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Sample records for pre-t cell receptor

  1. TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation

    NARCIS (Netherlands)

    Blom, B.; Verschuren, M. C.; Heemskerk, M. H.; Bakker, A. Q.; van Gastel-Mol, E. J.; Wolvers-Tettero, I. L.; van Dongen, J. J.; Spits, H.

    1999-01-01

    Recent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into

  2. Co-ordinate expression of the pre-T-cell receptor complex and a novel immature thymocyte-specific antigen, IMT-1, during thymocyte development.

    Science.gov (United States)

    Tong, J J; Kishi, H; Nagata, T; Muraguchi, A

    1999-01-01

    Previously we described a monoclonal antibody (mAb) that reacted with a cell-surface antigen, immature thymocyte antigen-1 (IMT-1), which is expressed on thymocytes of late CD4- CD8- (double negative) to early CD4+ CD8+ (double positive) differentiation stages. In this study, we investigated the expression of IMT-1 on various cell lineages in thymus as well as in peripheral lymphoid organs. We found that IMT-1 is expressed on T-cell receptor (TCR)-betalo and TCR-deltalo thymocytes, but not on TCR-betahi, TCR-deltahi or natural killer (NK)1.1+ thymocytes, or on peripheral alpha beta or gamma delta T cells. We also investigated the kinetics of expression of IMT-1 during fetal thymocyte development and compared it with the expression of the pre-TCR complex, comprising CD3, pre-TCR-alpha (pTalpha) and TCR-beta. We found that expression of both was similar, starting at day 14.5 of gestation, peaking on day 16.5 and gradually decreasing thereafter. Furthermore, the expression of both IMT-1 and pTalpha was drastically reduced when DN thymocytes in recombination activating gene (RAG)-2-/- mice were challenged in vivo with anti-CD3 mAb. These results indicate that IMT-1 is expressed on not only immature thymocytes of alpha beta T-cell lineage but also on those of gamma delta T-cell lineage, and that the expression of IMT-1 and the pre-TCR complex is co-ordinately regulated during the alpha beta lineage thymocyte development.

  3. Identification of human pre-T/NK cell-associated genes

    NARCIS (Netherlands)

    Ranes-Goldberg, M. G.; Hori, T.; Mohan-Peterson, S.; Spits, H.

    1993-01-01

    We have used a combination of subtractive cloning and differential screening techniques to identify genes preferentially expressed in early stages of human T/NK cell development compared with mature T and NK cells. A fetal liver-derived cytoplasmic (c) CD3+ membrane (m) CD3- clone, FL508, which

  4. Different role for mouse and human CD3delta/epsilon heterodimer in preT cell receptor (preTCR) function: human CD3delta/epsilon heterodimer restores the defective preTCR function in CD3gamma- and CD3gammadelta-deficient mice

    Czech Academy of Sciences Publication Activity Database

    Pan, Q.; Brodeur, J.F.; Drbal, Karel; Vibhuti, P.D.

    2006-01-01

    Roč. 43, č. 11 (2006), s. 1741-1750 ISSN 0161-5890 Institutional research plan: CEZ:AV0Z50520514 Keywords : CD3 * preTCR * receptor signaling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.768, year: 2006

  5. A transient receptor potential channel expressed in taste receptor cells.

    Science.gov (United States)

    Pérez, Cristian A; Huang, Liquan; Rong, Minqing; Kozak, J Ashot; Preuss, Axel K; Zhang, Hailin; Max, Marianna; Margolskee, Robert F

    2002-11-01

    We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.

  6. Expression of GABAergic receptors in mouse taste receptor cells.

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    Margaret R Starostik

    Full Text Available BACKGROUND: Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD for gamma-aminobutyric acid (GABA is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A and GABA(C or metabotropic receptors (GABA(B while it is terminated by the re-uptake of GABA through transporters (GATs. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcriptase-PCR (RT-PCR analysis, we investigated the expression of different GABA signaling molecules in the mouse taste system. Taste receptor cells (TRCs in the circumvallate papillae express multiple subunits of the GABA(A and GABA(B receptors as well as multiple GATs. Immunocytochemical analyses examined the distribution of the GABA machinery in the circumvallate papillae. Both GABA(A-and GABA(B- immunoreactivity were detected in the peripheral taste receptor cells. We also used transgenic mice that express green fluorescent protein (GFP in either the Type II taste cells, which can respond to bitter, sweet or umami taste stimuli, or in the Type III GAD67 expressing taste cells. Thus, we were able to identify that GABAergic receptors are expressed in some Type II and Type III taste cells. Mouse GAT4 labeling was concentrated in the cells surrounding the taste buds with a few positively labeled TRCs at the margins of the taste buds. CONCLUSIONS/SIGNIFICANCE: The presence of GABAergic receptors localized on Type II and Type III taste cells suggests that GABA is likely modulating evoked taste responses in the mouse taste bud.

  7. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  8. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore.......05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...

  9. Regulation of cell polarity by cell adhesion receptors.

    Science.gov (United States)

    Ebnet, Klaus; Kummer, Daniel; Steinbacher, Tim; Singh, Amrita; Nakayama, Masanori; Matis, Maja

    2017-07-22

    The ability of cells to polarize is an intrinsic property of almost all cells and is required for the devlopment of most multicellular organisms. To develop cell polarity, cells integrate various signals derived from intrinsic as well as extrinsic sources. In the recent years, cell-cell adhesion receptors have turned out as important regulators of cellular polarization. By interacting with conserved cell polarity proteins, they regulate the recruitment of polarity complexes to specific sites of cell-cell adhesion. By initiating intracellular signaling cascades at those sites, they trigger their specific subcellular activation. Not surprisingly, cell-cell adhesion receptors regulate diverse aspects of cell polarity, including apico-basal polarity in epithelial and endothelial cells, front-to-rear polarity in collectively migrating cells, and planar cell polarity during organ development. Here, we review the recent developments highlighting the central roles of cell-cell adhesion molecules in the development of cell polarity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

    Science.gov (United States)

    Capone, Myriam; Hockett, Richard D.; Zlotnik, Albert

    1998-01-01

    We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44+CD25+ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44−CD25+ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4+CD8+ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process. PMID:9770518

  11. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore.......05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...... studies of vitamin D's effect on TCC cells in vitro are necessary before the efficacy of treatment with vitamin D analogues in TCC can be evaluated in patients....

  12. RNase MRP cleaves pre-tRNASer-Met in the tRNA maturation pathway.

    Science.gov (United States)

    Saito, Yuichiro; Takeda, Jun; Adachi, Kousuke; Nobe, Yuko; Kobayashi, Junya; Hirota, Kouji; Oliveira, Douglas V; Taoka, Masato; Isobe, Toshiaki

    2014-01-01

    Ribonuclease mitochondrial RNA processing (RNase MRP) is a multifunctional ribonucleoprotein (RNP) complex that is involved in the maturation of various types of RNA including ribosomal RNA. RNase MRP consists of a potential catalytic RNA and several protein components, all of which are required for cell viability. We show here that the temperature-sensitive mutant of rmp1, the gene for a unique protein component of RNase MRP, accumulates the dimeric tRNA precursor, pre-tRNA(Ser-Met). To examine whether RNase MRP mediates tRNA maturation, we purified the RNase MRP holoenzyme from the fission yeast Schizosaccharomyces pombe and found that the enzyme directly and selectively cleaves pre-tRNA(Ser-Met), suggesting that RNase MRP participates in the maturation of specific tRNA in vivo. In addition, mass spectrometry-based ribonucleoproteomic analysis demonstrated that this RNase MRP consists of one RNA molecule and 11 protein components, including a previously unknown component Rpl701. Notably, limited nucleolysis of RNase MRP generated an active catalytic core consisting of partial mrp1 RNA fragments, which constitute "Domain 1" in the secondary structure of RNase MRP, and 8 proteins. Thus, the present study provides new insight into the structure and function of RNase MRP.

  13. Cloning the interleukin 1 receptor from human T cells

    International Nuclear Information System (INIS)

    Sims, J.E.; Acres, R.B.; Grubin, C.E.; McMahan, C.J.; Wignall, J.M.; March, C.J.; Dower, S.K.

    1989-01-01

    cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with K a values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells

  14. Adoptive T Cell Therapies: A Comparison of T Cell Receptors and Chimeric Antigen Receptors

    Science.gov (United States)

    Harris, Daniel T.; Kranz, David M.

    2016-01-01

    The tumor-killing properties of T cells provide tremendous opportunities to treat cancer. Adoptive T cell therapies have begun to harness this potential by endowing a functionally diverse repertoire of T cells with genetically modified, tumor-specific recognition receptors. Normally, this antigen recognition function is mediated by an αβ T cell receptor (TCR), but the dominant therapeutic forms currently in development are synthetic constructs called chimeric antigen receptors (CARs). While CAR-based adoptive cell therapies are already showing great promise, their basic mechanistic properties have been studied in less detail compared with those of αβ TCRs. In this review, we compare and contrast various features of TCRs versus CARs, with a goal of highlighting issues that need to be addressed to fully exploit the therapeutic potential of both. PMID:26705086

  15. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  16. Elementary Steps in T Cell Receptor Triggering

    OpenAIRE

    Dushek, Omer

    2012-01-01

    The mechanism by which antigen binding to the T cell antigen receptor (TCR) generates intracellular signaling, a process termed TCR triggering, is incompletely understood. A large body of experimental evidence has implicated multiple biophysical/biochemical effects and multiple molecules in the process of TCR triggering, which likely reflect the uniquely demanding role of the TCR in recognizing diverse antigenic ligands. In this perspective, I propose that breaking down the process of TCR tri...

  17. Recruitment of activation receptors at inhibitory NK cell immune synapses.

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    Nicolas Schleinitz

    2008-09-01

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  18. Andrographolide regulates epidermal growth factor receptor and transferrin receptor trafficking in epidermoid carcinoma (A-431) cells

    Science.gov (United States)

    Tan, Y; Chiow, KH; Huang, D; Wong, SH

    2010-01-01

    Background and purpose: Andrographolide is the active component of Andrographis paniculata, a plant used in both Indian and Chinese traditional medicine, and it has been demonstrated to induce apoptosis in different cancer cell lines. However, not much is known about how it may affect the key receptors implicated in cancer. Knowledge of how andrographolide affects receptor trafficking will allow us to better understand new mechanisms by which andrographolide may cause death in cancer cells. Experimental approach: We utilized the well-characterized epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) expressed in epidermoid carcinoma (A-431) cells as a model to study the effect of andrographolide on receptor trafficking. Receptor distribution, the total number of receptors and surface receptors were analysed by immunofluorescence, Western blot as well as flow-cytometry respectively. Key results: Andrographolide treatment inhibited cell growth, down-regulated EGFRs on the cell surface and affected the degradation of EGFRs and TfRs. The EGFR was internalized into the cell at an increased rate, and accumulated in a compartment that co-localizes with the lysosomal-associated membrane protein in the late endosomes. Conclusion and implications: This study sheds light on how andrographolide may affect receptor trafficking by inhibiting receptor movement from the late endosomes to lysosomes. The down-regulation of EGFR from the cell surface also indicates a new mechanism by which andrographolide may induce cancer cell death. PMID:20233216

  19. Andrographolide regulates epidermal growth factor receptor and transferrin receptor trafficking in epidermoid carcinoma (A-431) cells.

    Science.gov (United States)

    Tan, Y; Chiow, K H; Huang, D; Wong, S H

    2010-04-01

    Andrographolide is the active component of Andrographis paniculata, a plant used in both Indian and Chinese traditional medicine, and it has been demonstrated to induce apoptosis in different cancer cell lines. However, not much is known about how it may affect the key receptors implicated in cancer. Knowledge of how andrographolide affects receptor trafficking will allow us to better understand new mechanisms by which andrographolide may cause death in cancer cells. We utilized the well-characterized epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) expressed in epidermoid carcinoma (A-431) cells as a model to study the effect of andrographolide on receptor trafficking. Receptor distribution, the total number of receptors and surface receptors were analysed by immunofluorescence, Western blot as well as flow-cytometry respectively. Andrographolide treatment inhibited cell growth, down-regulated EGFRs on the cell surface and affected the degradation of EGFRs and TfRs. The EGFR was internalized into the cell at an increased rate, and accumulated in a compartment that co-localizes with the lysosomal-associated membrane protein in the late endosomes. This study sheds light on how andrographolide may affect receptor trafficking by inhibiting receptor movement from the late endosomes to lysosomes. The down-regulation of EGFR from the cell surface also indicates a new mechanism by which andrographolide may induce cancer cell death.

  20. Architecture and Folding Mechanism of the Azoarcus Group I Pre-tRNA

    Energy Technology Data Exchange (ETDEWEB)

    Rangan,P.; Masquida, B.; Westhof, E.; Woodson, S.

    2004-01-01

    Self-splicing RNAs must evolve to function in their specific exon context. The conformation of a group I pre-tRNA{sup ile} from the bacterium Azoarcus was probed by ribonuclease T1 and hydroxyl radical cleavage, and by native gel electrophoresis. Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains. Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3{prime}-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron. Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone. The dependence of the folding kinetics on Mg{sup 2+} and the concentration of urea, and RNase T1 experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing between residues in P9 and the tRNA. Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs. This could help prevent premature processing of the 5{prime} and 3{prime} ends of unspliced pre-tRNA.

  1. A novel method to generate T-cell receptor-deficient chimeric antigen receptor T cells.

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    Kamiya, Takahiro; Wong, Desmond; Png, Yi Tian; Campana, Dario

    2018-03-13

    Practical methods are needed to increase the applicability and efficacy of chimeric antigen receptor (CAR) T-cell therapies. Using donor-derived CAR-T cells is attractive, but expression of endogenous T-cell receptors (TCRs) carries the risk for graft-versus-host-disease (GVHD). To remove surface TCRαβ, we combined an antibody-derived single-chain variable fragment specific for CD3ε with 21 different amino acid sequences predicted to retain it intracellularly. After transduction in T cells, several of these protein expression blockers (PEBLs) colocalized intracellularly with CD3ε, blocking surface CD3 and TCRαβ expression. In 25 experiments, median TCRαβ expression in T lymphocytes was reduced from 95.7% to 25.0%; CD3/TCRαβ cell depletion yielded virtually pure TCRαβ-negative T cells. Anti-CD3ε PEBLs abrogated TCRαβ-mediated signaling, without affecting immunophenotype or proliferation. In anti-CD3ε PEBL-T cells, expression of an anti-CD19-41BB-CD3ζ CAR induced cytokine secretion, long-term proliferation, and CD19 + leukemia cell killing, at rates meeting or exceeding those of CAR-T cells with normal CD3/TCRαβ expression. In immunodeficient mice, anti-CD3ε PEBL-T cells had markedly reduced GVHD potential; when transduced with anti-CD19 CAR, these T cells killed engrafted leukemic cells. PEBL blockade of surface CD3/TCRαβ expression is an effective tool to prepare allogeneic CAR-T cells. Combined PEBL and CAR expression can be achieved in a single-step procedure, is easily adaptable to current cell manufacturing protocols, and can be used to target other T-cell molecules to further enhance CAR-T-cell therapies. © 2018 by The American Society of Hematology.

  2. Genetic engineering with T cell receptors.

    Science.gov (United States)

    Zhang, Ling; Morgan, Richard A

    2012-06-01

    In the past two decades, human gene transfer research has been translated from a laboratory technology to clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers. Published by Elsevier B.V.

  3. Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

    Science.gov (United States)

    Peñas-Cazorla, Raúl; Vilaró, M Teresa

    2015-11-01

    Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists.

  4. Metabotropic glutamate receptors in glial cells

    NARCIS (Netherlands)

    D'Antoni, Simona; Berretta, Antonio; Bonaccorso, Carmela Maria; Bruno, Valeria; Aronica, Eleonora; Nicoletti, Ferdinando; Catania, Maria Vincenza

    2008-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and exerts its actions via a number of ionotropic glutamate receptors/channels and metabotropic glutamate (mGlu) receptors. In addition to being expressed in neurons, glutamate receptors are expressed in

  5. Simultaneous Profiling of 194 Distinct Receptor Transcripts in Human Cells

    Science.gov (United States)

    Kang, Byong H.; Jensen, Karin J.; Hatch, Jaime A.; Janes, Kevin A.

    2013-01-01

    Many signal transduction cascades are initiated by transmembrane receptors with the presence or absence and abundance of receptors dictating cellular responsiveness. Here, we provide a validated array of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) reagents for high-throughput profiling of the presence and relative abundance of transcripts for 194 transmembrane receptors in the human genome. We found that the qRT-PCR array had greater sensitivity and specificity for the detected receptor transcript profiles compared to conventional oligonucleotide microarrays or exon microarrays. The qRT-PCR array also distinguished functional receptor presence versus absence more accurately than deep sequencing of adenylated RNA species, RNA-seq. By applying qRT-PCR-based receptor transcript profiling to 40 human cell lines representing four main tissues (pancreas, skin, breast, and colon), we identified clusters of cell lines with enhanced signaling capabilities and revealed a role for receptor silencing in defining tissue lineage. Ectopic expression of the interleukin 10 (IL-10) receptor encoding gene IL10RA in melanoma cells engaged an IL-10 autocrine loop not otherwise present in this cell type, which altered signaling, gene expression, and cellular responses to proinflammatory stimuli. Our array provides a rapid, inexpensive, and convenient means for assigning a receptor signature to any human cell or tissue type. PMID:23921087

  6. T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness

    NARCIS (Netherlands)

    C.H.J. Lamers (Cor); S. van Steenbergen-Langeveld (Sabine); M. van Brakel (Mandy); C.M. Groot-van Ruijven (Corrien); P.M.M.L. van Elzakker (Pascal); B.A. van Krimpen (Brigitte); S. Sleijfer (Stefan); J.E.M.A. Debets (Reno)

    2014-01-01

    textabstractTherapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T

  7. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  8. 5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

    DEFF Research Database (Denmark)

    Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

    2006-01-01

    gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... in the central nervous system (CNS). We have recently demonstrated that 5-HT4 receptor couples to G13 protein to induce RhoA-dependent gene transcription, neurite retraction, and neuronal cell rounding (Ponimaskin et al, 2002). Although multiple studies were focused on the function of the 5-HT4 receptor...

  9. Internalisation of gonadotrophin-receptor complex in ovarian luteal cells

    International Nuclear Information System (INIS)

    Conn, P.M.; Conti, M.; Harwood, J.P.; Dufau, M.L.; Catt, K.J.

    1978-01-01

    Following evidence that certain protein hormones can enter target cells the present investigation was undertaken which shows that gonadotrophin-induced receptor loss may occur by a process of internalisation of the hormone-receptor complex following the initial interaction of gonadotrophin with the cell surface. Localisation studies were carried out in 33-d old female rats previously treated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin (hCG) to induce ovarian luteinisation. Animals were injected with 125 I-hCG to label the ovarian receptors for luteinising hormone in vivo. Microscope autoradiographs demonstrating distribution of 125 I-hCG in ovaries at various times following injection are shown. The combined results from the autoradiographs and from solubilisation experiments were used to determine the location and nature of the hCG-receptor complex following occupancy and loss of receptors from the plasma membrane of luteinised ovarian cells. (U.K.)

  10. Functional somatostatin receptors on a rat pancreatic acinar cell line

    International Nuclear Information System (INIS)

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A.

    1988-01-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125 I-[Tyr 11 ]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/10 6 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125 I-[Tyr 11 ]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N i to inhibit adenylate cyclase

  11. Regulation of Mu Opioid Receptor Expression in Developing T Cells

    OpenAIRE

    Zhang, Lily; Belkowski, Judith Sliker; Briscoe, Tammi; Rogers, Thomas J.

    2012-01-01

    We have previously reported that functionally active μ-opioid receptors (MOR) are constitutively expressed at relatively low levels by developing T cells in the thymus. However, very little is known about the regulation of MOR expression by immature T cells. In this report, we first attempted to determine the effect of T cell receptor-induced T cell activation on the expression of MOR. We activated T cells with either the combination of anti-CD3 and CD28, or with superantigen, and observed a ...

  12. Chimeric antigen receptor T-cell therapy for solid tumors

    Directory of Open Access Journals (Sweden)

    Kheng Newick

    2016-01-01

    Full Text Available Chimeric antigen receptor (CAR T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias. This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment.

  13. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... lung cancer cell lines express the EGF receptor....... of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell...

  14. Prospects and limitations of T cell receptor gene therapy

    NARCIS (Netherlands)

    Jorritsma, Annelies; Schotte, Remko; Coccoris, Miriam; de Witte, Moniek A.; Schumacher, Ton N. M.

    2011-01-01

    Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining

  15. A response calculus for immobilized T cell receptor ligands

    DEFF Research Database (Denmark)

    Andersen, P S; Menné, C; Mariuzza, R A

    2001-01-01

    To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affini...

  16. Role of laminin receptor in tumor cell migration

    DEFF Research Database (Denmark)

    Wewer, U M; Taraboletti, G; Sobel, M E

    1987-01-01

    Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986...... but not on fibronectin. Synthetic peptide GRGDS corresponding to the fibronectin cell-binding domain inhibited haptotaxis on fibronectin but not on laminin. Both types of anti-laminin receptor antisera inhibited haptotaxis on laminin but not on fibronectin. Using immunohistochemistry, invading human carcinoma cells...

  17. A possible new target in lung-cancer cells: The orphan receptor, bombesin receptor subtype-3.

    Science.gov (United States)

    Moreno, Paola; Mantey, Samuel A; Lee, Suk H; Ramos-Álvarez, Irene; Moody, Terry W; Jensen, Robert T

    2018-03-01

    Human bombesin receptors, GRPR and NMBR, are two of the most frequently overexpressed G-protein-coupled-receptors by lung-cancers. Recently, GRPR/NMBR are receiving considerable attention because they act as growth factor receptors often in an autocrine manner in different lung-cancers, affect tumor angiogenesis, their inhibition increases the cytotoxic potency of tyrosine-kinase inhibitors reducing lung-cancer cellular resistance/survival and their overexpression can be used for sensitive tumor localization as well as to target cytotoxic agents to the cancer. The orphan BRS-3-receptor, because of homology is classified as a bombesin receptor but has received little attention, despite the fact that it is also reported in a number of studies in lung-cancer cells and has growth effects in these cells. To address its potential importance, in this study, we examined the frequency/relative quantitative expression of human BRS-3 compared to GRPR/NMBR and the effects of its activation on cell-signaling/growth in 13 different human lung-cancer cell-lines. Our results showed that BRS-3 receptor is expressed in 92% of the cell-lines and that it is functional in these cells, because its activation stimulates phospholipase-C with breakdown of phosphoinositides and changes in cytosolic calcium, stimulates ERK/MAPK and stimulates cell growth by EGFR transactivation in some, but not all, the lung-cancer cell-lines. These results suggest that human BRS-3, similar to GRPR/NMBR, is frequently ectopically-expressed by lung-cancer cells in which, it is functional, affecting cell signaling/growth. These results suggest that similar to GRPR/NMBR, BRS-3 should receive increased attention as possible approach for the development of novel treatments and/or diagnosis in lung-cancer. Published by Elsevier Inc.

  18. Atypical B cell receptor signaling: straddling immune diseases and cancer.

    Science.gov (United States)

    Faris, Mary

    2013-08-01

    The B-cell receptor (BCR) signaling pathway plays an essential role in the survival, proliferation, differentiation and trafficking of lymphocytic. Recent findings associate aberrant BCR signaling with specific disease pathologies, including B-cell malignancies and autoimmune disorders. Inhibition of the BCR signaling pathway may therefore provide promising new strategies for the treatment of B-cell diseases. This special issue of International Reviews of Immunology focuses on atypical B-cell receptor signaling, its role in immune diseases and cancer, and its implications for potential therapeutic intervention.

  19. Correlated receptor transport processes buffer single-cell heterogeneity.

    Science.gov (United States)

    Kallenberger, Stefan M; Unger, Anne L; Legewie, Stefan; Lymperopoulos, Konstantinos; Klingmüller, Ursula; Eils, Roland; Herten, Dirk-Peter

    2017-09-01

    Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR) trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.

  20. Correlated receptor transport processes buffer single-cell heterogeneity.

    Directory of Open Access Journals (Sweden)

    Stefan M Kallenberger

    2017-09-01

    Full Text Available Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.

  1. Dynamics of Corticosteroid Receptors: Lessons from Live Cell Imaging

    International Nuclear Information System (INIS)

    Nishi, Mayumi

    2011-01-01

    Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert numerous effects on the central nervous system that regulates the stress response, mood, learning and memory, and various neuroendocrine functions. Corticosterone (CORT) actions in the brain are mediated via two receptor systems: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). It has been shown that GR and MR are highly colocalized in the hippocampus. These receptors are mainly distributed in the cytoplasm without hormones and translocated into the nucleus after treatment with hormones to act as transcriptional factors. Thus the subcellular dynamics of both receptors are one of the most important issues. Given the differential action of MR and GR in the central nervous system, it is of great consequence to clarify how these receptors are trafficked between cytoplasm and nucleus and their interactions are regulated by hormones and/or other molecules to exert their transcriptional activity. In this review, we focus on the nucleocytoplasmic and subnuclear trafficking of GR and MR in neural cells and non-neural cells analyzed by using molecular imaging techniques with green fluorescent protein (GFP) including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET), and discuss various factors affecting the dynamics of these receptors. Furthermore, we discuss the future directions of in vivo molecular imaging of corticosteroid receptors at the whole brain level

  2. Estrogen receptor and progesterone receptor synthesis and degradation in target cells

    Energy Technology Data Exchange (ETDEWEB)

    Nardulli, A.M.

    1987-01-01

    It was the intent of this study to determine the turnover of the estrogen receptor (ER) and progesterone receptors (PR) in rat uterine and human breast cancer cells, respectively, and to examine the effect of estrogen and progestin on PR levels. The rates of synthesis and degradation of ER were determined in rat uterine cells in vitro and in vivo. The affinity labeling antiestrogen, (/sup 3/H)tamoxifen aziridine, was used in pulse chase experiments to show that the 65,000 molecular weight ER has a half-life of 3-4h in primary cultures of rat uterine cells in vitro and in the intact rat uterus in vivo. Density shift analyses using dense (/sup 15/N, /sup 13/C, /sup 2/H) amino acid incorporation corroborate the rapid turnover of ER in rat uterine cell cultures. The regulation of PR by progestins in T47D human breast cancer cells was examined using density shift-dense amino acid incorporation. When T47D cells, which normally maintain high PR levels, are exposed to progestin (R5020), PR levels decline. Receptor half-life, which is 21h in control cells, is reduced to 6h when cells are exposed to 20 nM (/sup 3/H)R5020. In addition, PR synthesis rate declines exponentially following R5020 exposure. The reduction in receptor level is thus due to dramatic increases in PR degradation as well as marked decreases in PR synthesis.

