investigated using microbeam or non-microbeam (broad beams) irradiation techniques. Identification of the targeted and non-targeted bystander cells is critical to our understanding of mechanisms underlying such effects. We developed a novel cell culture dish where the base consists of a thin CR-39 sheet grafted on a thin polyethylene terephthalate (PET) foil. The validity of this device in identifying not only irradiated cells, but also the cellular compartment traversed by the particle track is described. We have optimized track etch parameters that do not interfere with measurement of induced biological endpoints under normal incident irradiation. Thus the culture dishes can be used to determine distance distributions for the propagation of induced biological effects from a hit cell to bystander cells. We describe the computer code developed to determine the distance distributions of propagated biological stress responses in normal human fibroblast cells exposed to very low fluences of alpha particles

  6. Immunoreactivity for alpha-smooth muscle actin characterizes a potentially aggressive subgroup of little basal cell carcinomas

    Directory of Open Access Journals (Sweden)

    G Faa

    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.

  7. Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

    International Nuclear Information System (INIS)

    Paessler, Slobodan; Yun, Nadezhda E.; Judy, Barbara M.; Dziuba, Natallia; Zacks, Michele A.; Grund, Anna H.; Frolov, Ilya; Campbell, Gerald A.; Weaver, Scott C.; Estes, D. Mark

    2007-01-01

    We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (αβ) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta (γδ) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (μMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3 + T cells are required for protection

  8. Targeted alpha therapy in vivo: direct evidence for single cancer cell kill using 149Tb-rituximab

    International Nuclear Information System (INIS)

    Beyer, G.J.; Soloviev, D.; Buchegger, F.; Miederer, M.; Vranjes-Duric, S.; Comor, J.J.; Kuenzi, G.; Hartley, O.; Senekowitsch-Schmidtke, R.

    2004-01-01

    This study demonstrates high-efficiency sterilisation of single cancer cells in a SCID mouse model of leukaemia using rituximab, a monoclonal antibody that targets CD20, labelled with terbium-149, an alpha-emitting radionuclide. Radio-immunotherapy with 5.5 MBq labelled antibody conjugate (1.11 GBq/mg) 2 days after an intravenous graft of 5.10 6 Daudi cells resulted in tumour-free survival for >120 days in 89% of treated animals. In contrast, all control mice (no treatment or treated with 5 or 300 μg unlabelled rituximab) developed lymphoma disease. At the end of the study period, 28.4%±4% of the long-lived daughter activity remained in the body, of which 91.1% was located in bone tissue and 6.3% in the liver. A relatively high daughter radioactivity concentration was found in the spleen (12%±2%/g), suggesting that the killed cancer cells are mainly eliminated through the spleen. This promising preliminary in vivo study suggests that targeted alpha therapy with 149 Tb is worthy of consideration as a new-generation radio-immunotherapeutic approach. (orig.)

  9. 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...

  10. 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.

  11. The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis

    DEFF Research Database (Denmark)

    Danen, Erik H J; Sonneveld, Petra; Brakebusch, Cord

    2002-01-01

    We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels ...... receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.......We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels...... of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated...

  12. 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

  13. 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

  14. The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

    Science.gov (United States)

    Mizejewski, G J

    2016-09-01

    The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

  15. Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

    Science.gov (United States)

    Fischer, Walter; Gustafsson, Lotta; Mossberg, Ann-Kristin; Gronli, Janne; Mork, Sverre; Bjerkvig, Rolf; Svanborg, Catharina

    2004-03-15

    Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

  16. The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

    Science.gov (United States)

    Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

    1998-12-01

    To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

  17. 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

  18. Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

    Science.gov (United States)

    Liu, Hong; Xu, Yiyang; Xiang, Jingyi; Long, Li; Green, Shon; Yang, Zhiyuan; Zimdahl, Bryan; Lu, Jingwei; Cheng, Neal; Horan, Lucas H; Liu, Bin; Yan, Su; Wang, Pei; Diaz, Juan; Jin, Lu; Nakano, Yoko; Morales, Javier F; Zhang, Pengbo; Liu, Lian-Xing; Staley, Binnaz K; Priceman, Saul J; Brown, Christine E; Forman, Stephen J; Chan, Vivien W; Liu, Cheng

    2017-01-15

    The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP 158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01 + /AFP + while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP 158 -expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR. ©2016 American Association for Cancer Research.

  19. Alpha-fetoprotein-producing ovarian clear cell adenocarcinoma with fetal gut differentiation: a rare case report and literature review.

    Science.gov (United States)

    Chao, Wei-Ting; Liu, Chia-Hao; Lai, Chiung-Ru; Chen, Yi-Jen; Chuang, Chi-Mu; Wang, Peng-Hui

    2018-06-22

    Alpha-fetoprotein (AFP) is a useful tumor marker for ovarian germ cell tumors, particularly yolk sac tumor (YST). It is valuable for both diagnosis and further follow-up. Epithelial ovarian carcinoma (EOC) rarely secretes AFP, especially for clear cell type and in the postmenopausal women. Based on the limited knowledge about AFP-producing clear cell type EOC, a case and literature review on this topic is extensively reviewed. We report a 55-year-old postmenopausal woman experienced vaginal spotting for one month, and serum level of AFP was 60,721 ng/ml initially. Histological examination was clear cell type EOC. Tumor cells revealed strong immunoreactivity for glypican-3 (GPC3) and AFP and weak for hepatocyte nuclear factor-1 beta (HNF-1 beta), but negative for CD30, making the diagnosis of AFP-producing clear cell type EOC with fetal gut differentiation in focal areas, FIGO (International Federation of Gynecology and Obstetrics) IIIc. Although the patient underwent an intensive treatment, including optimal debulking surgery and multi-agent chemotherapy, the patient died of disease. To provide a better understanding of clinical and molecular characteristics of the AFP-producing clear cell type EOC, we conducted a systematic literature review. A total of three papers described the AFP-producing clear cell type EOC are available. The overall survival rate of these cases, including the current case is 50%. Although immunohistochemical examination is not always needed in routine for the diagnosis of clear cell type EOC, to distinguish from other tumors, especially germ cell tumors, or to provide the better way to monitor therapeutic response or to evaluate the disease status, immunostaining, including GPC3, HNF-1 beta, CD30, cytokeratin 7 or 20, and AFP is taken into account. Due to rarity, the appropriate chemotherapy regimen and the biological behavior of AFP-producing clear cell type EOC are still unclear.

  20. 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

  1. 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

  2. Cdc25A promotes cell survival by stimulating NF-{kappa}B activity through I{kappa}B-{alpha} phosphorylation and destabilization

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Hey-Young; Choi, Jiyeon [Department of Biochemistry, College of Natural Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 200-701 (Korea, Republic of); Cho, Young-Wook [Korea Basic Science Institute, Chuncheon Center, Gangwondaehak-gil 1, Chuncheon 200-701 (Korea, Republic of); Kim, Byung-Chul, E-mail: bckim@kangwon.ac.kr [Department of Biochemistry, College of Natural Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 200-701 (Korea, Republic of)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer We examine the antiapoptotic mechanisms of Cdc25A. Black-Right-Pointing-Pointer Smad7 decreases the phosphorylation of I{kappa}B-alpha at Ser-32. Black-Right-Pointing-Pointer Smad7 positively regulates NF-{kappa}B activity through I{kappa}B-alpha ubiquitination. -- Abstract: Cell division cycle 25A (Cdc25A), a dual specificity protein phosphatase, exhibits anti-apoptotic activity, but the underlying molecular mechanisms are poorly characterized. Here we report that Cdc25A inhibits cisplatin-induced apoptotic cell death by stimulating nuclear factor-kappa B (NF-{kappa}B) activity. In HEK-293 cells, Cdc25A decreased protein level of inhibitor subunit kappa B alpha (I{kappa}-B{alpha}) in association with increased serine 32-phosphorylation, followed by stimulation of transcriptional activity of NF-{kappa}B. Inhibition of NF-{kappa}B activity by chemical inhibitor or overexpression of I{kappa}-B{alpha} in Cdc25A-elevated cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Our data show for the first time that Cdc25A has an important physiological role in NF-{kappa}B activity regulation and it may be an important survival mechanism of cancer cells.

  3. A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

    Science.gov (United States)

    MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

    2007-06-01

    Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

  4. A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

    Directory of Open Access Journals (Sweden)

    Patrick E MacDonald

    2007-06-01

    Full Text Available Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+ responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+ signalling was blocked, but was reversed by low concentrations (1-20 muM of the ATP-sensitive K(+ (KATP channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM. Higher diazoxide concentrations (>/=30 muM decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM, glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+ (TTX and N-type Ca(2+ channels (omega-conotoxin, but not L-type Ca(2+ channels (nifedipine, prevented glucagon secretion. Both the N-type Ca(2+ channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

  5. Transcription regulation of the alpha-glucanase gene agn1 by cell separation transcription factor Ace2p in fission yeast

    NARCIS (Netherlands)

    Dekker, Nick; de Haan, Annett; Hochstenbach, Frans

    2006-01-01

    During the final stage of the cell division cycle in the fission yeast Schizosaccharomyces pombe, transcription factor Ace2p activates expression of genes involved in the separation of newly formed daughter cells, such as agn1+, which encodes the alpha-glucanase Agn1p. The agn1 promoter contains

  6. 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...

  7. 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.

  8. 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.

  9. Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

    Science.gov (United States)

    Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

    2010-01-01

    Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1. Copyright 2009 Elsevier Inc. All rights reserved.

  10. 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.

  11. Alpha-ketoglutarate enhances freeze-thaw tolerance and prevents carbohydrate-induced cell death of the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Bayliak, Maria M; Hrynkiv, Olha V; Knyhynytska, Roksolana V; Lushchak, Volodymyr I

    2018-01-01

    Stress resistance and fermentative capability are important quality characteristics of baker's yeast. In the present study, we examined protective effects of exogenous alpha-ketoglutarate (AKG), an intermediate of the tricarboxylic acid cycle and amino acid metabolism, against freeze-thaw and carbohydrate-induced stresses in the yeast Saccharomyces cerevisiae. Growth on AKG-supplemented medium prevented a loss of viability and improved fermentative capacity of yeast cells after freeze-thaw treatment. The cells grown in the presence of AKG had higher levels of amino acids (e.g., proline), higher metabolic activity and total antioxidant capacity, and higher activities of catalase, NADP-dependent glutamate dehydrogenase and glutamine synthase compared to control ones. Both synthesis of amino acids and enhancement of antioxidant system capacity could be involved in AKG-improved freeze-thaw tolerance in S. cerevisiae. Cell viability dramatically decreased under incubation of stationary-phase yeast cells in 2% glucose or fructose solutions (in the absence of the other nutrients) as compared with incubation in distilled water or in 10 mM AKG solution. The decrease in cell viability was accompanied by acidification of the medium, and decrease in cellular respiration, aconitase activity, and levels of total protein and free amino acids. The supplementation with 10 mM AKG effectively prevented carbohydrate-induced yeast death. Protective mechanisms of AKG could be associated with the intensification of respiration and prevention of decreasing protein level as well as with direct antioxidant AKG action.

  12. Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable. alpha. -amylases and pullulanases

    Energy Technology Data Exchange (ETDEWEB)

    Klingeberg, M [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Vorlop, K D [Technische Univ. Braunschweig (Germany, F.R.). Inst. fuer Technische Chemie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    For the production of cell-free thermostable {alpha}-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full was well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60deg C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10{sup 12} cells up to 700 U/10{sup 12} cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450 000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. (orig.).

  13. Critical roles of mucin-1 in sensitivity of lung cancer cells to tumor necrosis factor-alpha and dexamethasone.

    Science.gov (United States)

    Xu, Menglin; Wang, Xiangdong

    2017-08-01

    Lung cancer is the leading cause of death from cancer. Mucins are glycoproteins with high molecular weight, responsible for cell growth, differentiation, and signaling, and were proposed to be correlated with gene heterogeneity of lung cancer. Here, we report aberrant expression of mucin genes and tumor necrosis factor receptors in lung adenocarcinoma tissues compared with normal tissues in GEO datasets. Mucin-1 (MUC1) gene was selected and considered as the target gene; furthermore, the expression pattern of adenocarcinomic cells (A549, H1650, or H1299 cells) was validated under the stimulation with tumor necrosis factor-alpha (TNFα) or dexamethasone (DEX), separately. MUC1 gene interference was done to A549 cells to show its role in sensitivity of lung cancer cells to TNFα and DEX. Results of our experiments indicate that MUC1 may regulate the influence of inflammatory mediators in effects of glucocorticoids (GCs), as a regulatory target to improve therapeutics. It shows the potential effect of MUC1 and GCs in lung adenocarcinoma (LADC), which may help in LADC treatment in the future.

  14. 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

  15. Ovarian Sertoli-Leydig cell tumor with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review.

    Science.gov (United States)

    Horta, Mariana; Cunha, Teresa Margarida; Marques, Rita Canas; Félix, Ana

    2014-11-01

    Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker.

  16. Oligoclonal CD8+ T-cell expansion in patients with chronic hepatitis C is associated with liver pathology and poor response to interferon-alpha therapy.

    Science.gov (United States)

    Manfras, Burkhard J; Weidenbach, Hans; Beckh, Karl-Heinz; Kern, Peter; Möller, Peter; Adler, Guido; Mertens, Thomas; Boehm, Bernhard O

    2004-05-01

    The role of CD8(+) T lymphocytes in chronic hepatitis C virus (HCV) infection and in liver injury with subsequent development of fibrosis and cirrhosis is poorly understood. To address this question, we performed a follow-up study including 27 chronically HCV-infected individuals. We determined clonality and phenotypes of circulating CD8(+) T cells employing TCRBV spectratyping. Antigen specificity was tested by rMHC-peptide tetramer staining and stimulation with recombinant HCV antigens. In addition, T-cell clonality and phenotypes were followed during the variable clinical response of interferon- (IFN) alpha treatment. We could demonstrate that CD8(+) T-cell expansions were significantly associated with liver fibrosis and cirrhosis. Likewise, increased oligoclonality of circulating CD8(+) T cells in chronic HCV infection was identified as an indicator for poor clinical response to IFN-alpha therapy. Moreover, we also found that IFN-alpha therapy enhanced the differentiation of CD8(+) T cells towards a late differentiation phenotype (CD28(-) CD57(+)). In cases of virus elimination the disappearance of expanded terminally differentiated CD8(+) cells was observed. Thus, this study identifies an association of clonal expansions of circulating CD8(+) T cells with liver pathology and provides a possible explanation for the fact that response to IFN-alpha therapy diminishes with the duration of infection.

  17. In vitro secretion of TNF-{alpha} from bone marrow mononuclear cells incubated on amino group modified TiO{sub 2} nano-composite under ultrasound irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Furuzono, T., E-mail: furuzono@ri.ncvc.go.jp [Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565 (Japan); Masuda, M. [Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565 (Japan); Nitta, N.; Kaya, A.; Yamane, T. [Institute for Human Science and Biomedical Engineering, National Institute of Advanced Industrial Science and Technology, 1-2-1 Namiki, Tsukuba, Ibaraki, 305-8564 (Japan); Okada, M. [Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565 (Japan)

    2010-10-15

    It is recently known that titanium dioxide (TiO{sub 2}) can be excited by ultrasound and release of OH radicals on the surface. In this study, secretion of an indirect angiogenic factor, tumor necrosis factor-{alpha} (TNF-{alpha}), from bone marrow mononuclear cells (BM-MNC) incubated on amino group modified TiO{sub 2} nano-particles covalently coated on polyester fabric (TiO{sub 2}/PET) under ultrasonic irradiation was examined in vitro. The cell viability and TNF-{alpha} secretion were measured under ultrasound irradiation condition with 255 mW/cm{sup 2} of intensity, which is below the highest output (1 W/cm{sup 2}) specified in the safety standard for a medical ultrasonic diagnostic apparatus. The living cell number on the TiO{sub 2}/PET and original PET with/without continuous ultrasound irradiation was unchanged statistically by ANOVA test. TNF-{alpha} secretion level from BM-MNC remarkably increased on the TiO{sub 2}/PET under ultrasonic irradiation without cell damage. It was, therefore, thought that the high level of TNF-{alpha} secretion on the TiO{sub 2} nano-composite by ultrasound irradiation was due to oxidative stress induced from OH radicals on TiO{sub 2}.