  3. Human mast cell activation through Fc receptors and Toll-like receptors

    Directory of Open Access Journals (Sweden)

    Yoshimichi Okayama

    2004-01-01

    Full Text Available Mast cells express high-affinity IgE receptors (FcεRI on their surface and can be activated to secrete a variety of biologically active mediators by cross-linking of receptor-bound IgE. Recent studies in animal models indicate that mouse mast cells may play a protective role in host defense against bacteria through the production of tumor necrosis factor-α, mainly as a result of Toll-like receptor (TLR 4- or CD48-mediated activation. Moreover, several recent observations in animal models have indicated that mast cells may also play a pivotal role in coordinating the early phases of autoimmune diseases, particularly those involving auto-antibodies. We recently identified functional TLR4 and FcγRI on human mast cells, in which their expression had been upregulated by interferon-γ. We compared each of the receptor-mediated gene expression profiles with the FcεRI-mediated gene expression profile using high-density oligonucleotide probe arrays and discovered that human mast cells may modulate the immune system in a receptor-specific manner.

  4. Mast cell adenosine receptors function: a focus on the A3 adenosine receptor and inflammation

    Directory of Open Access Journals (Sweden)

    Noam eRudich

    2012-06-01

    Full Text Available Adenosine is a metabolite, which has long been implicated in a variety of inflammatory processes. Inhaled adenosine provokes bronchoconstriction in asthmatics or chronic obstructive pulmonary disease (COPD patients, but not in non-asthmatics. This hyper responsiveness to adenosine appears to be mediated by mast cell activation. These observations have marked the receptor that mediates the bronchoconstrictor effect of adenosine on mast cells, as an attractive drug candidate. Four subtypes (A1, A2a, A2b and A3 of adenosine receptors have been cloned and shown to display distinct tissue distributions and functions. Animal models have firmly established the ultimate role of the A3 adenosine receptor (A3R in mediating hyper responsiveness to adenosine in mast cells, although the influence of the A2b adenosine receptor was confirmed as well. In contrast, studies of the A3R in humans have been controversial. In this review, we summarize data on the role of different adenosine receptors in mast cell regulation of inflammation and pathology, with a focus on the common and distinct functions of the A3R in rodent and human mast cells. The relevance of mouse studies to the human is discussed.

  5. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    Science.gov (United States)

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; De Marinis, Marta; Tafuri, Domenico; Cinelli, Mariapia; Ammendola, Rosario

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we

  6. Attachment and Postattachment Receptors Important for Hepatitis C Virus Infection and Cell-to-Cell Transmission.

    Science.gov (United States)

    Fan, Huahao; Qiao, Luhua; Kang, Kyung-Don; Fan, Junfen; Wei, Wensheng; Luo, Guangxiang

    2017-07-01

    Hepatitis C virus (HCV) requires multiple receptors for its attachment to and entry into cells. Our previous studies found that human syndecan-1 (SDC-1), SDC-2, and T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) are HCV attachment receptors. Other cell surface molecules, such as CD81, Claudin-1 (CLDN1), Occludin (OCLN), SR-BI, and low-density lipoprotein receptor (LDLR), function mainly at postattachment steps and are considered postattachment receptors. The underlying molecular mechanisms of different receptors in HCV cell-free and cell-to-cell transmission remain elusive. In the present study, we used a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology, gene-specific small interfering RNAs, and a newly developed luciferase-based reporter system to quantitatively determine the importance of individual receptors in HCV cell-free and cell-to-cell transmission. Knockouts of SDC-1 and SDC-2 resulted in remarkable reductions of HCV infection and cell attachment, whereas SDC-3 and SDC-4 knockouts did not affect HCV infection. Defective HCV attachment to SDC-1 and/or SDC-2 knockout cells was completely restored by SDC-1 and SDC-2 but not SDC-4 expression. Knockout of the attachment receptors SDC-1, SDC-2, and TIM-1 also modestly decreased HCV cell-to-cell transmission. In contrast, silencing and knockout of the postattachment receptors CD81, CLDN1, OCLN, SR-BI, and LDLR greatly impaired both HCV cell-free and cell-to-cell transmission. Additionally, apolipoprotein E was found to be important for HCV cell-to-cell spread, but very-low-density lipoprotein (VLDL)-containing mouse serum did not affect HCV cell-to-cell transmission, although it inhibited cell-free infection. These findings demonstrate that attachment receptors are essential for initial HCV binding and that postattachment receptors are important for both HCV cell-free and cell-to-cell transmission. IMPORTANCE The importance and underlying molecular mechanisms

  7. The role of purinergic receptors in stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Constanze Kaebisch

    2015-01-01

    Full Text Available A major challenge modern society has to face is the increasing need for tissue regeneration due to degenerative diseases or tumors, but also accidents or warlike conflicts. There is great hope that stem cell-based therapies might improve current treatments of cardiovascular diseases, osteochondral defects or nerve injury due to the unique properties of stem cells such as their self-renewal and differentiation potential. Since embryonic stem cells raise severe ethical concerns and are prone to teratoma formation, adult stem cells are still in the focus of research. Emphasis is placed on cellular signaling within these cells and in between them for a better understanding of the complex processes regulating stem cell fate. One of the oldest signaling systems is based on nucleotides as ligands for purinergic receptors playing an important role in a huge variety of cellular processes such as proliferation, migration and differentiation. Besides their natural ligands, several artificial agonists and antagonists have been identified for P1 and P2 receptors and are already used as drugs. This review outlines purinergic receptor expression and signaling in stem cells metabolism. We will briefly describe current findings in embryonic and induced pluripotent stem cells as well as in cancer-, hematopoietic-, and neural crest-derived stem cells. The major focus will be placed on recent findings of purinergic signaling in mesenchymal stem cells addressed in in vitro and in vivo studies, since stem cell fate might be manipulated by this system guiding differentiation towards the desired lineage in the future.

  8. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    International Nuclear Information System (INIS)

    Levy, J.R.; Olefsky, J.M.

    1988-01-01

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

  9. NMDA Receptors in Glial Cells: Pending Questions

    Czech Academy of Sciences Publication Activity Database

    Džamba, Dávid; Honsa, Pavel; Anděrová, Miroslava

    2013-01-01

    Roč. 11, č. 3 (2013), s. 250-262 ISSN 1570-159X R&D Projects: GA ČR GA309/08/1381; GA ČR(CZ) GBP304/12/G069 Grant - others:GA UK(CZ) 604212 Institutional support: RVO:68378041 Keywords : astrocytes * ischemia * NMDA receptors Subject RIV: FH - Neurology Impact factor: 2.347, year: 2013

  10. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    Science.gov (United States)

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells

    OpenAIRE

    Stroncek, David F.; Lee, Daniel W.; Ren, Jiaqiang; Sabatino, Marianna; Highfill, Steven; Khuu, Hanh; Shah, Nirali N.; Kaplan, Rosandra N.; Fry, Terry J.; Mackall, Crystal L.

    2017-01-01

    Background Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. Methods Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We ...

  12. Chemokine receptor expression by inflammatory T cells in EAE

    DEFF Research Database (Denmark)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20...... immunofluorescence. Consistent with flow cytometry data some but not all CD4(+) T cells expressed CCR6 within infiltrates. CD4-negative CCR6(+) cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4(+) and CD8(+) T cells in the CNS of mice with peak EAE, and determined IFNγ...

  13. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    Science.gov (United States)

    Gonzalez-Rodriguez, David; Barakat, Abdul I.

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

  14. Killer Cell Immunoglobulin-like Receptors and their Ligands

    Directory of Open Access Journals (Sweden)

    Tajik N.

    2010-09-01

    Full Text Available The Natural killer (NK cells are a subset of lymphocytes comprising around 10% of total lymphocytes in peripheral blood. Due to their role in the innate response, NK cells provide a ‘first line of defense’ against infectious agents and cancer and are also thought to play a role in autoimmunity. The killer cell immunoglobulin-like receptors (KIR are regulatory surface molecules, found on NK cells and on a subset of T lymphocytes. The genes for KIR are present on chromosome 19 in the leukocyte receptor complex and show a major difference for both the type and number of KIR genes present among different ethnic groups. They have been divided into two groups of 2D or 3D, depending on the number of external immunoglobulin domains. The presence of a long cytoplasmic tail with two immune tyrosine-based inhibitory motifs (ITIM allows the transduction of inhibitory signals and characterizes the inhibitory KIRs (2DL and 3DL, whereas the presence of short cytoplasmic tails corresponds to the activating KIR receptors (2DS and 3DS.These polymorphic receptors interact with specific motifs on human leukocyte antigen (HLA class I molecules, modulate NK cytolytic activity. Some KIRs are known to interact with HLA-C molecules of target cells, HLA-Bw4 molecules and HLA-A3/11. For some KIRs the corresponding ligands are still unknown.

  15. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  16. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling....... In this study, we constructed a chimeric TCR-KIR receptor. We demonstrated that SHP-1 is recruited to the chimeric TCR-KIR receptor following T-cell stimulation with either anti-TCR monoclonal antibody (MoAb) or superantigen. However, in spite of this we could not detect any effect of SHP-1 on TCR signalling...

  17. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  18. Nonimmune cells equipped with T-cell-receptor-like signaling for cancer cell ablation.

    Science.gov (United States)

    Kojima, Ryosuke; Scheller, Leo; Fussenegger, Martin

    2018-01-01

    The ability to engineer custom cell-contact-sensing output devices into human nonimmune cells would be useful for extending the applicability of cell-based cancer therapies and for avoiding risks associated with engineered immune cells. Here we have developed a new class of synthetic T-cell receptor-like signal-transduction device that functions efficiently in human nonimmune cells and triggers release of output molecules specifically upon sensing contact with a target cell. This device employs an interleukin signaling cascade, whose OFF/ON switching is controlled by biophysical segregation of a transmembrane signal-inhibitory protein from the sensor cell-target cell interface. We further show that designer nonimmune cells equipped with this device driving expression of a membrane-penetrator/prodrug-activating enzyme construct could specifically kill target cells in the presence of the prodrug, indicating its potential usefulness for target-cell-specific, cell-based enzyme-prodrug cancer therapy. Our study also contributes to the advancement of synthetic biology by extending available design principles to transmit extracellular information to cells.

  19. Soluble Triggering Receptor Expressed on Myeloid Cells-1 as a ...

    African Journals Online (AJOL)

    Soluble Triggering Receptor Expressed on Myeloid Cells-1 as a marker to differentiate septic from aseptic meningitis in children: comparison with procalcitonin and ... Procalcitonin (PCT) was suggested by many researchers as a sensitive marker for early diagnosis of septic meningitis but with varying discriminative power.

  20. Toll-like receptor polymorphisms in allogeneic hematopoietic cell transplantation

    DEFF Research Database (Denmark)

    Kornblit, Brian; Enevold, Christian; Wang, Tao

    2014-01-01

    To assess the impact of the genetic variation in toll-like receptors (TLRs) on outcome after allogeneic myeloablative conditioning hematopoietic cell transplantation (HCT), we investigated 29 single nucleotide polymorphisms across 10 TLRs in 816 patients and donors. Only donor genotype of TLR8 rs...

  1. Regulation of HIV receptor expression in cervical epithelial cells by ...

    African Journals Online (AJOL)

    Background. Sexually transmitted infections (STIs) caused by the Gram-negative bacteria Chlamydia trachomatis and Neisseria gonorrhoeae are associated with an increased risk of HIV acquisition in South African women. HIV infection involves binding of the virus to CD4+ receptors on host cells and subsequent binding to ...

  2. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... nuclear factor-kappa B (NF-KB). This will result in the inhibition of the expression of cell surface adhesion molecules and the reduction in the synthesis and release of inflammatory cytokine, inhibiting neutrophil activation and extravasation in damaged tissue parts. Endothelial protein C receptor (EPCR) was ...

  3. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

    2015-01-01

    of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high....... e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point...... to a new, unique approach enabling to eliminate non-cycling uterine leiomyosarcoma cells providing that they over-express the AT2 receptor....

  4. Comparative genomics of natural killer cell receptor gene clusters.

    Directory of Open Access Journals (Sweden)

    James Kelley

    2005-08-01

    Full Text Available Many receptors on natural killer (NK cells recognize major histocompatibility complex class I molecules in order to monitor unhealthy tissues, such as cells infected with viruses, and some tumors. Genes encoding families of NK receptors and related sequences are organized into two main clusters in humans: the natural killer complex on Chromosome 12p13.1, which encodes C-type lectin molecules, and the leukocyte receptor complex on Chromosome 19q13.4, which encodes immunoglobulin superfamily molecules. The composition of these gene clusters differs markedly between closely related species, providing evidence for rapid, lineage-specific expansions or contractions of sets of loci. The choice of NK receptor genes is polarized in the two species most studied, mouse and human. In mouse, the C-type lectin-related Ly49 gene family predominates. Conversely, the single Ly49 sequence is a pseudogene in humans, and the immunoglobulin superfamily KIR gene family is extensive. These different gene sets encode proteins that are comparable in function and genetic diversity, even though they have undergone species-specific expansions. Understanding the biological significance of this curious situation may be aided by studying which NK receptor genes are used in other vertebrates, especially in relation to species-specific differences in genes for major histocompatibility complex class I molecules.

  5. Plant cell wall signalling and receptor-like kinases.

    Science.gov (United States)

    Wolf, Sebastian

    2017-02-15

    Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  6. Rapid internalization of the insulin receptor in rat hepatoma cells

    International Nuclear Information System (INIS)

    Backer, J.M.; White, M.F.; Kahn, C.R.

    1987-01-01

    The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4 0 C, stimulated with insulin at 37 0 C, and then cooled rapidly, trypsinized at 4 0 C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37 0 C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4 0 C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific [ 125 I]insulin binding measured at 4 0 C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways

  7. Receptor control in mesenchymal stem cell engineering

    Science.gov (United States)

    Dalby, Matthew J.; García, Andrés J.; Salmeron-Sanchez, Manuel

    2018-03-01

    Materials science offers a powerful tool to control mesenchymal stem cell (MSC) growth and differentiation into functional phenotypes. A complex interplay between the extracellular matrix and growth factors guides MSC phenotypes in vivo. In this Review, we discuss materials-based bioengineering approaches to direct MSC fate in vitro and in vivo, mimicking cell-matrix-growth factor crosstalk. We first scrutinize MSC-matrix interactions and how the properties of a material can be tailored to support MSC growth and differentiation in vitro, with an emphasis on MSC self-renewal mechanisms. We then highlight important growth factor signalling pathways and investigate various materials-based strategies for growth factor presentation and delivery. Integrin-growth factor crosstalk in the context of MSC engineering is introduced, and bioinspired material designs with the potential to control the MSC niche phenotype are considered. Finally, we summarize important milestones on the road to MSC engineering for regenerative medicine.

  8. A Comprehensive Nuclear Receptor Network for Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ralf Kittler

    2013-02-01

    Full Text Available In breast cancer, nuclear receptors (NRs play a prominent role in governing gene expression, have prognostic utility, and are therapeutic targets. We built a regulatory map for 24 NRs, six chromatin state markers, and 14 breast-cancer-associated transcription factors (TFs that are expressed in the breast cancer cell line MCF-7. The resulting network reveals a highly interconnected regulatory matrix where extensive crosstalk occurs among NRs and other breast -cancer-associated TFs. We show that large numbers of factors are coordinately bound to highly occupied target regions throughout the genome, and these regions are associated with active chromatin state and hormone-responsive gene expression. This network also provides a framework for stratifying and predicting patient outcomes, and we use it to show that the peroxisome proliferator-activated receptor delta binds to a set of genes also regulated by the retinoic acid receptors and whose expression is associated with poor prognosis in breast cancer.

  9. T cell antigen receptor activation and actin cytoskeleton remodeling

    Science.gov (United States)

    Kumari, Sudha; Curado, Silvia; Mayya, Viveka

    2013-01-01

    T cells constitute a crucial arm of the adaptive immune system and their optimal function is required for a healthy immune response. After the initial step of T cell-receptor (TCR) triggering by antigenic peptide complexes on antigen presenting cell (APC), the T cell exhibits extensive cytoskeletal remodeling. This cytoskeletal remodeling leads to formation of an “immunological synapse” [1] characterized by regulated clustering, segregation and movement of receptors at the interface. Synapse formation regulates T cell activation and response to antigenic peptides and proceeds via feedback between actin cytoskeleton and TCR signaling. Actin polymerization participates in various events during the synapse formation, maturation, and eventually its disassembly. There is increasing knowledge about the actin effectors that couple TCR activation to actin rearrangements [2, 3], and how defects in these effectors translate into impairment of T cell activation. In this review we aim to summarize and integrate parts of what is currently known about this feedback process. In addition, in light of recent advancements in our understanding of TCR triggering and translocation at the synapse, we speculate on the organizational and functional diversity of microfilament architecture in the T cell. PMID:23680625

  10. Integrating signals from the T-cell receptor and the interleukin-2 receptor.

    Directory of Open Access Journals (Sweden)

    Tilo Beyer

    2011-08-01

    Full Text Available T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells.

  11. Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line

    International Nuclear Information System (INIS)

    Androlewicz, M.J.; Straus, D.S.; Brandenburg, D.F.

    1989-01-01

    Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125 I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degree C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation

  12. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling....... In this study, we constructed a chimeric TCR-KIR receptor. We demonstrated that SHP-1 is recruited to the chimeric TCR-KIR receptor following T-cell stimulation with either anti-TCR monoclonal antibody (MoAb) or superantigen. However, in spite of this we could not detect any effect of SHP-1 on TCR signalling...

  13. NMDA Receptors Contribute to Retrograde Synaptic Transmission from Ganglion Cell Photoreceptors to Dopaminergic Amacrine Cells

    Directory of Open Access Journals (Sweden)

    Lei-Lei Liu

    2017-09-01

    Full Text Available Recently, a line of evidence has demonstrated that the vertebrate retina possesses a novel retrograde signaling pathway. In this pathway, phototransduction is initiated by the photopigment melanopsin, which is expressed in a small population of retinal ganglion cells. These ganglion cell photoreceptors then signal to dopaminergic amacrine cells (DACs through glutamatergic synapses, influencing visual light adaptation. We have previously demonstrated that in Mg2+-containing solution, α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA receptors mediate this glutamatergic transmission. Here, we demonstrate that removing extracellular Mg2+ enhances melanopsin-based DAC light responses at membrane potentials more negative than −40 mV. Melanopsin-based responses in Mg2+-free solution were profoundly suppressed by the selective N-methyl-D-aspartate (NMDA receptor antagonist D-AP5. In addition, application of NMDA to the retina produced excitatory inward currents in DACs. These data strongly suggest that DACs express functional NMDA receptors. We further found that in the presence of Mg2+, D-AP5 reduced the peak amplitude of melanopsin-based DAC responses by ~70% when the cells were held at their resting membrane potential (−50 mV, indicating that NMDA receptors are likely to contribute to retrograde signal transmission to DACs under physiological conditions. Moreover, our data show that melanopsin-based NMDA-receptor-mediated responses in DACs are suppressed by antagonists specific to either the NR2A or NR2B receptor subtype. Immunohistochemical results show that NR2A and NR2B subunits are expressed on DAC somata and processes. These results suggest that DACs express functional NMDA receptors containing both NR2A and NR2B subunits. Collectively, our data reveal that, along with AMPA receptors, NR2A- and NR2B-containing NMDA receptors mediate retrograde signal transmission from ganglion cell photoreceptors to DACs.

  14. Renal cells activate the platelet receptor CLEC-2 through podoplanin

    Science.gov (United States)

    Christou, Charita M.; Pearce, Andrew C.; Watson, Aleksandra A.; Mistry, Anita R.; Pollitt, Alice Y.; Fenton-May, Angharad E.; Johnson, Louise A.; Jackson, David G.; Watson, Steve P.; O'Callaghan, Chris A.

    2009-01-01

    We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin, rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing human immunodeficiency virus type 1 (HIV-1). This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of this study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to 293T cells in which the HIV can be grown. Further, 293T cells activate both platelets and CLEC-2-transfected DT-40 B cells. The transmembrane protein podoplanin was identified on 293T cells and demonstrated to mediate both binding of 293T cells to CLEC-2 and 293T cell activation of CLEC-2-transfected DT-40 B cells. Podoplanin is expressed on renal cells (podocytes). Further, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5 ± 3.7μM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells. PMID:18215137

  15. Fungal pattern-recognition receptors and tetraspanins: partners on antigen-presenting cells.

    NARCIS (Netherlands)

    Figdor, C.G.; Spriel, A.B. van

    2010-01-01

    Fungal pattern-recognition receptors (F-PRRs), including C-type lectins, Toll-like receptors, scavenger receptors and Fc/complement receptors, are crucial for inducing anti-fungal immune responses by antigen-presenting cells. The recent identification of specific F-PRR interactions with tetraspanins

  16. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR and chimeric antigen receptor (CAR modified T cells. On July 1, 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the beneficiaries of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical-clinical studies, effectiveness and drawbacks of this strategy.

  17. Monoclonal T-cell receptors: new reagents for cancer therapy.

    Science.gov (United States)

    Stauss, Hans J; Cesco-Gaspere, Michela; Thomas, Sharyn; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; Wright, Graham; Perro, Mario; Little, Ann-Margaret; Pospori, Constantina; King, Judy; Morris, Emma C

    2007-10-01

    Adoptive transfer of antigen-specific T lymphocytes is an effective form of immunotherapy for persistent virus infections and cancer. A major limitation of adoptive therapy is the inability to isolate antigen-specific T lymphocytes reproducibly. The demonstration that cloned T-cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. TCR gene-modified lymphocytes display antigen-specific function in vitro, and were shown to protect against virus infection and tumor growth in animal models. A recent trial in humans demonstrated that TCR gene-modified T cells persisted in all and reduced melanoma burden in 2/15 patients. In future trials, it may be possible to use TCR gene transfer to equip helper and cytotoxic T cells with new antigen-specificity, allowing both T-cell subsets to cooperate in achieving improved clinical responses. Sequence modifications of TCR genes are being explored to enhance TCR surface expression, while minimizing the risk of pairing between introduced and endogenous TCR chains. Current T-cell transduction protocols that trigger T-cell differentiation need to be modified to generate "undifferentiated" T cells, which, upon adoptive transfer, display improved in vivo expansion and survival. Both, expression of only the introduced TCR chains and the production of naïve T cells may be possible in the future by TCR gene transfer into stem cells.

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

    Directory of Open Access Journals (Sweden)

    Francesco Marras

    2011-01-01

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

  19. Chimeric antigen receptor engineered stem cells: a novel HIV therapy.

    Science.gov (United States)

    Zhen, Anjie; Carrillo, Mayra A; Kitchen, Scott G

    2017-03-01

    Despite the success of combination antiretroviral therapy (cART) for suppressing HIV and improving patients' quality of life, HIV persists in cART-treated patients and remains an incurable disease. Financial burdens and health consequences of lifelong cART treatment call for novel HIV therapies that result in a permanent cure. Cellular immunity is central in controlling HIV replication. However, HIV adopts numerous strategies to evade immune surveillance. Engineered immunity via genetic manipulation could offer a functional cure by generating cells that have enhanced antiviral activity and are resistant to HIV infection. Recently, encouraging reports from several human clinical trials using an anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy for treating B-cell malignancies have provided valuable insights and generated remarkable enthusiasm in engineered T-cell therapy. In this review, we discuss the development of HIV-specific chimeric antigen receptors and the use of stem cell based therapies to generate lifelong anti-HIV immunity.

  20. Role of CD3 gamma in T cell receptor assembly

    DEFF Research Database (Denmark)

    Dietrich, J; Neisig, A; Hou, X

    1996-01-01

    The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC......), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were....... In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta...

  1. Histamine receptor 2 modifies dendritic cell responses to microbial ligands.

    Science.gov (United States)

    Frei, Remo; Ferstl, Ruth; Konieczna, Patrycja; Ziegler, Mario; Simon, Tunde; Rugeles, Tulia Mateus; Mailand, Susanne; Watanabe, Takeshi; Lauener, Roger; Akdis, Cezmi A; O'Mahony, Liam

    2013-07-01

    The induction of tolerance and protective immunity to microbes is significantly influenced by host- and microbiota-derived metabolites, such as histamine. We sought to identify the molecular mechanisms for histamine-mediated modulation of pattern recognition receptor signaling. Human monocyte-derived dendritic cells (MDDCs), myeloid dendritic cells, and plasmacytoid dendritic cells were examined. Cytokine secretion, gene expression, and transcription factor activation were measured after stimulation with microbial ligands and histamine. Histamine receptor 2 (H₂R)-deficient mice, histamine receptors, and their signaling pathways were investigated. Histamine suppressed MDDC chemokine and proinflammatory cytokine secretion, nuclear factor κB and activator protein 1 activation, mitogen-activated protein kinase phosphorylation, and T(H)1 polarization of naive lymphocytes, whereas IL-10 secretion was enhanced in response to LPS and Pam3Cys. Histamine also suppressed LPS-induced myeloid dendritic cell TNF-α secretion and suppressed CpG-induced plasmacytoid dendritic cell IFN-α gene expression. H₂R signaling through cyclic AMP and exchange protein directly activated by cyclic AMP was required for the histamine effect on LPS-induced MDDC responses. Lactobacillus rhamnosus, which secretes histamine, significantly suppressed Peyer patch IL-2, IL-4, IL-5, IL-12, TNF-α, and GM-CSF secretion in wild-type but not H₂R-deficient animals. Both host- and microbiota-derived histamine significantly alter the innate immune response to microbes through H₂R. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  2. A logical model provides insights into T cell receptor signaling.

    Directory of Open Access Journals (Sweden)

    Julio Saez-Rodriguez

    2007-08-01

    Full Text Available Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.

  3. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Department of Gastroenterology, The Tenth People’s Hospital of Shanghai, Tongji University, Shanghai 200072 (China); Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yang, Yong, E-mail: yyang@houstonmethodist.org [Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Medicine, Weill Cornell Medical College, New York, NY 10065 (United States)

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  4. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    International Nuclear Information System (INIS)

    Wang, Feng; Yang, Yong

    2014-01-01

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers

  5. Natural killer cells and their receptors in multiple sclerosis.

    Science.gov (United States)

    Kaur, Gurman; Trowsdale, John; Fugger, Lars

    2013-09-01

    The immune system has crucial roles in the pathogenesis of multiple sclerosis. While the adaptive immune cell subsets, T and B cells, have been the main focus of immunological research in multiple sclerosis, it is now important to realize that the innate immune system also has a key involvement in regulating autoimmune responses in the central nervous system. Natural killer cells are innate lymphocytes that play vital roles in a diverse range of infections. There is evidence that they influence a number of autoimmune conditions. Recent studies in multiple sclerosis and its murine model, experimental autoimmune encephalomyelitis, are starting to provide some understanding of the role of natural killer cells in regulating inflammation in the central nervous system. Natural killer cells express a diverse range of polymorphic cell surface receptors, which interact with polymorphic ligands; this interaction controls the function and the activation status of the natural killer cell. In this review, we discuss evidence for the role of natural killer cells in multiple sclerosis and experimental autoimmune encephalomyelitis. We consider how a change in the balance of signals received by the natural killer cell influences its involvement in the ensuing immune response, in relation to multiple sclerosis.

  6. Expression of histamine receptor genes Hrh3 and Hrh4 in rat brain endothelial cells.

    Science.gov (United States)

    Karlstedt, K; Jin, C; Panula, P

    2013-09-01

    Brain vascular endothelial cells express histamine H1 and H2 receptors, which regulate brain capillary permeability. We investigated whether H3 and H4 receptors are also expressed in these cells and may thus play a role in permeability regulation. An immortalized rat brain endothelial cell line RBE4 was used to assess the presence of H3 and H4 receptors. Reverse transcription-PCR (RT-PCR) and sequencing were used to identify the receptor mRNAs. The receptors were stimulated with histamine and immepip, and specific inverse agonists/antagonists ciproxifan and JNJ 7777120 were used to block H3 and H4 receptors, respectively. RT-PCR of mRNA extracted from cultured immortalized RBE4 cells revealed two rat H4 receptor gene (Hrh4) transcripts, one full-length (coding sequence 1173 bp), and one with a 164 bp deletion. Also, two rat H3 receptor gene (Hrh3) isoform mRNAs were expressed in RBE4 cells, and sequencing showed they were the full-length H3 receptor and the 144 bp deletion form. Both histamine and immepip (H3 and H4 receptor agonists) activated the Erk1/2 MAPK pathway in the RBE4 cells and in vivo in brain blood vessels by activating H4 receptors, as the H4 receptor-specific inverse agonists/antagonist JNJ 7777120, but not ciproxifan, H3 receptor antagonist, dose-dependently blocked this effect in RBE4 cells. Both Hrh3 and Hrh4 receptors are expressed in rat brain endothelial cells, and activation of the histamine H4 receptor activates the Erk1/2 cascade. H3 and H4 receptors in endothelial cells are potentially important for regulation of blood-brain barrier permeability, including trafficking of immunocompetent cells. © 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.

  7. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  8. The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1985-01-01

    Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (Kd between 15 and 450 nM) and different off-rates of the cAMP-receptor complex (t½ between 0.7 and 150 s). The association of cAMP to the receptor and the

  9. T-cell receptor gamma delta bearing cells in normal human skin

    NARCIS (Netherlands)

    Bos, J. D.; Teunissen, M. B.; Cairo, I.; Krieg, S. R.; Kapsenberg, M. L.; Das, P. K.; Borst, J.

    1990-01-01

    T-cell antigen receptors (TCR) are divided into common alpha beta and less common gamma delta types. In the murine skin, TCR gamma delta+ cells have been reported to form the great majority of epidermal T lymphocytes. We have examined the relative contribution of TCR alpha beta+ and TCR gamma delta+

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

    Science.gov (United States)

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

  11. The shape of the lymphocyte receptor repertoire: lessons from the B cell receptor

    Directory of Open Access Journals (Sweden)

    Katherine J. L. Jackson

    2013-09-01

    Full Text Available Both the B cell receptor (BCR and the T cell receptor (TCR repertoires are generated through essentially identical processes of V(DJ recombination, exonuclease trimming of germline genes and the random addition of non-template encoded nucleotides. The naïve TCR repertoire is constrained by thymic selection, and TCR repertoire studies have therefore focused strongly on the diversity of MHC-binding CDR3. The process of somatic point mutations has given B cell studies a major focus on variable (IGHV, IGLV and IGKV genes. This in turn has influenced how both the naïve and memory BCR repertoires have been studied. Diversity (D genes are also more easily identified in BCR VDJ rearrangements than in TCR VDJ rearrangements, and this has allowed the processes and elements that contribute to the incredible diversity of the immunoglobulin heavy chain CDR3 to be analyzed in detail. This diversity can be contrasted with that of the light chain where a small number of polypeptide sequences dominate the repertoire. Biases in the use of different germline genes, in gene processing and in the addition of non-template encoded nucleotides appear to be intrinsic to the recombination process, imparting ‘shape’ to the repertoire of rearranged genes as a result of differences spanning many orders of magnitude in the probabilities that different BCRs will be generated. This may function to increase the precursor frequency of naïve B cells with important specificities, and the likely emergence of such B cell lineages upon antigen exposure is discussed with reference to public and private T cell clonotypes.

  12. BAFF controls neural cell survival through BAFF receptor.

    Directory of Open Access Journals (Sweden)

    Satoru Tada

    Full Text Available Various neuroprotective factors have been shown to help prevention of neuronal cell death, which is responsible for the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS. However, most of these therapeutic potentials have been tested by administration of recombinant proteins, transgenic expression or virus vector-mediated gene transfer. Therefore, it remains to be clarified whether any endogenous factors has advantage for neuroprotection in a pathological nervous system. Here we show the role of BAFF-R signaling pathway in the control of neural cell survival. Both B cell-activating factor (BAFF and its receptor (BAFF-R are expressed in mouse neurons and BAFF-R deficiency reduces the survival of primary cultured neurons. Although many studies have so far addressed the functional role of BAFF-R on the differentiation of B cells, impaired BAFF-R signaling resulted in accelerated disease progression in an animal model of inherited ALS. We further demonstrate that BAFF-R deficient bone marrow cells or genetic depletion of B cells does not affect the disease progression, indicating that BAFF-mediated signals on neurons, not on B cells, support neural cell survival. These findings suggest opportunities to improve therapeutic outcome for patients with neurodegenerative diseases by synthesized BAFF treatment.

  13. Preliminary study on mononuclear cells insulin receptor in porcine blood

    International Nuclear Information System (INIS)

    Dalimunthe, D.; Hara, Hitoshi; Hayashi, Hirofumi; Mito, Kazuyo; Kawate, Ryoso

    1980-01-01

    Insulin receptor of porcine mononuclear cells was investigated. After passaging over a Boyum method gradient, mononuclear cells from freshly collected heparinized blood were isolated and 1 x 10 7 /ml mononuclear cells were incubated for 24 hrs at 4 0 C in Tris-salt buffer pH 8.0 with 125 I-insulin (2.2 ng/ml) and a range of native insulin concentration from 0 to 1 x 10 3 ng/ml. γ-globulin-polyethylene glycol was used to separate the unbound 125 I-insulin from the incubation mixture. After centrifugation the supernatant was discarded, the radioactivity of the cell pellets were counted in a gammacounter and the percentage of 125 I-insulin bound could be determined. The result demonstrated that circulating porcine mononuclear cells prepared from Ficoll-Conray gradient readily bound with 125 I-insulin. The binding of 125 I-insulin to porcine mononuclear cells was rapid and reversible process, and could be inhibited by physiological amount of native insulin. 125 I-insulin binding to porcine mononuclear cells was a linear function of cell concentration and a saturable ability of native insulin to displace the binding of 125 I-insulin. (author)

  14. Palindromic nucleotide analysis in human T cell receptor rearrangements.

    Directory of Open Access Journals (Sweden)

    Santosh K Srivastava

    Full Text Available Diversity of T cell receptor (TCR genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3 of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8(+ and CD4(+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8(+ naïve T cells. The naïve CD8(+ T cell clones with P nucleotides are more highly expanded.

  15. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    Science.gov (United States)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

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

    Directory of Open Access Journals (Sweden)

    Pan Zhongzong

    2009-01-01

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

  17. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    Kolko, Miriam; de Turco, Elena B; Diemer, Nils Henrik

    2002-01-01

    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H...... neuronal cell death. We conclude that glutamatergic synaptic activity modulates sPLA -induced neuronal cell death....

  18. Modeling multivalent ligand-receptor interactions with steric constraints on configurations of cell surface receptor aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Monine, Michael [Los Alamos National Laboratory; Posner, Richard [TRANSLATION GENOMICS RESAEARCH INSTITUTE; Savage, Paul [BYU; Faeder, James [UNIV OF PITTSBURGH; Hlavacek, William S [UNM

    2008-01-01

    Signal transduction generally involves multivalent protein-protein interactions, which can produce various protein complexes and post-translational modifications. The reaction networks that characterize these interactions tend to be so large as to challenge conventional simulation procedures. To address this challenge, a kinetic Monte Carlo (KMC) method has been developed that can take advantage of a model specification in terms of reaction rules for molecular interactions. A set of rules implicitly defines the reactions that can occur as a result of the interactions represented by the rules. With the rule-based KMC method, explicit generation of the underlying chemical reaction network implied by rules is avoided. Here, we apply and extend this method to characterize the interactions of a trivalent ligand with a bivalent cell-surface receptor. This system is also studied experimentally. We consider the following kinetic models: an equivalent-site model, an extension of this model, which takes into account steric constraints on the configurations of receptor aggregates, and finally, a model that accounts for cyclic receptor aggregates. Simulation results for the equivalent-site model are consistent with an equilibrium continuum model. Using these models, we investigate the effects of steric constraints and the formation of cyclic aggregates on the kinetics and equilibria of small and large aggregate formation and the percolation phase transition that occurs in this system.

  19. Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil

    2014-10-01

    Full Text Available Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

  20. Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

    Science.gov (United States)

    Veettil, Mohanan Valiya; Bandyopadhyay, Chirosree; Dutta, Dipanjan; Chandran, Bala

    2014-01-01

    Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry. PMID:25341665

  1. Methods for quantifying T cell receptor binding affinities and thermodynamics

    Science.gov (United States)

    Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

  2. Characterization of LH/hCG receptor by enzyme receptor assay in human ovarian cancer cell line 3AO

    International Nuclear Information System (INIS)

    Xing Yan; Liao Ying; Chen Manling

    2005-01-01

    To establish an enzyme receptor assay (ERA) for LH/hCG receptor on intact culture cells and study the characteristics of LH/hCG receptor of ovarian cancer cell line 3AO by ERA, the enzyme-ligand conjugate was prepared by the oxidation method of NaIO 4 . An ERA of marker ligand with embedded cells was established in suitable conditions, and applied to determine the maximum binding and affinity of LH/hCG receptor on 3AO cells as well as on an ovarian cancer cell line SKOV-3 serving as the negative control. The results showed that the B max and K d values of LH/hCG receptor were 3.65 mmol/g protein and 0.41 nmol/L, respectively. For competitive inhibition, the IC 50 of unlabelled hCG against HRP-hCG binding was 0.92 μmol/L. Our conclusion is that by competitive inhibition assay, saturation binding assay and Scatchard analysis, LH/hCG receptors with high affinity are present on 3AO cells. (authors)

  3. Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors.

    Science.gov (United States)

    Chen, Jin; Chaurio, Ricardo A; Maueröder, Christian; Derer, Anja; Rauh, Manfred; Kost, Andriy; Liu, Yi; Mo, Xianming; Hueber, Axel; Bilyy, Rostyslav; Herrmann, Martin; Zhao, Yi; Muñoz, Luis E

    2017-01-01

    Many antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells. Cultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS) in the presence of dead and dying cells, their supernatants (SNs), or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo . The stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment. Inosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

  4. Platelet-Activating Factor Receptor Ligands Protect Tumor Cells from Radiation-Induced Cell Death

    Directory of Open Access Journals (Sweden)

    Ildefonso Alves da Silva-Junior

    2018-02-01

    Full Text Available Irradiation generates oxidized phospholipids that activate platelet-activating factor receptor (PAFR associated with pro-tumorigenic effects. Here, we investigated the involvement of PAFR in tumor cell survival after irradiation. Cervical cancer samples presented higher levels of PAF-receptor gene (PTAFR when compared with normal cervical tissue. In cervical cancer patients submitted to radiotherapy (RT, the expression of PTAFR was significantly increased. Cervical cancer-derived cell lines (C33, SiHa, and HeLa and squamous carcinoma cell lines (SCC90 and SCC78 express higher levels of PAFR mRNA and protein than immortalized keratinocytes. Gamma radiation increased PAFR expression and induced PAFR ligands and prostaglandin E2 (PGE2 in these tumor cells. The blocking of PAFR with the antagonist CV3938 before irradiation inhibited PGE2 and increased tumor cells death. Similarly, human carcinoma cells transfected with PAFR (KBP were more resistant to radiation compared to those lacking the receptor (KBM. PGE2 production by irradiated KBP cells was also inhibited by CV3988. These results show that irradiation of carcinoma cells generates PAFR ligands that protect tumor cells from death and suggests that the combination of RT with a PAFR antagonist could be a promising strategy for cancer treatment.

  5. Role of histamine and its receptor subtypes in stimulation of conjunctival goblet cell secretion.

    Science.gov (United States)

    Hayashi, Densen; Li, Dayu; Hayashi, Chisato; Shatos, Marie; Hodges, Robin R; Dartt, Darlene A

    2012-05-17

    The purpose of this study was to determine the effect of histamine and its receptors on goblet cell secretion. Cultured rat and human goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugate was measured by an enzyme-linked lectin assay. Cultured rat goblet cells were homogenized and either RNA was isolated for RT-PCR or proteins were isolated for Western blot analysis for presence of histamine receptors subtypes H₁ through H₄. The localization of these receptors was determined in rat and human goblet cells by immunofluorescence microscopy. Histamine stimulated goblet cell secretion in a concentration- and time-dependent manner. All four histamine receptors were present in cultured rat and human goblet cells. Use of agonists specific to individual histamine receptor subtypes indicated that the rank order of agonist stimulation was H₁ = H₃ > H₄ > H₂. Using antagonists specific to individual histamine receptor subtypes determined that H₂ and H₃, but not the H₁ and H₄, antagonists, inhibited histamine-stimulated conjunctival goblet cell secretion. Rat and human conjunctival goblet cells are a direct target of histamine, which induces secretion. All four histamine receptors are present in rat and human conjunctiva and are active in rat conjunctival goblet cells. These findings suggest that all four histamine receptor subtypes are important for conjunctival goblet cell secretion. Blockage of histamine receptor subtypes could prevent the excess mucus production associated with ocular allergy.

  6. Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

    International Nuclear Information System (INIS)

    Cameron, W.; Doyle, K.; Rocklin, R.E.

    1986-01-01

    A single, specific binding site for [ 3 H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes was documented. The binding of the radioligand to its receptor is reversible with cold H 1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), the authors calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity for [ 3 H]pyrilamine, followed by T helper cells, B cells and T suppressor cells (K/sub D/ = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H 1 receptors per cell followed by B cells, T helper cells, and monocytes. The binding affinity for [ 3 H]pyrilamine increased over a 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [ 3 H]pyrilamine decreased over the 48-hr period. Although the function of H 1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in the modulating the immune response

  7. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak-Wismann, Martin; Schjerling, Peter

    2010-01-01

    Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering...... led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR...... signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation....

  8. The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

    Science.gov (United States)

    Foley, Bree; Felices, Martin; Cichocki, Frank; Cooley, Sarah; Verneris, Michael R; Miller, Jeffrey S

    2014-03-01

    Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

    Science.gov (United States)

    Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

    2018-04-03

    The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

  10. Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

    Science.gov (United States)

    Ng, KY; Yeung, BHS; Wong, YH; Wise, H

    2013-01-01

    Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

  11. CSF-1 receptor signalling is governed by pre-requisite EHD1 mediated receptor display on the macrophage cell surface.

    Science.gov (United States)

    Cypher, Luke R; Bielecki, Timothy Alan; Huang, Lu; An, Wei; Iseka, Fany; Tom, Eric; Storck, Matthew D; Hoppe, Adam D; Band, Vimla; Band, Hamid

    2016-09-01

    Colony stimulating factor-1 receptor (CSF-1R), a receptor tyrosine kinase (RTK), is the master regulator of macrophage biology. CSF-1 can bind CSF-1R resulting in receptor activation and signalling essential for macrophage functions such as proliferation, differentiation, survival, polarization, phagocytosis, cytokine secretion, and motility. CSF-1R activation can only occur after the receptor is presented on the macrophage cell surface. This process is reliant upon the underlying macrophage receptor trafficking machinery. However, the mechanistic details governing this process are incompletely understood. C-terminal Eps15 Homology Domain-containing (EHD) proteins have recently emerged as key regulators of receptor trafficking but have not yet been studied in the context of macrophage CSF-1R signalling. In this manuscript, we utilize primary bone-marrow derived macrophages (BMDMs) to reveal a novel function of EHD1 as a regulator of CSF-1R abundance on the cell surface. We report that EHD1-knockout (EHD1-KO) macrophages cell surface and total CSF-1R levels are significantly decreased. The decline in CSF-1R levels corresponds with reduced downstream macrophage functions such as cell proliferation, migration, and spreading. In EHD1-KO macrophages, transport of newly synthesized CSF-1R to the macrophage cell surface was reduced and was associated with the shunting of the receptor to the lysosome, which resulted in receptor degradation. These findings reveal a novel and functionally important role for EHD1 in governing CSF-1R signalling via regulation of anterograde transport of CSF-1R to the macrophage cell surface. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Ikaros limits follicular B cell activation by regulating B cell receptor signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Heizmann, Beate [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Sellars, MacLean [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Macias-Garcia, Alejandra [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Institute for Medical Engineering and Science at MIT, Cambridge, MA 02139 (United States); Chan, Susan, E-mail: scpk@igbmc.fr [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Kastner, Philippe, E-mail: scpk@igbmc.fr [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Faculté de Médecine, Université de Strasbourg, Strasbourg (France)

    2016-02-12

    The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.

  13. Ikaros limits follicular B cell activation by regulating B cell receptor signaling pathways

    International Nuclear Information System (INIS)

    Heizmann, Beate; Sellars, MacLean; Macias-Garcia, Alejandra; Chan, Susan; Kastner, Philippe

    2016-01-01

    The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.

  14. Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells

    DEFF Research Database (Denmark)

    Egerod, Kristoffer L; Engelstoft, Maja S; Lund, Mari L

    2015-01-01

    characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly...

  15. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells

    Directory of Open Access Journals (Sweden)

    Gin-Wen Chang

    2016-05-01

    Full Text Available Natural killer (NK cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

  16. Desensitization of parathyroid hormone receptors on cultured bone cells

    International Nuclear Information System (INIS)

    Pun, K.K.; Ho, P.W.; Nissenson, R.A.; Arnaud, C.D.

    1990-01-01

    Administration of excessive amounts of parathyroid hormone (PTH) in the treatment of osteoporosis can reverse the beneficial effects of a low-dose, intermittent regime. To investigate the direct actions and the possible cellular mechanisms of PTH in inducing desensitization of PTH receptors, we studied the effects of desensitization on rat osteoblastic UMR-106 cells. When the osteoblasts were preincubated with bPTH-(1-34), complete refractoriness to a subsequent challenge with the hormone developed within 1 h and at hormone concentrations as low as 5 nM. When osteoblasts thus desensitized were incubated in hormone-free medium, recovery of the cAMP responses began within 2 h and reached maximum after 16 h. Cycloheximide did not affect the process of desensitization. [Nle8,Nle18,Tyr34]bPTH-(3-34)amide significantly impaired the desensitization process by PTH-(1-34) but did not have stimulatory effect on cAMP responses. No significant heterologous desensitization was obvious after preincubation with isoprenaline (50 microM), prostaglandin E1 (50 microM), or prostaglandin E2 (50 microM) for 2 h. Binding experiments with [125I]PLP-(1-36)amide after desensitization revealed that there was an approximate twofold decrease in receptor affinities as analyzed by Scatchard analysis, showing that the decrease in affinity was prominent in the process of desensitization. When the cells were treated with monensin during desensitization, PTH challenge after desensitization produced significantly lower cyclic AMP responses. Recovery after desensitization occurred over a period of 16 h. Inclusion of monensin, but not cycloheximide, impaired the recovery. The results show that homologous desensitization of rat osteoblasts to PTH is brought about by the occupancy of receptors by PTH-(1-34) but not by cAMP generation itself

  17. Desensitization of parathyroid hormone receptors on cultured bone cells

    Energy Technology Data Exchange (ETDEWEB)

    Pun, K.K.; Ho, P.W.; Nissenson, R.A.; Arnaud, C.D. (Univ. of Hong Kong (Hong Kong))

    1990-12-01

    Administration of excessive amounts of parathyroid hormone (PTH) in the treatment of osteoporosis can reverse the beneficial effects of a low-dose, intermittent regime. To investigate the direct actions and the possible cellular mechanisms of PTH in inducing desensitization of PTH receptors, we studied the effects of desensitization on rat osteoblastic UMR-106 cells. When the osteoblasts were preincubated with bPTH-(1-34), complete refractoriness to a subsequent challenge with the hormone developed within 1 h and at hormone concentrations as low as 5 nM. When osteoblasts thus desensitized were incubated in hormone-free medium, recovery of the cAMP responses began within 2 h and reached maximum after 16 h. Cycloheximide did not affect the process of desensitization. (Nle8,Nle18,Tyr34)bPTH-(3-34)amide significantly impaired the desensitization process by PTH-(1-34) but did not have stimulatory effect on cAMP responses. No significant heterologous desensitization was obvious after preincubation with isoprenaline (50 microM), prostaglandin E1 (50 microM), or prostaglandin E2 (50 microM) for 2 h. Binding experiments with (125I)PLP-(1-36)amide after desensitization revealed that there was an approximate twofold decrease in receptor affinities as analyzed by Scatchard analysis, showing that the decrease in affinity was prominent in the process of desensitization. When the cells were treated with monensin during desensitization, PTH challenge after desensitization produced significantly lower cyclic AMP responses. Recovery after desensitization occurred over a period of 16 h. Inclusion of monensin, but not cycloheximide, impaired the recovery. The results show that homologous desensitization of rat osteoblasts to PTH is brought about by the occupancy of receptors by PTH-(1-34) but not by cAMP generation itself.

  18. Beta-Adrenergic Receptor Expression in Muscle Cells

    Science.gov (United States)

    Young, Ronald B.; Bridge, K.; Vaughn, J. R.

    1999-01-01

    beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

  19. Regulation of Innate Lymphoid Cells by Aryl Hydrocarbon Receptor

    Directory of Open Access Journals (Sweden)

    Shiyang Li

    2018-01-01

    Full Text Available With striking similarity to their adaptive T helper cell counterparts, innate lymphoid cells (ILCs represent an emerging family of cell types that express signature transcription factors, including T-bet+ Eomes+ natural killer cells, T-bet+ Eomes− group 1 ILCs, GATA3+ group 2 ILCs, RORγt+ group 3 ILCs, and newly identified Id3+ regulatory ILC. ILCs are abundantly present in barrier tissues of the host (e.g., the lung, gut, and skin at the interface of host–environment interactions. Active research has been conducted to elucidate molecular mechanisms underlying the development and function of ILCs. The aryl hydrocarbon receptor (Ahr is a ligand-dependent transcription factor, best known to mediate the effects of xenobiotic environmental toxins and endogenous microbial and dietary metabolites. Here, we review recent progresses regarding Ahr function in ILCs. We focus on the Ahr-mediated cross talk between ILCs and other immune/non-immune cells in host tissues especially in the gut. We discuss the molecular mechanisms of the action of Ahr expression and activity in regulation of ILCs in immunity and inflammation, and the interaction between Ahr and other pathways/transcription factors in ILC development and function with their implication in disease.

  20. Differences of IL-1β Receptors Expression by Immunocompetent Cells Subsets in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Alina A. Alshevskaya

    2015-01-01

    Full Text Available IL-1β is involved in the induction and maintenance of chronic inflammation in rheumatoid arthritis (RA. Its activity is regulated and induced by soluble and membrane-bound receptors, respectively. The effectiveness of the cytokine depends not only on the percentage of receptor-positive cells in an immunocompetent subset but also on the density of receptor expression. The objective of this study was to investigate the expression of IL-1β membrane-bound receptors (IL-1R1 and IL-1R2 in terms of the percentage of receptor-positive cells and the number of receptors per cell in different subsets of immune cells in RA patients before and after a course of basic (excluding anticytokine therapy and in healthy individuals. The resulting data indicate differences in the expression of IL-1β receptors among T cells, B cells, and monocytes in healthy volunteers and in rheumatoid arthritis patients. The importance of determining both the relative percentage of cells expressing receptors to immunomodulatory cytokines and the number of membrane-bound receptors per cell is highlighted by evidence of unidirectional or multidirectional changing of these parameters according to cell subset and health status.

  1. Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

    Science.gov (United States)

    Janssen-Bienhold, Ulrike; Schultz, Konrad; Cimiotti, Kerstin; Weiler, Reto; Willecke, Klaus; Dedek, Karin

    2013-01-01

    In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input

  2. Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

    International Nuclear Information System (INIS)

    Scott, C.D.; Baxter, R.C.

    1987-01-01

    Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

  3. Chemokine receptor expression on B cells and effect of interferon-beta in multiple sclerosis

    DEFF Research Database (Denmark)

    Sørensen, Torben Lykke; Roed, Hanne; Sellebjerg, Finn

    2002-01-01

    We investigated the B-cell expression of chemokine receptors CXCR3, CXCR5 and CCR5 in the blood and cerebrospinal fluid (CSF) from patients in relapse of multiple sclerosis (MS) and in neurological controls. Chemokine receptor expression was also studied in interferon-beta-treated patients with r......, and chemokine receptor expression was not affected by interferon-beta treatment....

  4. Expression and role of Fc- and complement-receptors on human dendritic cells.

    Science.gov (United States)

    Bajtay, Zsuzsa; Csomor, Eszter; Sándor, Noémi; Erdei, Anna

    2006-04-15

    Dendritic cells (DCs) are professional antigen presenting cells, which take up pathogens/foreign structures in peripheral tissues, then migrate to secondary lymphoid organs where they initiate adaptive immune responses by activating naive T-cells. In the early phase of antigen uptake pattern recognition receptors (including mannose-, scavenger- and toll-like receptors) that recognize pathogen-associated molecular patterns play an important role. Later receptors binding opsonized antigen are also involved in phagocytosis. These cell membrane molecules include various Fc-receptors, recognizing different isotypes of antibodies and various complement-receptors, such as CR3, CR4 and the C1q-binding complex of calreticulin and CD91. Here we aim to summarize how these immunecomplex binding receptors are involved in the initiation of DC maturation, and how they influence antigen presentation as well as some additional functions of these cells.

  5. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...

  6. Functional importance of GLP-1 receptor species and expression levels in cell lines.

    Science.gov (United States)

    Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

    2012-04-10

    Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  8. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    International Nuclear Information System (INIS)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    2015-01-01

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  9. Protease-activated receptor-2 turnover stimulated independently of receptor activation in porcine coronary endothelial cells

    Science.gov (United States)

    Hamilton, Justin R; Chow, Jean M; Cocks, Thomas M

    1999-01-01

    Protease-activated receptors (PARs) are activated by an irreversible proteolytic mechanism which renders cleaved receptors unresponsive to subsequent challenges with activating enzymes. Non-specific proteolysis of PARs downstream of the activation site also prevents subsequent enzymic activation. Therefore, we investigated the effects of non-activating amino-terminal proteolysis with the bacterial protease thermolysin on PAR-mediated relaxation of porcine coronary artery ring preparations contracted with the thromboxane A2 mimetic U46619 (1–10 nM). Treatment of contracted artery ring segments with thermolysin (0.01–1 u ml−1, 20 min) caused no response, but abolished endothelium-dependent relaxations induced by the enzymic activators of PAR-1 and PAR-2, thrombin (0.01–0.3 u ml−1) and trypsin (0.003–0.1 u ml−1) respectively. The same treatment, however, did not affect similar responses to the proteolysis-independent PAR-1 and PAR-2 activating peptides, SFLLRN-NH2 and SLIGRL-NH2 respectively (0.1–10 μM). The inhibition of responsiveness to trypsin after thermolysin treatment recovered in a time-dependent manner, with maximal recovery (77.3±8.0% of time controls) occurring 150 min after thermolysin treatment. No recovery of responsiveness to thrombin after thermolysin treatment was observed within this time, however, the thrombin response returned to control levels after 20 h. The recovery of responsiveness to trypsin was inhibited by the translation inhibitor cycloheximide (100 μM; 17.3±4.7%) and the protein trafficking inhibitor brefeldin A (10 μM; 12.1±4.8%) but was unaffected by the transcription inhibitor actinomycin D (2 μM; 65.1±3.6%), which did, however, abolish upregulation of B1-kinin receptors in this preparation. In conclusion, our findings indicate that activation-independent amino-terminal proteolysis of PARs stimulates selective recovery of endothelial cell PAR-2 responsiveness, which appears to be

  10. Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

    Science.gov (United States)

    Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

    2015-11-19

    Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor

  11. Targeting Gallium to Cancer Cells through the Folate Receptor

    Directory of Open Access Journals (Sweden)

    Nerissa Viola-Villegas

    2008-01-01

    Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG ‘spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

  12. Targeting Gallium to Cancer Cells through the Folate Receptor

    Directory of Open Access Journals (Sweden)

    Nerissa Viola-Villegas

    2008-01-01

    Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG 'spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N, N′, N′, N′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

  13. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    Science.gov (United States)

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma.