  18. Addition of interferon-alpha to a standard maturation cocktail induces CD38 up-regulation and increases dendritic cell function

    DEFF Research Database (Denmark)

    Trepiakas, Redas; Pedersen, Anders Elm; Met, Ozcan

    2009-01-01

    Monocyte-derived dendritic cells (DCs) are used as adjuvant cells in cancer immunotherapy and have shown promising results. In order to obtain full functional capacity, these DCs need to be maturated, and the current "gold standard" for this process is maturation with TNF-alpha, IL-1beta, IL-6...... a functional relationship between CD38, IFN-alpha and TLR3. Thus, CD38 appear to be a relevant marker for activation by TLR3 or IFN-alpha. Addition of IFN-alpha to the sDC cocktail results in up-regulation of both CD38 and CD83 and improved capacity for induction of autologous T-cell responses despite few...... other changes in DC phenotype and cytokine secretion. Our observations suggest that IFN-alpha could be included in maturation protocols for clinical grade DCs used for immunotherapy against cancer and should be included if DCs are used for CD8+ T-cell stimulation in vitro....

  19. 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

  20. 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.

  1. 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.

  2. The combination of MLN2238 (ixazomib) with interferon-alpha results in enhanced cell death in melanoma.

    Science.gov (United States)

    Suarez-Kelly, Lorena P; Kemper, Gregory M; Duggan, Megan C; Stiff, Andrew; Noel, Tiffany C; Markowitz, Joseph; Luedke, Eric A; Yildiz, Vedat O; Yu, Lianbo; Jaime-Ramirez, Alena Cristina; Karpa, Volodymyr; Zhang, Xiaoli; Carson, William E

    2016-12-06

    The ubiquitin-proteasome signaling pathway is critical for cell cycle regulation and neoplastic growth. Proteasome inhibition can activate apoptotic pathways. Bortezomib, a selective proteasome inhibitor, has anti-melanoma activity. MLN2238 (ixazomib), an oral proteasome inhibitor, has improved pharmacotherapeutic parameters compared to bortezomib. Interferon-alpha (IFN-α), an immune boosting agent, is FDA-approved for treatment of melanoma. In this study in vitro and in vivo evaluation of the antitumor potential of ixazomib and combination treatments with ixazomib and IFN-α were performed. Apoptosis induced by ixazomib was first observed at 12 hours and was maximal at 48 hours with similar levels of cell death compared to bortezomib. IFN-α alone had little effect on cell viability in vitro. However, the combination of ixazomib with IFN-α significantly enhanced ixazomib's ability to induce apoptotic cell death in BRAF V600E mutant and BRAF wild-type human melanoma tumor cells. The combination of ixazomib and IFN-α also enhanced inhibition of cell proliferation in BRAF V600E mutant melanoma tumor cells; however, this was not seen in BRAF wild-type cells. Ixazomib-induced apoptosis was associated with processing of the pro-apoptotic proteins procaspase-3, -7, -8, and -9, and cleavage of poly-ADP-ribose polymerase (PARP). In an in vivo xenograft model of human melanoma, combination treatment with IFN-α-2b and ixazomib demonstrated a significant reduction in tumor volume when compared to vehicle (p = 0.005) and single therapy ixazomib (p = 0.017) and IFN-α-2b (p = 0.036). These pre-clinical results support further evaluation of combination treatment with ixazomib and IFN-α for the treatment of advanced BRAF V600E mutant melanoma.

  3. Evaluation of polymer-coated CsI:Tl as an alpha/beta pulse shape discriminating flow-cell

    International Nuclear Information System (INIS)

    Branton, S.D.; Fjeld, R.A.; DeVol, T.A.

    1996-01-01

    A pulse shape discriminating flow-cell radiation detection system constructed with polymer coated CsI:Tl was evaluated for simultaneous gross alpha/gross beta quantification. The CsI:TI scintillator was crushed and sieved to 63-90 μm particle size and microencapsulated with Parylene C to reduce its rate of dissolution. Averaged over the first hour of use, the pulse shape discrimination figure-of-merit was 1.4 and the detection efficiencies were 64.9 ± 5.7 %, 52.5 ± 4.5 % and 4.5 ± 0.2 % for 233 U, 90 Sr/ 90 Y and 14 C , respectively. The typical background count rate in the alpha and beta pulse shape window was 0.17 and 0.004 cps, respectively. The resultant minimum detectable activity for a 30 second count time was calculated to be 0.19 ± 0.01 Bq, 0.9 ± 0.1 Bq and 11.4 ± 0.6 Bq for 233 U, 90 Sr/ 90 Y and 14 C, respectively. Although the 3 μm thick microencapsulation reduced CsI:Tl dissolution, the detection efficiency declined by a factor of two after 4.8 hours while the pulse shape resolution degraded slightly

  4. Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

    Science.gov (United States)

    Conacci-Sorrell, Maralice; Ngouenet, Celine; Eisenman, Robert N

    2010-08-06

    The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation. Copyright 2010 Elsevier Inc. All rights reserved.

  5. 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.

  6. Effects of alpha fetoprotein on escape of Bel 7402 cells from attack of lymphocytes

    International Nuclear Information System (INIS)

    Li, Mengsen; Liu, Xinhua; Zhou, Sheng; Li, Pingfeng; Li, Gang

    2005-01-01

    Involvement of AFP against apoptosis of tumor cell has been implicated in its evasion of immune surveillance. However, the molecular events of immune escape mechanisms are still unknown. The major observations reported here relate to a possible mechanism by which heptoloma Bel 7402 cells escape immune surveillance in vitro. Western blotting and a well-characterized cofocal scanning image were performed to analyze the expression of Fas/FasL and caspase-3 in co-cultured Bel 7402 and Jurkat cells. After co-culture with Jurkat cells, up-regulated Fas and reduced FasL expression could be observed. Treatment with AFP could remarkably inhibit the elevated Fas and, whereas, induce the FasL expression in co-cultured Bel 7402 cells. Cells co-culture could induce the expression of caspase-3 in both cells line. The elevated caspase-3 in Bel 7402 cells was abolished following the treatment of AFP. The expression of caspase-3 was elevated in co-cultured Jurkat cells treated with AFP. No detectable change on the expression of survivin was examined in both cells line. Monoclonal antibody against AFP treatment alone did not obviously influence the growth of cells, as well as the expression of Fas/FasL and caspase-3. However, the effect of AFP could be blocked by antibody. our results provide evidence that AFP could promote the escape of liver cancer cells from immune surveillance through blocking the caspase signal pathway of tumor cells and triggering the Fas/FasL interaction between tumor cells and lymphocytes

  7. 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.

  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. Alpha-fetoprotein is a biomarker of unfolded protein response and altered proteostasis in hepatocellular carcinoma cells exposed to sorafenib.

    Science.gov (United States)

    Houessinon, Aline; Gicquel, Albane; Bochereau, Flora; Louandre, Christophe; Nyga, Rémy; Godin, Corinne; Degonville, James; Fournier, Emma; Saidak, Zuzana; Drullion, Claire; Barbare, Jean-Claude; Chauffert, Bruno; François, Catherine; Pluquet, Olivier; Galmiche, Antoine

    2016-01-28

    Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. 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.

  11. Alpha2,3-sialyltransferase III knockdown sensitized ovarian cancer cells to cisplatin-induced apoptosis.

    Science.gov (United States)

    Wang, Xiaoyu; Zhang, Yiting; Lin, Haiyingjie; Liu, Yan; Tan, Yi; Lin, Jie; Gao, Fenze; Lin, Shaoqiang

    2017-01-22

    Emerging evidence indicates that β-galactoside-α2,3-sialyltransferase III (ST3Gal3) involves in development, inflammation, neoplastic transformation, and metastasis. However, the role of ST3Gal3 in regulating cancer chemoresistance remains elusive. Herein, we investigated the functional effects of ST3Gal3 in cisplatin-resistant ovarian cancer cells. We found that the levels of ST3Gal3 mRNA differed significantly among ovarian cancer cell lines. HO8910PM cells that have high invasive and metastatic capacity express elevated ST3Gal3 mRNA and are resistant to cisplatin, comparing to SKOV3 cells that have a lower level of ST3Gal3 expression and are more chemosensitive to cisplatin. We found that the expression of ST3Gal3 has reverse correlation with the dosage of cisplatin used in both SKOV3 and HO8910PM cells, and high dose of cisplatin could down-regulate ST3Gal3 expression. We then examined the functional effects of ST3Gal3 knockdown in cancer cell lines using FACS analysis. The number of apoptotic cells was much higher in cells if ST3Gal3 expression was knocked down by siRNA and/or by treating cells with higher dosage of cisplatin in comparison to control cells. Interestingly, in HO8910PM cells with ST3Gal3 knockdown, the levels of caspase 8 and caspase 3 proteins increased, which was more obvious in cells treated with both ST3Gal3 knockdown and cisplatin, suggesting that ST3Gal3 knockdown synergistically enhanced cisplatin-induced apoptosis in ovarian cancer cells. Taken together, these results uncover an alternative mechanism of cisplatin-resistance through ST3Gal3 and open a window for effective prevention of chemoresistance and relapse of ovarian cancer by targeting ST3Gal3. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. 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...

  13. 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 .

  14. 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.

  15. 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.

  16. Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

    Science.gov (United States)

    Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

    1992-08-05

    The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

  17. 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

    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...... CD14 and reduced CD1a on the cell surface. These VD3-treated DCs exert a long-lasting inefficient T cell stimulation and induce T cell hyporesponsiveness with regulatory potential. Importantly, such VD3-treated DCs were readily distinguishable from untreated DCs by low levels of interleukin-23...

  18. Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors.

    Science.gov (United States)

    Bkaily, Ghassan; El-Bizri, Nesrine; Bui, Michel; Sukarieh, Rami; Jacques, Danielle; Fu, Michael L X

    2003-03-01

    The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.

  19. 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

    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......, which is characterized by significant inflammation and neuroglial damage. We have recently shown that the exogenous administration of the antioxidant protein zinc-metallothionein-II (Zn-MT-II) significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE....... 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...

  20. Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

    International Nuclear Information System (INIS)

    Tachibana, Keisuke; Takeuchi, Kentaro; Inada, Hirohiko; Yamasaki, Daisuke; Ishimoto, Kenji; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Doi, Takefumi

    2009-01-01

    Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial β-oxidation. The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that plays an important role in the regulation of β-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPARα and found that PPARα induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPARα regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

  1. C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

    Science.gov (United States)

    Robertson, Danielle M; Ho, Su-Inn; Cavanagh, H Dwight

    2010-08-01

    In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

  2. Maternal and fetal mechanisms of B cell regulation during pregnancy: human Chorionic Gonadotropin stimulates B cells to produce IL-10 while alpha-fetoprotein drives them into apoptosis

    Directory of Open Access Journals (Sweden)

    Franziska Fettke

    2016-12-01

    Full Text Available Maternal immune tolerance towards the fetus is an essential requisite for pregnancy. While T cell functions are well documented, little is known about the participation of B cells. We have previously suggested that IL-10 producing B cells are involved in pregnancy tolerance in mice and humans. By employing murine and human systems, we report now that fetal trophoblasts positively regulate the generation of IL-10 producing B cells. We next studied the participation of hormones produced by the placenta as well as the fetal protein alpha-fetoprotein (AFP in B cell modulation. Human Chorionic Gonadotropin (hCG, but not progesterone, estrogen or a combination of both, was able to promote changes in B cell phenotype and boost their IL-10 production, which was abolished after blocking hCG. The hCG-induced B cell phenotype was not associated with augmented galactosylation, sialylation or fucosylation of IgG subclasses in their Fc. In vitro, hCG induced the synthesis of asymmetrically glycosylated antibodies in their Fab region. Interestingly, AFP had dual effects depending on the concentration. At concentrations corresponding to maternal serum levels, it did not modify the phenotype or IL-10 secretion of B cells. At fetal concentrations, however, AFP was able to drive B cells into apoptosis, which may indicate a protective mechanism to avoid maternal B cells to reach the fetus.Our data suggests that the fetus secrete factors that promote a pregnancy-friendly B cell phenotype, unraveling interesting aspects of B cell function and modulation by pregnancy hormones and fetal proteins.

  3. Melatonin suppresses activation of hepatic stellate cells through ROR alpha-mediated inhibition of 5-lipoxygenase

    NARCIS (Netherlands)

    Shajari, Shiva; Laliena, Almudena; Heegsma, Janette; Jesus Tunon, Maria; Moshage, Han; Faber, Klaas Nico

    2015-01-01

    Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to

  4. Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

    Science.gov (United States)

    Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

    2017-06-01

    To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

  5. Dismantling of an alpha contaminated hot cell at the Marcoule Pilot Plant

    International Nuclear Information System (INIS)

    Tachon, M.

    1988-01-01

    For the remodeling of Marcoule Pilot Plant, the cell 82: old unit for plutonium solution purification by extraction, was dismantled. About 42 tons of wastes were evacuated. Some wastes wen decontaminated by mechanical means other wastes with higher residual activity were stored for subsequent processing. The operation shows that dismantling of a hot cell is possible even if incorporated in an operating plant [fr

  6. Interferon-alpha subtype 11 activates NK cells and enables control of retroviral infection.

    Directory of Open Access Journals (Sweden)

    Kathrin Gibbert

    Full Text Available The innate immune response mediated by cells such as natural killer (NK cells is critical for the rapid containment of virus replication and spread during acute infection. Here, we show that subtype 11 of the type I interferon (IFN family greatly potentiates the antiviral activity of NK cells during retroviral infection. Treatment of mice with IFN-α11 during Friend retrovirus infection (FV significantly reduced viral loads and resulted in long-term protection from virus-induced leukemia. The effect of IFN-α11 on NK cells was direct and signaled through the type I IFN receptor. Furthermore, IFN-α11-mediated activation of NK cells enabled cytolytic killing of FV-infected target cells via the exocytosis pathway. Depletion and adoptive transfer experiments illustrated that NK cells played a major role in successful IFN-α11 therapy. Additional experiments with Mouse Cytomegalovirus infections demonstrated that the therapeutic effect of IFN-α11 is not restricted to retroviruses. The type I IFN subtypes 2 and 5, which bind the same receptor as IFN-α11, did not elicit similar antiviral effects. These results demonstrate a unique and subtype-specific activation of NK cells by IFN-α11.

  7. A gut-homing, oligoclonal CD4+ T cell population in severe-combined immunodeficient mice expressing a rearranged, transgenic class I-restricted alpha beta T cell receptor

    DEFF Research Database (Denmark)

    Reimann, J; Rudolphi, A; Spiess, S

    1995-01-01

    We studied the peripheral T cell compartment of H-2b severe combined immunodeficient (scid) mice that express a transgenic (tg) alpha beta T cell receptor (TcR) specific for the H-Y (male) epitope presented by the H-2 class I Db molecule. Large populations of CD3+ NK1.1-TCR beta T+ T cells were...

  8. Free fatty acids induce ER stress and block antiviral activity of interferon alpha against hepatitis C virus in cell culture

    Directory of Open Access Journals (Sweden)

    Gunduz Feyza

    2012-08-01

    Full Text Available Abstract Background Hepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C (CHC patients. The mechanism of response to interferon-alpha (IFN-α therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV replication and IFN-α antiviral response in a cell culture model. Methods Sub-genomic replicon (S3-GFP and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate and unsaturated (oleate long-chain free fatty acids (FFA. Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-α antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR. Results FFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP cell line. FFA treatment also partially blocked IFN-α response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-β promoter activation. We show that FFA treatment induces endoplasmic reticulum (ER stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response. Conclusion These results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN

  9. Protective effect of an alpha 7 nicotinic acetylcholine receptor agonist against enterovirus 71 infection in neuronal cells.