  14. Radiometallated receptor-avid peptide conjugates for specific in vivo targeting of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Hoffman, T.J.; Quinn, T.P.; Volkert, W.A. E-mail: VolkertW@health.missouri.edu

    2001-07-01

    New receptor-avid radiotracers are being developed for site-specific in vivo targeting of a myriad of receptors expressed on cancer cells. This review exemplifies strategies being used to design radiometallated peptide conjugates that maximize uptake in tumors and optimize their in vivo pharmacokinetic properties. Efforts to produce synthetic peptide analogues that target the following three receptor systems are highlighted: Gastrin releasing peptide (GRP), alpha-melanocyte stimulating hormone ({alpha}-MSH), and guanylate cyclase-C (GC-C) receptors.

  15. Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT1 receptor with neuronal and glial markers

    Directory of Open Access Journals (Sweden)

    McMillan Catherine R

    2004-10-01

    Full Text Available Abstract Background In order to optimize the potential benefits of neural stem cell (NSC transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different in vitro conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs. Results RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF, brain-derived neurotrophic factor (BDNF and nerve growth factor (NGF in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (β-tubulin III positive and glial (GFAP positive progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours. Conclusions The phenotypic characteristics of C17.2 cells suggest that they are

  16. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1 into Diverse Memory T-Cell Populations.

    Directory of Open Access Journals (Sweden)

    Drew C Deniger

    Full Text Available T cells modified with chimeric antigen receptors (CARs targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1 is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28 or CD137 (designated ROR1RCD137 and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC, which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

  17. Cheiradone: a vascular endothelial cell growth factor receptor antagonist

    Directory of Open Access Journals (Sweden)

    Ahmed Nessar

    2008-01-01

    Full Text Available Abstract Background Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing and pathological conditions (tumour development. Vascular endothelial growth factor (VEGF, fibroblast growth factor-2 (FGF-2 and epidermal growth factor (EGF are the major angiogenic regulators. We have identified a natural product (cheiradone isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation and in vivo (the chick chorioallantoic membrane models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC50 was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated. Results Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC50 values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC50 values of 2.9 and 0.61 μM respectively. Conclusion Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.

  18. Identifying specificity groups in the T cell receptor repertoire.

    Science.gov (United States)

    Glanville, Jacob; Huang, Huang; Nau, Allison; Hatton, Olivia; Wagar, Lisa E; Rubelt, Florian; Ji, Xuhuai; Han, Arnold; Krams, Sheri M; Pettus, Christina; Haas, Nikhil; Arlehamn, Cecilia S Lindestam; Sette, Alessandro; Boyd, Scott D; Scriba, Thomas J; Martinez, Olivia M; Davis, Mark M

    2017-07-06

    T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

  19. Human nicotinic receptors in chromaffin cells: characterization and pharmacology.

    Science.gov (United States)

    Albillos, Almudena; McIntosh, J Michael

    2018-01-01

    During the last 10 years, we have been working on human chromaffin cells obtained from the adrenal gland of organ donors that suffered encephalic or cardiac death. We first electrophysiologically characterized the nicotinic acetylcholine receptors (nAChRs) activated by acetylcholine, and their contribution to the exocytosis of chromaffin vesicles and release of catecholamines. We have shown that these cells possess an adrenergic phenotype. This phenotype may contribute to an increased expression of α7 nAChRs in these cells, allowing for recording of α7 nAChR currents, something that had previously not been achieved in non-human species. The use of α-conotoxins allowed us to characterize non-α7 nAChR subtypes and, together with molecular biology experiments, conclude that the predominant nAChR subtype in human chromaffin cells is α3β4* (asterisk indicates the posible presence of additional subunits). In addition, there is a minor population of αxβ2 nAChRs. Both α7 and non-α7 nAChR subtypes contribute to the exocytotic process. Exocytosis mediated by nAChRs could be as large in magnitude as that elicited by calcium entry through voltage-dependent calcium channels. Finally, we have also investigated the effect of nAChR-targeted tobacco cessation drugs on catecholamine release in chromaffin cells. We have concluded that at therapeutic concentrations, varenicline alone does not increase the frequency of action potentials evoked by ACh. However, varenicline in the presence of nicotine does increase this frequency, and thus, in the presence of both drugs, the probability of increased catecholamine release in human chromaffin cells is high.

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

    Directory of Open Access Journals (Sweden)

    Blandine C Mercier

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

  1. Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag*

    Science.gov (United States)

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

    2008-01-01

    GABAB 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, GABAB 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 GABAB 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, α-bungarotoxin. By using the α-bungarotoxin binding site-tagged GABAB R1a subunit (R1aBBS), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, α-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 GABAB 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. PMID:18812318

  2. 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.

  3. Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis.

    Directory of Open Access Journals (Sweden)

    Lea Weber

    Full Text Available The analysis and functional characterization of ectopically expressed human olfactory receptors (ORs is becoming increasingly important, as many ORs have been identified in several healthy and cancerous tissues. OR activation has been demonstrated to have influence on cancer cell growth and progression. Here, ORs were identified using RNA-Seq analyses and RT-PCR. We demonstrated the OR protein localization in HCT116 cells using immunocytochemistry (IHC. In order to analyze the physiological role of OR51B4, we deorphanized the receptor by the use of CRE-Luciferase assays, conducted calcium imaging experiments as well as scratch- and proliferation assays. Furthermore, western blot analyses revealed the involvement of different protein kinases in the ligand-dependent signaling pathway. Receptor knockdown via shRNA was used to analyze the involvement of OR51B4. We identified OR51B4, which is highly expressed in the colon cancer cell line HCT116 and in native human colon cancer tissues. We deorphanized the receptor and identified Troenan as an effective ligand. Troenan stimulation of HCT116 cells has anti-proliferative, anti-migratory and pro-apoptotic effects, mediated by changes in the intracellular calcium level upon PLC activation. These effects cause changes in the phosphorylation levels of p38, mTor and Akt kinases. Knockdown of the receptor via shRNA confirmed the involvement of OR51B4. This study emphasizes the importance of ectopically expressed ORs in the therapy for several diseases. The findings provide the basis for alternative treatments of colorectal cancer.

  4. Racial differences in B cell receptor signaling pathway activation.

    Science.gov (United States)

    Longo, Diane M; Louie, Brent; Mathi, Kavita; Pos, Zoltan; Wang, Ena; Hawtin, Rachael E; Marincola, Francesco M; Cesano, Alessandra

    2012-06-06

    Single-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males]. Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune

  5. Racial differences in B cell receptor signaling pathway activation

    Directory of Open Access Journals (Sweden)

    Longo Diane M

    2012-06-01

    Full Text Available Abstract Background Single-cell network profiling (SCNP is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. Methods The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs, 5 European Americans (36-56 yrs, all males]. Results Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt, the MAPK pathways (Erk and p38, and the NF-κB pathway (NF-κB. In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. Conclusions SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from

  6. Molecular characterization of the di-leucine-based internalization motif of the T cell receptor

    DEFF Research Database (Denmark)

    Dietrich, J; Hou, X; Wegener, A M

    1996-01-01

    Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinases. We have recently shown that both phosphorylation of Ser-126 and the presence of the di-leucine sequence Leu-131 and Leu-132 in CD3 gamma are required f...... are important. 2) Recognition of phosphorylated CD3 gamma by molecules involved in receptor internalization. In this process Ser(P)-126, Asp-127, Leu-131, and Leu-132 are important....

  7. Copy number and nucleotide variation of the LILR family of myelomonocytic cell activating and inhibitory receptors

    OpenAIRE

    López-Álvarez, María R.; Jones, Des C.; Jiang, Wei; Traherne, James A.; Trowsdale, John

    2013-01-01

    Leukocyte immunoglobulin-like receptors (LILR) are cell surface molecules that regulate the activities of myelomonocytic cells through the balance of inhibitory and activation signals. LILR genes are located within the leukocyte receptor complex (LRC) on chromosome 19q13.4 adjacent to KIR genes, which are subject to allelic and copy number variation (CNV). LILRB3 (ILT5) and LILRA6 (ILT8) are highly polymorphic receptors with similar extracellular domains. LILRB3 contains inhibitory ITIM motif...

  8. Muscarinic receptors in separate populations of noradrenaline- and adrenaline-containing chromaffin cells

    International Nuclear Information System (INIS)

    Michelena, P.; Moro, M.A.; Castillo, C.J.; Garcia, A.G.

    1991-01-01

    We have performed binding experiments of (a)[3H]quinuclidinyl benzilate to partially purified membranes from noradrenaline- and adrenaline-containing chromaffin cells and (b) [3H]N-methyl-quinuclidinyl benzilate to acutely isolated, or 48-h cultured, chromaffin cells subpopulations. Using this approach, we obtained enough evidence to conclude (1st) that muscarinic receptors are present in both noradrenaline- and adrenaline containing cells; (2nd) that noradrenaline cells contain in fact 2-3 fold higher density of those receptors; and (3rd) that those receptors undergo plastic changes upon chronic culturing of the cells

  9. A novel member of the interferon receptor family complements functionality of the murine interferon gamma receptor in human cells.

    Science.gov (United States)

    Hemmi, S; Böhni, R; Stark, G; Di Marco, F; Aguet, M

    1994-03-11

    Expression of the human interferon gamma receptor (IFN-gamma R) in mouse cells is not sufficient to confer biological responsiveness to human IFN-gamma and vice versa. An additional species-specific component is required for signal transduction. We identified this cofactor by expression cloning in simian COS cells stably transfected with the nonfunctional murine IFN-gamma R and a IFN-gamma-inducible reporter construct encoding the human Tac antigen (interleukin-2 receptor alpha chain, CD25). A cDNA clone was obtained that, upon stable transfection, rendered human HEp-2 cells expressing the murine IFN-gamma R fully responsive to murine IFN-gamma. This cDNA encodes a novel 332 amino acid type I transmembrane protein that belongs to the IFN receptor family and that we designate IFN-gamma R beta chain.

  10. Neural cell adhesion molecule differentially interacts with isoforms of the fibroblast growth factor receptor

    DEFF Research Database (Denmark)

    Christensen, Claus; Berezin, Vladimir; Bock, Elisabeth

    2011-01-01

    The fibroblast growth factor receptor (FGFR) can be activated through direct interactions with various fibroblast growth factors or through a number of cell adhesion molecules, including the neural cell adhesion molecule (NCAM). We produced recombinant proteins comprising the ligand...

  11. Toll-like receptor 3 signalling up-regulates expression of the HIV co-receptor G-protein coupled receptor 15 on human CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Miriam Kiene

    Full Text Available BACKGROUND: Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4(+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. RESULTS: Here, we show that GPR15 expression in CD4(+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4(+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF and was more prominent on gut-homing compared to lymph node-homing CD4(+ T cells. CONCLUSION: These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4(+ T cells to HIV infection and target cell availability in the gut in some infected individuals.

  12. Regulation of dopamine D2 receptors in a novel cell line (SUP1)

    International Nuclear Information System (INIS)

    Ivins, K.J.; Luedtke, R.R.; Artymyshyn, R.P.; Molinoff, P.B.

    1991-01-01

    A prolactin-secreting cell line, SUP1, has been established from rat pituitary tumor 7315a. In radioligand binding experiments, the D2 receptor antagonist (S)-(-)-3- 125 I iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2- pyrrolidinyl)methyl]benzamide ( 125 I IBZM) labeled a single class of sites in homogenates of SUP1 cells (Kd = 0.6 nM; Bmax = 45 fmol/mg of protein). The sites displayed a pharmacological profile consistent with that of D2 receptors. Inhibition of the binding of 125 I IBZM by dopamine was sensitive to GTP, suggesting that D2 receptors in SUP1 cells are coupled to guanine nucleotide-binding protein(s). In the presence of isobutylmethylxanthine, dopamine decreased the level of cAMP accumulation in SUP1 cells. Dopamine also inhibited prolactin secretion from SUP1 cells. Both the inhibition of cAMP accumulation and the inhibition of prolactin secretion were blocked by D2 receptor antagonists, suggesting that these effects of dopamine were mediated by an interaction with D2 receptors. The regulation of D2 receptors in SUP1 cells by D2 receptor agonists was investigated. Exposure of SUP1 cells to dopamine or to the D2 receptor agonist N-propylnorapomorphine led to increased expression of D2 receptors, with no change in the affinity of the receptors for 125 I IBZM. An increase in the density of D2 receptors in SUP1 cells was evident within 7 hr of exposure to dopamine. Spiroperidol, a D2 receptor antagonist, blocked the effect of dopamine on receptor density. These results suggest that exposure of D2 receptors in SUP1 cells to agonists leads to an up-regulation of D2 receptors. Dopamine retained the ability to inhibit cAMP accumulation in SUP1 cells exposed to dopamine for 24 hr, suggesting that D2 receptors in SUP1 cells are not desensitized by prolonged exposure to agonist

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

    Lifescience Database Archive (English)

    Full Text Available 7744810 Signals and receptors involved in recruitment of inflammatory cells. Ben-Ba...ruch A, Michiel DF, Oppenheim JJ. J Biol Chem. 1995 May 19;270(20):11703-6. (.png) (.svg) (.html) (.csml) Show Signals... and receptors involved in recruitment of inflammatory cells. PubmedID 7744810 Title Signals and r

  14. Purkinje cell NMDA receptors assume a key role in synaptic gain control in the mature cerebellum

    NARCIS (Netherlands)

    C. Piochon (Claire); C. Levenes (Carole); G. Ohtsuki (Gen); C.R.W. Hansel (Christian)

    2010-01-01

    textabstractA classic view in cerebellar physiology holds that Purkinje cells do not express functional NMDA receptors and that, therefore, postsynaptic NMDA receptors are not involved in the induction of long-term depression (LTD) at parallel fiber (PF) to Purkinje cell synapses. Recently, it has

  15. Urokinase receptor forms in serum from non-small cell lung cancer patients

    DEFF Research Database (Denmark)

    Almasi, Charlotte Elberling; Christensen, Ib Jarle; Høyer-Hansen, Gunilla

    2011-01-01

    To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients.......To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients....

  16. Molecular characterization of the di-leucine-based internalization motif of the T cell receptor

    DEFF Research Database (Denmark)

    Dietrich, J; Hou, X; Wegener, A M

    1996-01-01

    Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinases. We have recently shown that both phosphorylation of Ser-126 and the presence of the di-leucine sequence Leu-131 and Leu-132 in CD3 gamma are required...

  17. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry

    2011-01-01

    RNA analysis (24 women). Expression of Androgen Receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in FF, were correlated to the expression of FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor2 (AMHR2) mRNA in the granulosa cells and to the FF....... This suggests that follicular sensitivity towards FSH stimulation may be augmented by stimulation of androgens via the AR....

  19. Coevolution of paired receptors in Xenopus carcinoembryonic antigen-related cell adhesion molecule families suggests appropriation as pathogen receptors.

    Science.gov (United States)

    Zimmermann, Wolfgang; Kammerer, Robert

    2016-11-16

    In mammals, CEACAM1 and closely related members represent paired receptors with similar extracellular ligand-binding regions and cytoplasmic domains with opposing functions. Human CEACAM1 and CEACAM3 which have inhibitory ITIM/ITSM and activating ITAM-like motifs, respectively, in their cytoplasmic regions are such paired receptors. Various bacterial pathogens bind to CEACAM1 on epithelial and immune cells facilitating both entry into the host and down-regulation of the immune response whereas interaction with granulocyte-specific CEACAM3 leads to their uptake and destruction. It is unclear whether paired CEACAM receptors also exist in other vertebrate clades. We identified more than 80 ceacam genes in Xenopus tropicalis and X. laevis. They consist of two subgroups containing one or two putative paired receptor pairs each. Analysis of genomic sequences of paired receptors provide evidence that their highly similar ligand binding domains were adjusted by recent gene conversion events. In contrast, selection for diversification is observed among inhibitory receptor orthologs of the two frogs which split some 60 million years ago. The allotetraploid X. laevis arose later by hybridization of two closely related species. Interestingly, despite the conservation of the genomic landscape surrounding the homeologous ceacam loci only one locus resembles the one found in X. tropicalis. From the second X. laevis locus more than 80 % of the ceacam genes were lost including 5 of the 6 paired receptor genes. This suggests that once the gene for one of the paired receptors is lost the remaining gene cluster degrades rapidly probably due to lack of selection pressure exerted by pathogens. The presence of paired receptors and selection for diversification suggests that also in amphibians CEACAM1-related inhibitory proteins are or were used as pathogen receptors.

  20. On the use of cells or membranes for receptor binding: growth hormone secretagogues.

    Science.gov (United States)

    Pinyot, A; Nikolovski, Z; Bosch, J; Segura, J; Gutiérrez-Gallego, R

    2010-04-15

    Receptor binding techniques have been widely used in different biochemical applications, with isolated membranes being the most used receptor preparation in this type of assays. In this study, intact cells were compared with isolated membranes as receptor support for radioligand receptor binding assay. The growth hormone secretagogue receptor 1a (GHSR-1a) expressed in human embryonic kidney 293 (HEK293) cells was used as a model of G-protein-coupled receptors. Differences between using intact cells in suspension and using isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation, and competition using different agonists. Intact cells are more prone to variability. Although under optimized settings both preparations were equivalent, the K(d) value for intact cells was three times higher than that using isolated membranes. However, no significant differences were observed in competition assays obtaining practically identical K(i) values for all ligands tested. For the GHSR-1a, isolated membranes are the better choice if particular incubation conditions are required (less variability), whereas intact cells yield easy, fast, and physiological conditions for receptor binding assays. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction

    NARCIS (Netherlands)

    Klarenbeek, Paul L.; Doorenspleet, Marieke E.; Esveldt, Rebecca E. E.; van Schaik, Barbera D. C.; Lardy, Neubury; van Kampen, Antoine H. C.; Tak, Paul P.; Plenge, Robert M.; Baas, Frank; de Bakker, Paul I. W.; de Vries, Niek

    2015-01-01

    Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+

  2. Targeting Multiple Tumors Using T-Cells Engineered to Express a Natural Cytotoxicity Receptor 2-Based Chimeric Receptor

    Directory of Open Access Journals (Sweden)

    Vasyl Eisenberg

    2017-09-01

    Full Text Available Recent developments in cancer treatment are demonstrating the increasing and powerful potential of immunotherapeutic strategies. In this regard, the adoptive transfer of tumor-specific T-lymphocytes approaches can lead to tumor regression in cancer patients. More recently, the use of T-cells genetically engineered to express cancer-specific receptors such as the anti-CD19 chimeric antigen receptor (CAR continues to show promise for the treatment of hematological malignancies. Still, there is a crucial need to develop efficient CAR-T cell approaches for the treatment of solid tumors. It has been shown that other lymphocytes such as natural killer (NK cells can demonstrate potent antitumor function—nonetheless, their use in immunotherapy is rather limited due to difficulties in expanding these cells to therapeutically relevant numbers and to suppression by endogenous inhibitory mechanisms. Cancer recognition by NK cells is partly mediated by molecules termed natural cytotoxicity receptors (NCRs. In the present study, we hypothesize that it is possible to endow T-cells with an NK recognition pattern, providing them with a mean to recognize tumor cells, in a non-MHC restricted way. To test this, we genetically modified human T-cells with different chimeric receptors based on the human NCR2 molecule and then assessed their antitumor activity in vitro and in vivo. Our results show that expression in primary lymphocytes of an NCR2-derived CAR, termed s4428z, confers T-cells with the ability to specifically recognize heterogeneous tumors and to mediate tumor cytotoxicity in a mouse model. This study demonstrates the benefit of combining tumor recognition capability of NK cells with T cell effectiveness to improve cancer immunotherapy.

  3. Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells

    International Nuclear Information System (INIS)

    Backer, J.M.; Kahn, C.R.; White, M.F.

    1989-01-01

    The relation between insulin-stimulated autophosphorylation of the insulin receptor and internalization of the receptor was studied in Fao rat hepatoma cells. Treatment of Fao cells with 2,4-dinitrophenol for 45 min depleted cellular ATP by 80% and equally inhibited insulin-stimulated receptor autophosphorylation, as determined by immunoprecipitation of surface-iodinated or [ 32 P]phosphate-labeled cells with anti-phosphotyrosine antibody. In contrast, internalization of the insulin receptor and internalization and degradation of 125 I-labeled insulin by 2,4-dinitrophenol-treated cells were normal. These data show that autophosphorylation of the insulin receptor is not required for the receptor-mediated internalization of insulin in Fao cells and suggest that insulin receptor recycling is independent of autophosphorylation

  4. Rat insulinoma cells express both a 115-kDa growth hormone receptor and a 95-kDa prolactin receptor structurally related to the hepatic receptors

    DEFF Research Database (Denmark)

    Møldrup, Annette; Billestrup, N; Nielsen, Jens Høiriis

    1990-01-01

    Insulin-producing rat islet RIN-5AH tumor cells express multiple binding sites for human growth hormone (hGH). The effect of rat growth hormone (rGH), rat prolactin (rPRL), and human placental lactogen (hPL) on the binding of 125I-labeled hGH (125I-hGH) to RIN-5AH cells revealed the presence...... of both lactogen and somatogen receptor populations. Covalent cross-linking of 125I-hGH, 125I-rGH, and 125I-rPRL to the RIN cells identified a 115-kDa somatogen receptor protein that binds hGH and rGH but not rPRL and hPL, and a 95-kDa lactogen receptor protein that binds hGH, rPRL, and hPL but not r...

  5. T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

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    Kostas Patas

    2018-02-01

    Full Text Available While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR repertoire in MDD. For this cross-sectional case–control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20, who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20. T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/− cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.

  6. 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

  7. Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies

    Directory of Open Access Journals (Sweden)

    Jennifer D. Stone

    2013-08-01

    Full Text Available Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional alpha-beta T cell receptor (TCR against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR consisting of a single-chain antibody as an Fv fragment (scFv linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the alpha-beta TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.

  8. The BAFF receptor TACI controls IL-10 production by regulatory B cells and CLL B cells

    Science.gov (United States)

    Saulep-Easton, Damien; Vincent, Fabien B.; Quah, Pin Shie; Wei, Andrew; Ting, Stephen B.; Croce, Carlo M.; Tam, Constantine; Mackay, Fabienne

    2015-01-01

    Interleukin (IL)-10-producing B cells (B10 cells) have emerged as important regulatory players with immunosuppressive roles. Chronic lymphocytic leukemia (CLL) B cells also secrete IL-10 and share features of B10 cells, suggesting a possible contribution of CLL B cells to immunosuppression in CLL patients. Factors controlling the emergence of B10 cells are not known. B cell-activating factor of the tumour necrosis factor (TNF) family (BAFF) is critical for B cell maturation and survival, and is implicated in the development and progression of CLL. We sought to investigate the role of BAFF in the emergence of IL-10-producing B regulatory cells in healthy donors and CLL patients. Here, we report that BAFF signaling promotes IL-10 production by CLL B cells in a mouse model of CLL and in CLL patients. Moreover, BAFF-mediated IL-10 production by normal and CLL B cells is mediated via its receptor TACI. Our work uncovered a major targetable pathway important for the generation of regulatory B cells that is detrimental to immunity in CLL. PMID:26139429

  9. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

    Directory of Open Access Journals (Sweden)

    Georgia Schäfer

    2015-05-01

    Full Text Available Currently, seven viruses, namely Epstein-Barr virus (EBV, Kaposi’s sarcoma-associated herpes virus (KSHV, high-risk human papillomaviruses (HPVs, Merkel cell polyomavirus (MCPyV, hepatitis B virus (HBV, hepatitis C virus (HCV and human T cell lymphotropic virus type 1 (HTLV-1, have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

  10. Regulation of B cell functions by Toll-like receptors and complement.

    Science.gov (United States)

    Kremlitzka, Mariann; Mácsik-Valent, Bernadett; Erdei, Anna

    2016-10-01

    B cell functions triggered by the clonally-rearranged antigen-specific B cell receptor (BCR) are regulated by several germ-line encoded receptors - including Toll-like receptors (TLRs) and complement receptors (CRs). Simultaneous or sequential engagement of these structures expressed either on the cell membrane or intracellularly, may fundamentally alter and fine tune activation, antibody and cytokine production of B cells. Here we review the expression and function of TLRs and various C3 fragment binding CRs on B cells, emphasizing their role in different human B cell subsets under physiological and pathological conditions. Studies underlining the importance of the crosstalk between TLRs and CRs in regulating B cell functions are also highlighted. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  11. [Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells].

    Science.gov (United States)

    Kaji, Yuichi

    2005-11-01

    Corneal endothelial cell loss is a change that occurs with age, but its mechanism is still unclear. We postulated that interaction between advanced glycation end product(AGE) and its receptors is implicated in the corneal endothelial cell loss with age. We investigated the expression of AGE receptors: receptors for AGE(RAGE) and galectin-3 in bovine corneal endothelial cells by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry. In addition, we investigated the effect of AGE on the cultured corneal endothelial cells. Expression of RAGE and galectin-3 was detected in bovine corneal endothelial cells. Galectin-3 was important in the internalization of AGE. In contrast, RAGE was important in the generation of reactive oxygen species and induction of apoptosis. Based on these data, the interaction of AGE in aqueous humor and AGE receptors expressed on the corneal endothelial cells was speculated to have a role in the corneal endothelial cell loss with age.

  12. Regulation of C3a receptor signaling in human mast cells by G protein coupled receptor kinases.

    Directory of Open Access Journals (Sweden)

    Qiang Guo

    Full Text Available The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.We utilized lentivirus short hairpin (shRNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca(2+ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.This study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.

  13. Platelet-activating factor (PAF) receptor as a promising target for cancer cell repopulation after radiotherapy

    Science.gov (United States)

    da Silva-Jr, I A; Chammas, R; Lepique, A P; Jancar, S

    2017-01-01

    A major drawback of radiotherapy is the accelerated growth of the surviving tumor cells. Radiotherapy generates a variety of lipids that bind to the receptor for platelet-activating factor, expressed by cells in the tumor microenvironment. In the present study, using the TC-1 tumor cell line, we found that irradiation induced a twofold increase in receptor expression and generated agonists of receptor. Irradiated cells induced a 20-fold increase in live TC-1 proliferation in vitro. Furthermore, subcutaneous co-injection of irradiated TC-1 cells with TC-1 expressing luciferase (TC-1 fluc+) markedly increased TC-1 fluc+ proliferation in a receptor-dependent way. Moreover we used a human carcinoma cell line not expressing the PAF receptor (KBM) and the same cell transfected with the receptor gene (KBP). Following co-injection of live KBP cells with irradiated KBM in RAG mice, the tumor growth was significantly increased compared with tumor formed following co-injection of live KBM with irradiated KBM. This tumor cell repopulation correlated with increased infiltration of tumor-promoting macrophages (CD206+). We propose that receptor represents a possible target for improving the efficacy of radiotherapy through inhibition of tumor repopulation. PMID:28134937

  14. Morphine stimulates cell migration of oral epithelial cells by delta-opioid receptor activation.

    Directory of Open Access Journals (Sweden)

    Nada Charbaji

    Full Text Available Oral mucositis is one of the most common side effects of chemoradiation regimens and manifestation can be dose-limiting for the therapy, can impair the patient's nutritional condition and quality of life due to severe pain. The therapeutic options are limited; often only an alleviation of the symptoms such as pain reduction by using systemic opioids is possible. Stimulating opioid receptors on peripheral neurons and dermal tissue, potent analgesic effects are induced e.g. in skin grafted patients. Advantageous effects on the cell migration and, thus, on the wound healing process are described, too. In this study, we investigated whether opioid receptors are also expressed on oral epithelial cells and if morphine can modulate their cell migration behavior. The expression of the opioid receptors MOR, DOR and KOR on primary human oral epithelial cells was verified. Furthermore, a significantly accelerated cell migration was observed following incubation with morphine. The effect even slightly exceeded the cell migration stimulating effect of TGF-ß: After 14 h of morphine treatment about 86% of the wound area was closed, whereas TGF-ß application resulted in a closed wound area of 80%. With respect to morphine stimulated cell migration we demonstrate that DOR plays a key role and we show the involvement of the MAPK members Erk 1/2 and p38 using Western blot analysis.Further studies in more complex systems in vitro and in vivo are required. Nevertheless, these findings might open up a new therapeutic option for the treatment of oral mucositis.

  15. Glutamate receptors in glia: new cells, new inputs and new functions.

    Science.gov (United States)

    Gallo, V; Ghiani, C A

    2000-07-01

    Functional glutamate receptors are expressed on the majority of glial cell types in the developing and mature brain. Although glutamate receptors on glia are activated by glutamate released from neurons, their physiological role remains largely unknown. Potential roles for these receptors in glia include regulation of proliferation and differentiation, and modulation of synaptic efficacy. Recent anatomical and functional evidence indicates that glutamate receptors on immature glia are activated through direct synaptic inputs. Therefore, glutamate and its receptors appear to be involved in a continuous crosstalk between neurons and glia during development and also in the mature brain.

  16. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells

    DEFF Research Database (Denmark)

    Engelstoft, Maja S; Park, Won-Mee; Sakata, Ichiro

    2013-01-01

    /o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi...... ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin...

  17. Toll-Like Receptor 5 Signaling in Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Sabine M. Ivison

    2010-01-01

    Full Text Available Background. Bacterial flagellin triggers inflammation in mammalian cells via Toll-like receptor (TLR 5. Release of the chemokine IL-8 in response to flagellin involves NF-κB, p38 MAP kinase, and phosphatidylinositol 3-kinase (PI3K. However, PI3K has been reported to be either pro- or anti-inflammatory in different model systems. We hypothesized that this could be due to different activities of the p110α and β isoforms of PI3K. Results. PI3K and Akt were rapidly activated in Caco-2 colon carcinoma cells by flagellin. Using a plasmid-based shRNA delivery system and novel p110 isoform-specific inhibitors, we found that flagellin-induced IL-8 production was dependent on both p110α and p110β. However in the mouse, inhibition of p110β but not p110α reduced the increase of serum IL-6 levels induced by intraperitoneal injection of flagellin. Conclusions. These data demonstrate that the p110α and β isoforms of class IA PI3K are both required for the proinflammatory response to flagellin.

  18. Migration and chemokine receptor pattern of colitis-preventing DX5+NKT cells.

    Science.gov (United States)

    Hornung, Matthias; Werner, Jens M; Farkas, Stefan; Schlitt, Hans J; Geissler, Edward K

    2011-11-01

    DX5(+)NKT cells are a subpopulation of NKT cells expressing both T cell receptor and NK cell markers that show an immune-regulating function. Transferred DX5(+)NKT cells from immune competent Balb/c mice can prevent or reduce induced colitis in severe combined immunodeficient (SCID) mice. Here, we investigated the in vivo migration of DX5(+)NKT cells and their corresponding chemokine receptor patterns. DX5(+)NKT cells were isolated from spleens of Balb/c mice and transferred into Balb/c SCID mice. After 2 and 8 days, in vivo migration was examined using in vivo microscopy. In addition, the chemokine receptor pattern was analyzed with fluorescence-activated cell sorting (FACS) and the migration assay was performed. Our results show that labeled DX5(+)NKT cells were primarily detectable in mesenteric lymph nodes and spleen after transfer. After 8 days, DX5(+)NKT cells were observed in the colonic tissues, especially the appendix. FACS analysis of chemokine receptors in DX5(+)NKT cells revealed expression of CCR3, CCR6, CCR9, CXCR3, CXCR4, and CXCR6, but no CCR5, CXCR5, or the lymphoid homing receptor CCR7. Stimulation upregulated especially CCR7 expression, and chemokine receptor patterns were different between splenic and liver DX5(+)NKT cells. These data indicate that colitis-preventing DX5(+)NKT cells need to traffic through lymphoid organs to execute their immunological function at the site of inflammation. Furthermore, DX5(+)NKT cells express a specific chemokine receptor pattern with an upregulation of the lymphoid homing receptor CCR7 after activation.

  19. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  20. Core Fucosylation of the T Cell Receptor Is Required for T Cell Activation.

    Science.gov (United States)

    Liang, Wei; Mao, Shanshan; Sun, Shijie; Li, Ming; Li, Zhi; Yu, Rui; Ma, Tonghui; Gu, Jianguo; Zhang, Jianing; Taniguchi, Naoyuki; Li, Wenzhe

    2018-01-01

    CD4 + T cell activation promotes the pathogenic process of systemic lupus erythematosus (SLE). T cell receptor (TCR) complex are highly core fucosylated glycoproteins, which play important roles in T cell activation. In this study, we found that the core fucosylation of CD4 + T cells was significantly increased in SLE patients. Loss of core fucosyltransferase (Fut8), the sole enzyme for catalyzing the core fucosylation of N-glycan, significantly reduced CD4 + T cell activation and ameliorated the experimental autoimmune encephalomyelitis-induced syndrome in Fut8 -/- mice. T cell activation with OVA 323-339 loaded major histocompatibility complex II (pMHC-II) on B cell was dramatically attenuated in Fut8 -/- OT-II CD4 + T cells compared with Fut8 +/+ OT-II CD4 + T cells. Moreover, the phosphorylation of ZAP-70 was significantly reduced in Fut8 +/+ OT-II CD4 + T cells by the treatment of fucosidase. Our results suggest that core fucosylation is required for efficient TCR-pMHC-II contacts in CD4 + T cell activation, and hyper core fucosylation may serve as a potential novel biomarker in the sera from SLE patients.

  1. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    International Nuclear Information System (INIS)

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline; Sun, Jianmin; Jögi, Annika; Neumann, Drorit; Rönnstrand, Lars; Påhlman, Sven

    2014-01-01

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα + ) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells

  2. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Sun, Jianmin [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Jögi, Annika [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Neumann, Drorit [Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rönnstrand, Lars [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Påhlman, Sven, E-mail: sven.pahlman@med.lu.se [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel)

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  3. C-X-C chemokine receptor type 4 (CXCR4) is a key receptor for chicken primordial germ cell migration.

    Science.gov (United States)

    Lee, Jeong Hyo; Park, Jeong-Woong; Kim, Si Won; Park, Joonghoon; Park, Tae Sub

    2017-12-15

    In mammals, germ cells originate outside of the developing gonads and follow a unique migration pattern through the embryonic tissue toward the genital ridges. Many studies have attempted to identify critical receptors and factors involved in germ cell migration. However, relatively few reports exist on germ cell receptors and chemokines that are involved in germ cell migration in avian species. In the present study, we investigated the specific migratory function of C-X-C chemokine receptor type 4 (CXCR4) in chicken primordial germ cells (PGCs). We induced loss-of-function via a frameshift mutation in the CXCR4 gene in chicken PGCs using clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. The migratory capacity of CXCR4 knockout PGCs was significantly reduced in vivo after transplantation into recipient embryos. However, CXCR4-expressing somatic cell lines, such as chicken DT40 and DF1, failed to migrate into the developing gonads, suggesting that another key factor(s) is necessary for targeting and settlement of PGCs into the genital ridges. In conclusion, we show that CXCR4 plays a critical role in the migration of chicken germ cells.

  4. Potential cellular receptors involved in hepatitis C virus entry into cells

    Directory of Open Access Journals (Sweden)

    Muellhaupt Beat

    2005-04-01

    Full Text Available Abstract Hepatitis C virus (HCV infects hepatocytes and leads to permanent, severe liver damage. Since the genomic sequence of HCV was determined, progress has been made towards understanding the functions of the HCV-encoded proteins and identifying the cellular receptor(s responsible for adsorption and penetration of the virus particle into the target cells. Several cellular receptors for HCV have been proposed, all of which are associated with lipid and lipoprotein metabolism. This article reviews the cellular receptors for HCV and suggests a general model for HCV entry into cells, in which lipoproteins play a crucial role.

  5. Role of Triggering Receptor Expressed on Myeloid Cells in the Activation of Innate Immunity

    Directory of Open Access Journals (Sweden)

    V. G. Matveyeva

    2011-01-01

    Full Text Available The innate immune system plays a key role in triggering a systemic inflammatory response (SIR. The triggering receptor expressed on myeloid cells (TREM-1, which is located on neutrophils and monocytes, is involved in SIR, by regulating the effector mechanisms of innate immunity. Hyperproduction of proinflammatory cytokines is a pathogenetic component of the hyperergic phase of acute systemic inflammation. The simultaneous activation of Toll-like receptors and TREM-1 increases the production of cytokines manifold. This is compensatory and adaptive, however, resulting in damage to organs and tissues during excessive production of cytokines. Key words: triggering receptor expressed on myeloid cells, Toll-like receptors, cytokines, inflammation.

  6. Mast cells down-regulate CD4+CD25+ T regulatory cell suppressor function via histamine H1 receptor interaction.

    Science.gov (United States)

    Forward, Nicholas A; Furlong, Suzanne J; Yang, Yongjun; Lin, Tong-Jun; Hoskin, David W

    2009-09-01

    Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (T(reg)) cell function. In this study, we show for the first time that the capacity of murine CD4(+)CD25(+) T(reg) cells to suppress in vitro proliferation by CD4(+)CD25(-) T responder (T(resp)) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcepsilonR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited T(reg) cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored T(reg) cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of T(reg) cells with loratadine, but not famotidine, rescued T(reg) cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited T(reg) cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both T(reg) cells and T(resp) cells expressed H1 receptors. Exposure to histamine caused T(reg) cells to express lower levels of CD25 and the T(reg) cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited T(reg) cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in T(reg) cell suppressor function, thereby enhancing the development of protective immunity.

  7. Prevention of B cell antigen receptor-induced apoptosis by ligation of CD40 occurs downstream of cell cycle regulation

    NARCIS (Netherlands)

    Mackus, Wendelina J. M.; Lens, Susanne M. A.; Medema, René H.; Kwakkenbos, Mark J.; Evers, Ludo M.; Oers, Marinus H. J. van; van Lier, René A. W.; Eldering, Eric

    2002-01-01

    Cross-linking of the B cell antigen receptor (BCR) on germinal center B cells can induce growth arrest and apoptosis, thereby eliminating potentially autoreactive B cells. Using the Burkitt lymphoma cell line Ramos as a model, we studied the commitment to apoptosis following growth arrest, as well

  8. TGFβ activated kinase 1 (TAK1 at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

    Directory of Open Access Journals (Sweden)

    Dániel Szili

    Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

  9. Synaptic connections of starburst amacrine cells and localization of acetylcholine receptors in primate retinas.

    Science.gov (United States)

    Yamada, Elizabeth S; Dmitrieva, Nina; Keyser, Kent T; Lindstrom, Jon M; Hersh, Louis B; Marshak, David W

    2003-06-16

    Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways. Copyright 2003 Wiley-Liss, Inc.

  10. Cell Receptor-Basement Membrane Interactions in Health and Disease: a Kidney-Centric View

    Science.gov (United States)

    Borza, Corina M.; Chen, Xiwu; Zent, Roy; Pozzi, Ambra

    2016-01-01

    Cell-extracellular matrix (ECM) interactions are essential for tissue development, homeostasis, and response to injury. Basement membranes (BMs) are specialized ECMs that separate epithelial or endothelial cells from stromal components and interact with cells via cellular receptors, including integrins and discoidin domain receptors. Disruption of cell-BM interactions due to either injury or genetic defects in either the ECM components or cellular receptors often lead to irreversible tissue injury and loss of organ function. Animal models that lack specific BM components or receptors either globally or in selective tissues have been used to help with our understanding of the molecular mechanisms whereby cell-BM interactions regulate organ function in physiological and pathological conditions. We review recently published work on animal models that explore how cell-BM interactions regulate kidney homeostasis in both health and disease. PMID:26610916

  11. Analysis of 125I-[Tyr3] octreotide receptors of NCI-H466 cell line

    International Nuclear Information System (INIS)

    Sun Junjie; Fan Wo; Xu Yujie; Zhang Youjiu; Zhu Ran

    2002-01-01

    Objective: To study the affinity of small cell lung carcinoma to [Tyr 3 ] octreotide (TOC). Methods: Taking 125 I-[Tyr 3 ] octreotide (labeled by chloramine-T method), as the ligand, small cell lung carcinoma NCI-H466 cell line was inspected for the receptor-binding points and affinity constant. Results: The radio-chemical purity of 125 I-TOC purified through sephadex G-10 was higher than 95%. Receptor analysis study showed that the expression of somatostatin receptors on NCI-H446 cells was numerous (Bmax = 1.17 x 10 5 /cell) with strong affinity to 125 I-TOC (Kd = 0.56 nM). Conclusion: Labeled TOC could be used for small cell lung carcinoma receptor imaging and radio-pharmaceutical therapy

  12. Muscarinic acetylcholine receptor subtype expression in avian vestibular hair cells, nerve terminals and ganglion cells.

    Science.gov (United States)

    Li, G Q; Kevetter, G A; Leonard, R B; Prusak, D J; Wood, T G; Correia, M J

    2007-04-25

    Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and peripheral nervous system and play an important role in modulating the cell activity and function. We have shown that the cholinergic agonist carbachol reduces the pigeon's inwardly rectifying potassium channel (pKir2.1) ionic currents in native vestibular hair cells. We have cloned and sequenced pigeon mAChR subtypes M2-M5 and we have studied the expression of all five mAChR subtypes (M1-M5) in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular nerve fibers and the vestibular (Scarpa's) ganglion using tissue immunohistochemistry (IH), dissociated single cell immunocytochemistry (IC) and Western blotting (WB). We found that vestibular hair cells, nerve fibers and ganglion cells each expressed all five (M1-M5) mAChR subtypes. Two of the three odd-numbered mAChRs (M1, M5) were present on the hair cell cilia, supporting cells and nerve terminals. And all three odd numbered mAChRs (M1, M3 and M5) were expressed on cuticular plates, myelin sheaths and Schwann cells. Even-numbered mAChRs were seen on the nerve terminals. M2 was also shown on the cuticular plates and supporting cells. Vestibular efferent fibers and terminals were not identified in our studies. Results from WB of the dissociated vestibular epithelia, nerve fibers and vestibular ganglia were consistent with the results from IH and IC. Our findings suggest that there is considerable co-expression of the subtypes on the neural elements of the labyrinth. Further electrophysiological and pharmacological studies should delineate the mechanisms of action of muscarinic acetylcholine receptors on structures in the labyrinth.

  13. DMPD: Toll-like receptors: paving the path to T cell-driven autoimmunity? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17888644 Toll-like receptors: paving the path to T cell-driven autoimmunity? Marsla... Toll-like receptors: paving the path to T cell-driven autoimmunity? PubmedID 17888644 Title Toll-like recep...tors: paving the path to T cell-driven autoimmunity? Authors Marsland BJ, Kopf M.

  14. Characterization of the recognition of tumor cells by the natural cytotoxicity receptor, NKp44.

    NARCIS (Netherlands)

    Hershkovitz, O.; Jivov, S.; Bloushtain, N.; Zilka, A.; Landau, G.; Bar-Ilan, A.; Lichtenstein, R.G.; Campbell, K.S.; Kuppevelt, A.H.M.S.M. van; Porgador, A.

    2007-01-01

    NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent

  15. Pharmacological targeting of the KIT growth factor receptor: a therapeutic consideration for mast cell disorders

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Akin, C; Gilfillan, A M

    2008-01-01

    KIT is a member of the tyrosine kinase family of growth factor receptors which is expressed on a variety of haematopoietic cells including mast cells. Stem cell factor (SCF)-dependent activation of KIT is critical for mast cell homeostasis and function. However, when KIT is inappropriately activa...

  16. Glucagon-Like Peptide-1 Receptor Agonists: Beta-Cell Protection or Exhaustion?

    Science.gov (United States)

    van Raalte, Daniël H; Verchere, C Bruce

    2016-07-01

    Glucagon-like peptide (GLP)-1 receptor agonists enhance insulin secretion and may improve pancreatic islet cell function. However, GLP-1 receptor (GLP-1R) agonist treatment may have more complex, and sometimes deleterious, effects on beta cells. We discuss the concepts of beta cell protection versus exhaustion for different GLP-1R agonists based on recent data. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

    Science.gov (United States)

    Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

    2006-02-17

    Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

  18. Dual effects of muscarinic M2 acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists

    Czech Academy of Sciences Publication Activity Database

    Michal, Pavel; Lysíková, Michaela; Tuček, Stanislav

    2001-01-01

    Roč. 132, č. 6 (2001), s. 1217-1228 ISSN 0007-1188 R&D Projects: GA ČR GA309/99/0214; GA AV ČR IAA7011910 Institutional research plan: CEZ:AV0Z5011922 Keywords : cyclic AMP * muscarinic receptors * CHO cells Subject RIV: ED - Physiology Impact factor: 3.502, year: 2001

  19. Different processing of LH/hCG receptors in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1)

    International Nuclear Information System (INIS)

    Kellokumpu, S.

    1987-01-01

    The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with [ 125 I]hCG indicated that the bound hormone was lost much more rapidly from the tumor cells than from the luteal cells. The tumor cells were also found to internalize and degrade the hormone more effectively than the luteal cells. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-[ 125 I]hCG complexes (M/sub r/ 96,000 and 74,000) into the medium, although their amount was negligible in MLTC-1 cells. Possibly due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the M/sub r/ 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalized receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumor cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface

  20. Distinct angiotensin II receptor in primary cultures of glial cells from rat brain

    International Nuclear Information System (INIS)

    Raizada, M.K.; Phillips, M.I.; Crews, F.T.; Sumners, C.

    1987-01-01

    Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Agn-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of 125 I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of 125 I-Ang-II binding was determined. Scatchard analysis revealed a homogeneous population of high-affinity binding sites with a B/sub max/ of 110 fmol/mg of protein. Light-microscopic autoradiography of 125 I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence [ 3 H] norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors

  1. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

    Directory of Open Access Journals (Sweden)

    Callihan Phillip

    2008-12-01

    Full Text Available Abstract Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA and Sphingosine-1-phosphate (S1P receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

  2. Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

    Science.gov (United States)

    Schreiner, Jens; Thommen, Daniela S.; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A.; Umana, Pablo; Pisa, Pavel; von Bergwelt-Baildon, M.; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

    2016-01-01

    ABSTRACT T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction. PMID:27057429

  3. Gastrin-Releasing Peptide Receptor Mediates Activation of the Epidermal Growth Factor Receptor in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sufi Mary Thomas

    2005-04-01

    Full Text Available Gastrin-releasing peptide receptor (GRPR and the epidermal growth factor receptor (EGFR are expressed in several cancers including non-small cell lung carcinoma (NSCLC. Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP, in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-α or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18μM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.

  4. The Role of Prolactin Receptor in GH Signaling in Breast Cancer Cells

    OpenAIRE

    Xu, Jie; Sun, Dongmei; Jiang, Jing; Deng, Luqin; Zhang, Yue; Yu, Hao; Bahl, Deepti; Langenheim, John F.; Chen, Wen Y.; Fuchs, Serge Y.; Frank, Stuart J.

    2012-01-01

    GH and prolactin (PRL) are structurally related hormones that exert important effects in disparate target tissues. Their receptors (GHR and PRLR) reside in the cytokine receptor superfamily and share signaling pathways. In humans, GH binds both GHR and PRLR, whereas PRL binds only PRLR. Both hormones and their receptors may be relevant in certain human and rodent cancers, including breast cancer. GH and PRL promote signaling in human T47D breast cancer cells that express both GHR and PRLR. Fu...

  5. BDNF and its receptors in human myasthenic thymus: implications for cell fate in thymic pathology.

    Science.gov (United States)

    Berzi, Angela; Ayata, C Korcan; Cavalcante, Paola; Falcone, Chiara; Candiago, Elisabetta; Motta, Teresio; Bernasconi, Pia; Hohlfeld, Reinhard; Mantegazza, Renato; Meinl, Edgar; Farina, Cinthia

    2008-07-15

    Here we show that in myasthenic thymus several cell types, including thymic epithelial cells (TEC) and immune cells, were the source and the target of the neurotrophic factor brain-derived growth factor (BDNF). Interestingly, many actively proliferating medullary thymocytes expressed the receptor TrkB in vivo in involuted thymus, while this population was lost in hyperplastic or neoplastic thymuses. Furthermore, in hyperplastic thymuses the robust coordinated expression of BDNF in the germinal centers together with the receptor p75NTR on all proliferating B cells strongly suggests that this factor regulates germinal center reaction. Finally, all TEC dying of apoptosis expressed BDNF receptors, indicating that this neurotrophin is involved in TEC turnover. In thymomas both BDNF production and receptor expression in TEC were strongly hindered. This may represent an attempt of tumour escape from cell death.

  6. Cell specific effects of PCB 126 on aryl hydrocarbone receptors in follicular cells of porcine ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Wojtowicz, A.; Augustowska, K.; Gregoraszczuk, E. [Lab. of Physiology and Toxicology of Reproduction, Dept. of Animal Physiology, Inst. of Zoology, Jagiellonian Univ., Krakow (Poland)

    2004-09-15

    Polychlorinated biphenyles (PCBs) like other endocrine disrupters could interfere with natural hormones by binding to their receptors and thus mimicking the cellular response to them. They are known to possess either estrogenic or antiestrogenic properties. In our previous papers we demonstrated that PCBs are able to disrupt ovarian steroidogenesis. We found that the coplanar PCB 126 caused the decrease in estradiol secretion in whole cultured pig ovarian follicles. PCB 126 congener is structurally related to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since TCDD effects are known to be mediated by aryl hydrocarbone receptors (AhRs), we decided to determine if PCB 126 affects signal transduction pathway activated by these receptors. It has been reported that the functional AhR is present in ovary including oocytes, granulosa and theca cells of rat, mouse, rhesus monkey and human ovary. Moreover, the expression of AhR in the rat ovary appeared to be estrous cycle-dependent, thus suggesting that AhR expression may be regulated by fluctuating hormone levels. This study was designed to investigate the effects of the non-ortho-substituted 3,3',4,4',5-pentachlorobiphenyl (PCB126) on the AhR activation, localization and protein level in pig ovarian follicle cells.

  7. Gene expression of muscarinic, tachykinin and purinergic receptors in porcine bladder: comparison with cultured cells

    Directory of Open Access Journals (Sweden)

    Forough eBahadory

    2013-11-01

    Full Text Available Urothelial cells, myofibroblasts, and smooth muscle cells are important cell types contributing to bladder function. Multiple receptors including muscarinic (M3/M5, tachykinin (NK1/NK2 and purinergic (P2X1/P2Y6 receptors are involved in bladder motor and sensory actions. Using female pig bladder, our aim was to differentiate between various cell types in bladder by genetic markers. We compared the molecular expression pattern between the fresh tissue layers and their cultured cell counterparts. We also examined responses to agonists for these receptors in cultured cells. Urothelial, suburothelial (myofibroblasts and smooth muscle cells isolated from pig bladder were cultured (10-14 days and identified by marker antibodies. Gene (mRNA expression level was demonstrated by real-time PCR. The receptor expression pattern was very similar between suburothelium and detrusor, and higher than urothelium. The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells. Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A and β,γ-MeATP, up to concentrations of 0.1 and 1 mM. The significant reduction of M3, NK2 and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists. These results suggest that under culture conditions, bladder cells lose the receptors that are involved in contraction, as this function is no longer required. The study provides further evidence that cultured cells do not necessarily mimic the actions exerted by intact tissues.

  8. Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth

    DEFF Research Database (Denmark)

    Green, M R; Basketter, D A; Couchman, J R

    1983-01-01

    markedly with age. This decrease in receptor number is similar in trend to the known drop in basal cell [3H]thymidine labelling index which occurs over the same time period. The data suggest that the distribution of EGF receptors and EGF cell surface receptor number in skin are important in the spatial......, and keratinisation when injected into neonatal mice (S. Cohen and G.A. Elliott, 1963, J. Invest. Dermatol, 40, 1-5). We have determined the distribution of the available receptors for epidermal growth factor in rat skin using autoradiography following incubation of explants with 125I-labelled mouse EGF. EGF...... receptors are detected on the epithelial cells overlying the basement membranes of the epidermis, sebaceous gland, and regions of the hair follicle all of which have proliferative capacity. In marked contrast, tissues which have started to differentiate and lost their growth potential, carry either...

  9. Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

    International Nuclear Information System (INIS)

    Matozaki, T.; Sakamoto, C.; Nagao, M.; Nishisaki, H.; Konda, Y.; Nakano, O.; Matsuda, K.; Wada, K.; Suzuki, T.; Kasuga, M.

    1991-01-01

    The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125 I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors

  10. Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor

    DEFF Research Database (Denmark)

    Ellis, V; Behrendt, N; Danø, K

    1991-01-01

    importance for the efficient activation of Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell surface constitutes......The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by u.......67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact...