    Science.gov (United States)

    Song, Feng Xia; Zhao, Lin Qing; Zhu, Ru Nan; Song, Qin Wei; Deng, Jie; Tian, Run; Wang, Fang; Qian, Yuan

    2018-01-01

    Enterovirus 71, as one of the dominant pathogens associated with severe hand, foot, and mouth disease, has been well reported to trigger severe neurological symptoms among young children over the last decade, particularly among children in the Asia-Pacific region. To date, no effective antiviral agent has been developed for the treatment of severe enterovirus 71 infection. PNU-282987, a selective alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist, has been reported to have a neuroprotective effect by participating in inflammatory regulation in previous studies. Therefore, in the present study, we aimed to assess the cell-protective effect of PNU-282987 against enterovirus 71 infection in neuronal cells, and to discuss potential mechanisms underlying this cell-protective effect in order to elucidate the potential impact of such agonists in the treatment of neurotropic viral infection. We observed that treatment with PNU-282987 improved cell viability and inhibited viral replication in enterovirus 71-infected SH-SY5Y cells. Further investigation revealed that inhibition of enterovirus 71 production by PNU-282987 is likely associated with events of RNA replication, and that increased levels of INF mRNA and its downstream antiviral proteins stimulated by the JAK-STAT2 pathway may contribute to the antiviral effect of PNU-282987. Moreover, our findings suggest that both the antiviral and anti-inflammatory effects of PNU-282987 may contribute to the neural protective effect of the drug in enterovirus 71-infected cells. Taken together, the results suggest that selective α7nAChR agonists may represent viable candidates for future therapeutic treatment of severe enterovirus 71 infection, and for other cases of neurotropic viral infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Alpha-particles induce preneoplastic transformation of rat tracheal epithelial cells in culture

    International Nuclear Information System (INIS)

    Thomassen, D.G.; Seiler, F.A.; Shyr, L.-J.; Griffith, W.C.

    1990-01-01

    To characterize the potential role of high-l.e.t. radiation in respiratory carcinogenesis, the cytotoxic and transforming potency of 5.5 MeV α-particles from electroplated sources of 238 Pu were determined using primary cultures of rat tracheal epithelial cells. RBE for cell killing by α-particles versus X-rays varied with dose, and ranged between 4 and 1.5 for α doses in the range 0.2-4 Gy. At equally toxic doses (relative survival 0.18-0.2), all three agents induced similar frequencies of preneoplastic transformation. For preneoplastic transformation induced by doses of α- and X-radiations giving 80 per cent toxicity, an α RBE of 2.4 was derived. The similar RBEs for cell killing and for preneoplastic transformation suggest an association between the type or degree of radiation-induced damage responsible for both cell killing and cell transformation. (author)

  11. Interferon-alpha administration enhances CD8+ T cell activation in HIV infection.

    Directory of Open Access Journals (Sweden)

    Maura Manion

    Full Text Available Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006. These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy. As plasma HIV levels fell with interferon therapy, this was correlated with a "paradoxical" increase in CD8+ T cell activation (p<0.001.Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection.

  12. Interferon-alpha triggers B cell effector 1 (Be1 commitment.

    Directory of Open Access Journals (Sweden)

    Marie-Ghislaine de Goër de Herve

    Full Text Available B-cells can contribute to the pathogenesis of autoimmune diseases not only through auto-antibody secretion but also via cytokine production. Therapeutic depletion of B-cells influences the functions and maintenance of various T-cell subsets. The mechanisms governing the functional heterogeneity of B-cell subsets as cytokine-producing cells are poorly understood. B-cells can differentiate into two functionally polarized effectors, one (B-effector-1-cells producing a Th-1-like cytokine pattern and the other (Be2 producing a Th-2-like pattern. IL-12 and IFN-γ play a key role in Be1 polarization, but the initial trigger of Be1 commitment is unclear. Type-I-interferons are produced early in the immune response and prime several processes involved in innate and adaptive responses. Here, we report that IFN-α triggers a signaling cascade in resting human naive B-cells, involving STAT4 and T-bet, two key IFN-γ gene imprinting factors. IFN-α primed naive B-cells for IFN-γ production and increased IFN-γ gene responsiveness to IL-12. IFN-γ continues this polarization by re-inducing T-bet and up-regulating IL-12Rβ2 expression. IFN-α and IFN-γ therefore pave the way for the action of IL-12. These results point to a coordinated action of IFN-α, IFN-γ and IL-12 in Be1 polarization of naive B-cells, and may provide new insights into the mechanisms by which type-I-interferons favor autoimmunity.

  13. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes.

    Science.gov (United States)

    Ackermann, Amanda M; Wang, Zhiping; Schug, Jonathan; Naji, Ali; Kaestner, Klaus H

    2016-03-01

    Although glucagon-secreting α-cells and insulin-secreting β-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and exhibit overlapping transcriptomes and epigenomes. Notably, destruction of β-cells can stimulate repopulation via transdifferentiation of α-cells, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both α- and β-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and β-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in α- and β-cell specification and plasticity. We sorted human α- and β-cells and performed the "Assay for Transposase-Accessible Chromatin with high throughput sequencing" (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions. We identified numerous transcripts with either α-cell- or β-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The "group specific protein" (GC; or vitamin D binding protein) was restricted to α-cells, while CHODL (chondrolectin) immunoreactivity was only present in β-cells. Furthermore, α-cell- and β-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes. We have determined the genetic landscape of

  14. Human alpha-defensin-1 protects cells from intoxication with Clostridium perfringens iota toxin.

    Science.gov (United States)

    Fischer, Stephan; Popoff, Michel R; Barth, Holger

    2018-03-01

    Iota toxin is produced by Clostridium perfringens type E strains and associated with diarrhea in cattle and lambs. This binary protein toxin comprises the enzyme component iota a (Ia), which ADP-ribosylates G-actin, and the separate transport component iota b (Ib), which delivers Ia into the cytosol of target cells. Ib binds to cell receptors and forms biologically active toxin complexes with Ia, which cause rounding of adherent cells due to the destruction of the actin cytoskeleton. Here, we report that the human peptide α-defensin-1 protects cultured cells including human colon cells from intoxication with iota toxin. In contrast, the related ß-defensin-1 had no effect, indicating a specific mode of action. The α-defensin-1 did not inhibit ADP-ribosylation of actin by Ia in vitro. Pretreatment of Ib with α-defensin-1 prior to addition of Ia prevented intoxication. Additionally, α-defensin-1 protected cells from cytotoxic effects mediated by Ib in the absence of Ia, implicating that α-defensin-1 interacts with Ib to prevent the formation of biologically active iota toxin on cells. In conclusion, the findings contribute to a better understanding of the functions of α-defensin-1 and suggest that this human peptide might be an attractive starting point to develop novel pharmacological options to treat/prevent diseases associated with iota toxin-producing Clostridium perfringens strains.

  15. Intraperitoneal implant of recombinant encapsulated cells overexpressing alpha-L-iduronidase partially corrects visceral pathology in mucopolysaccharidosis type I mice.

    Science.gov (United States)

    Baldo, Guilherme; Mayer, Fabiana Quoos; Martinelli, Barbara; Meyer, Fabiola Schons; Burin, Maira; Meurer, Luise; Tavares, Angela Maria Vicente; Giugliani, Roberto; Matte, Ursula

    2012-08-01

    Mucopolysaccharidosis type I (MPS I) is characterized by deficiency of the enzyme alpha-L-iduronidase (IDUA) and storage of glycosaminoglycans (GAG) in several tissues. Current available treatments present limitations, thus the search for new therapies. Encapsulation of recombinant cells within polymeric structures combines gene and cell therapy and is a promising approach for treating MPS I. We produced alginate microcapsules containing baby hamster kidney (BHK) cells overexpressing IDUA and implanted these capsules in the peritoneum of MPS I mice. An increase in serum and tissue IDUA activity was observed at early time-points, as well as a reduction in GAG storage; however, correction in the long term was only partially achieved, with a drop in the IDUA activity being observed a few weeks after the implant. Analysis of the capsules obtained from the peritoneum revealed inflammation and a pericapsular fibrotic process, which could be responsible for the reduction in IDUA levels observed in the long term. In addition, treated mice developed antibodies against the enzyme. The results suggest that the encapsulation process is effective in the short term but improvements must be achieved in order to reduce the immune response and reach a stable correction.

  16. Genomic Profiling of a Human Leukemic Monocytic Cell-Line (THP-1 Exposed to Alpha Particle Radiation

    Directory of Open Access Journals (Sweden)

    Vinita Chauhan

    2012-01-01

    Full Text Available This study examined alpha (α- particle radiation effects on global changes in gene expression in human leukemic monocytic cells (THP-1 for the purposes of mining for candidate biomarkers that could be used for the development of a biological assessment tool. THP-1 cells were exposed to α-particle radiation at a dose range of 0 to 1.5 Gy. Twenty-four hours and three days after exposure gene expression was monitored using microarray technology. A total of 16 genes were dose responsive and classified as early onset due to their expression 24 h after exposure. Forty-eight transcripts were dose responsive and classified as late-onset as they were expressed 72 h after exposure. Among these genes, 6 genes were time and dose responsive and validated further using alternate technology. These transcripts were upregulated and associated with biological processes related to immune function, organelle stability and cell signalling/communication. This panel of genes merits further validation to determine if they are strong candidate biomarkers indicative of α-particle exposure.

  17. The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

    Science.gov (United States)

    Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

    2000-03-01

    Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

  18. MicroRNA-143 Downregulates Interleukin-13 Receptor Alpha1 in Human Mast Cells

    Directory of Open Access Journals (Sweden)

    Jianqiu Cheng

    2013-08-01

    Full Text Available MicroRNA-143 (miR-143 was found to be downregulated in allergic rhinitis, and bioinformatics analysis predicted that IL-13Rα1 was a target gene of miR-143. To understand the molecular mechanisms of miR-143 involved in the pathogenesis of allergic inflammation, recombinant miR-143 plasmid vectors were constructed, and human mast cell-1(HMC-1 cells which play a central role in the allergic response were used for study. The plasmids were transfected into HMC-1 cells using a lentiviral vector. Expression of IL-13Rα1 mRNA was then detected by reverse transcriptase polymerase chain reaction (RT-PCR and Western Blotting. The miR-143 lentiviral vector was successfully stably transfected in HMC-1 cells for target gene expression. Compared to the control, the target gene IL-13Rα1 was less expressed in HMC-1 transfected with miR-143 as determined by RT-PCR and Western Blotting (p < 0.05; this difference in expression was statistically significant and the inhibition efficiency was 71%. It indicates that miR-143 directly targets IL-13Rα1 and suppresses IL-13Rα1 expression in HMC-1 cells. Therefore, miR-143 may be associated with allergic reaction in human mast cells.

  19. MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Wei [Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Sa, Ke-Di; Zhang, Xiang; Jia, Lin-Tao; Zhao, Jing [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Yang, An-Gang [State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, 710032 Xi' an (China); Zhang, Rui, E-mail: ruizhang@fmmu.edu.cn [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Fan, Jing, E-mail: jingfan@fmmu.edu.cn [Department of Vascular and Endocrine Surgery, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Bian, Ka, E-mail: kakamax85@hotmail.com [State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, 710032 Xi' an (China); Department of Otolaryngology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)

    2015-08-07

    The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealed that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells.

  20. MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

    International Nuclear Information System (INIS)

    Hua, Wei; Sa, Ke-Di; Zhang, Xiang; Jia, Lin-Tao; Zhao, Jing; Yang, An-Gang; Zhang, Rui; Fan, Jing; Bian, Ka

    2015-01-01

    The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealed that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells

  1. A soluble form of IL-13 receptor alpha 1 promotes IgG2a and IgG2b production by murine germinal center B cells.

    Science.gov (United States)

    Poudrier, J; Graber, P; Herren, S; Gretener, D; Elson, G; Berney, C; Gauchat, J F; Kosco-Vilbois, M H

    1999-08-01

    A functional IL-13R involves at least two cell surface proteins, the IL-13R alpha 1 and IL-4R alpha. Using a soluble form of the murine IL-13R alpha 1 (sIL-13R), we reveal several novel features of this system. The sIL-13R promotes proliferation and augmentation of Ag-specific IgM, IgG2a, and IgG2b production by murine germinal center (GC) B cells in vitro. These effects were enhanced by CD40 signaling and were not inhibited by an anti-IL4R alpha mAb, a result suggesting other ligands. In GC cell cultures, sIL-13R also promoted IL-6 production, and interestingly, sIL-13R-induced IgG2a and IgG2b augmentation was absent in GC cells isolated from IL-6-deficient mice. Furthermore, the effects of the sIL-13R molecule were inhibited in the presence of an anti-IL-13 mAb, and preincubation of GC cells with IL-13 enhanced the sIL-13R-mediated effects. When sIL-13R was injected into mice, it served as an adjuvant-promoting production to varying degrees of IgM and IgG isotypes. We thus propose that IL-13R alpha 1 is a molecule involved in B cell differentiation, using a mechanism that may involve regulation of IL-6-responsive elements. Taken together, our data reveal previously unknown activities as well as suggest that the ligand for the sIL-13R might be a component of the IL-13R complex or a counterstructure yet to be defined.

  2. TNF{alpha} and IL-1{beta} are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Wenwen; Zheng, Xuexing [College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, Jilin Province (China); Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, FL 33136 (United States); Liu, Shue [Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, FL 33136 (United States); Ouyang, Hongsheng [College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, Jilin Province (China); Levitt, Roy C.; Candiotti, Keith A. [Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, FL 33136 (United States); Hao, Shuanglin, E-mail: shao@med.miami.edu [Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, FL 33136 (United States)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer LPS induces proinflammatory cytokine release in HAPI cells. Black-Right-Pointing-Pointer JNK pathway is dependent on TLR4 signaling to release cytokines. Black-Right-Pointing-Pointer NF-{kappa}B pathway is dependent on Nod1 signaling to release cytokines. -- Abstract: A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-{kappa}B (pNF-{kappa}B), TNF{alpha} and IL-1{beta}. Silencing TLR4 with siRNA reduced the expression of pJNK, TNF{alpha} and IL-1{beta}, but not pNF-{kappa}B in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNF{alpha} and IL-1{beta}. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-{kappa}B. Inhibition of NF-{kappa}B also reduced the expression of TNF{alpha} and IL-1{beta}. Nod1 ligand, DAP induced the upregulation of pNF-{kappa}B which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-{kappa}B is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-{kappa}B pathways is involved in the expression of TNF{alpha} and IL-1{beta}.

  3. Mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines.

    Science.gov (United States)

    Gelsomino, L; Panza, S; Giordano, C; Barone, I; Gu, G; Spina, E; Catalano, S; Fuqua, S; Andò, S

    2018-04-24

    The detection of recurrent mutations affecting the hormone binding domain (HBD) of estrogen receptor alpha (ERα/ESR1) in endocrine therapy-resistant and metastatic breast cancers has prompted interest in functional characterization of these genetic alterations. Here, we explored the role of HBD-ESR1 mutations in influencing the behavior of breast cancer stem cells (BCSCs), using various BC cell lines stably expressing wild-type or mutant (Y537 N, Y537S, D538G) ERα. Compared to WT-ERα clones, mutant cells showed increased CD44 + /CD24 - ratio, mRNA levels of stemness genes, Mammosphere Forming Efficiency (MFE), Self-Renewal and migratory capabilities. Mutant clones exhibited high expression of NOTCH receptors/ligands/target genes and blockade of NOTCH signaling reduced MFE and migratory potential. Mutant BCSC activity was dependent on ERα phosphorylation at serine 118, since its inhibition decreased MFE and NOTCH4 activation only in mutant cells. Collectively, we demonstrate that the expression of HBD-ESR1 mutations may drive BC cells to acquire stem cell traits through ER/NOTCH4 interplay. We propose the early detection of HBD-ESR1 mutations as a challenge in precision medicine strategy, suggesting the development of tailored-approaches (i.e. NOTCH inhibitors) to prevent disease development and metastatic spread in BC mutant-positive patients. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells

    International Nuclear Information System (INIS)

    Kobayashi, M.; Shimada, K.; Ozawa, T.

    1990-01-01

    Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation

  5. Combined cytotoxic effects of tumor necrosis factor-alpha with various cytotoxic agents in tumor cell lines that are drug resistant due to mutated p53

    NARCIS (Netherlands)

    Sleijfer, S; Le, T. K. P.; de Jong, S.; Timmer-Bosscha, H; Withoff, S; Mulder, NH

    Several studies suggest that tumor necrosis factor-alpha (TNF) is able to overcome drug resistance in tumors. Whether TNF is able to do so in tumor cell lines that are drug resistant due to a mutation in the tumor suppressor gene p53 is unclear. Therefore, we studied the in vitro cytotoxic effects

  6. An in vitro model for cytogenetic conversion in CML. Interferon-alpha preferentially inhibits the outgrowth of malignant stem cells preserved in long-term culture

    NARCIS (Netherlands)

    J.J. Cornelissen (Jan); R.E. Ploemacher (Robert); B.W. Wognum; A. Borsboom; J.C. Kluin-Nelemans (Hanneke); A. Hagemeijer (Anne); B. Löwenberg (Bob)

    1998-01-01

    textabstractIFN-alpha has been shown to prolong survival in chronic myeloid leukemia patients, but its mechanism of action is still not understood. The human cobblestone area-forming cell (CAFC) assay allows for the measurement of the concentration of normal as well

  7. Transforming growth factor alpha (TGFα regulates granulosa cell tumor (GCT cell proliferation and migration through activation of multiple pathways.