  11. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

    International Nuclear Information System (INIS)

    Xu, Ling; Hausmann, Martin; Dietmaier, Wolfgang; Kellermeier, Silvia; Pesch, Theresa; Stieber-Gunckel, Manuela; Lippert, Elisabeth; Klebl, Frank; Rogler, Gerhard

    2010-01-01

    Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab

  12. Eph receptor interclass cooperation is required for the regulation of cell proliferation

    International Nuclear Information System (INIS)

    Jurek, Aleksandra; Genander, Maria; Kundu, Parag; Catchpole, Timothy; He, Xiao; Strååt, Klas; Sabelström, Hanna; Xu, Nan-Jie; Pettersson, Sven; Henkemeyer, Mark; Frisén, Jonas

    2016-01-01

    Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells. - Highlights: • We demonstrate that ephrin-B1 and ephrin-B2 have largely non-overlapping functions in the intestinal stem cell niche. • Ephrin-B1 regulates cell positioning and ephrin-B2 regulates cell proliferation in the intestinal stem cell niche. • EphA4/B2 receptor cooperation in response to ephrin-B2 binding is obligatory to convey mitogenic signals in the intestine. • EphA4 facilitates EphB2 phosphorylation in response to ephrin-B2 in SW480 adenocarcinoma cells. • Ephrin-B1 and ephrin-B2 induce phosphorylation and degradation of the EphB2 receptor with different kinetics.

  13. Eph receptor interclass cooperation is required for the regulation of cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Jurek, Aleksandra; Genander, Maria [Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Kundu, Parag [Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Lee Kong Chian School of Medicine, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Catchpole, Timothy [Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas TX 75390 (United States); He, Xiao; Strååt, Klas; Sabelström, Hanna [Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Xu, Nan-Jie [Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas TX 75390 (United States); Pettersson, Sven [Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Lee Kong Chian School of Medicine, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); The National Cancer Centre, Singapore General Hospital (Singapore); Henkemeyer, Mark [Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas TX 75390 (United States); Frisén, Jonas, E-mail: jonas.frisen@ki.se [Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden)

    2016-10-15

    Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells. - Highlights: • We demonstrate that ephrin-B1 and ephrin-B2 have largely non-overlapping functions in the intestinal stem cell niche. • Ephrin-B1 regulates cell positioning and ephrin-B2 regulates cell proliferation in the intestinal stem cell niche. • EphA4/B2 receptor cooperation in response to ephrin-B2 binding is obligatory to convey mitogenic signals in the intestine. • EphA4 facilitates EphB2 phosphorylation in response to ephrin-B2 in SW480 adenocarcinoma cells. • Ephrin-B1 and ephrin-B2 induce phosphorylation and degradation of the EphB2 receptor with different kinetics.

  14. Rapid desensitization of the histamine H2 receptor on the human monocytic cell line U937

    NARCIS (Netherlands)

    Smit, M J; Leurs, R; Shukrula, S R; Bast, A; Timmerman, H

    1994-01-01

    In the present study we have subjected the histamine H2 receptor on the monocytic cell line U937 to a thorough pharmacological characterization using a series of selective histamine H1, H2 and H3 receptor agonists and antagonists. Recent reports have demonstrated the existence of a histamine H2

  15. PHARMACOLOGICAL PROPERTIES OF CLONED MUSCARINIC RECEPTORS EXPRESSED IN A9 L CELLS - COMPARISON WITH INVITRO MODELS

    NARCIS (Netherlands)

    BODDEKE, HWGM; BUTTINI, M

    1991-01-01

    The effects of a series of muscarinic agonists and antagonists at cloned ml and m3 muscarinic receptors expressed in mouse fibroblast A9 L cells have been compared with their effects in in vitro models of M1 (rat superior cervical ganglion) and M3 (guinea-pig ileum) muscarinic receptors. A good

  16. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    NARCIS (Netherlands)

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria,

  17. Functional analysis of synthetic insectatachykinin analogs on recombinant neurokinin receptor expressing cell lines

    NARCIS (Netherlands)

    Torfs, H.; Akerman, K.E.; Nachman, R.J.; Oonk, H.B.; Detheux, M.; Poels, J.; Loy, van T.; Loof, A.; Meloen, R.H.; Vassart, G.; Parmentier, M.; Broeck, van den J.

    2002-01-01

    The activity of a series of synthetic tachykinin-like peptide analogs was studied by means of microscopic calcium imaging on recombinant neurokinin receptor expressing cell lines. A C-terminal pentapeptide (FTGMRa) is sufficient for activation of the stomoxytachykinin receptor (STKR) expressed in

  18. The P2X7 Receptor Supports Both Life and Death in Fibrogenic Pancreatic Stellate Cells

    DEFF Research Database (Denmark)

    Haanes, Kristian; Schwab, Albrecht; Novak, Ivana

    2012-01-01

    inhibitor az10606120. The P2X7 receptor-pore inhibitor A438079 partially prevented cell death induced by millimolar ATP concentrations. This study shows that ATP and P2X7 receptors are important regulators of PSC proliferation and death, and therefore might be potential targets for treatments of pancreatic...

  19. A dual immunocytochemical assay for oestrogen and epidermal growth factor receptors in tumour cell lines

    NARCIS (Netherlands)

    A.K. Sharma (Anisha K.); J.H. Horgan; R.L. McClelland (Robyn); A.G. Douglas-Jones (A.); T. van Agthoven (Ton); L.C.J. Dorssers (Lambert); R.I. Nicholson (R.)

    1994-01-01

    textabstractA new dual immunocytochemical assay for oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) has been developed. It has been tested in a variety of conditions using cell culture lines and the results correlate well with those obtained from single immunocytochemical assays.

  20. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Rønne, E; Solberg, H.

    1992-01-01

    The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associat...

  1. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    Science.gov (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  2. The nuclear receptor NR4A1 induces a form of cell death dependent on autophagy in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Jimena Bouzas-Rodríguez

    Full Text Available The control of cell death is a biological process essential for proper development, and for preventing devastating pathologies like cancer and neurodegeneration. On the other hand, autophagy regulation is essential for protein and organelle degradation, and its dysfunction is associated with overlapping pathologies like cancer and neurodegeneration, but also for microbial infection and aging. In the present report we show that two evolutionarily unrelated receptors--Neurokinin 1 Receptor (NK(1R, a G-protein coupled receptor, and Insulin-like Growth Factor 1 Receptor (IGF1R, a tyrosine kinase receptor--both induce non-apoptotic cell death with autophagic features and requiring the activity of the autophagic core machinery proteins PI3K-III, Beclin-1 and Atg7. Remarkably, this form of cell death occurs in apoptosis-competent cells. The signal transduction pathways engaged by these receptors both converged on the activation of the nuclear receptor NR4A1, which has previously been shown to play a critical role in some paradigms of apoptosis and in NK(1R-induced cell death. The activity of NR4A1 was necessary for IGF1R-induced cell death, as well as for a canonical model of cell death by autophagy induced by the presence of a pan-caspase inhibitor, suggesting that NR4A1 is a general modulator of this kind of cell death. During cell death by autophagy, NR4A1 was transcriptionally competent, even though a fraction of it was present in the cytoplasm. Interestingly, NR4A1 interacts with the tumor suppressor p53 but not with Beclin-1 complex. Therefore the mechanism to promote cell death by autophagy might involve regulation of gene expression, as well as protein interactions. Understanding the molecular basis of autophagy and cell death mediation by NR4A1, should provide novel insights and targets for therapeutic intervention.

  3. Increased angiotensin II type 1 receptor expression in temporal arteries from patients with giant cell arteritis

    DEFF Research Database (Denmark)

    Dimitrijevic, Ivan; Malmsjö, Malin; Andersson, Christina

    2009-01-01

    PURPOSE: Currently, giant cell arteritis (GCA) is primarily treated with corticosteroids or immunomodulating agents, but there is interest in identifying other noncorticosteroid alternatives. Similarities exist in the injury pathways between GCA and atherosclerosis. Angiotensin II is a vasoactive......, internal elastic lamina degeneration, and band-shaped infiltrates of inflammatory cells, including lymphocytes, histocytes, and multinucleated giant cells. AT(1) receptor staining was primarily observed in the medial layer of the temporal arteries and was higher in the patients with GCA than in the control...... patients. This was a result of increased AT(1) receptor immunostaining of both vascular smooth muscle cells and infiltrating inflammatory cells. Only faint immunostaining was seen for AT(2) receptors, primarily in the endothelial cells, and to a lesser extent on the smooth muscle cells. Immunostaining...

  4. Expression and function of β-adrenergic receptors in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Maeki, T.; Andersson, L.C.; Kontula, K.K.

    1992-01-01

    We investigated the expression and functional characteristics of β-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [ 125 I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of β-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimala or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of β-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32 P-labelled β 2 -adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their β-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage. (au)

  5. NOVEL CHARACTERIZATION OF bEnd.3 CELLS THAT EXPRESS LYMPHATIC VESSEL ENDOTHELIAL HYALURONAN RECEPTOR-1

    OpenAIRE

    Yuen, D.; Leu, R.; Tse, J.; Wang, S.; Chen, L.L.; Chen, L.

    2014-01-01

    Murine bEnd.3 endothelioma cell line has been widely used in vascular research and here we report the novel finding that bEnd.3 cells express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR-3). Moreover, these cells express progenitor cell markers of Sca-1 and CD133. Upon stimulation with tumor necrosis factor-alpha (TNF-α), the bEnd.3 cells demonstrate enhanced formation of capillary-type tubes, which express LYVE-1. As the...

  6. Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking

    Directory of Open Access Journals (Sweden)

    Rocio Flores-Fernández

    2016-01-01

    Full Text Available Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE. In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease.

  7. Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking

    Science.gov (United States)

    Flores-Fernández, Rocio; Blanco-Favela, Francisco; Fuentes-Pananá, Ezequiel M.; Chávez-Sánchez, Luis; Gorocica-Rosete, Patricia; Pizaña-Venegas, Alberto; Chávez-Rueda, Adriana Karina

    2016-01-01

    Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease. PMID:27314053

  8. Role of ErbB receptors in cancer cell migration and invasion

    Directory of Open Access Journals (Sweden)

    Aline eAppert-Collin

    2015-11-01

    Full Text Available Growth factors mediate their diverse biologic responses (regulation of cellular proliferation, differentiation, migration and survival by binding to and activating cell-surface receptors with intrinsic protein kinase activity named Receptor Tyrosine Kinases (RTKs. About 60 RTKs have been identified and can be classified into more than 16 different receptor families. Their activity is normally tightly controlled and regulated. Overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal stimulation by autocrine growth factor loops contribute to constitutive RTK signaling, resulting in alterations in the physiological activities of cells. The ErbB receptor family of RTKs comprises four distinct receptors: the EGFR (also known as ErbB1/HER1, ErbB2 (neu, HER2, ErbB3 (HER3 and ErbB4 (HER4. ErbB family members are often overexpressed, amplified, or mutated in many forms of cancer, making them important therapeutic targets. EGFR has been found to be amplified in gliomas and non-small-cell lung carcinoma while ErbB2 amplifications are seen in breast, ovarian, bladder, non-small-cell lung carcinoma, as well as several other tumor types. Several data have shown that ErbB receptor family and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion by modulating extracellular matrix components. Recent findings indicate that extracellular matrix components such as matrikines bind specifically to EGF receptor and promote cell invasion. In this review, we will present an in-depth overview of the structure, mechanisms, cell signaling, and functions of ErbB family receptors in cell adhesion and migration. Furthermore, we will describe in a last part the new strategies developed in anti-cancer therapy to inhibit ErbB family receptor activation.

  9. Modeling Starburst cells' GABA(B) receptors and their putative role in motion sensitivity.

    Science.gov (United States)

    Grzywacz, Norberto M; Zucker, Charles L

    2006-07-15

    Neal and Cunningham (Neal, M. J., and J. R. Cunningham. 1995. J. Physiol. (Lond.). 482:363-372) showed that GABA(B) agonists and glycinergic antagonists enhance the light-evoked release of retinal acetylcholine. They proposed that glycinergic cells inhibit the cholinergic Starburst amacrine cells and are in turn inhibited by GABA through GABA(B) receptors. However, as recently shown, glycinergic cells do not appear to have GABA(B) receptors. In contrast, the Starburst amacrine cell has GABA(B) receptors in a subpopulation of its varicosities. We thus propose an alternate model in which GABA(B)-receptor activation reduces the release of ACh from some dendritic compartments onto a glycinergic cell, which then feeds back and inhibits the Starburst cell. In this model, the GABA necessary to make these receptors active comes from the Starburst cell itself, making them autoreceptors. Computer simulations of this model show that it accounts quantitatively for the Neal and Cunningham data. We also argue that GABA(B) receptors could work to increase the sensitivity to motion over other stimuli.

  10. Modeling Starburst Cells' GABAB Receptors and Their Putative Role in Motion Sensitivity

    Science.gov (United States)

    Grzywacz, Norberto M.; Zucker, Charles L.

    2006-01-01

    Neal and Cunningham (Neal, M. J., and J. R. Cunningham. 1995. J. Physiol. (Lond.). 482:363–372) showed that GABAB agonists and glycinergic antagonists enhance the light-evoked release of retinal acetylcholine. They proposed that glycinergic cells inhibit the cholinergic Starburst amacrine cells and are in turn inhibited by GABA through GABAB receptors. However, as recently shown, glycinergic cells do not appear to have GABAB receptors. In contrast, the Starburst amacrine cell has GABAB receptors in a subpopulation of its varicosities. We thus propose an alternate model in which GABAB-receptor activation reduces the release of ACh from some dendritic compartments onto a glycinergic cell, which then feeds back and inhibits the Starburst cell. In this model, the GABA necessary to make these receptors active comes from the Starburst cell itself, making them autoreceptors. Computer simulations of this model show that it accounts quantitatively for the Neal and Cunningham data. We also argue that GABAB receptors could work to increase the sensitivity to motion over other stimuli. PMID:16648160

  11. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Directory of Open Access Journals (Sweden)

    Yue Yu

    Full Text Available Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood.Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels.Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1 and interlukin-6 (IL-6 by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion.Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  12. Utilization of two different T cell receptors by T cell acute lymphoblastic lymphoma and leukemia.

    Science.gov (United States)

    Gouttefangeas, C; Bensussan, A; Boumsell, L

    1990-01-01

    We show further differences between two clinically related entities, T cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LL), by using several monoclonal antibodies (mAb) reacting either with constant or variable regions of T cell receptors (TcR) alpha beta and gamma delta or with various CD molecules. We analysed a panel of 15 T-ALL and 15 T-LL selected for their cell surface expression of the CD3 molecules. The results indicate that TcR gamma delta is more frequently used than TcR alpha beta in T-ALL (10 out of the 15 patients tested). This is in contrast to the results obtained with T-LL where the vast majority expressed TcR alpha beta (13 out of the 15 patients). These findings suggest that the leukemic cells could have a different origin in these two diseases. In addition analysis of TcR variable regions expressed by the leukemic blasts showed that in most cases they had rearranged functional V delta 1 gene segments (8 out of 11 patients) whereas in a unique case V delta 2 gene segment was used. Taken together these results and those indicating that T-ALL cell coexpress the CD1a,b and c molecules strengthen the possibility that even though these leukemic cells express the CD3-TcR complex at their cell surface their normal counterparts are not found in peripheral blood.

  13. Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells

    International Nuclear Information System (INIS)

    Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

    1989-01-01

    Epidermal growth factor receptors (EGF-R) have been shown to be associated with the detergent-insoluble cytoskeleton of A431 cells, where they retained both a functional ligand-binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF-R. The tyrosine kinase activity of the cytoskeletally associated EGF-R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF-R associated with cytoskeletons of EGF-treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent-insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF-stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF-R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF-R. Saturable binding of 125I-EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High-affinity EGF-R were preferentially retained upon detergent extraction of adherent cells, whereas both low- and high-affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF-R to that solubilized in adherent cells, indicating that EGF-R can reversibly associate with the structural elements of the cell

  14. Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma

    OpenAIRE

    Long Zheng; Peisheng Hu; Brandon Wolfe; Caryn Gonsalves; Luqing Ren; Leslie A. Khawli; Harvey R. Kaslow; Alan L. Epstein

    2017-01-01

    T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas...

  15. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

  16. Expression of Ricinus communis receptors on epithelial cells in oral carcinomas and oral wounds.

    Science.gov (United States)

    Dabelsteen, E; Mackenzie, I C

    1978-12-01

    The histological distribution of receptors for Ricinus communis Fraction 1 (RCA1) in oral carcinomas and in oral epithelial cells during wound healing has been studied by use of fluorescein-tagged RCA1. Biopsies from 15 human oral carcinomas and adjacent normal mucosa showed RCA1 receptors at the cell membranes in the basal and spinous layer of the normal epithelium, whereas receptors could not be demonstrated in invading islands of the tumors. In healing oral wounds from eight humans and three monkeys, RCA1 receptors were demonstrated both in normal epithelium adjacent to the wounds and in the epithelial outgrowth from the wound margin. Titrations, however, showed that the epithelial outgrowth reacted more weakly than did the normal adjacent epithelium. These results support previous in vitro studies showing changes in carbohydrate composition of moving normal cells and of malignant cells, a finding that may be of interest in relation to formation of metastases.

  17. Ligands, cell-based models, and readouts required for Toll-like receptor action.

    LENUS (Irish Health Repository)

    Dellacasagrande, Jerome

    2012-02-01

    This chapter details the tools that are available to study Toll-like receptor (TLR) biology in vitro. This includes ligands, host cells, and readouts. The use of modified TLRs to circumvent some technical problems is also discussed.

  18. Deficiency of leptin receptor in myeloid cells disrupts hypothalamic metabolic circuits and causes body weight increase

    Directory of Open Access Journals (Sweden)

    Yuanqing Gao

    2018-01-01

    Conclusions: Myeloid cell leptin receptor deficient mice partially replicate the db/db phenotype. Leptin signaling in hypothalamic microglia is important for microglial function and a correct formation of the hypothalamic neuronal circuit regulating metabolism.

  19. Truncation of CXCL12 by CD26 reduces its CXC chemokine receptor 4- and atypical chemokine receptor 3-dependent activity on endothelial cells and lymphocytes

    DEFF Research Database (Denmark)

    Janssens, Rik; Mortier, Anneleen; Boff, Daiane

    2017-01-01

    The chemokine CXCL12 or stromal cell-derived factor 1/SDF-1 attracts hematopoietic progenitor cells and mature leukocytes through the G protein-coupled CXC chemokine receptor 4 (CXCR4). In addition, it interacts with atypical chemokine receptor 3 (ACKR3 or CXCR7) and glycosaminoglycans. CXCL12 ac...

  20. Can Selective MHC Downregulation Explain the Specificity and Genetic Diversity of NK Cell Receptors?

    NARCIS (Netherlands)

    Carrillo-Bustamante, Paola; Kesmir, Can; de Boer, Rob J

    2015-01-01

    Natural killer (NK) cells express inhibiting receptors (iNKRs), which specifically bind MHC-I molecules on the surface of healthy cells. When the expression of MHC-I on the cell surface decreases, which might occur during certain viral infections and cancer, iNKRs lose inhibiting signals and the

  1. Current perspectives on natural killer cell education and tolerance: emerging roles for inhibitory receptors

    Directory of Open Access Journals (Sweden)

    Thomas LM

    2015-03-01

    Full Text Available L Michael Thomas Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA Abstract: Natural killer (NK cells are regulated through the coordinated functions of activating and inhibitory receptors. These receptors can act during the initial engagement of an NK cell with a target cell, or in subsequent NK cell engagements to maintain tolerance. Notably, each individual possesses a sizable minority-population of NK cells that are devoid of inhibitory receptors that recognize the surrounding MHC class I (ie, self-MHC. Since these NK cells cannot perform conventional inhibition, they are rendered less responsive through the process of NK cell education (also known as licensing in order to reduce the likelihood of auto-reactivity. This review will delineate current views on NK cell education, clarify various misconceptions about NK cell education, and, lastly, discuss the relevance of NK cell education in anti-cancer therapies. Keywords: natural killer cell education, natural killer cell inhibitory receptors, immunotherapy, cancer

  2. Lack of NMDA receptor subunit exchange alters Purkinje cell dendritic morphology in cerebellar slice cultures

    NARCIS (Netherlands)

    Metzger, F; Pieri, [No Value; Eisel, ULM; Pieri, Isabelle

    2005-01-01

    Early postnatal developmental changes in N-methyl-D-aspartate (NMDA) receptor (NR) subunits regulate cerebellar granule cell maturation and potentially Purkinje cell development. We therefore investigated Purkinje cell morphology in slice cultures from mice with genetic subunit exchange from NR2C to

  3. Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yong-Xin Sun

    2015-01-01

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs are considered as the most promising cells source for bone engineering. Cannabinoid (CB receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

  4. Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Todd M Brusko

    2010-07-01

    Full Text Available Therapies directed at augmenting regulatory T cell (Treg activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition.To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff activity as determined by tumor cell growth and luciferase reporter-based imaging.These results support the

  5. Purkinje cell NMDA receptors assume a key role in synaptic gain control in the mature cerebellum

    Science.gov (United States)

    Piochon, Claire; Levenes, Carole; Ohtsuki, Gen; Hansel, Christian

    2010-01-01

    A classic view in cerebellar physiology holds that Purkinje cells do not express functional N-methyl-D-aspartate (NMDA) receptors and that, therefore, postsynaptic NMDA receptors are not involved in the induction of long-term depression (LTD) at parallel fiber (PF) to Purkinje cell synapses. Recently, it has been demonstrated that functional NMDA receptors are postsynaptically expressed at climbing fiber (CF) to Purkinje cell synapses in mice, reaching full expression levels at about 2 months after birth. Here, we show that in the mature mouse cerebellum LTD (induced by paired PF and CF activation), but not long-term potentiation (LTP; PF stimulation alone) at PF to Purkinje cell synapses is blocked by bath application of the NMDA receptor antagonist D-APV. A blockade of LTD, but not LTP, was also observed when the non-competitive NMDA channel blocker MK-801 was added to the patch-pipette saline, suggesting that postsynaptically expressed NMDA receptors are required for LTD induction. Using confocal calcium imaging, we show that CF-evoked calcium transients in dendritic spines are reduced in the presence of D-APV. This observation confirms that NMDA receptor signaling occurs at CF synapses, and suggests that NMDA receptor-mediated calcium transients at the CF input site might contribute to LTD induction. Finally, we performed dendritic patch-clamp recordings from rat Purkinje cells. Dendritically recorded CF responses were reduced when D-APV was bath-applied. Together, these data suggest that the late developmental expression of postsynaptic NMDA receptors at CF synapses onto Purkinje cells is associated with a switch towards an NMDA receptor-dependent LTD induction mechanism. PMID:21068337

  6. B cell activating factor (BAFF) and BAFF receptors: fakes and facts.

    Science.gov (United States)

    Ferraccioli, G; Gremese, E

    2017-12-01

    Analysis of B cell activating factor (BAFF) receptors before and after B cell depletion therapy (BCDT) might offer a clue to the understanding of whether some B cell subsets may represent useful biomarkers of biological and clinical responses. Among the BAFF receptors in a cohort of rheumatoid arthritis (RA) patients, the AA have shown, by fluorescence activated cell sorter (FACS) analysis of median fluorescence intensity (MFI), that transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA) do not change, whereas the most important, BAFF receptor 3 (BR3), appears to be decreased before as well as after BCDT in all B cell subsets but not in plasmablasts, the most important subset, depleted by BCDT. © 2017 British Society for Immunology.

  7. Identification of androgen receptors in normal human osteoblast-like cells

    International Nuclear Information System (INIS)

    Colvard, D.S.; Eriksen, E.F.; Keeting, P.E.; Riggs, B.L.; Spelsberg, T.C.; Wilson, E.M.; Lubahn, D.B.; French, F.S.

    1989-01-01

    The sex steroids, androgens and estrogens, are major regulators of bone metabolism. However, whether these hormones act on bone cells through direct or indirect mechanisms has remained unclear. A nuclear binding assay recently used to demonstrate estrogen receptors in bone was used to identify specific nuclear binding of a tritiated synthetic androgen, [ 3 H]R1881 (methyltrienolone), in 21 of 25 (84%) human osteoblast-like cell strains and a concentration of bound steroid receptors of 821 ± 140 molecules per cell nucleus. Binding was saturable and steroid-specific. Androgen receptor gene expression in osteoblasts was confirmed by RNA blot analysis. Relative concentrations of androgen and estrogen receptors were compared by measuring specific nuclear estrogen binding. Nuclear binding of [ 3 H]estradiol was observed in 27 of 30 (90%) cell strains; the concentration of bound estradiol receptor was 1537 ± 221 molecules per cell nucleus. The concentrations of nuclear binding sites were similar in males and females for both [ 3 H]R1881 and [ 3 H]estradiol. The authors conclude that both androgens and estrogens act directly on human bone cells through their respective receptor-mediated mechanisms

  8. Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells

    International Nuclear Information System (INIS)

    Vale, R.D.; Ignatius, M.J.; Shooter, E.M.

    1985-01-01

    Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125 I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin

  9. Atomic force microscopic study of folate receptors in live cells with functionalized tips

    Science.gov (United States)

    Girish, C. M.; Binulal, N. S.; Anitha, V. C.; Nair, Shantikumar; Mony, U.; Prasanth, R.

    2009-11-01

    Membrane associated folate receptors (FR) is gaining importance in cancer research. Understanding the FR density, distribution, and the strength of its interaction with ligands is crucial in cancer diagnostics and therapeutics. Here we reported the enhanced phase contrast image of FR by scanning with properly functionalized atomic force microscope (AFM) tips over live cell lines. The choice of the ligand was made for better interaction of tip with FR, expressed in the cell lines. The selectively enhanced force of interaction at the receptor molecule produced a considerable enhancement in the phase contrast between a receptor site and a nonreceptor site.

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

    Science.gov (United States)

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

    1999-01-16

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

  11. Netupitant and palonosetron trigger NK1 receptor internalization in NG108-15 cells.

    Science.gov (United States)

    Thomas, Ajit G; Stathis, Marigo; Rojas, Camilo; Slusher, Barbara S

    2014-08-01

    Current therapy for chemotherapy-induced nausea and vomiting includes the use of both 5-HT3 and NK1 receptor antagonists. Acute emesis has largely been alleviated with the use of 5-HT3 receptor antagonists, while an improvement in preventing delayed emesis has been achieved with NK1 receptor antagonists. Delayed emesis, however, remains a problem with a significant portion of cancer patients receiving highly emetogenic chemotherapy. Like other drugs in its class, palonosetron, a 5-HT3 receptor antagonist, has shown efficacy against acute emesis. However, palonosetron has also shown consistent improvement in the suppression of delayed emesis. Since both 5-HT3 and NK1 receptor antagonists are often simultaneously administered to patients, the question remains if palonosetron's effect on delayed emesis would remain distinct when co-administered with an NK1 receptor antagonist. Recent mechanistic studies using NG108-15 cells have shown that palonosetron and netupitant, an NK1 receptor antagonist currently in phase 3 clinical trials, exhibited synergistic effects when inhibiting the substance P response. The present studies showed that both netupitant and palonosetron-induced NK1 receptor internalization in NG108-15 cells and that when used together receptor internalization was additive. Palonosetron-induced NK1 receptor internalization was dependent on the presence of the 5-HT3 receptor. Results provide a possible explanation for palonosetron's enhancement of the inhibition of the SP response and suggest that the effect of palonosetron and NK1 receptor antagonists on prevention of delayed emesis could be additive.

  12. Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Eun Hyang; Jang, Soon Young; Cho, In-Hye [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Hong, Darong [Department of Life and Nanopharmaceutical Science, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Jung, Bom; Park, Min-Ju [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Kim, Jong-Ho, E-mail: jonghokim@khu.ac.kr [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of)

    2015-08-07

    Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy. - Highlights: • Hispolon decreased ERα expression at both mRNA and protein levels. • Hispolon decreased ERα transcriptional activity. • Hispolon treatment inhibited expression of ERα target gene pS2. • Shikonin is a candidate chemotherapeutic target in the treatment of human breast cancer.

  13. Growth hormone action in rat insulinoma cells expressing truncated growth hormone receptors

    DEFF Research Database (Denmark)

    Møldrup, Annette; Allevato, G; Dyrberg, Thomas

    1991-01-01

    Transfection of the insulin-producing rat islet tumor cell line RIN-5AH with a full length cDNA of the rat hepatic growth hormone (GH) receptor (GH-R1-638) augments the GH-responsive insulin synthesis in these cells. Using this functional system we analyzed the effect of COOH-terminal truncation...... of the GH receptor. Two mutated cDNAs encoding truncated GH receptors, GH-R1-294 and GH-R1-454, respectively, were generated by site-directed mutagenesis and transfected into the RIN cells. Both receptor mutants were expressed on the cell surface and displayed normal GH binding affinity. Whereas GH-R1......-638 had a molecular mass of about 110 kDa, GH-R1-294 and GH-R1-454 showed molecular masses of 49 and 80 kDa, respectively. Cells expressing GH-R1-454 internalized GH to a similar extent as cells transfected with the full length receptor and the parent cell line, but GH-R1-294-expressing cells showed...

  14. Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells.

    Directory of Open Access Journals (Sweden)

    Yutaka Maruyama

    Full Text Available Recently, we reported that calcium-sensing receptor (CaSR is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca(2+ concentration ([Ca(2+](i in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca(2+. CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami.

  15. Intracellular Ca2+ and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells

    Science.gov (United States)

    Huang, Yijen A; Roper, Stephen D

    2010-01-01

    ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca2+ and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca2+ release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca2+ has a dual role: (1) Ca2+ opens TRPM5 channels to depolarize receptor cells and (2) Ca2+ plus membrane depolarization opens ATP-permeable gap junction hemichannels. PMID:20498227

  16. Intracellular Ca(2+) and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells.

    Science.gov (United States)

    Huang, Yijen A; Roper, Stephen D

    2010-07-01

    ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca(2+) and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca(2+) release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca(2+) has a dual role: (1) Ca(2+) opens TRPM5 channels to depolarize receptor cells and (2) Ca(2+) plus membrane depolarization opens ATP-permeable gap junction hemichannels.

  17. Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin

    DEFF Research Database (Denmark)

    Wewer, U M; Liotta, L A; Jaye, M

    1986-01-01

    of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were...... bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells....... This suggests that the amount of laminin receptor mRNA may be a rate-limiting control step in the biosynthesis of the laminin receptor, and hence in the regulation of cellular attachment to basement membranes via laminin....

  18. Nuclear receptor 4a3 (nr4a3 regulates murine mast cell responses and granule content.

    Directory of Open Access Journals (Sweden)

    Gianni Garcia-Faroldi

    Full Text Available Nuclear receptor 4a3 (Nr4a3 is a transcription factor implicated in various settings such as vascular biology and inflammation. We have recently shown that mast cells dramatically upregulate Nuclear receptor 4a3 upon activation, and here we investigated the functional impact of Nuclear receptor 4a3 on mast cell responses. We show that Nuclear receptor 4a3 is involved in the regulation of cytokine/chemokine secretion in mast cells following activation via the high affinity IgE receptor. Moreover, Nuclear receptor 4a3 negatively affects the transcript and protein levels of mast cell tryptase as well as the mast cell's responsiveness to allergen. Together, these findings identify Nuclear receptor 4a3 as a novel regulator of mast cell function.

  19. Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors.

    Science.gov (United States)

    Dzamba, David; Honsa, Pavel; Valny, Martin; Kriska, Jan; Valihrach, Lukas; Novosadova, Vendula; Kubista, Mikael; Anderova, Miroslava

    2015-11-01

    Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP(+)) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP(+) glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP(+) glial cells and that its expression is increased after ischemia. In situ and in vitro Ca(2+) imaging revealed that Ca(2+) elevations evoked by the application of NMDA were diminished in GFAP/EGFP(+) glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP(+) glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.

  20. Chimeric Antigen Receptor T-Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia

    Directory of Open Access Journals (Sweden)

    Ciprian Tomuleasa

    2018-02-01

    Full Text Available Chimeric antigen receptor (CAR T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects.

  1. Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Nørgaard, P; Abrahamsen, N

    1999-01-01

    Transforming growth factor beta (TGF-beta) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-beta due to lack of type II receptor (RII) has been described in some cancer types including small cell lung...... cancer (SCLC). The purpose of this study was to examine the cause of absent RII expression in SCLC cell lines. Northern blot analysis showed that RII RNA expression was very weak in 16 of 21 cell lines. To investigate if the absence of RII transcript was due to mutations, we screened the poly-A tract...... for mutations, but no mutations were detected. Additional screening for mutations of the RII gene revealed a GG to TT base substitution in one cell line, which did not express RII. This mutation generates a stop codon resulting in predicted synthesis of a truncated RII of 219 amino acids. The nature...

  2. A license to kill : The evolution of NK cell receptors

    NARCIS (Netherlands)

    Carrillo Bustamante, N.P.

    2015-01-01

    Natural killer (NK) cells innate immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells must be tightly regulated. Unlike B and T cells, NK cell do not undergo DNA

  3. The Natural Selection of Herpesviruses and Virus-Specific NK Cell Receptors

    Directory of Open Access Journals (Sweden)

    Joseph C. Sun

    2009-10-01

    Full Text Available During the co-evolution of cytomegalovirus (CMV and natural killer (NK cells, each has evolved specific tactics in an attempt to prevail. CMV has evolved multiple immune evasion mechanisms to avoid detection by NK cells and other immune cells, leading to chronic infection. Meanwhile, the host has evolved virus-specific receptors to counter these evasion strategies. The natural selection of viral genes and host receptors allows us to observe a unique molecular example of "survival of the fittest", as virus and immune cells try to out-maneuver one another or for the virus to achieve détente for optimal dissemination in the population.

  4. Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells

    Science.gov (United States)

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Backert, Steffen

    2013-01-01

    Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin α5β1 receptor. Other targeted membrane-based receptors include the integrins αvβ3, αvβ5, and β2 (CD18), RPTP-α/β, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed. PMID:24280762

  5. Estrogen stimulates adenosine receptor expression subtypes in human breast cancer MCF-7 cell line.

    Science.gov (United States)

    Mohamadi, Azam; Aghaei, Mahmoud; Panjehpour, Mojtaba

    2018-02-01

    Estrogen is a steroid hormone that plays a key role in the development and regulation of reproductive system. It has been shown that estrogen is related to breast cancer development through binding to its receptors. In order to uncover the estrogen effects on adenosine receptor expression, estrogen-positive MCF-7 cells were used to treat with agonist and antagonist of estrogen and then the mRNA expression of adenosine receptor subtypes were evaluated. Estrogen-positive MCF-7 cells were treated with various concentrations of 17β estradiol (E2) as an estrogen agonist, and ICI 182,780 as an estrogen antagonist. The gene expression of adenosine receptor subtypes were detected by real time RT-PCR. The results of MTT assay showed that E2 increased cell viability in a dose dependent manner. The expression pattern of all adenosine receptor subtypes are as follow; A2b > A1 > A2a > A3 in untreated MCF-7 cells. Obtained results showed that E2 incubation at 0.001-0.01 μM led to up-regulation of A1ARs, A2aARs and A3ARs dose dependently. E2 at 0.001 μM also had no significant effect on A2bARs expression but, at higher doses induced a considerable decrease in mRNA A2bARs expression. Treatment with antagonist confirmed that up-regulation of these receptors is mediated by estrogen receptor. Taken together, our results indicate that treatment of MCF-7 cells with E2 led to up-regulation of adenosine receptors. However, these effects were partially restored by treatment with antagonist suggesting that such effects are mediated by estrogen receptors.

  6. Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

    Science.gov (United States)

    Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

    2014-01-01

    Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

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

    NARCIS (Netherlands)

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

    1997-01-01

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

  8. Cell type specificity of signaling: view from membrane receptors distribution and their downstream transduction networks.

    Science.gov (United States)

    He, Ying; Yu, Zhonghao; Ge, Dongya; Wang-Sattler, Rui; Thiesen, Hans-Jürgen; Xie, Lu; Li, Yixue

    2012-09-01

    Studies on cell signaling pay more attention to spatial dynamics and how such diverse organization can relate to high order of cellular capabilities. To overview the specificity of cell signaling, we integrated human receptome data with proteome spatial expression profiles to systematically investigate the specificity of receptors and receptor-triggered transduction networks across 62 normal cell types and 14 cancer types. Six percent receptors showed cell-type-specific expression, and 4% signaling networks presented enriched cell-specific proteins induced by the receptors. We introduced a concept of "response context" to annotate the cell-type dependent signaling networks. We found that most cells respond similarly to the same stimulus, as the "response contexts" presented high functional similarity. Despite this, the subtle spatial diversity can be observed from the difference in network architectures. The architecture of the signaling networks in nerve cells displayed less completeness than that in glandular cells, which indicated cellular-context dependent signaling patterns are elaborately spatially organized. Likewise, in cancer cells most signaling networks were generally dysfunctional and less complete than that in normal cells. However, glioma emerged hyper-activated transduction mechanism in malignant state. Receptor ATP6AP2 and TNFRSF21 induced rennin-angiotensin and apoptosis signaling were found likely to explain the glioma-specific mechanism. This work represents an effort to decipher context-specific signaling network from spatial dimension. Our results indicated that although a majority of cells engage general signaling response with subtle differences, the spatial dynamics of cell signaling can not only deepen our insights into different signaling mechanisms, but also help understand cell signaling in disease.

  9. CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

    Directory of Open Access Journals (Sweden)

    János Haskó

    2014-05-01

    Full Text Available During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB. The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2; therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A, GPR18 (transcriptional variant 1 and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A, GPR18 (transcriptional variants 1 and 2, GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.

  10. A feedback mechanism controlling SCRAMBLED receptor accumulation and cell-type pattern in Arabidopsis.

    Science.gov (United States)

    Kwak, Su-Hwan; Schiefelbein, John

    2008-12-23

    Cellular pattern formation in the root epidermis of Arabidopsis occurs in a position-dependent manner, generating root-hair (H) cells contacting two underlying cortical cells and nonhair (N) cells contacting one cortical cell. SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase (LRR-RLK), mediates this process through its effect on a downstream transcription factor regulatory network. After perception of a positional cue, the SCM signaling pathway is proposed to preferentially repress WEREWOLF (WER) transcription factor expression in H cells and thereby bias the outcome of mutual lateral inhibition acting between H and N cells. However, the molecular mechanism responsible for this preferential SCM signaling is unknown. Here, we analyze the distribution of the SCM receptor and the biological effect of altering its accumulation pattern. We find that SCM expression and accumulation in the epidermal cell layer is necessary and sufficient to direct the cell-type pattern. Further, SCM preferentially accumulates in H cells, and this accumulation pattern is dependent on the downstream transcription factors. Thus, SCM participates in an autoregulatory feedback loop, enabling cells engaged in SCM signaling to maintain high levels of SCM receptor, which provides a simple mechanism for reinforcing a bias in receptor-mediated signaling to ensure robust pattern formation.

  11. Identifying plant cell-surface receptors: combining 'classical' techniques with novel methods.

    Science.gov (United States)

    Uebler, Susanne; Dresselhaus, Thomas

    2014-04-01

    Cell-cell communication during development and reproduction in plants depends largely on a few phytohormones and many diverse classes of polymorphic secreted peptides. The peptide ligands are bound at the cell surface of target cells by their membranous interaction partners representing, in most cases, either receptor-like kinases or ion channels. Although knowledge of both the extracellular ligand and its corresponding receptor(s) is necessary to describe the downstream signalling pathway(s), to date only a few ligand-receptor pairs have been identified. Several methods, such as affinity purification and yeast two-hybrid screens, have been used very successfully to elucidate interactions between soluble proteins, but most of these methods cannot be applied to membranous proteins. Experimental obstacles such as low concentration and poor solubility of membrane receptors, as well as instable transient interactions, often hamper the use of these 'classical' approaches. However, over the last few years, a lot of progress has been made to overcome these problems by combining classical techniques with new methodologies. In the present article, we review the most promising recent methods in identifying cell-surface receptor interactions, with an emphasis on success stories outside the field of plant research.

  12. Overexpression of functional TrkA receptors after internalisation in human airway smooth muscle cells.

    Science.gov (United States)

    Freund-Michel, Véronique; Frossard, Nelly

    2008-10-01

    Trafficking of the TrkA receptor after stimulation by NGF is of emerging importance in structural cells in the context of airway inflammatory diseases. We have recently reported the expression of functional TrkA receptors in human airway smooth muscle cells (HASMC). We have here studied the TrkA trafficking mechanisms in these cells. TrkA disappearance from the cell membrane was induced within 5 min of NGF (3pM) stimulation. Co-immunoprecipitation of clathrin-TrkA was revealed, and TrkA internalisation inhibited either by clathrin inhibitors or by siRNA inducing downregulation of endogenous clathrin. TrkA internalised receptors were totally degraded in lysosomes, with no recycling phenomenon. Newly synthesized TrkA receptors were thereafter re-expressed at the cell membrane within 10 h. TrkA re-synthesis was inhibited by blockade of clathrin-dependent internalisation, but not of TrkA receptors lysosomal degradation. Finally, we observed that NGF multiple stimulations progressively increased TrkA expression in HASMC, which was associated with an increase in NGF/TrkA-dependent proliferation. In conclusion, we show here the occurrence of clathrin-dependent TrkA internalisation and lysosomal degradation in the airway smooth muscle, followed by upregulated re-synthesis of functional TrkA receptors and increased proliferative effect in the human airway smooth muscle. This may have pathophysiological consequences in airway inflammatory diseases.

  13. Natural killer cell subsets and receptor expression in peripheral blood mononuclear cells of a healthy Korean population: Reference range, influence of age and sex, and correlation between NK cell receptors and cytotoxicity.

    Science.gov (United States)

    Phan, Minh-Trang; Chun, Sejong; Kim, Sun-Hee; Ali, Alaa Kassim; Lee, Seung-Hwan; Kim, Seokho; Kim, Soo-Hyun; Cho, Duck

    2017-02-01

    The purpose of this study was to identify CD56 bright and CD56 dim natural killer (NK) cell subsets and analyze their receptors expression in a healthy Korean population, and to determine whether receptor expression correlates with age, sex, and cytotoxicity. We performed multicolor flow cytometry assays to analyze the expression of various NK cell receptors (CD16, NKG2A, NKG2C, NKG2D, CD57, DNAM-1, CD8a, CD62L, NKp30, and NKp46) on both CD3 - /CD56 dim and CD3 - /CD56 bright NK cells in whole-blood samples from 122 healthy donors. The expression of these receptors was compared according to age (60years, n=27) and gender (male, n=61, female, n=61). NK cell cytotoxicity assays were performed with peripheral blood mononuclear cells (PBMCs) from 18 individuals. The results were compared to the expression levels of NKp30 and NKp46 receptors. A normal reference range for NK cell receptor expression in two NK cell subsets was established. NKp46 and NKG2D expression gradually decreased with age (pcytotoxicity was found to positively correlate with NCR expression (p=0.02), but not NK cell proportion (p=0.80). We have established a profile of NK cell surface receptors for a Korean population, and revealed that age and gender have an effect on the expression of NK cell receptors in the population. Our data might explain why neither NK cell numbers nor proportions correlate with NK cell cytotoxicity. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  14. GLP-1/Exendin-4 induces β-cell proliferation via the epidermal growth factor receptor.

    Science.gov (United States)

    Fusco, Joseph; Xiao, Xiangwei; Prasadan, Krishna; Sheng, Qingfeng; Chen, Congde; Ming, Yung-Ching; Gittes, George

    2017-08-22

    Exendin-4 is a long acting glucagon-like peptide 1 (GLP-1) analogue that is an agonist for the GLP-1 receptor, a G-protein coupled receptor (GPCR). Exendin-4 is used to clinically improve glucose tolerance in diabetic patients due to its ability to enhance insulin secretion. In rodents, and possibly in humans, exendin-4 can stimulate β-cell proliferation. The exact mechanism of action to induce β-cell proliferation is not well understood. Here, using a β-cell specific epidermal growth factor receptor (EGFR) null mouse, we show that exendin-4 induced an increase in proliferation and β-cell mass through EGFR. Thus, our study sheds light on the role of EGFR signaling in the effects of exendin-4 on the control of blood glucose metabolism and β-cell mass.

  15. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    Science.gov (United States)

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.

  16. Urokinase receptor derived peptides as potent inhibitors of the formyl peptide receptor type 1-triggered cell migration.

    Science.gov (United States)

    Yousif, Ali Munaim; Ingangi, Vincenzo; Merlino, Francesco; Brancaccio, Diego; Minopoli, Michele; Bellavita, Rosa; Novellino, Ettore; Carriero, Maria Vincenza; Carotenuto, Alfonso; Grieco, Paolo

    2018-01-01

    The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration. We and others have previously documented that the uPAR(84-95) sequence, interacts with the formyl peptide receptors (FPR)s, henceforth inducing cell migration of several cell lines, including leukocytes, and the synthetic shorter peptide (Ser 88 -Arg-Ser-Arg-Tyr 92 , SRSRY) retains chemotactic activity in vitro and in vivo. Recently, we have developed the head-to-tail cyclic analog [SRSRY], a new potent and stable inhibitor of monocyte trafficking. This prompted us to develop novel cyclic and linear analogs of [SRSRY] with the aim to broaden the knowledge about structure-activity relationships of peptide [SRSRY]. Herein we report their synthesis, effects on cell migration, conformational and docking analyses which served to envisage a new pharmacophore model for inhibitors of FPR1-triggered cell migration. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor

    Science.gov (United States)

    Wu, Chia-Yung; Roybal, Kole T.; Puchner, Elias M.; Onuffer, James; Lim, Wendell A.

    2016-01-01

    There is growing promise in using engineered cells as therapeutic agents. For example, synthetic Chimeric Antigen Receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, excessive activity and poor control over such engineered T cells can cause severe toxicities. We present the design of “ON-switch” CARs that enable small molecule-control over T cell therapeutic functions, while still retaining antigen specificity. In these split receptors, antigen binding and intracellular signaling components only assemble in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate both cell autonomous recognition and user control. PMID:26405231

  18. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.

    Science.gov (United States)

    Wu, Chia-Yung; Roybal, Kole T; Puchner, Elias M; Onuffer, James; Lim, Wendell A

    2015-10-16

    There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control. Copyright © 2015, American Association for the Advancement of Science.

  19. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Guescini, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Leo, G.; Genedani, S. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Carone, C. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); IRCCS San Camillo Lido, Venezia (Italy); Pederzoli, F. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Ciruela, F. [Departament Patologia i Terapeutica Experimental, Universitat de Barcelona (Spain); Guidolin, D. [Department of Human Anatomy and Physiology, University of Padua (Italy); Stocchi, V.; Mantuano, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Borroto-Escuela, D.O.; Fuxe, K. [Department of Neuroscience, Karolinska Institutet, Stockholm (Sweden); Agnati, L.F., E-mail: luigiagnati@tin.it [IRCCS San Camillo Lido, Venezia (Italy)

    2012-03-10

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  20. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    International Nuclear Information System (INIS)

    Guescini, M.; Leo, G.; Genedani, S.; Carone, C.; Pederzoli, F.; Ciruela, F.; Guidolin, D.; Stocchi, V.; Mantuano, M.; Borroto-Escuela, D.O.; Fuxe, K.; Agnati, L.F.

    2012-01-01

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A 2A and D 2 receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D 2 R-CFP or A 2A R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D 2 R-CFP and A 2A R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A 2A R positive MVs were treated with the adenosine A 2A receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A 2A Rs were functionally competent in target cells. These findings demonstrate that A 2A receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  1. Estrogen receptor-α and aryl hydrocarbon receptor involvement in the actions of botanical estrogens in target cells

    Science.gov (United States)

    Gong, Ping; Madak-Erdogan, Zeynep; Flaws, Jodi A.; Shapiro, David J.; Katzenellenbogen, John A.; Katzenellenbogen, Benita S.

    2018-01-01

    Botanical estrogen (BE) dietary supplements are consumed by women as substitutes for loss of endogenous estrogens at menopause. To examine the roles of estrogen receptor α (ERα) and aryl hydrocarbon receptor (AhR) and their crosstalk in the actions of BEs, we studied gene regulation and proliferation responses to four widely used BEs, genistein, daidzein, and S-equol from soy, and liquiritigen from licorice root in breast cancer and liver cells. BEs and estradiol (E2), acting through ERα, stimulated proliferation, ERα chromatin binding and target-gene expression. BEs but not E2, acting through AhR, bound to xenobiotic response element-containing chromatin sites and enhanced AhR target-gene expression (CYP1A1, CYP1B1). While E2 and TCDD acted quite selectively through their respective receptors, BEs acted via both receptors, with their AhR activity moderated by negative crosstalk through ERα. Both ERα and AhR should be considered as mediators of the biology and pharmacology of BEs. PMID:27543265

  2. Slow Receptor Dissociation Kinetics Differentiate Macitentan from Other Endothelin Receptor Antagonists in Pulmonary Arterial Smooth Muscle Cells

    Science.gov (United States)

    Gatfield, John; Mueller Grandjean, Celia; Sasse, Thomas; Clozel, Martine; Nayler, Oliver

    2012-01-01

    Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP1) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with Kb values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt1/2) compared to bosentan and ambrisentan (ROt1/2∶17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP1 assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt1/2 rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with

  3. Slow receptor dissociation kinetics differentiate macitentan from other endothelin receptor antagonists in pulmonary arterial smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    John Gatfield

    Full Text Available Two endothelin receptor antagonists (ERAs, bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH, a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC. The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP(1 assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K(b values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt(1/2 compared to bosentan and ambrisentan (ROt(1/2:17 minutes versus 70 seconds and 40 seconds, respectively. Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP(1 assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1 concentrations. However, prolongation of the ET-1 stimulation time beyond ROt(1/2 rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive

  4. Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides.

    Science.gov (United States)

    Citro, G; Perrotti, D; Cucco, C; D'Agnano, I; Sacchi, A; Zupi, G; Calabretta, B

    1992-01-01

    Exposure of human leukemia HL-60 cells to an oligodeoxynucleotide complementary to an 18-base sequence (codons 2-7) of c-myb-encoded mRNA has previously been shown to result in inhibition of cell proliferation. Because HL-60 cells express high levels of transferrin receptor we adapted a DNA delivery system based on receptor-mediated endocytosis to introduce myb oligomers complexed with a transferrin-polylysine conjugate into those cells. A DNA.RNA duplex resistant to S1 nuclease digestion was detected as early as 12 hr after culture of HL-60 cells in the presence of the myb antisense/transferrin-polylysine complex. Exposure of HL-60 cells to the myb antisense/transferrin-polylysine complex resulted in rapid and profound inhibition of proliferation and loss of cell viability much more pronounced than that occurring in cells exposed to free myb antisense oligodeoxynucleotides. The transferrin-polylysine/myb sense complex or the transferrin-polylysine conjugate alone had no effect on HL-60 cell proliferation and viability. These findings indicate that myb synthetic oligodeoxynucleotides enter efficiently into HL-60 by transferrin receptor-mediated endocytosis and exert a profound biological effect. Such a delivery system could exploit other ligand-receptor interactions for the selective delivery of oncogene-targeted antisense oligodeoxynucleotides. Images PMID:1495997

  5. Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

    International Nuclear Information System (INIS)

    Raufman, Jean-Pierre; Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng

    2011-01-01

    Highlights: ► Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. ► Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. ► Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers – this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding

  6. Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States); Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre

  7. Autoregulation of enterochromaffin-like cell histamine secretion via the histamine 3 receptor subtype.

    OpenAIRE

    Kidd, M.; Tang, L. H.; Miu, K.; Lawton, G. P.; Sandor, A.; Modlin, I. M.

    1996-01-01

    INTRODUCTION: The neuroendocrine histamine-secreting cell of the gastric fundus, the enferochromaffin-like cell, is the principal regulator of parietal cell acid secretion. We have proposed that histamine may regulate its own synthesis and release via an autocrine mechanism. The purpose of this study was to evaluate the role of the histamine receptor subtypes H1, H2 and H3 in the regulation of this phenomenon. METHODS: Purified ECL cells were isolated by pronase digestion and EDTA exposure of...

  8. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

    DEFF Research Database (Denmark)

    Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali

    2014-01-01

    Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination...... of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells....

  9. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    Science.gov (United States)

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  10. Regulation of 1,25-dihydroxyvitamin D, receptors by [3H]-1,25-dihydroxyvitamin D3 in cultured cells (T-47D): evidence for receptor upregulation

    International Nuclear Information System (INIS)

    Reinhardt, T.A.; Horst, R.L.

    1986-01-01

    The authors examined the effect of 1,25-(OH) 2 D 3 on receptor concentration in cultured cells (T-47D). Two days prior to experiment, cells were fed with RPMI 1640 + 10% serum and 24-32 hours prior to experiment the media was replaced with RPMI 1640 + 25 mM Hepes + 1% serum. [ 3 H]-1,25-(OH) 2 D 3 +/- 100-fold molar excess cold hormone was used to treat the cells. Occupied receptors were measured in freshly prepared cytosols. Total receptors were measured following a 16-hour incubation of cytosols in the presence of 0.6 nM [ 3 H]-1,25-(OH) 2 D 3 +/- 100-fold molar excess of cold hormone at 4 0 C. Treatment of cell cultures for 16-18 hours with 0.5-1.0 nM [ 3 H]-1,25-(OH) 2 D 3 resulted in a 30-40% receptor occupancy by the hormone and a 2- to 3-fold increase in total cell receptor as compared to vehicle-treated controls. Time course studies showed a rapid increase in total receptors up to 16 hours post-treatment in the face of declining receptor occupancy. Actinomycin D blocked the [ 3 H]-1,25-(OH) 2 D 3 -dependent rise in cell receptor. The physiological significance of this receptor upregulation is not known nor is it known whether upregulation results from synthesis of new receptors and/or is the result of the activation of preformed receptors by a inducible activator protein

  11. Neurotransmitter receptors as signaling platforms in anterior pituitary cells

    Czech Academy of Sciences Publication Activity Database

    Zemková, Hana; Stojilkovic, S. S.