    Directory of Open Access Journals (Sweden)

    Cheng Wang

    Full Text Available Granulosa cell tumors (GCTs are the most common ovarian estrogen producing tumors, leading to symptoms of excessive estrogen such as endometrial hyperplasia and endometrial adenocarcinoma. These tumors have malignant potential and often recur. The etiology of GCT is unknown. TGFα is a potent mitogen for many different cells. However, its function in GCT initiation, progression and metastasis has not been determined. The present study aims to determine whether TGFα plays a role in the growth of GCT cells. KGN cells, which are derived from an invasive GCT and have many features of normal granulosa cells, were used as the cellular model. Immunohistochemistry, Western blot and RT-PCR results showed that the ErbB family of receptors is expressed in human GCT tissues and GCT cell lines. RT-PCR results also indicated that TGFα and EGF are expressed in the human granulosa cells and the GCT cell lines, suggesting that TGFα might regulate GCT cell function in an autocrine/paracrine manner. TGFα stimulated KGN cell DNA synthesis, cell proliferation, cell viability, cell cycle progression, and cell migration. TGFα rapidly activated EGFR/PI3K/Akt and mTOR pathways, as indicated by rapid phosphorylation of Akt, TSC2, Rictor, mTOR, P70S6K and S6 proteins following TGFα treatment. TGFα also rapidly activated the EGFR/MEK/ERK pathway, and P38 MAPK pathways, as indicated by the rapid phosphorylation of EGFR, MEK, ERK1/2, P38, and CREB after TGFα treatment. Whereas TGFα triggered a transient activation of Akt, it induced a sustained activation of ERK1/2 in KGN cells. Long-term treatment of KGN cells with TGFα resulted in a significant increase in cyclin D2 and a decrease in p27/Kip1, two critical regulators of granulosa cell proliferation and granulosa cell tumorigenesis. In conclusion, TGFα, via multiple signaling pathways, regulates KGN cell proliferation and migration and may play an important role in the growth and metastasis of GCTs.

  8. The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells

    DEFF Research Database (Denmark)

    Muñoz, A; Höppner, W; Sap, J

    1990-01-01

    To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon...

  9. Glucose decouples intracellular Ca2+ activity from glucagon secretion in mouse pancreatic islet alpha-cells.

    Directory of Open Access Journals (Sweden)

    Sylvain J Le Marchand

    Full Text Available The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca(2+](i and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca(2+](i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K(ATP channel activity but not on tetrodotoxin-sensitive Na(+ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca(2+](i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K(ATP channel activity or reduction in α-cell [Ca(2+](i. Our results demonstrate that glucose uncouples the positive relationship between [Ca(2+](i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.

  10. A synthetic peptide derived from alpha-fetoprotein inhibits the estradiol-induced proliferation of mammary tumor cells in culture through the modulation of p21.

    Science.gov (United States)

    Sierralta, Walter D; Epuñan, María J; Reyes, José M; Valladares, Luis E; Pino, Ana M

    2008-01-01

    A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.

  11. An alpha-adrenergic receptor mechanism controlling potassium permeability in the rat lacrimal gland acinar cell

    International Nuclear Information System (INIS)

    Parod, R.J.; Putney, J.W. Jr.

    1978-01-01

    Rat lacrimal gland slices, incubated in a balanced, buffered salt solution, were found to be physiologically stable for up to 2 hr with respect to 0 2 consumption, extracellular space, and water and ion content. The release of 86 Rb serves as a good substitute for 42 K in monitoring the movement of K through the cell membrane. Adrenaline appears to increase membrane permeability to K as evidenced by an increase in the rate of 86 Rb efflux. This response to adrenaline was blocked by phentolamine but not by propranolol and was mimicked by phenylephrine but not by isoprenaline. The magnitude of the 86 Rb release indicates that it is being released, at least in part, from the lacrimal gland acinar cell. It is concluded that the lacrimal gland acinar cell has an α-adrenergic receptor, activation of which leads to an increase in membrane permeability to K. (author)

  12. 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

  13. 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

  14. Application of alpha microdosimetry in possible differentiation of inactivation effect from malignant cell transformation

    International Nuclear Information System (INIS)

    Sedlak, A.

    1984-01-01

    The model is described with a certain threshold value of specific energy z 0 independent of the linear transfer of energy (LTE) whose exceedance would result in the inactivation of the cell. The survival curves may be fitted to distribution function F(z 0 ,D). The final relationship is described by the linear dependence of quotient L 3 /D 37 on L 2 . The survival curves in low and high LTE are given. It may be assumed that at high LTE there exists a certain probability of survival even after passage of a densely ionizing particle through the cell nucleus. (E.S.)

  15. Alpha 4 integrin directs virus-activated CD8+ T cells to sites of infection

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Andersson, E C; Scheynius, A

    1995-01-01

    This article examines the role of VLA-4 in directing lymphocytes to sites of viral infection using the murine lymphocytic choriomeningitis virus infection (LCMV) as the model system. This virus by itself induces little or no inflammation, but in most mouse/virus strain combinations a potent T cell...... response is induced, which is associated with marked CD8+ cell-mediated inflammation. Two expressions of LCMV-induced inflammation were studied: meningitis induced by intracerebral infection and adoptive transfer of virus-specific delayed-type hypersensitivity. Our previous studies have shown that LCMV...

  16. The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

    Science.gov (United States)

    Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C

    2009-04-01

    To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

  17. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

    Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha\\/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  18. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

    Directory of Open Access Journals (Sweden)

    Sanjeev Gupta

    2010-07-01

    Full Text Available Endoplasmic reticulum (ER stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR. Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  19. Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Philipp Gierok

    Full Text Available Staphylococcus aureus alpha-toxin (Hla is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o- under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml of recombinant Hla (rHla in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o- cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.

  20. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes

    Directory of Open Access Journals (Sweden)

    Amanda M. Ackermann

    2016-03-01

    Conclusions: We have determined the genetic landscape of human α- and β-cells based on chromatin accessibility and transcript levels, which allowed for detection of novel α- and β-cell signature genes not previously known to be expressed in islets. Using fine-mapping of open chromatin, we have identified thousands of potential cis-regulatory elements that operate in an endocrine cell type-specific fashion.

  1. Alpha-Tocopherol alters transcription activities that modulate tumor necrosis factor alpha (TNF-¿)-induced inflammatory response in bovine cells

    Science.gov (United States)

    To further investigate the potential role of '-tocopherol in maintaining immuno-homeostasis in bovine cells (Madin-Darby bovine kidney epithelial cell line), we undertook in vitro experiments using recombinant TNF-a as an immuno-stimulant to simulate inflammation response in cells with and without '...

  2. Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells.

    Science.gov (United States)

    Montgomery, M D; Bylund, D B

    2010-02-01

    The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

  3. Increased glucose metabolism and alpha-glucosidase inhibition in Cordyceps militaris water extract-treated HepG2 cells

    Science.gov (United States)

    Kim, Dae Jung; Kang, Yun Hwan; Kim, Kyoung Kon; Kim, Tae Woo; Park, Jae Bong

    2017-01-01

    BACKGROUND/OBJECTIVES Recent living condition improvements, changes in dietary habits, and reductions in physical activity are contributing to an increase in metabolic syndrome symptoms including diabetes and obesity. Through such societal developments, humankind is continuously exposed to metabolic diseases such as diabetes, and the number of the victims is increasing. This study investigated Cordyceps militaris water extract (CMW)-induced glucose uptake in HepG2 cells and the effect of CMW treatment on glucose metabolism. MATERIALS/METHODS Colorimetric assay kits were used to determine the glucokinase (GK) and pyruvate dehydrogenase (PDH) activities, glucose uptake, and glycogen content. Either RT-PCR or western blot analysis was performed for quantitation of glucose transporter 2 (GLUT2), hepatocyte nuclear factor 1 alpha (HNF-1α), phosphatidylinositol 3-kinase (PI3k), protein kinase B (Akt), phosphorylated AMP-activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase, GK, PDH, and glycogen synthase kinase 3 beta (GSK-3β) expression levels. The α-glucosidase inhibitory activities of acarbose and CMW were evaluated by absorbance measurement. RESULTS CMW induced glucose uptake in HepG2 cells by increasing GLUT2 through HNF-1α expression stimulation. Glucose in the cells increased the CMW-induced phosphorylation of AMPK. In turn, glycolysis was stimulated, and glyconeogenesis was inhibited. Furthermore, by studying the mechanism of action of PI3k, Akt, and GSK-3β, and measuring glycogen content, the study confirmed that the glucose was stored in the liver as glycogen. Finally, CMW resulted in a higher level of α-glucosidase inhibitory activity than that from acarbose. CONCLUSION CMW induced the uptake of glucose into HepG2 cells, as well, it induced metabolism of the absorbed glucose. It is concluded that CMW is a candidate or potential use in diabetes prevention and treatment. PMID:28584574

  4. Melatonin promotes cardiomyogenesis of embryonic stem cells via inhibition of HIF-1 alpha stabilization

    Czech Academy of Sciences Publication Activity Database

    Kudová, Jana; Vašíček, Ondřej; Číž, Milan; Kubala, Lukáš

    2016-01-01

    Roč. 61, č. 4 (2016), s. 493-503 ISSN 0742-3098 R&D Projects: GA MŠk(CZ) LD14030 Institutional support: RVO:68081707 Keywords : prostate -cancer cells * increased vascular-permeability * reperfusion injury Subject RIV: BO - Biophysics Impact factor: 10.391, year: 2016

  5. Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

    Science.gov (United States)

    Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

    2009-01-01

    Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

  6. 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

    D(3) the active form of Vitamin D(3) (D(3)) in combination with dexamethasone (Dex) has a synergistic effect on LPS-induced maturation of DC. Monocyte-derived DCs cultured with D(3) and Dex during LPS-induced maturation have a low stimulatory effect on allogeneic T cells comparable...

  7. Buffett's Alpha

    DEFF Research Database (Denmark)

    Frazzini, Andrea; Kabiller, David; Heje Pedersen, Lasse

    Berkshire Hathaway has realized a Sharpe ratio of 0.76, higher than any other stock or mutual fund with a history of more than 30 years, and Berkshire has a significant alpha to traditional risk factors. However, we find that the alpha becomes insignificant when controlling for exposures to Betting...

  8. Alpha Blockers

    Science.gov (United States)

    ... quickly, but their effects last only a few hours. Long-acting medications take longer to work, but their effects last longer. Which alpha blocker is best for you depends on your health and the condition being treated. Alpha blockers are ...

  9. Enhancement of Mucosal Immunogenicity of Viral Vectored Vaccines by the NKT Cell Agonist Alpha-Galactosylceramide as Adjuvant

    Directory of Open Access Journals (Sweden)

    Shailbala Singh

    2014-10-01

    Full Text Available Gene-based vaccination strategies, specifically viral vectors encoding vaccine immunogens are effective at priming strong immune responses. Mucosal routes offer practical advantages for vaccination by ease of needle-free administration, and immunogen delivery at readily accessible oral/nasal sites to efficiently induce immunity at distant gut and genital tissues. However, since mucosal tissues are inherently tolerant for induction of immune responses, incorporation of adjuvants for optimal mucosal vaccination strategies is important. We report here the effectiveness of alpha-galactosylceramide (α-GalCer, a synthetic glycolipid agonist of natural killer T (NKT cells, as an adjuvant for enhancing immunogenicity of vaccine antigens delivered using viral vectors by mucosal routes in murine and nonhuman primate models. Significant improvement in adaptive immune responses in systemic and mucosal tissues was observed by including α-GalCer adjuvant for intranasal immunization of mice with vesicular stomatitis virus vector encoding the model antigen ovalbumin and adenoviral vectors expressing HIV env and Gag antigens. Activation of NKT cells in systemic and mucosal tissues along with significant increases in adaptive immune responses were observed in rhesus macaques immunized by intranasal and sublingual routes with protein or adenovirus vectored antigens when combined with α-GalCer adjuvant. These results support the utility of α-GalCer adjuvant for enhancing immunogenicity of mucosal vaccines delivered using viral vectors.

  10. DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

    OpenAIRE

    Couldrey, Christine; Wells, David N.

    2013-01-01

    Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT bla...

  11. Structural requirement of carboxyl-terminal globular domains of laminin alpha 3 chain for promotion of rapid cell adhesion and migration by laminin-5.

    Science.gov (United States)

    Hirosaki, T; Mizushima, H; Tsubota, Y; Moriyama, K; Miyazaki, K

    2000-07-21

    The basement membrane protein laminin-5, a heterotrimer of laminin alpha3, beta3, and gamma2 chains, potently promotes cellular adhesion and motility. It has been supposed that the carboxyl-terminal globular region of the alpha3 chain consisting of five distinct domains (G1 to G5) is important for its interaction with integrins. To clarify the function of each G domain, we transfected cDNAs for the full-length (wild type (WT)) and five deletion derivatives (DeltaGs) of the alpha3 chain into human fibrosarcoma cell line HT1080, which expressed and secreted the laminin beta3 and gamma2 chains but not the alpha3 chain. The transfectants with the alpha3 chain cDNAs lacking G5 (DeltaG(5)), G4-5 (DeltaG(4-5)), G3-5 (DeltaG(3-5)), and G2-5 (DeltaG(2-5)) secreted laminin-5 variants at levels comparable to that with WT cDNA. However, the transfectant with the cDNA without any G domains (DeltaG(1-5)) secreted little laminin-5, suggesting that the G domains are essential for the efficient assembly and secretion of the heterotrimer alpha3beta3gamma2. The transfectants with WT, DeltaG(5), and DeltaG(4-5) cDNAs survived in serum-free medium longer than those with DeltaG(3-5), DeltaG(2-5), and DeltaG(1-5) cDNAs. The transfectants with WT, DeltaG(5), and DeltaG(4-5) cDNAs secreted apparently the same size of laminin-5, which lacked G4 and G5 due to proteolytic cleavage between G3 and G4, and these laminin-5 forms potently promoted integrin alpha(3)beta(1)-dependent cell adhesion and migration. However, the laminin-5 forms of DeltaG(3-5) and DeltaG(2-5) hardly promoted the cell adhesion and motility. These findings demonstrate that the G3 domain, but not the G4 and G5 domains, of the alpha3 chain is essential for the potent promotion of cell adhesion and motility by laminin-5.

  12. Construction of Expression Vector for Anti-Alpha-Fetoprotein Gene and Its Inhibition Effects on Alpha-Fetoprotein Positive Hepg2 Cells

    Science.gov (United States)

    Wang, Ze; Zhang, Hui

    As research previously demonstrated, suppression of AFP expression or its biological activities might inhibit the proliferation of AFP positive human hepatocellular carcinoma cells. In this study, we constructed an anti-AFP gene vector and transfected it to HepG2 cells. RT-PCR showed AFP gene expression in the transfected cells was reduced. MTT assay suggested the proliferation of the transfected cells was also inhibited comparing with the untransfected cells. This result provides a new insight into AFP as the target for preventing and treating hepatocellular carcinoma.

  13. Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines.

    Science.gov (United States)

    Campbell, Sharon E; Stone, William L; Lee, Steven; Whaley, Sarah; Yang, Hongsong; Qui, Min; Goforth, Paige; Sherman, Devin; McHaffie, Derek; Krishnan, Koyamangalath

    2006-01-17

    Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-gamma-tocopherol isoform is found primarily in the US diet, while RRR-alpha-tocopherol is highest in the plasma. The effectiveness of RRR-alpha- and RRR-gamma-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-alpha- or RRR-gamma-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. Treatment with RRR-gamma-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-alpha-tocopherol did not. Further, RRR-gamma-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-gamma-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-gamma-tocopherol to induce cell death. This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-gamma-tocopherol without damage to

  14. Choline kinase-alpha by regulating cell aggressiveness and drug sensitivity is a potential druggable target for ovarian cancer

    OpenAIRE

    Granata, A; Nicoletti, R; Tinaglia, V; De Cecco, L; Pisanu, M E; Ricci, A; Podo, F; Canevari, S; Iorio, E; Bagnoli, M; Mezzanzanica, D

    2013-01-01

    Background: Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). Methods: To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profi...

  15. Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

    International Nuclear Information System (INIS)

    Schwarz, Margaret A.; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

    2005-01-01

    Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function

  16. Induction of Programmed Cell Death by Parvovirus H-1 in U937 Cells: Connection with the Tumor Necrosis Factor Alpha Signalling Pathway

    Science.gov (United States)

    Rayet, Béatrice; Lopez-Guerrero, José-Antonio; Rommelaere, Jean; Dinsart, Christiane

    1998-01-01

    The human promonocytic cell line U937 undergoes apoptosis upon treatment with tumor necrosis factor alpha (TNF-α). This cell line has previously been shown to be very sensitive to the lytic effect of the autonomous parvovirus H-1. Parvovirus infection leads to the activation of the CPP32 ICE-like cysteine protease which cleaves the enzyme poly(ADP-ribose)polymerase and induces morphologic changes that are characteristic of apoptosis in a way that is similar to TNF-α treatment. This effect is also observed when the U937 cells are infected with a recombinant H-1 virus which expresses the nonstructural (NS) proteins but in which the capsid genes are replaced by a reporter gene, indicating that the induction of apoptosis can be assigned to the cytotoxic nonstructural proteins in this cell system. The c-Myc protein, which is overexpressed in U937 cells, is rapidly downregulated during infection, in keeping with a possible role of this product in mediating the apoptotic cell death induced by H-1 virus infection. Interestingly, four clones (designated RU) derived from the U937 cell line and selected for their resistance to H-1 virus (J. A. Lopez-Guerrero et al., Blood 89:1642–1653, 1997) failed to decrease c-Myc expression upon treatment with differentiation agents and also resisted the induction of cell death after TNF-α treatment. Our data suggest that the RU clones have developed defense strategies against apoptosis, either by their failure to downregulate c-Myc and/or by activating antiapoptotic factors. PMID:9765434

  17. Expression of the chicken GDNF family receptor alpha-1 as a marker of spermatogonial stem cells

    Czech Academy of Sciences Publication Activity Database

    Mucksová, J.; Kalina, J.; Bakst, M.; Yan, H.; Brillard, J.-P.; Benešová, B.; Fafílek, Bohumil; Hejnar, Jiří; Trefil, P.

    2013-01-01

    Roč. 142, 1-2 (2013), s. 75-83 ISSN 0378-4320 R&D Projects: GA ČR GAP502/11/2207 Grant - others:GA MŠk(CZ) ME10104 Institutional support: RVO:68378050 Keywords : GFRα1 * chicken spermatogonial stem cell * male germ line transplantation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.581, year: 2013

  18. Trichostatin A enhances estrogen receptor-alpha repression in MCF-7 breast cancer cells under hypoxia

    International Nuclear Information System (INIS)

    Noh, Hyunggyun; Park, Joonwoo; Shim, Myeongguk; Lee, YoungJoo

    2016-01-01

    Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERα protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia. - Highlights: • TSA augments ESR1 gene repression at the transcriptional level under hypoxia. • TSA downregulates ERα protein expression under hypoxia. • TSA-induced ERα regulation under hypoxia is essential for understanding the behavior and progression of breast cancer.

  19. Trichostatin A enhances estrogen receptor-alpha repression in MCF-7 breast cancer cells under hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Hyunggyun; Park, Joonwoo; Shim, Myeongguk; Lee, YoungJoo, E-mail: yjlee@sejong.ac.kr

    2016-02-12

    Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERα protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia. - Highlights: • TSA augments ESR1 gene repression at the transcriptional level under hypoxia. • TSA downregulates ERα protein expression under hypoxia. • TSA-induced ERα regulation under hypoxia is essential for understanding the behavior and progression of breast cancer.

  20. Staurosporine, but not Ro 31-8220, induces interleukin 2 production and synergizes with interleukin 1alpha in EL4 thymoma cells.

    Science.gov (United States)

    Mahon, T M; Matthews, J S; O'Neill, L A

    1997-07-01

    Protein kinase C (PKC) has been implicated in interleukin 1 (IL1) signal transduction in a number of cellular systems, either as a key event in IL1 action or as a negative regulator. Here we have examined the effects of two PKC inhibitors, staurosporine and the more selective agent Ro 31-8220, on IL1 responses in the murine thymoma line EL4.NOB-1. A 1 h pulse of staurosporine was found to strongly potentiate the induction of IL2 by IL1alpha in these cells. In contrast, neither a pulse nor prolonged incubation with Ro 31-8220 affected the response to IL1alpha. Both agents blocked the response to PMA, however. A 1 h pulse of staurosporine was also found to induce IL2 production on its own, activate the transcription factor nuclear factor kappaB (NFkappaB) and increase the expression of a NFkappaB-linked reporter gene. It synergized with IL1alpha in all of these responses. Ro 31-8220 was again without effect, although both staurosporine and Ro 31-8220 blocked the activation of NFkappaB by PMA. Finally, staurosporine caused the translocation of PKC-alpha and -epsilon, and to a lesser extent PKC-beta, but not PKC-θ or -zeta, from the cytosol to the membrane, although a similar effect was observed with Ro 31-8220. The results suggest that PKC is not involved in IL1alpha signalling in EL4 cells. Furthermore, the potentiating effect of staurosporine on IL1alpha action does not involve PKC inhibition, and is likely to be at the level of NFkappaB activation.

  1. Mitochondrial Effects of PGC-1alpha Silencing in MPP+ Treated Human SH-SY5Y Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Qinyong Ye

    2017-05-01

    Full Text Available The dopaminergic neuron degeneration and loss that occurs in Parkinson’s disease (PD has been tightly linked to mitochondrial dysfunction. Although the aged-related cause of the mitochondrial defect observed in PD patients remains unclear, nuclear genes are of potential importance to mitochondrial function. Human peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α is a multi-functional transcription factor that tightly regulates mitochondrial biogenesis and oxidative capacity. The goal of the present study was to explore the potential pathogenic effects of interference by the PGC-1α gene on N-methyl-4-phenylpyridinium ion (MPP+-induced SH-SY5Y cells. We utilized RNA interference (RNAi technology to probe the pathogenic consequences of inhibiting PGC-1α in the SH-SY5Y cell line. Remarkably, a reduction in PGC-1α resulted in the reduction of mitochondrial membrane potential, intracellular ATP content and intracellular H2O2 generation, leading to the translocation of cytochrome c (cyt c to the cytoplasm in the MPP+-induced PD cell model. The expression of related proteins in the signaling pathway (e.g., estrogen-related receptor α (ERRα, nuclear respiratory factor 1 (NRF-1, NRF-2 and Peroxisome proliferator-activated receptor γ (PPARγ also decreased. Our finding indicates that small interfering RNA (siRNA interference targeting the PGC-1α gene could inhibit the function of mitochondria in several capacities and that the PGC-1α gene may modulate mitochondrial function by regulating the expression of ERRα, NRF-1, NRF-2 and PPARγ. Thus, PGC-1α can be considered a potential therapeutic target for PD.

  2. Spaceflight Activates Protein Kinase C Alpha Signaling and Modifies the Developmental Stage of Human Neonatal Cardiovascular Progenitor Cells.

    Science.gov (United States)

    Baio, Jonathan; Martinez, Aida F; Bailey, Leonard; Hasaniya, Nahidh; Pecaut, Michael J; Kearns-Jonker, Mary

    2018-02-12

    Spaceflight impacts cardiovascular function in astronauts; however, its impact on cardiac development and the stem cells that form the basis for cardiac repair is unknown. Accordingly, further research is needed to uncover the potential relevance of such changes to human health. Using simulated microgravity (SMG) generated by two-dimensional clinorotation and culture aboard the International Space Station (ISS), we assessed the effects of mechanical unloading on human neonatal cardiovascular progenitor cell (CPC) developmental properties and signaling. Following 6-7 days of SMG and 12 days of ISS culture, we analyzed changes in gene expression. Both environments induced the expression of genes that are typically associated with an earlier state of cardiovascular development. To understand the mechanism by which such changes occurred, we assessed the expression of mechanosensitive small RhoGTPases in SMG-cultured CPCs and observed decreased levels of RHOA and CDC42. Given the effect of these molecules on intracellular calcium levels, we evaluated changes in noncanonical Wnt/calcium signaling. After 6-7 days under SMG, CPCs exhibited elevated levels of WNT5A and PRKCA. Similarly, ISS-cultured CPCs exhibited elevated levels of calcium handling and signaling genes, which corresponded to protein kinase C alpha (PKCα), a calcium-dependent protein kinase, activation after 30 days. Akt was activated, whereas phosphorylated extracellular signal-regulated kinase levels were unchanged. To explore the effect of calcium induction in neonatal CPCs, we activated PKCα using hWnt5a treatment on Earth. Subsequently, early cardiovascular developmental marker levels were elevated. Transcripts induced by SMG and hWnt5a-treatment are expressed within the sinoatrial node, which may represent embryonic myocardium maintained in its primitive state. Calcium signaling is sensitive to mechanical unloading and directs CPC developmental properties. Further research both in space and on Earth

  3. Eosinophils Regulate Interferon Alpha Production in Plasmacytoid Dendritic Cells Stimulated with Components of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Skrzeczynska-Moncznik, Joanna; Zabieglo, Katarzyna; Bossowski, Jozef P; Osiecka, Oktawia; Wlodarczyk, Agnieszka; Kapinska-Mrowiecka, Monika; Kwitniewski, Mateusz; Majewski, Pawel; Dubin, Adam; Cichy, Joanna

    2017-03-01

    Eosinophils constitute an important component of helminth immunity and are not only associated with various allergies but are also linked to autoinflammatory disorders, including the skin disease psoriasis. Here we demonstrate the functional relationship between eosinophils and plasmacytoid dendritic cells (pDCs) as related to skin diseases. We previously showed that pDCs colocalize with neutrophil extracellular traps (NETs) in psoriatic skin. Here we demonstrate that eosinophils are found in psoriatic skin near neutrophils and NETs, suggesting that pDC responses can be regulated by eosinophils. Eosinophils inhibited pDC function in vitro through a mechanism that did not involve cell contact but depended on soluble factors. In pDCs stimulated by specific NET components, eosinophil-conditioned media attenuated the production of interferon α (IFNα) but did not affect the maturation of pDCs as evidenced by the unaltered expression of the costimulatory molecules CD80 and CD86. As pDCs and IFNα play a key role in autoimmune skin inflammation, these data suggest that eosinophils may influence autoinflammatory responses through their impact on the production of IFNα by pDCs.

  4. Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors.

    Science.gov (United States)

    Iwamoto, K; Mata, D; Linn, D M; Linn, C L

    2013-05-01

    Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 μM ACh, 100 μM nicotine or 100 nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double

  5. Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of aspergillus niger

    NARCIS (Netherlands)

    Kaaij, van der Rachel; Yuan, X.-L.; Franken, A.; Ram, A. F. J.; Punt, P. J.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be

  6. Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

    Science.gov (United States)

    Hennemann, B; Tam, Y K; Tonn, T; Klingemann, H G

    1999-07-01

    Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.

  7. Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

    Science.gov (United States)

    Mellor, J R; Wisden, W; Randall, A D

    2000-07-10

    Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

  8. Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells.

    Science.gov (United States)

    Sozio, Margaret S; Lu, Changyue; Zeng, Yan; Liangpunsakul, Suthat; Crabb, David W

    2011-10-01

    AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) are critical regulators of short-term and long-term fatty acid oxidation, respectively. We examined whether the activities of these molecules were coordinately regulated. H4IIEC3 cells were transfected with PPAR-α and PPAR-γ expression plasmids and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid. The cells were treated with PPAR agonists (WY-14,643 and rosiglitazone), AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin, and the AMPK inhibitor compound C. Both AICAR and metformin decreased basal and WY-14,643-stimulated PPAR-α activity; compound C increased agonist-stimulated reporter activity and partially reversed the effect of the AMPK activators. Similar effects on PPAR-γ were seen, with both AICAR and metformin inhibiting PPRE reporter activity. Compound C increased basal PPAR-γ activity and rosiglitazone-stimulated activity. In contrast, retinoic acid receptor-α (RAR-α), another nuclear receptor that dimerizes with retinoid X receptor (RXR), was largely unaffected by the AMPK activators. Compound C modestly increased AM580 (an RAR agonist)-stimulated activity. The AMPK activators did not affect PPAR-α binding to DNA, and there was no consistent correlation between effects of the AMPK activators and inhibitor on PPAR and the nuclear localization of AMPK-α subunits. Expression of either a constitutively active or dominant negative AMPK-α inhibited basal and WY-14,643-stimulated PPAR-α activity and basal and rosiglitazone-stimulated PPAR-γ activity. We concluded that the AMPK activators AICAR and metformin inhibited transcriptional activities of PPAR-α and PPAR-γ, whereas inhibition of AMPK with compound C activated both PPARs. The effects of AMPK do not appear to be mediated through effects on RXR or on PPAR/RXR binding to DNA. These effects are independent of kinase activity and instead appear to

  9. Generation of a human immunodeficiency virus type 1 chronically infected monkey B cell line expressing low levels of endogenous TRIM5alpha.

    Science.gov (United States)

    Ridolfi, Barbara; Catone, Stefania; Sgarbanti, Marco; Sernicola, Leonardo; Battistini, Angela; Parolin, Cristina; Titti, Fausto; Borsetti, Alessandra

    2009-12-01

    Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5alpha has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5alpha expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis.

  10. Dexamethasone/1alpha-25-dihydroxyvitamin D3-treated dendritic cells suppress colitis in the SCID T-cell transfer model

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Schmidt, Esben Gjerløff Wedebye; Gad, Monika

    2008-01-01

    severe combined immunodeficient (SCID) mice adoptively transferred with CD4(+) CD25(-) T cells from the development of wasting disease and colitis. We therefore established an in vitro test that could predict the in vivo function of DCs and improve strategies for the preparation of immunomodulatory DCs...... in this model. Based on these in vitro findings, we here evaluate three methods for DC generation including short-term and long-term IL-10 exposure or DC exposure to dexamethasone in combination with vitamin D3 (Dex/D3). All DCs resulted in lower CD4(+) CD25(-) T-cell enteroantigen-specific responses in vitro...

  11. 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.

  12. Human T-cell lymphotropic virus type 1-infected T lymphocytes impair catabolism and uptake of glutamate by astrocytes via Tax-1 and tumor necrosis factor alpha.

    Science.gov (United States)

    Szymocha, R; Akaoka, H; Dutuit, M; Malcus, C; Didier-Bazes, M; Belin, M F; Giraudon, P

    2000-07-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of a chronic progressive myelopathy called tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). In this disease, lesions of the central nervous system (CNS) are associated with perivascular infiltration by lymphocytes. We and others have hypothesized that these T lymphocytes infiltrating the CNS may play a prominent role in TSP/HAM. Here, we show that transient contact of human or rat astrocytes with T lymphocytes chronically infected by HTLV-1 impairs some of the major functions of brain astrocytes. Uptake of extracellular glutamate by astrocytes was significantly decreased after transient contact with infected T cells, while the expression of the glial transporters GLAST and GLT-1 was decreased. In two-compartment cultures avoiding direct cell-to-cell contact, similar results were obtained, suggesting possible involvement of soluble factors, such as cytokines and the viral protein Tax-1. Recombinant Tax-1 and tumor necrosis factor alpha (TNF-alpha) decreased glutamate uptake by astrocytes. Tax-1 probably acts by inducing TNF-alpha, as the effect of Tax-1 was abolished by anti-TNF-alpha antibody. The expression of glutamate-catabolizing enzymes in astrocytes was increased for glutamine synthetase and decreased for glutamate dehydrogenase, the magnitudes of these effects being correlated with the level of Tax-1 transcripts. In conclusion, Tax-1 and cytokines produced by HTLV-1-infected T cells impair the ability of astrocytes to manage the steady-state level of glutamate, which in turn may affect neuronal and oligodendrocytic functions and survival.

  13. Pertussis toxin-sensitive G-protein mediates the alpha 2-adrenergic receptor inhibition of melatonin release in photoreceptive chick pineal cell cultures

    International Nuclear Information System (INIS)

    Pratt, B.L.; Takahashi, J.S.

    1988-01-01

    The avian pineal gland is a photoreceptive organ that has been shown to contain postjunctional alpha 2-adrenoceptors that inhibit melatonin synthesis and/or release upon receptor activation. Physiological response and [32P]ADP ribosylation experiments were performed to investigate whether pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) were involved in the transduction of the alpha 2-adrenergic signal. For physiological response studies, the effects of pertussis toxin on melatonin release in dissociated cell cultures exposed to norepinephrine were assessed. Pertussis toxin blocked alpha 2-adrenergic receptor-mediated inhibition in a dose-dependent manner. Pertussis toxin-induced blockade appeared to be noncompetitive. One and 10 ng/ml doses of pertussis toxin partially blocked and a 100 ng/ml dose completely blocked norepinephrine-induced inhibition. Pertussis toxin-catalyzed [32P]ADP ribosylation of G-proteins in chick pineal cell membranes was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Membranes were prepared from cells that had been pretreated with 0, 1, 10, or 100 ng/ml pertussis toxin. In the absence of pertussis toxin pretreatment, two major proteins of 40K and 41K mol wt (Mr) were labeled by [32P]NAD. Pertussis toxin pretreatment of pineal cells abolished [32P] radiolabeling of the 40K Mr G-protein in a dose-dependent manner. The norepinephrine-induced inhibition of both cAMP efflux and melatonin release, as assessed by RIA of medium samples collected before membrane preparation, was also blocked in a dose-dependent manner by pertussis toxin. Collectively, these results suggest that a pertussis toxin-sensitive 40K Mr G-protein labeled by [32P]NAD may be functionally associated with alpha 2-adrenergic signal transduction in chick pineal cells

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

    International Nuclear Information System (INIS)

    Miettinen, Johanna A.; Pietilae, Mika; Salonen, Riikka J.; Ohlmeier, Steffen; Ylitalo, Kari; Huikuri, Heikki V.; Lehenkari, Petri

    2011-01-01

    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-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α 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-α 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-α 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-α exposure, which might influence MSC differentiation stage and capacity.

  15. Interferon-gamma and tumor necrosis factor-alpha sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis

    DEFF Research Database (Denmark)

    Fluhr, Herbert; Krenzer, Stefanie; Stein, Gerburg M

    2007-01-01

    The subtle interaction between the implanting embryo and the maternal endometrium plays a pivotal role during the process of implantation. Human endometrial stromal cells (ESCs) express Fas and the implanting trophoblast cells secrete Fas ligand (FASLG, FasL), suggesting a possible role for Fas......-mediated signaling during early implantation. Here we show that ESCs are primarily resistant to Fas-mediated apoptosis independently of their state of hormonal differentiation. Pre-treatment of ESCs with interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha sensitizes them to become apoptotic upon stimulation...... of Fas by an agonistic anti-Fas antibody. Incubation of ESCs with the early embryonic signal human chorionic gonadotropin (hCG, CGB) does not influence their reaction to Fas stimulation. The sensitizing effect of IFN-gamma and TNF-alpha was accompanied by a significant upregulation of Fas and FLICE...

  16. Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha recruits bone marrow-derived cells to the murine pulmonary vasculature.

    Directory of Open Access Journals (Sweden)

    Daniel J Angelini

    2010-06-01

    Full Text Available Pulmonary hypertension (PH is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo.We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP(+ transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (approximately 20 microm capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP(+ BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP(+ cells that localized to the pulmonary vasculature were alpha-smooth muscle actin(+ and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs. The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner.These results demonstrate HIMF-dependent recruitment of BMD

  17. Studies of the Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene and the relationship to beta-cell function during an OGTT in glucose-tolerant women with and without previous gestational diabetes mellitus

    DEFF Research Database (Denmark)

    Lauenborg, J; Damm, P; Ek, J

    2004-01-01

    In pregnancies complicated by gestational diabetes mellitus (GDM) an increased demand for insulin is not met due to beta-cell dysfunction. An Ala/Val polymorphism at codon 98 of the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene has been associated with decreased serum insulin and C-peptide r...

  18. Diagnostic value of (99m)Tc-3PRGD2 scintimammography for differentiation of malignant from benign breast lesions: Comparison of visual and semi-quantitative analysis.

    Science.gov (United States)

    Chen, Qianqian; Xie, Qian; Zhao, Min; Chen, Bin; Gao, Shi; Zhang, Haishan; Xing, Hua; Ma, Qingjie

    2015-01-01

    To compare the diagnostic value of visual and semi-quantitative analysis of technetium-99m-poly-ethylene glycol, 4-arginine-glycine-aspartic acid ((99m)Tc-3PRGD2) scintimammography (SMG) for better differentiation of benign from malignant breast masses, and also investigate the incremental role of semi-quantitative index of SMG. A total of 72 patients with breast lesions were included in the study. Technetium-99m-3PRGD2 SMG was performed with single photon emission computed tomography (SPET) at 60 min after intravenous injection of 749 ± 86MBq of the radiotracer. Images were evaluated by visual interpretation and semi-quantitative indices of tumor to non-tumor (T/N) ratios, which were compared with pathology results. Receiver operating characteristics (ROC) curve analyses were performed to determine the optimal visual grade, to calculate cut-off values of semi-quantitative indices, and to compare visual and semi-quantitative diagnostic values. Among the 72 patients, 89 lesions were confirmed by histopathology after fine needle aspiration biopsy or surgery, 48 malignant and 41 benign lesions. The mean T/N ratio of (99m)Tc-3PRGD2 SMG in malignant lesions was significantly higher than that in benign lesions (Pvalue for the detection of primary breast cancer, the sensitivity, specificity and accuracy were 81.3%, 70.7%, and 76.4%, respectively. When a T/N ratio of 2.01 was used as cut-off value, the sensitivity, specificity and accuracy were 79.2%, 75.6%, and 77.5%, respectively. According to ROC analysis, the area under the curve for semi-quantitative analysis was higher than that for visual analysis, but the statistical difference was not significant (P=0.372). Compared with visual analysis or semi-quantitative analysis alone, the sensitivity, specificity and accuracy of visual analysis combined with semi-quantitative analysis in diagnosing primary breast cancer were higher, being: 87.5%, 82.9%, and 85.4%, respectively. The area under the curve was 0.891. Results of

  19. 17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

    Science.gov (United States)

    Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

    2015-01-05

    Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. Waste reduction efforts through the evaluation and procurement of a digital camera system for the Alpha-Gamma Hot Cell Facility at Argonne National Laboratory-East

    International Nuclear Information System (INIS)

    Bray, T. S.; Cohen, A. B.; Tsai, H.; Kettman, W. C.; Trychta, K.

    1999-01-01

    The Alpha-Gamma Hot Cell Facility (AGHCF) at Argonne National Laboratory-East is a research facility where sample examinations involve traditional photography. The AGHCF documents samples with photographs (both Polaroid self-developing and negative film). Wastes generated include developing chemicals. The AGHCF evaluated, procured, and installed a digital camera system for the Leitz metallograph to significantly reduce labor, supplies, and wastes associated with traditional photography with a return on investment of less than two years

  1. Influence of mycotoxin zearalenone and its derivatives (alpha and beta zearalenol on apoptosis and proliferation of cultured granulosa cells from equine ovaries

    Directory of Open Access Journals (Sweden)

    Minoia Paolo

    2006-11-01

    Full Text Available Abstract Background The mycotoxin zearalenone (ZEA and its derivatives, alpha and beta-zearalenol (alpha and beta-ZOL, synthesized by genera Fusarium, often occur as contaminants in cereal grains and animal feeds. The importance of ZEA on reproductive disorders is well known in domestic animals species, particularly in swine and cattle. In the horse, limited data are available to date on the influence of dietary exposure to ZEA on reproductive health and on its in vitro effects on reproductive cells. The aim of this study was to evaluate the effects of ZEA and its derivatives, alpha and beta-ZOL, on granulosa cells (GCs from the ovaries of cycling mares. Methods The cell proliferation was evaluated by using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT test after 3 days exposure at different concentrations of ZEA and its derivatives (from 1 × 10-7 to 0.1 microM. The apoptosis induction was evaluated after 1 day exposure, by DNA analysis using flow cytometry. Results An increase in cell proliferation with respect to the control was observed in the presence of ZEA at 1 × 10-3 and 1 × 10-4 microM and apoptosis was induced by all mycotoxins at different concentrations. Conclusion The simultaneous presence of apoptosis and proliferation in GC cultures treated with zearalenones could indicate that these mycotoxins could be effective in inducing follicular atresia. These effects of zearalenones may result from both direct interaction with oestrogen-receptors as well as interaction with the enzymes 3alpha (beta-hydroxysteroid dehydrogenase (HSD, involved in the synthesis and metabolism of endogenous steroid hormones. These cellular disturbances, described for the first time in equine GCs cultured in vitro, could be hypothesized as referred to reproductive failures of unknown ethiology in the mare.

  2. The effects of TNF-alpha and inhibitors of arachidonic acid metabolism on human colon HT-29 cells depend on differentiation status

    Czech Academy of Sciences Publication Activity Database

    Kovaříková, Martina; Hofmanová, Jiřina; Souček, Karel; Kozubík, Alois

    2004-01-01

    Roč. 72, č. 1 (2004), s. 23-31 ISSN 0301-4681 R&D Projects: GA ČR GA525/01/0419; GA ČR GP524/02/P051; GA AV ČR IBS5004009 Institutional research plan: CEZ:AV0Z5004920 Keywords : colon cancer * cell differentiation * TNF-alpha Subject RIV: BO - Biophysics Impact factor: 4.481, year: 2004

  3. Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

    Science.gov (United States)

    Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

    2005-03-01

    In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of

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

    National Research Council Canada - National Science Library

    Huang, Qiaojia

    2003-01-01

    ...-alpha treatment, and MEKK3 is an essential component of this process. I constructed expression vectors that express dominant negative forms of MEKK3 that will be used to block the MEKK3-down stream cascades...

  5. Alcohol depletes coenzyme-Q10 associated with increased TNF-alpha secretion to induce cytotoxicity in HepG2 cells

    International Nuclear Information System (INIS)

    Vidyashankar, Satyakumar; Nandakumar, Krishna S.; Patki, Pralhad S.

    2012-01-01

    Highlights: ► Ethanol induced cytotoxicity in HepG2 cells in absence of lipogenesis. ► Ethanol inhibited HMG-CoA reductase activity. ► Ethanol induced HMG-CoA reductase inhibition is due to decreased cell viability. ► Incubation with mevalonate could not increase the cholesterol. ► Cytotoxicity brought about by CoQ10 depletion and increased TNF-alpha. -- Abstract: Alcohol consumption has been implicated to cause severe hepatic steatosis which is mediated by alcohol dehydrogenase (ADH) activity and CYP 450 2E1 expression. In this context, the effect of ethanol was studied for its influence on lipogenesis in HepG2 cell which is deficient of ADH and does not express CYP 450 2E1. The results showed that ethanol at 100 mM concentration caused 40% cytotoxicity at 72 h as determined by MTT assay. The incorporation of labeled [2- 14 C] acetate into triacylglycerol and phospholipid was increased by 40% and 26% respectively upon 24 h incubation, whereas incorporation of labeled [2- 14 C] acetate into cholesterol was not significantly increased. Further, ethanol inhibited HMG-CoA reductase which is a rate-limiting enzyme in the cholesterol biosynthesis. It was observed that, HMG-CoA reductase inhibition was brought about by ethanol as a consequence of decreased cell viability, since incubation of HepG2 cells with mevalonate could not increase the cholesterol content and increase the cell viability. Addition of ethanol significantly increased TNF-alpha secretion and depleted mitochondrial coenzyme-Q 10 which is detrimental for cell viability. But vitamin E (10 mM) could partially restore coenzyme-Q 10 and glutathione content with decreased TNF-alpha secretion in ethanol treated cells. Further, lipid peroxidation, glutathione peroxidase and superoxide dismutase enzyme activities remained unaffected. Ethanol decreased glutathione content while, GSH/GSSG ratio was significantly higher compared to other groups showing cellular pro-oxidant and antioxidant balance remained

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

    Science.gov (United States)

    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.

  7. Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas.

    Science.gov (United States)

    Hafian, Hilal; Venteo, Lydie; Sukhanova, Alyona; Nabiev, Igor; Lefevre, Benoît; Pluot, Michel

    2004-06-01

    Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

  8. The Ikaros transcription factor regulates responsiveness to IL-12 and expression of IL-2 receptor alpha in mature, activated CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Eric T Clambey

    Full Text Available The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

  9. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B

    1999-01-01

    in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug......-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack...... of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant...

  10. Immunoprecipitation assay of alpha-fetoprotein synthesis by cultured mouse hepatoma cells treated with estrogens and glucocorticoids.

    Science.gov (United States)

    Rosebrock, J A; Parker, C L; Kute, T E

    1981-01-01

    This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.

  11. Amelioration of hypertriglyceridemia with hypo-alpha-cholesterolemia in LPL deficient mice by hematopoietic cell-derived LPL.

    Directory of Open Access Journals (Sweden)

    Yinyuan Ding

    Full Text Available BACKGROUND: Macrophage-derived lipoprotein lipase (LPL has been shown uniformly to promote atherosclerotic lesion formation while the extent to which it affects plasma lipid and lipoprotein levels varies in wild-type and hypercholesterolemic mice. It is known that high levels of LPL in the bulk of adipose tissue and skeletal muscle would certainly mask the contribution of macrophage LPL to metabolism of plasma lipoprotein. Therefore, we chose LPL deficient (LPL⁻/⁻ mice with severe hypertriglyceridemia as an alternative model to assess the role of macrophage LPL in plasma lipoprotein metabolism via bone marrow transplant, through which LPL will be produced mainly by hematopoietic cell-derived macrophages. METHODS AND RESULTS: Hypertriglyceridemic LPL⁻/⁻ mice were lethally irradiated, then transplanted with bone marrow from wild-type (LPL⁺/⁺ or LPL⁻/⁻ mice, respectively. Sixteen weeks later, LPL⁺/⁺ →LPL⁻/⁻ mice displayed significant reduction in plasma levels of triglyceride and cholesterol (408±44.9 vs. 2.7±0.5×10³ and 82.9±7.1 vs. 229.1±30.6 mg/dl, p<0.05, respectively, while a 2.7-fold increase in plasma high density lipoprotein- cholesterol (p<0.01 was observed, compared with LPL⁻/⁻→LPL⁻/⁻ control mice. The clearance rate for the oral fat load test in LPL⁺/⁺ →LPL⁻/⁻ mice was faster than that in LPL⁻/⁻→LPL⁻/⁻ mice, but slower than that in wild-type mice. Liver triglyceride content in LPL⁺/⁺→LPL⁻/⁻ mice was also significantly increased, compared with LPL⁻/⁻→LPL⁻/⁻ mice (6.8±0.7 vs. 4.6±0.5 mg/g wet tissue, p<0.05, n = 6. However, no significant change was observed in the expression levels of genes involved in hepatic lipid metabolism between the two groups. CONCLUSIONS: Hematopoietic cell-derived LPL could efficiently ameliorate severe hypertriglyceridemia and hypo-alpha-cholesterolemia at the compensation of increased triglyceride content of liver in LPL

  12. ArF excimer laser modulation of TNF-alpha and gelatinase B in NIH 3T3 cells; Modulation de l`expression du TNF-alpha et de la gelatinase B, apres irradiation de fibroblastes NIH 3T3 par un laser a excimeres a 193 NM

    Energy Technology Data Exchange (ETDEWEB)

    Naudy-Vives, C.; Courant, D.; Perot, J.C.; Garcia, J.; Fretier, P.; Court, L.; Dormont, D.

    1995-12-31

    The effects on TNF-alpha and gelatinase B activity in mammalian cells induced by 193 nm argon fluoride excimer laser have been investigated. The data show that a secretion of 92 kDa type IV collagenase and TNF-alpha were increased in cell culture supernatants. Moreover, the 193 nm laser radiation produces a decrease of cell proliferation and an increase of cell activation 8 hours after irradiation. The total protein amount increases with the delivered dose. Same, but less effects were obtained after exposure to a conventional UV lamp at 254 nm. (author). 8 refs.

  13. PRODUCTION OF PROINFLAMMATORY CYTOKINES AND ALPHA-2-MACROGLOBULIN BY PERIPHERAL BLOOD CELLS IN THE PATIENTS WITH COLORECTAL CANCER

    Directory of Open Access Journals (Sweden)

    V. N. Zorina

    2016-01-01

    Full Text Available Colorectal cancer (CRC is the third most common cancer worldwide, being quite complicated, with respect to diagnostics and postoperative prognosis. Proinflammatory cytokines are shown to be involved into CRC pathogenesis. However, the changes in alpha-2-macroglobulin (α2-MG, a known regulator of cytokine production, still remain unclear. The aim of this work was to compare contents and production of a2-MG and several pro-inflammatory cytokines in blood serum and supernates from short-term blood cell cultures. The samples were taken from the patients with CRC at initial terms and after surgical removal of the tumor.Studies of cytokines and a2-MG concentrations in serum and supernates of 24-h blood cell cultures from the patients with verified CRC (stages T2-3N0-1M0 and T4N0-1M0 have shown some sufficient differences from healthy volunteers (control group. Pre-surgery IL-6 and TNFα contents in blood of CRC patients was significantly increased agains healthy controls (respectively, 29.9±5.4 and 3.4±1.5 pg/mL versus control group (1.0±0.3 and 0 pg/mL, respectively. Following surgical treatment, the cytokine levels were decreased by 40- 60% after the operation, however, without significant differences from initial values.The supernates of blood cultures stimulated with polyclonal mitogens exhibited significant reduction of IFNγ levels prior to surgery (273±123 pg/ml versus 804±154 pg/mL, and elevated IL-6 levels (14412±2570 pg/mL versus 1970±457 pg/mL. The mean α2-MG concentrations before CRC surgery comprised 1.96±0.11 g/L for blood serum, 0.0304±0.0047 g/L, for non-stimulated blood cell cultures, and 0.0300±0.0052 g/L in mitogen-induced cultures. These parameters did not significantly differ from control values (2.21±0.17 g/L, 0.0328±0.0018 g/L, and 0.0314±0.0019 g/L, respectively. Similar results have been yielded with the samples obtained after surgical treatment of the CRC patients.The obtained data indicate that surgical

  14. Influence of High Aspect Ratio Vessel Cell Culture on TNF-Alpha, Insulin Secretion and Glucose Homeostasis in Pancreatic Islets of Langerhans from Wistar Furth Rats

    Science.gov (United States)

    Tobin, Brian W.a; Leeper-Woodford, Sandra K.

    1999-01-01

    The present studies were carried out to determine the influence of a ground based microgravity paradigm, utilizing the High Aspect Ratio Vessel (HARV) cell culture upon lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha (TNF-alpha) production of pancreatic islets of Langerhans. An additional aim was to elucidate alterations in insulin secretion and glucose utilization using the HARV low shear, gravity averaged vector, cell culture technique. Islets were isolated (1726 +/- 117, 150 micron islet equivalent units) from Wistar Furth rats and assigned to four treatment groups: 1) HARV, 2) HARV plus LPS, 3) static culture, 4) static culture plus LPS. Following 48 hours of culture, insulin concentration was increased in both HARV and static cultures (palpha (L929 cytotoxicity assay) and was measured at selected time points for 48 hours. TNF-alpha was significantly increased in LPS-induced HARV and static cultures, yet the increase was more pronounced in the static culture group (palpha is associated with a decreased insulin secretion is intriguing, both as it relates to in-flight investigations, and as it may provide insight into the pathophysiology of Type I and Type 11 diabetes. Glucose concentration in islet medium was lesser throughout the experiment in static cultures, suggesting a decreased reliance upon glucose as a metabolic substrate in the islets cultured in HARVS. In conclusion, the present studies demonstrate alterations in LPS induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF production in the microgravity HARV paradigm. Additionally, alterations in fuel homeostasis may be promulgated by HARV culture. The clinical and physiological significance of these observations remains to be determined.

  15. CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents

    Directory of Open Access Journals (Sweden)

    Feng Zhang

    2017-12-01

    Full Text Available Paeoniflorin-6′-O-benzene sulfonate (code: CP-25 was the chemistry structural modifications of Paeoniflorin (Pae. CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF or Tumor necrosis factor alpha (TNF-alpha. CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19+ B cells, CD19+CD20+ B cells, CD19+CD27+ B cells and CD19+CD20+CD27+ B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

  16. CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

    Science.gov (United States)

    Zhang, Feng; Shu, Jin-Ling; Li, Ying; Wu, Yu-Jing; Zhang, Xian-Zheng; Han, Le; Tang, Xiao-Yu; Wang, Chen; Wang, Qing-Tong; Chen, Jing-Yu; Chang, Yan; Wu, Hua-Xun; Zhang, Ling-Ling; Wei, Wei

    2017-01-01

    Paeoniflorin-6'- O -benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19 + B cells, CD19 + CD20 + B cells, CD19 + CD27 + B cells and CD19 + CD20 + CD27 + B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

  17. Differential Recognition of CD1d-[alpha]-Galactosyl Ceramide by the V[beta]8.2 and V[beta]7 Semi-invariant NKT T Cell Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Pellicci, Daniel G.; Patel, Onisha; Kjer-Nielsen, Lars; Pang, Siew Siew; Sullivan, Lucy C.; Kyparissoudis, Konstantinos; Brooks, Andrew G.; Reid, Hugh H.; Gras, Stephanie; Lucet, Isabelle S.; Koh, Ruide; Smyth, Mark J.; Mallevaey, Thierry; Matsuda, Jennifer L.; Gapin, Laurent; McCluskey, James; Godfrey, Dale I.; Rossjohn, Jamie; PMCI-A; Monash; UCHSC; Melbourne

    2009-09-02

    The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR{alpha} chain is typically invariant, the {beta} chain expression is more diverse, where three V{beta} chains are commonly expressed in mice. We report the structures of V{alpha}14-V{beta}8.2 and V{alpha}14-V{beta}7 NKT TCRs in complex with CD1d-{alpha}-galactosylceramide ({alpha}-GalCer) and the 2.5 {angstrom} structure of the human NKT TCR-CD1d-{alpha}-GalCer complex. Both V{beta}8.2 and V{beta}7 NKT TCRs and the human NKT TCR ligated CD1d-{alpha}-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V{beta} domains of the V{beta}8.2 and V{beta}7 NKT TCR-CD1d complexes resulted in altered TCR{beta}-CD1d-mediated contacts and modulated recognition mediated by the invariant {alpha} chain. Mutagenesis studies revealed the differing contributions of V{beta}8.2 and V{beta}7 residues within the CDR2{beta} loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V{beta} usage in NKT cells.

  18. Neonatal screening for sickle cell disease, Glucose-6-PhosphateDehydrogenase deficiency and Alpha-Thalassemia in Qatif and Al-Hasa

    International Nuclear Information System (INIS)

    Nasserullah, Z.; Srair, Hussain Abu; Al-Jame, A.; Mokhtar, M.; Al-Qatari, G.; Al-Naim, S.; Al-Aqib, A.

    1998-01-01

    Screening programs to determine the frequency of sickle cell,glucose-6-phosphate dehydrogenase deficiency and alpha-thalassemia gene areavailable in Saudi Arabia, although not used frequently. Greater use of theseprograms will decrease the morbidity and mortality of Saudi children affectedby these disorders. Neonatal hemoglobin electrophoresis andglucose-6-dehydrogenase fluorescent spot tests were performed on new bornbabies delivered between December 1992 and December 1993 at the Qatif CentralHospital and at the King Fahd Hospital in Al-Hasa. Cord blood samples werecollected from babies born in these two hospitals. Babies born in otherhospitals had blood collected in their first visit to Qatif primary carecenters at the time of vaccination. All specimens were sent to Dammam CentralLaboratory. The diagnosis of sickle cell and alpha-thalassemia was based oncellulose acetate electrophoresis and confirmed by agar gel electrophoresisand glucose-6-phosphate dehydrgenase was confirmed by fluorescent spot test.A total of 12,220 infants, including 11,313 Saudis (92.6%), were screenedover a 12-month period. The common phenotype detected in these infantsincluded AF, SFA, SFA Bart's, FS and FS Bart's. In Saudi infants, homozygoussickle cell disease was detected in 2.35% and 1.08% in Qatif and Al-Hasa,respectively. The frequencies of sickle cell gene were 0.1545% and 0.1109% inQatif and Al-Hasa. Alpha-thalassemia genes based on an elevated level of HbBart's were 28% and 16.3% in Qatif and Al-Hasa. The screening for G6PDdeficiency revealed a high prevalence of 30.6% and 14.7% in Qatif andAl-Hasa. In the non-Saudi infants the frequencies were low. The outcome ofthis study indicates that the Saudi populations in Qatif and Al-Hasa are atrisk for hemoglobinopathies and G6PD. Neonatal screening programs areessential and cost effective and should be maintained as a routine practice.(author)

  19. The contraction induced increase in gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1alpha), mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in primary rat skeletal muscle cells is dependent on reactive oxygen species

    DEFF Research Database (Denmark)

    Silveira, Leonardo R.; Pilegaard, Henriette; Kusuhara, Keiko

    2006-01-01

    We evaluated the role of reactive oxygen species (ROS) for the contraction induced increase in expression of PGC-1alpha, HKII and UCP3 mRNA. Rat skeletal muscle cells were subjected to acute or repeated electrostimulation in the presence and absence of antioxidants. Contraction of muscle cells lead...... to an increased H2O2 formation, as measured by oxidation of H2HFF. Acute contraction of the muscle cells lead to a transient increase in PGC-1alpha and UCP3 mRNA by 172 and 65%, respectively (pantioxidants. Repeated contraction sessions induced...... a sustained elevation in PGC-1alpha and UCP3 mRNA and a transient increase in HKII (pantioxidant cocktail or with GPX+GSH. Incubation of cells for 10 days with ROS produced by xanthine oxidase/xanthine increased the level of PGC-1...

  20. Bid integrates intrinsic and extrinsic signaling in apoptosis induced by alpha-tocopheryl succinate in human gastric carcinoma cells

    Czech Academy of Sciences Publication Activity Database

    Zhao, Y.; Li, R.; Xia, W.; Neužil, Jiří; Lu, Y.; Zhang, H.; Zhao, X.; Zhang, X.; Sun, C.; Wu, K.

    2010-01-01

    Roč. 288, č. 1 (2010), s. 42-49 ISSN 0304-3835 R&D Projects: GA ČR(CZ) GA204/08/0811 Institutional research plan: CEZ:AV0Z50520701 Keywords : Alpha-tocopheryl succinate * signaling * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.864, year: 2010

  1. Alpha-Tocopheryl succinate induces cytostasis and apoptosis in osteosarcoma cells: the role of E2F1

    Czech Academy of Sciences Publication Activity Database

    Alleva, R.; Benassi, M.S.; Tomasetti, M.; Gellert, N.; Ponticelli, F.; Borghi, B.; Picci, P.; Neužil, Jiří

    2005-01-01

    Roč. 331, č. 4 (2005), s. 1515-1521 ISSN 0006-291X Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z5052915 Keywords : osteosarcoma * alpha-tocopheryl succinate * E2F1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.000, year: 2005

  2. Suppressor of cytokine signalling-3 inhibits Tumor necrosis factor-alpha induced apoptosis and signalling in beta cells

    DEFF Research Database (Denmark)

    Bruun, Christine; Heding, Peter E; Rønn, Sif G

    2009-01-01

    Tumor necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine involved in the pathogenesis of several diseases including type 1 diabetes mellitus (T1DM). TNFalpha in combination with interleukin-1-beta (IL-1beta) and/or interferon-gamma (IFNgamma) induces specific destruction...

  3. Innovations in Los Alamos alpha box design

    International Nuclear Information System (INIS)

    Ledbetter, J.M.; Dowler, K.E.; Cook, J.H.

    1985-01-01

    Destructive examinations of irradiated fuel pins containing plutonium fuel must be performed in shielded hot cells with strict provisions for containing the plutonium. Alpha boxes provide containment for the plutonium, toxic fission products, and other hazardous highly radioactive materials. The alpha box contains windows for viewing and a variety of transfer systems specially designed to allow transfers in and out of the alpha box without spread of the hazardous materials that are contained in the box. Alpha boxes have been in use in the Wing 9 hot cells at Los Alamos National Laboratory for more than 20 years. Features of the newly designed alpha boxes are presented

  4. A preliminary investigation of the enzymatic inhibition of 5alpha-reduction and growth of prostatic carcinoma cell line LNCap-FGC by natural astaxanthin and Saw Palmetto lipid extract in vitro.

    Science.gov (United States)

    Anderson, Mark L

    2005-01-01

    Inhibition of 5alpha-reductase has been reported to decrease the symptoms of benign prostate hyperplasia (BPH) and possibly inhibit or help treat prostate cancer. Saw Palmetto berry lipid extract (SPLE) is reported to inhibit 5alpha-reductase and decrease the clinical symptoms of BPH. Epidemiologic studies report that carotenoids such as lycopene may inhibit prostate cancer. In this investigation the effect of the carotenoid astaxanthin, and SPLE were examined for their effect on 5alpha-reductase inhibition as well as the growth of prostatic carcinoma cells in vitro. These studies support patent #6,277,417 B1. The results show astaxanthin demonstrated 98% inhibition of 5alpha-reductase at 300 microg/mL in vitro. Alphastat, the combination of astaxanthin and SPLE, showed a 20% greater inhibition of 5alpha-reductase than SPLE alone n vitro. A nine day treatment of prostatic carcinoma cells with astaxanthin in vitro produced a 24% decrease in growth at 0.1 mcg/mL and a 38% decrease at 0.01 mcg/mL. SPLE showed a 34% decrease at 0.1 mcg/mL. Low levels of carotenoid astaxanthin inhibit 5alpha-reductase and decrease the growth of human prostatic cancer cells in vitro. Astaxanthin added to SPLE shows greater inhibition of 5alpha-reductase than SPLE alone in vitro.

  5. Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

    International Nuclear Information System (INIS)

    Hanot, Maite; Hoarau, Jim; Carriere, Marie; Angulo, Jaime F.; Khodja, Hicham

    2009-01-01

    Purpose: Free radicals are believed to play an active role in the bystander response. This study investigated their origin as well as their temporal and spatial impacts in the bystander effect. Methods and Materials: We employed a precise alpha-particle microbeam to target a small fraction of subconfluent osteoblastic cells (MC3T3-E1). γH2AX-53BP1 foci, oxidative metabolism changes, and micronuclei induction in targeted and bystander cells were assessed. Results: Cellular membranes and mitochondria were identified as two distinct reactive oxygen species producers. The global oxidative stress observed after irradiation was significantly attenuated after cells were treated with filipin, evidence for the primal role of membrane in the bystander effect. To determine the membrane's impact at a cellular level, micronuclei yield was measured when various fractions of the cell population were individually targeted while the dose per cell remained constant. Induction of micronuclei increased in bystander cells as well as in targeted cells and was attenuated by filipin treatment, demonstrating a role for bystander signals between irradiated cells in an autocrine/paracrine manner. Conclusions: A complex interaction of direct irradiation and bystander signals leads to a membrane-dependent amplification of cell responses that could influence therapeutic outcomes in tissues exposed to low doses or to environmental exposure.

  6. Reoxygenation of human coronary smooth muscle cells suppresses HIF-1{alpha} gene expression and augments radiation-induced growth delay and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Grumann, T.; Arab, A.; Bode, C.; Hehrlein, C. [Dept. of Cardiology, Univ. Clinic of Freiburg (Germany); Guttenberger, R. [Dept. of Radiotherapy, Univ. Clinic of Freiburg (Germany)

    2006-01-01

    Background and Purpose: Catheter-based coronary brachytherapy with {beta}- and {gamma}-radiation is an evidence-based method to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) and stent implantation, but the outcome may be PTCA are hypoxic. A lack of oxygen decreases the effect of low LET (linear energy transfer) irradiation. The authors assumed that reoxygenation of hypoxic human coronary smooth muscle cells (HCSMCs) improves the results of coronary brachytherapy. The expression of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) gene, and the rates of growth and apoptosis of hypoxic and reoxygenated HCSMCs after {gamma}-iradiation were therefore analyzed. Material and Methods: An in vitro model of megacolonies of HCSMCs was developed. After exposure to chronic hypoxia the HCSMCs were irradiated with graded doses of 2, 4, 8, and 16 Gy using a {sup 60}Co source either under hypoxia (pO{sub 2}<3 mmHg) or after reoxygenation (pO{sub 2}{approx}150 mmHg). RT-PCR (reverse transcription-polymerase chain reaction) analysis was used to quantify HIF-1{alpha} gene expression and the growth of HCSMC megacolonies was measured serially. The oxygen enhancement ratio (OER) was calculate from the specific growth delay. Apoptosis of HCSMCs was quantified by counting cells with specific DNA strand breaks using the TUNEL assy. Results: HIF-1{alpha} gene expression was markedly suppressed in reoxygenated cells versus hypoxic cells 30 min after {gamma}-irradiation at all radiation doses (158{+-}46% vs. 1,675{+-}1,211%; p<0.01). Apoptosis was markedly increased in reoxygenated HCSMCs. The OER was 1.8(95% CI[confidence interval]1.3-2.4). Therefore, reoxygenated HCSMCs require 44% less radiation dose to achieve the equivalent biological radiation effect compared to hypoxic HCSMCs. Conclusion: Reoxygenation of coronary smooth muscle cells should be considered an option to increase efficacy of coronary brachytherapy. This could be used to reduce radiation dose

  7. Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers

    NARCIS (Netherlands)

    Almeida, Afonso R. M.; Legrand, Nicolas; Papiernik, Martine; Freitas, António A.

    2002-01-01

    We show that the lymphoid hyperplasia observed in IL-2Ralpha- and IL-2-deficient mice is due to the lack of a population of regulatory cells essential for CD4 T cell homeostasis. In chimeras reconstituted with bone marrow cells from IL-2Ralpha-deficient donors, restitution of a population of

  8. SM22{alpha}-induced activation of p16{sup INK4a}/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of {gamma}-radiation and doxorubicin in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Tae Rim; Lee, Hee Min; Lee, So Yong; Kim, Eun Jin; Kim, Kug Chan [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Paik, Sang Gi [Department of Biology, School of Biosciences and Biotechnology, Chungnam National University, Daejeon (Korea, Republic of); Cho, Eun Wie, E-mail: ewcho@kribb.re.kr [Daejeon-KRIBB-FHCRC Cooperation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Kim, In Gyu, E-mail: igkim@kaeri.re.kr [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2010-09-10

    Research highlights: {yields} SM22{alpha} overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of {gamma}-radiation or doxorubicin promotes cellular senescence. {yields} SM22{alpha} overexpression elevates p16{sup INK4a} followed by pRB activation, but there are no effects on p53/p21{sup WAF1/Cip1} pathway. {yields} SM22{alpha}-induced MT-1G activates p16{sup INK4a}/pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22{alpha}) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22{alpha} overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22{alpha} overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of {gamma}-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 {mu}g/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21{sup WAF1/Cip1} induction or p16{sup INK4a}/retinoblastoma protein (pRB) activation. SM22{alpha} overexpression in HepG2 cells elevated p16{sup INK4a} followed by pRB activation, but did not activate the p53/p21{sup WAF1/Cip1} pathway. Moreover, MT-1G, which is induced by SM22{alpha} overexpression, was involved in the activation of the p16{sup INK4a}/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22{alpha} modulates cellular senescence caused by damaging agents via regulation of the p16{sup INK4a}/pRB pathway in HepG2 cells and that these effects of SM22{alpha} are partially mediated by MT-1G.

  9. Discovery, SAR, and Radiolabeling of Halogenated Benzimidazole Carboxamide Antagonists as Useful Tools for (alpha)4(beta)1 Integrin Expressed on T- and B-cell Lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Carpenter, R D; Natarajan, A; Lau, E Y; Andrei, M; Solano, D M; Lightstone, F C; DeNardo, S J; Lam, K S; Kurth, M J

    2010-02-08

    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin is an attractive yet poorly understood target for selective diagnosis and treatment of T- and B-cell lymphomas. This report focuses on the rapid microwave preparation of medicinally pertinent benzimidazole heterocycles, structure-activity relationships (SAR) of novel halobenzimidazole carboxamide antagonists 3-6, and preliminary biological evaluation of radioiodinated agents 7, 8, and 18. The I-125 derivative 18 had good tumor uptake (12 {+-} 1% ID/g at 24 h; 4.5 {+-} 1% ID/g at 48 h) and tumor:kidney ratio ({approx}4:1 at 24 h; 2.5:1 at 48 h) in xenograft murine models of B-cell lymphoma. Molecular homology models of {alpha}{sub 4}{beta}{sub 1} integrin have predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. These high affinity ({approx} pM binding) halogenated ligands are attractive tools for medicinal and biological use; the fluoro and iodo derivatives are potential radiodiagnostic ({sup 18}F) or radiotherapeutic ({sup 131}I) agents, whereas the chloro and bromo analogues could provide structural insight into integrin-ligand interactions through photoaffinity cross-linking/mass spectroscopy experiments, as well as co-crystallization X-ray studies.

  10. Triple composite tumor of stomach: A rare combination of alpha fetoprotein positive hepatoid adenocarcinoma, tubular adenocarcinoma and large cell neuroendocrine carcinoma

    Directory of Open Access Journals (Sweden)

    Lipika Lipi

    2014-01-01

    Full Text Available A 50-year-old male patient presented with pain abdomen of 6 months duration. Computed tomography scan revealed a large mass in the stomach occluding the lumen. Histopathology revealed a triple composite tumor comprising of tubular adenocarcinoma arising on a background of high-grade dysplasia, hepatoid adenocarcinoma (positive for Hep Par-1 and alpha fetoprotein and large cell neuroendocrine carcinoma (positive for synaptophysin and chromogranin with nodal metastasis.Triple composite tumors are distinctly rare with few reports in literature.

  11. Microwave-assisted synthesis of N-pyrazole ureas and the p38alpha inhibitor BIRB 796 for study into accelerated cell ageing.

    Science.gov (United States)

    Bagley, Mark C; Davis, Terence; Dix, Matthew C; Widdowson, Caroline S; Kipling, David

    2006-11-21

    Microwave irradiation of substituted hydrazines and beta-ketoesters gives 5-aminopyrazoles in excellent yield, which can be transformed to the corresponding N-carbonyl derivatives by treatment with an isocyanate or chloroformate. Derivatization of 4-nitronaphth-1-ol using predominantly microwave heating methods and reaction with an N-pyrazole carbamate provides a rapid route to the N-pyrazole urea BIRB 796 in high purity, as a potent and selective inhibitor of p38alpha mitogen-activated protein kinase for the study of accelerated ageing in Werner syndrome cells.

  12. Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. I. Recognition by two alpha beta T cell receptors

    DEFF Research Database (Denmark)

    Rognan, D; Stryhn, A; Fugger, L

    2000-01-01

    dynamics. Next, three-dimensional models of two different T cell receptors (TCRs) both specific for the Ha255-262/Kk complex were generated based on previously published TCR X-ray structures. Finally, guided by the recently published X-ray structures of ternary TCR/peptide/MHC-I complexes, the TCR models...... the models. They were found to account well for the experimentally obtained data, lending considerable support to the proposed models and suggesting a universal docking mode for alpha beta TCRs to MHC-peptide complexes. Such models may also be useful in guiding future rational experimentation....

  13. Cell death triggered by alpha-emitting 213Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death

    International Nuclear Information System (INIS)

    Seidl, Christof; Schroeck, Hedwig; Seidenschwang, Sabine; Beck, Roswitha; Schwaiger, Markus; Senekowitsch-Schmidtke, Reingard; Schmid, Ernst; Abend, Michael; Becker, Karl-Friedrich; Apostolidis, Christos; Nikula, Tuomo K.; Kremmer, Elisabeth

    2005-01-01

    Radioimmunotherapy with α-particle-emitting nuclides, such as 213 Bi, is a promising concept for the elimination of small tumour nodules or single disseminated tumour cells. The aim of this study was to investigate cellular damage and the mode of cell death triggered by 213 Bi-immunoconjugates. Human gastric cancer cells (HSC45-M2) expressing d9-E-cadherin were incubated with different levels of activity of 213 Bi-d9MAb targeting d9-E-cadherin and 213 Bi-d8MAb, which does not bind to d9-E-cadherin. Micronucleated (M) cells, abnormal (A) cells and apoptotic (A) [(MAA)] cells were scored microscopically in the MAA assay following fluorescent staining of nuclei and cytoplasm. Chromosomal aberrations were analysed microscopically following Giemsa staining. The effect of z-VAD-fmk, known to inhibit apoptosis, on the prevention of cell death was investigated following treatment of HSC45-M2 cells with sorbitol as well as 213 Bi-d9MAb. Activation of caspase 3 after incubation of HSC45-M2 cells with both sorbitol and 213 Bi-d9MAb was analysed via Western blotting. Following incubation of HSC45-M2 human gastric cancer cells expressing d9-E-cadherin with 213 Bi-d9MAb the number of cells killed increased proportional to the applied activity concentration. Microscopically visible effects of α-irradiation of HSC45-M2 cells were formation of micronuclei and severe chromosomal aberrations. Preferential induction of these lesions with specific 213 Bi-d9MAb compared with unspecific 213 Bi-d8MAb (not targeting d9-E-cadherin) was not observed if the number of floating, i.e. unbound 213 Bi-immunoconjugates per cell exceeded 2 x 10 4 , most likely due to intense crossfire. In contrast to sorbitol-induced cell death, cell death triggered by 213 Bi-immunoconjugates was independent of caspase 3 activation and could not be inhibited by z-VAD-fmk, known to suppress the apoptotic pathway. 213 Bi-immunoconjugates seem to induce a mode of cell death different from apoptosis in HSC45-M2 cells

  14. The Almahata Sitta Polymict Ureilite from the University of Khartoum Collection: Classification, Distribution of Clast Types in the Strewn Field, New Meteorite Types, and Implications for the Structure of Asteroid 2008 TC3

    Science.gov (United States)

    Goodrich, C. A.; Fioretti, A. M.; Zolensky, M.; Ross, Daniel K.; Shaddad, M.; Ross, D. K.; Kohl, I.; Young, E.; Kita, N.; Hiroi, T.; hide

    2018-01-01

    The Almahata Sitta (AhS) polymict ureilite fell in 2008 when asteroid 2008 TC3 impacted over Sudan]. It is the first meteorite to originate from an asteroid that had been tracked and studied in space (with spectral classification) before impact, and provides a unique opportunity to correlate properties of meteorites with those of their parent asteroid. More than 700 monolithologic stones from the AhS fall were collected. Of those previously studied, approx. 70% were ureilites and approx. 30% were chondrites. It has been inferred that 2008 TC3 was loosely aggregated and porous and disintegrated in the atmosphere, with only its most coherent clasts falling as stones. However, understanding the structure of this asteroid is limited by incomplete study of the heterogeneous stones, and the loss of most of the mass of the asteroid. The University of Khartoum (UOK) AhS collection contains over >600 AhS stones with find coordinates. We are studying this collection to determine: 1) the proportion of ureilitic to various non-ureilitic stones; 2) the distribution of types of stones in the strewn field; and 3) the compositional and physical structure of 2008 TC3. We report on 61 new stones, including a unique sample that may represent the bulk of the material lost from 2008 TC3.

  15. Potential of Burkholderia seminalis TC3.4.2R3 as Biocontrol Agent Against Fusarium oxysporum Evaluated by Mass Spectrometry Imaging

    Science.gov (United States)

    Araújo, Francisca Diana da Silva; Araújo, Welington Luiz; Eberlin, Marcos Nogueira

    2017-05-01

    Species of genus Burkholderia display different interaction profiles in the environment, causing either several diseases in plants and animals or being beneficial to some plants, promoting their growth, and suppressing phytopathogens. Burkholderia spp. also produce many types of biomolecules with antimicrobial activity, which may be commercially used to protect crops of economic interest, mainly against fungal diseases. Herein we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate secondary metabolites produced by B. seminalis TC3.4.2R3 in monoculture and coculture with plant pathogen Fusarium oxysporum. The siderophore pyochelin and the rhamnolipid Rha-Rha-C15-C14 were detected in wild-type B. seminalis strain, and their productions were found to vary in mutant strains carrying disruptions in gene clusters associated with antimicrobial compounds. Two mycotoxins were detected in F. oxysporum. During coculture with B. seminalis, metabolites probably related to defense mechanisms of these microorganisms were observed in the interspecies interaction zone. Our findings demonstrate the effective application of MALDI-MSI in the detection of bioactive molecules involved in the defense mechanism of B. seminalis, and these findings suggest the potential use of this bacterium in the biocontrol of plant diseases caused by F. oxysporum.

  16. Alpha particle emitters in medicine

    International Nuclear Information System (INIS)

    Fisher, D.R.

    1989-09-01

    Radiation-induced cancer of bone, liver and lung has been a prominent harmful side-effect of medical applications of alpha emitters. In recent years, however, the potential use of antibodies labeled with alpha emitting radionuclides against cancer has seemed promising because alpha particles are highly effective in cell killing. High dose rates at high LET, effectiveness under hypoxic conditions, and minimal expectancy of repair are additional advantages of alpha emitters over antibodies labeled with beta emitting radionuclides for cancer therapy. Cyclotron-produced astatine-211 ( 211 At) and natural bismuth-212 ( 212 Bi) have been proposed and are under extensive study in the United States and Europe. Radium-223 ( 223 Ra) also has favorable properties as a potential alpha emitting label, including a short-lived daughter chain with four alpha emissions. The radiation dosimetry of internal alpha emitters is complex due to nonuniformly distributed sources, short particle tracks, and high relative specific ionization. The variations in dose at the cellular level may be extreme. Alpha-particle radiation dosimetry, therefore, must involve analysis of statistical energy deposition probabilities for cellular level targets. It must also account fully for nonuniform distributions of sources in tissues, source-target geometries, and particle-track physics. 18 refs., 4 figs

  17. Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

    Science.gov (United States)

    Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

    2010-10-01

    We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

  18. GnRH signalling pathways and GnRH-induced homologous desensitization in a gonadotrope cell line (alphaT3-1).

    Science.gov (United States)

    Poulin, B; Rich, N; Mas, J L; Kordon, C; Enjalbert, A; Drouva, S V

    1998-07-25

    Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on

  19. Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

    Science.gov (United States)

    Capmany, G; Querol, S; Cancelas, J A; García, J

    1999-08-01

    The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.

  20. [Neonatal screening of hemoglobinopathies and G6PD deficiency in Catalonia (Spain). Molecular study of sickle cell disease associated with alpha thalassemia and G6PD deficiency].

    Science.gov (United States)

    Mañú Pereira, María Del Mar; Cabot, Anna; Martínez González, Ana; Sitjà Navarro, Eulalia; Cararach, Vicent; Sabrià, Josep; Boixaderas, Jordi; Teixidor, Roser; Bosch, Albert; López Vílchez, M Angeles; Martín Ibáñez, Itziar; Carrión, Teresa; Plaja, Pere; Sánchez, Mario; Vives Corrons, José Luis

    2007-06-30

    The prevalence of hemoglobinopathies and glucose-6-phosphate dehidrogenase (G6PD) deficiency in the Catalan neonatal population is increasing due to immigration. Coinheritance of more than a single RBC genetic defect is becoming more frequent and diagnostic pitfalls are also increasing. We intended to demonstrate the need to perform an early diagnosis of sickle cell disease (SCD) by means of neonatal screening, to establish the prevalence of SCD associated with alpha thalassemia and G6PD deficiency and to identify genotypes associated with sickle cell disease and G6PD deficiency. 4,020 blood samples from newborns were screened. For the screening of hemoglobinopathies the high performance liquid chromatography method was used and for G6PD deficiency the fluorescent spot test was employed. We studied the association between betaS gene and alpha thalassaemia del-3.7 Kb. SCD and G6PD deficiency genotypes were established. Prevalence of SCD in population at risk was 1/475 newborns. Prevalence of G6PD deficiency in population at risk was 1/43, and in autochthonous population was 1/527 newborns. In all the cases, sickle hemoglobin was confirmed by ARMS (amplification refractory mutation system). Association between betaS gene and alpha thalassaemia del-3.7 Kb was found in 32.2% of the samples, and an association between betaS gene and G6PD deficiency was observed in 7% of the samples. This study confirms the high prevalence of SCD and G6PD deficiency in population at risk as well as their genetic and clinical heterogeneity. The study of genotype/phenotype relationships allows a better knowledge of molecular mechanism and is useful to establish suitable criteria of diagnosis.

  1. 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