    2018-01-01

    Roč. 463, C (2018), s. 49-64 ISSN 0303-7207 R&D Projects: GA ČR(CZ) GA16-12695S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : pituitary * ligand-gated receptor channels * G protein-coupled receptors * neurotransmitters * action potentials * calcium signaling * hormone secretion Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 3.754, year: 2016

  12. Selective prostacyclin receptor agonism augments glucocorticoid-induced gene expression in human bronchial epithelial cells.

    Science.gov (United States)

    Wilson, Sylvia M; Shen, Pamela; Rider, Christopher F; Traves, Suzanne L; Proud, David; Newton, Robert; Giembycz, Mark A

    2009-11-15

    Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to

  13. Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

    International Nuclear Information System (INIS)

    Rojas, Olga Lucia; Gonzalez, Ana Maria; Gonzalez, Rosabel; Perez-Schael, Irene; Greenberg, Harry B.; Franco, Manuel A.; Angel, Juana

    2003-01-01

    Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4 + and CD8 + T cells secreting INFγ, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFγ-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4 + and CD8 + T cell subsets, IFNγ-secreting RV-specific CD8 + but not CD4 + T cells were detected in recently infected children. Using the same approach, both CD4 + and CD8 + RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFγ-secreting CD4 + T cells from adult volunteers preferentially express the intestinal homing receptor α4β7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFγ-secreting CD4 + T cells preferentially express L-selectin but not α4β7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFγ is independent of the expression of these homing receptors

  14. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

    DEFF Research Database (Denmark)

    Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M

    1998-01-01

    receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot...... analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...

  15. CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells.

    Science.gov (United States)

    Ziegler, Sabrina; Weiss, Esther; Schmitt, Anna-Lena; Schlegel, Jan; Burgert, Anne; Terpitz, Ulrich; Sauer, Markus; Moretta, Lorenzo; Sivori, Simona; Leonhardt, Ines; Kurzai, Oliver; Einsele, Hermann; Loeffler, Juergen

    2017-07-21

    Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

  16. Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Tonatiuh Barrios-García

    2016-06-01

    Full Text Available Tristetraprolin (TTP is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα, which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR, glucocorticoid receptor (GR and androgen receptor (AR. In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.

  17. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    International Nuclear Information System (INIS)

    Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

    1986-01-01

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [ 32 P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

  18. Pathogen sensing pathways in human embryonic stem cell derived-endothelial cells: role of NOD1 receptors.

    Directory of Open Access Journals (Sweden)

    Daniel M Reed

    Full Text Available Human embryonic stem cell-derived endothelial cells (hESC-EC, as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR Toll-like receptor (TLR-4 and nucleotide-binding oligomerisation domain-containing protein (NOD-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC. HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC, and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.

  19. Functional expression of the extracellular-Ca2+-sensing receptor in mouse taste cells.

    Science.gov (United States)

    Bystrova, Marina F; Romanov, Roman A; Rogachevskaja, Olga A; Churbanov, Gleb D; Kolesnikov, Stanislav S

    2010-03-15

    Three types of morphologically and functionally distinct taste cells operate in the mammalian taste bud. We demonstrate here the expression of two G-protein-coupled receptors from the family C, CASR and GPRC6A, in the taste tissue and identify transcripts for both receptors in type I cells, no transcripts in type II cells and only CASR transcripts in type III cells, by using the SMART-PCR RNA amplification method at the level of individual taste cells. Type I taste cells responded to calcimimetic NPS R-568, a stereoselective CASR probe, with Ca(2+) transients, whereas type I and type II cells were not specifically responsive. Consistent with these findings, certain amino acids stimulated PLC-dependent Ca(2+) signaling in type III cells, but not in type I and type II cells, showing the following order of efficacies: Phe~Glu>Arg. Thus, CASR is coupled to Ca(2+) mobilization solely in type III cells. CASR was cloned from the circumvallate papilla into a pIRES2-EGFP plasmid and heterologously expressed in HEK-293 cells. The transfection with CASR enabled HEK-293 cells to generate Ca(2+) transients in response to the amino acids, of which, Phe was most potent. This observation and some other facts favor CASR as the predominant receptor subtype endowing type III cells with the ability to detect amino acids. Altogether, our results indicate that type III cells can serve a novel chemosensory function by expressing the polymodal receptor CASR. A role for CASR and GPRC6A in physiology of taste cells of the type I remains to be unveiled.

  20. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy); Campagnolo, Luisa, E-mail: campagno@med.uniroma2.it [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy)

    2009-11-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75{sup NTR}), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75{sup NTR} and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75{sup NTR} and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75{sup NTR}/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75{sup NTR} or TrkA. Interestingly, immunoreactivity to anti-p75{sup NTR} antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75{sup NTR}, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75{sup NTR} is turned on.

  1. Pathway of Toll-like receptor 7/B cell activating factor/B cell activating factor receptor plays a role in immune thrombocytopenia in vivo.

    Directory of Open Access Journals (Sweden)

    Qing Yang

    Full Text Available Immune thrombocytopenia (ITP is an autoimmune disorder characterized by anti-platelet autoantibody-mediated platelet destruction. Antigen-presenting cell (APC dysfunction is considered to play crucial roles in ITP. However, how APC affects autoreactive B cells in ITP is still unknown. Using a mouse model of immune thrombocytopenia, we demonstrated an increase in levels of TLR7 in splenic mononuclear cells (SMCs. Using both TLR7 agonist and TLR7 silencing lentivirus, we found stimulation of TLR7 decreased platelet counts and increased levels of platelet-associated IgG (PAIgG in ITP mice, which correlates TLR7 with platelet destruction by autoantibodies. Levels of serum BAFF increased significantly in ITP mice and stimulation of TLR7 promoted secretion of BAFF. Among the three BAFF receptors, only BAFF receptor (BAFF-R increased in ITP mice. However, activation of TLR7 showed no effect on the expression of BAFF receptors. These findings indicate that upregulation of TLR7 may augment BAFF secretion by APC and through ligation of BAFF-R promote autoreactive B cell survival and thus anti-platelet autoantibody production. The pathway of TLR7/BAFF/BAFF-R provides us with an explanation of how activation of APC affects autoantibody production by B cells in ITP and thus might provide a reasonable therapeutic strategy for ITP.

  2. Characterization of muscarinic receptor subtypes in primary cultures of cerebellar granule cells using specific muscarinic receptor antagonists

    International Nuclear Information System (INIS)

    McLeskey, S.W.

    1989-01-01

    In cerebellar granule cell cultures, two muscarinic receptor mediated responses were observed: inhibition of adenylate cyclase (M-AC) and stimulation of phosphoinositide hydrolysis (M-PI). These responses were antagonized by three purported specific muscarinic antagonists: pirenzipine and (-)QNX (specific for M-PI) and methoctramine (specific for M-AC). However, the specificity for the three antagonists in blocking these responses is not comparable to the specificity observed in binding studies on these cells or to that quoted in the literature. Two peaks of molecular sizes were found in these cells corresponding to the two molecular sizes of muscarinic receptive proteins reported in the literature. Muscarinic receptive proteins were alkylated with 3 H-propylbenzilylcholine mustard followed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Pirenzipine and (-)QNX were able to block alkylation of the high molecular size peak, which corresponds to the receptive protein m 3 reported in the literature. Methoctramine was able to block alkylation of a portion of the lower molecular size peak, possibly corresponding to the m 2 and/or m 4 receptive proteins reported in the literature. Studies attempting to show the presence of receptor reserve for either of the two biochemical responses present in these cells by alkylation of the receptive protein with nonradiolabeled propylbenzilylcholine mustard (PBCM) were confounded by specificity of this agent for the lower molecular weight peak of muscarinic receptive protein. Thus the muscarinic receptive proteins coupled to M-AC were alkylated preferentially over the ones coupled to M-PI

  3. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...... and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

  4. Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling*

    Science.gov (United States)

    Moreno, Estefanía; Andradas, Clara; Medrano, Mireia; Caffarel, María M.; Pérez-Gómez, Eduardo; Blasco-Benito, Sandra; Gómez-Cañas, María; Pazos, M. Ruth; Irving, Andrew J.; Lluís, Carme; Canela, Enric I.; Fernández-Ruiz, Javier; Guzmán, Manuel; McCormick, Peter J.; Sánchez, Cristina

    2014-01-01

    The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology. PMID:24942731

  5. Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

    International Nuclear Information System (INIS)

    Takahashi, Tadanobu; Moriyama, Yusuke; Ikari, Akira; Sugatani, Junko; Suzuki, Takashi; Miwa, Masao

    2008-01-01

    Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism

  6. [The origin and possible role of microvesicles in olfactory receptor cells].

    Science.gov (United States)

    Bakhtin, E K

    1975-08-01

    Microvesicles and spherical particles have been described in the bulbs of receptor olfactory cells of Acipenser ruthenus. Two pathways of the origin of the above vesicles have been followed. These structures derive at the stage of differentiation from non-ciliary to ciliary cell type. The first of the pathways involves the autolysis of microfibril bundles produced during the regression of microvilli. The other one includes micropinocytosis induced on the basis of regressing microvilli. Taking into account the genesis of the microvesicles of the receptor cell bulb, it is concluded that they cannot contain a mediator able to modify membrane ion permeability in response to the specific stimulus of the odorant.

  7. Exploiting natural killer group 2D receptors for CAR T-cell therapy.

    Science.gov (United States)

    Demoulin, Benjamin; Cook, W James; Murad, Joana; Graber, David J; Sentman, Marie-Louise; Lonez, Caroline; Gilham, David E; Sentman, Charles L; Agaugue, Sophie

    2017-08-01

    Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

  8. Putative thyroid hormone receptors in red blood cells of some reptiles.

    Science.gov (United States)

    Wong, C C; Chiu, K W

    1987-06-01

    Putative triiodothyronine (T3) receptors have been detected in the nuclei of red blood cells (RBC) in a number of reptile species. The binding characteristics of T3 receptors in vitro were dissociation constant (Kd) 9.1 to 28.58, 36.8 and 40, and 11.12 and 11.36 pM, and binding capacity (Bmax) 0.12 to 0.37, 0.17 and 0.24, and 0.19 and 0.28 fmol per million cells in the rat snake (Ptyas korros), soft-shelled turtle (Trionyx sinensis), and tokay gecko (Gekko gecko), respectively. These data were obtained in all species using in vitro incubation of whole cell according to current receptor studies on living cells. With modified technique in subsequent experiments, these values of the binding characteristics were seemingly low. The discrepancy was ascribed to the assessment of "free" fraction of hormone which would be used in subsequent calculation.

  9. Generation and functional characterization of anti-clonotype antibodies to human T-cell receptors

    NARCIS (Netherlands)

    Steenbakkers, PGA; Boots, AMH; Rijnders, AWM

    1997-01-01

    Monoclonal antibodies (mAb) directed against the clonotypic structure of the T-cell receptor (TCR) may be useful reagents in the study and therapy of T-cell-mediated diseases. In contrast to several reports concerning the generation of anti-clonotype mAb to mouse TCR, only very limited numbers of

  10. C-type lectin receptors in the control of T helper cell differentiation

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.; Gringhuis, Sonja I.

    2016-01-01

    Pathogen recognition by C-type lectin receptors (CLRs) expressed by dendritic cells is important not only for antigen presentation, but also for the induction of appropriate adaptive immune responses via T helper (TH) cell differentiation. CLRs act either by themselves or in cooperation with other

  11. T Cell Receptors that Recognize the Tyrosinase Tumor Antigen | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute, Surgery Branch, Tumor Immunology Section, is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize T Cells Attacking Cancer: T Cell Receptors that Recognize the Tyrosinase Tumor Antigen

  12. Endothelin-Type A receptors mediate pain in a mouse model of sickle cell disease.

    Science.gov (United States)

    Lutz, Brianna Marie; Wu, Shaogen; Gu, Xiyao; Atianjoh, Fidelis E; Li, Zhen; Fox, Brandon M; Pollock, David M; Tao, Yuan-Xiang

    2018-03-15

    Sickle cell disease is associated with acute painful episodes and chronic intractable pain. Endothelin-1, a known pain inducer, is elevated in the blood plasma of both sickle cell patients and mouse models of sickle cell disease. We show here that the levels of endothelin-1 and its endothelin type A receptor are increased in the dorsal root ganglia of a mouse model of sickle cell disease. Pharmacologic inhibition or neuron-specific knockdown of endothelin type A receptors in primary sensory neurons of dorsal root ganglion alleviated basal and post-hypoxia evoked pain hypersensitivities in sickle cell mice. Mechanistically, endothelin type A receptors contribute to sickle cell disease-associated pain likely through the activation of NF-κB-induced Nav1.8 channel upregulation in primary sensory neurons of sickle cell mice. Our findings suggest that endothelin type A receptor is a potential target for the management of sickle cell disease-associated pain, although this expectation needs to be further verified in clinic settings. Copyright © 2018, Ferrata Storti Foundation.

  13. Toll-like receptors in the pathogenesis of human B cell malignancies

    NARCIS (Netherlands)

    Isaza-Correa, Johana M.; Liang, Zheng; van den Berg, Anke; Diepstra, Arjan; Visser, Lydia

    2014-01-01

    Toll-like receptors (TLRs) are important players in B-cell activation, maturation and memory and may be involved in the pathogenesis of B-cell lymphomas. Accumulating studies show differential expression in this heterogeneous group of cancers. Stimulation with TLR specific ligands, or agonists of

  14. Effects of estradiol and progestogens on human breast cells: Regulation of sex steroid receptors

    Directory of Open Access Journals (Sweden)

    Fang-Ping Chen

    2013-09-01

    Conclusions: The combination of E2 and various progestogens resulted in diverging effects on ERs and PRs expressions, which induced different effects on MCF-7 cell growth. Compared with P4, aberrant hormone and biological activity of synthetic progestin, by way of altered receptor expression, may be an important factor in affecting breast cell growth.

  15. Expression of the growth hormone receptor gene in insulin producing cells

    DEFF Research Database (Denmark)

    Møldrup, Annette; Billestrup, N; Nielsen, Jens Høiriis

    1990-01-01

    -T2-clone B was studied. The binding characteristics with regard to specificity for the native 22 kDa hGH, and the 20 kDa variant were similar to that reported on rat adipocytes. Normal rat islet cells showed a similar affinity for hGH. The RIN cells express GH receptors similar to the cloned liver...

  16. Stat3 activates the receptor tyrosine kinase like orphan receptor-1 gene in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Ping Li

    Full Text Available BACKGROUND: The receptor tyrosine kinase like orphan receptor (ROR-1 gene is overexpressed in chronic lymphocytic leukemia (CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors gamma-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1. METHODOLOGY/PRINCIPAL FINDINGS: Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells. CONCLUSION/SIGNIFICANCE: Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.

  17. The effects of black cohosh on the regulation of estrogen receptor (ERα) and progesterone receptor (PR) in breast cancer cells.

    Science.gov (United States)

    Szmyd, Monica; Lloyd, Victoria; Hallman, Kelly; Aleck, Katie; Mladenovik, Viktoria; McKee, Christina; Morse, Mia; Bedgood, Tyler; Dinda, Sumi

    2018-01-01

    The North American plant Cimicifuga racemosa , also known as black cohosh (BC), is a herb that recently has gained attention for its hormonal effects. As the usage of hormone replacement therapy is declining due to its adverse effects in women with cancer, many are turning to herbal remedies like BC to treat menopausal symptoms. It is crucial to determine whether the effects of BC involve estrogen receptor-alpha (ERα). Previous studies from our laboratory have shown ERα to be a possible molecular target for BC. In this study, we examined the effects of BC (8% triterpene glycosides) alone and in combination with hormones and antihormones on the cellular viability, expression of ERα and progesterone receptor (PR)-A/B, and cytolocalization of ERα in ER (+) and PR-A/B (+) T-47D breast cancer cells. Cells were cultured and proteins were extracted and quantified. Western blot analysis revealed alterations in the expression of ERα and PR after treatment with BC (5-100 µM). BC induced a concentration-dependent decrease in ERα and PR protein levels when compared to the control. Image cytometric analysis with propidium iodide staining was used to enumerate changes in T-47D cell number and viability. A decrease in T-47D cell viability was observed upon treatment with 5-100 µM BC. The ideal concentration of BC (100 µM) was used in combination with hormones and antihormones in an effort to further understand the possible similarities between this compound and other known effectors of ERα and PR. After a 24-hour concomitant treatment with and/or in combination of BC, estradiol, ICI 182, 780, and Tamoxifen, downregulation of ERα and PR protein levels was observed. Delineating the role of BC in the regulation of ERα, PR, as well as its mechanisms of action, may be important in understanding the influence of BC on hormone receptors in breast cancer.

  18. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  19. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

    Directory of Open Access Journals (Sweden)

    Prabhat S Kunwar

    2003-12-01

    Full Text Available In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR, Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

  20. Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines.

    Science.gov (United States)

    Kouzel, Ivan U; Pohlentz, Gottfried; Schmitz, Julia S; Steil, Daniel; Humpf, Hans-Ulrich; Karch, Helge; Müthing, Johannes

    2017-10-25

    Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and lipid raft association. In this study we identified Gb3Cer and Gb4Cer lipoforms of serum-free cultivated Caco-2 and HCT-8 cells, chiefly harboring ceramide moieties composed of sphingosine (d18:1) and C16:0, C22:0 or C24:0/C24:1 fatty acid. The most significant difference between the two cell lines was the prevalence of Gb3Cer with C16 fatty acid in HCT-8 and Gb4Cer with C22-C24 fatty acids in Caco-2 cells. Lipid compositional analysis of detergent-resistant membranes (DRMs), which were used as lipid raft -equivalents, indicated slightly higher relative content of Stx receptor Gb3Cer in DRMs of HCT-8 cells when compared to Caco-2 cells. Cytotoxicity assays revealed substantial sensitivity towards Stx2a for both cell lines, evidencing little higher susceptibility of Caco-2 cells versus HCT-8 cells. Collectively, Caco-2 and HCT-8 cells express a plethora of different receptor lipoforms and are susceptible towards Stx2a exhibiting somewhat lower sensitivity when compared to Vero cells.

  1. Neurotransmitters activate T-cells and elicit crucial functions via neurotransmitter receptors.

    Science.gov (United States)

    Levite, Mia

    2008-08-01

    Neurotransmitters are traditionally viewed as nerve-secreted molecules that trigger or inhibit neuronal functions. Yet, neurotransmitters bind also their neurotransmitter receptors in T-cells and directly activate or suppress T-cell functions. This review focuses only on the activating effects of neurotransmitters on T-cells, primarily naïve/resting cells, and covers dopamine, glutamate, serotonin, and few neuropeptides: GnRH-I, GnRH-II, substance P, somatostatin, CGRP, and neuropeptide Y. T-cells express many neurotransmitter receptors. These are regulated by TCR-activation, cytokines, or the neurotransmitters themselves, and are upregulated/downregulated in some human diseases. The context - whether the T-cells are naïve/resting or antigen/mitogen/cytokine-activated, the T-cell subset (CD4/CD8/Th1/Th2/Teff/Treg), neurotransmitter dose (low/optimal or high/excess), exact neurotransmitter receptors expressed, and the cytokine milieu - is crucial, and can determine either activation or suppression of T-cells by the same neurotransmitter. T-cells also produce many neurotransmitters. In summary, neurotransmitters activate vital T-cell functions in a direct, potent and specific manner, and may serve for communicating between the brain and the immune system to elicit an effective and orchestrated immune function, and for new therapeutic avenues, to improve T-cell eradication of cancer and infectious organisms.

  2. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    International Nuclear Information System (INIS)

    Kuwasako, Kenji; Sekiguchi, Toshio; Nagata, Sayaka; Jiang, Danfeng; Hayashi, Hidetaka; Murakami, Manabu; Hattori, Yuichi; Kitamura, Kazuo; Kato, Johji

    2016-01-01

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM 1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM 1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM 1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [ 125 I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β 2 -adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM 1 receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  3. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

    2016-02-19

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  4. Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells.

    Science.gov (United States)

    Shimizu, Kanako; Shinga, Jun; Yamasaki, Satoru; Kawamura, Masami; Dörrie, Jan; Schaft, Niels; Sato, Yusuke; Iyoda, Tomonori; Fujii, Shin-Ichiro

    2015-01-01

    Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

  5. T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

    DEFF Research Database (Denmark)

    Dietrich, J; Geisler, C

    1998-01-01

    The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosph......The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently...... to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating...... that the leucine-based motif in these complexes was inactive. In contrast, the CD4/CD3gamma chimeras did not associate with TCRzeta, and the leucine-based motif in these chimeras was constitutively active resulting in a high spontaneous internalization rate and low expression of the chimeras at the cell surface...

  6. Death Receptor-Mediated Cell Death and Proinflammatory Signaling in Nonalcoholic SteatohepatitisSummary

    Directory of Open Access Journals (Sweden)

    Petra Hirsova

    2015-01-01

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is becoming a public health problem worldwide. A subset of patients develop an inflammatory disease, nonalcoholic steatohepatitis (NASH, characterized by steatosis, hepatocellular death, macrophage and neutrophil accumulation, and varying stages of fibrosis. Hepatocyte cell death triggers the cellular inflammatory response, therefore reducing cell death may be salutary in the steatohepatitis disease process. Recently, a better understanding of hepatocyte apoptosis in NASH has been obtained and new information regarding other cell death modes such as necroptosis and pyroptosis has been reported. Hepatocyte lipotoxicity is often triggered by death receptors. In addition to causing apoptosis, death receptors have been shown to mediate proinflammatory signaling, suggesting that apoptosis in this context is not an immunologically silent process. Here, we review recent developments in our understanding of hepatocyte cell death by death receptors and its mechanistic link to inflammation in NASH. We emphasize how proapoptotic signaling by death receptors may induce the release of proinflammatory extracellular vesicles, thereby recruiting and activating macrophages and promoting the steatohepatitis process. Potential therapeutic strategies are discussed based on this evolving information. Keywords: Apoptosis, Caspase Inhibitor, Cell Death, Death Receptors, Exosomes, Extracellular Vesicles, Fibrosis, Inflammation, Inflammasome, Microvesicles, Necroptosis, Pyroptosis

  7. Characterization of the receptor for endothelial cell growth factor (ECGF) by affinity cross-linking

    Energy Technology Data Exchange (ETDEWEB)

    Friesel, R.; Burgess, W.H.; Mehlman, T.; Maciag, T.

    1986-05-01

    The authors have demonstrated high affinity receptors for ECGF on endothelial cells by covalent cross-linking of (/sup 125/I)-ECGF with disuccinimidyl suberate and observe a single chain cross-linked polypeptide species with an apparent M/sub r/ of 170K. The M/sub r/ 170K species represents (/sup 125/I)-ECGF bound to its receptor since (i) excess unlabeled ECGF inhibits the cross-linking of (/sup 125/I)-ECGF, (ii) labeling of the M/sub r/170K species does not take place in the absence of cross-linker, (iii) cells previously shown to be refractory to ECGF and lack ECGF receptors do not yield a cross-linked species, (iv) the cross-linked species can be immunoprecipitated with anti-ECGF antibodies, and (v) preincubation of cells with ECGF at 37/sup 0/C significantly reduces cross-linking while incubation at 4/sup 0/C does not. These data demonstrate that ECGF induced cell proliferation occurs through the occupancy of a specific cell surface polypeptide receptor with an apparent M/sub r/ of 150K, and suggests that internalization of the receptor-ligand complex may be relevant to ECGF-induced signal transduction.

  8. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

    Directory of Open Access Journals (Sweden)

    L.F.S. Sampaio

    2005-04-01

    Full Text Available The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5, while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM blocked the response to acetylcholine (3.0 nM-2.0 µM only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

  9. Receptors responsive to protein breakdown products in G-cells and D-cells of mouse, swine and human

    Directory of Open Access Journals (Sweden)

    Désirée Christine Haid

    2012-04-01

    Full Text Available Monitoring the luminal content in the stomach is of vital importance for adjusting the gastric activities, including the release of gastric hormones such as gastrin. Our previous studies have shown that in mice the gastrin-secreting G-cells express receptor types which are responsive to amino acids. Since the pig is considered as more suitable model for studying gastro-physiological aspects relevant for men, in this study we have analysed the distribution of G-cells and D-cells in the gastric antrum of men, swine and mouse and the expression of receptor types which may render these cells responsiveness to protein breakdown products. The results indicate that the number of G-cells per antral invagination was significantly higher in swine and human compared to mice and also the distribution pattern for G-cells differed between the species. The molecular phenotyping revealed that the receptors GPRC6A and CaSR were also expressed in G- and D-cells from swine and men. In the course of this study, an additional receptor type was found to be expressed in G- and D-cells, the peptone-receptor GPR92. This receptor type may be particular suitable for sensing protein breakdown products and thus be a key element to adjust the activity of G-cells and D-cells according to the progress of the digestive processes in the stomach. In search for elements of an intracellular signaling cascade it was found that G-cells express the G-protein subunits Gαq and Gαi2, as well as the phospholipase C subtype PLCβ3. In contrast, D-cells expressed the subtype PLCβ2 and neither Gαq nor Gαi2. These results indicate that there are significant species differences concerning the number and distribution pattern of gastric endocrine cells. However, the molecular phenotype of G-cells and D-cells appears to be similar in the three species.

  10. Segregation of receptor-ligand complexes in cell adhesion zones: Phase diagrams and role of thermal membrane roughness

    OpenAIRE

    Rozycki, Bartosz; Lipowsky, Reinhard; Weikl, Thomas R.

    2010-01-01

    The adhesion zone of immune cells, the 'immunological synapse', exhibits characteristic domains of receptor-ligand complexes. The domain formation is likely caused by a length difference of the receptor-ligand complexes, and has been investigated in experiments in which T cells adhere to supported membranes with anchored ligands. For supported membranes with two types of anchored ligands, MHCp and ICAM1, that bind to the receptors TCR and LFA1 in the cell membrane, the coexistence of domains ...

  11. Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

    Directory of Open Access Journals (Sweden)

    Sebastian König

    Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

  12. Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

    Science.gov (United States)

    Yamamoto, H; Karasawa, J; Sagi, N; Takahashi, S; Horikomi, K; Okuyama, S; Nukada, T; Sora, I; Yamamoto, T

    2001-08-03

    Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In

  13. Metabotropic glutamate receptor 5 activation enhances tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor and NMDA-induced cell death in hippocampal cultured neurons.

    Science.gov (United States)

    Takagi, Norio; Besshoh, Shintaro; Marunouchi, Tetsuro; Takeo, Satoshi; Tanonaka, Kouichi

    2012-01-01

    The activation of group I metabotropic glutamate receptors (mGluRs), which are coupled with Gq-protein, initiates a variety physiological responses in different types of cells. While Gq-protein-coupled receptors can upregulate N-methyl-D-aspartate (NMDA) receptor function, group I mGluR-mediated regulations of NMDA receptor function are not fully understood. To determine biochemical roles of group I mGluRs in the regulation of the NMDA receptor, we have investigated changes in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B induced by a selective mGluR5 agonist, (RS)-chloro-5-hydroxyphenylglycine (CHPG) in hippocampal neuronal cultures. Activation of mGluR5 by CHPG increased active-forms of Src. CHPG also enhanced tyrosine phosphorylation of NR2A and NR2B in hippocampal neuronal cultures. In addition, NMDA-induced cell death was enhanced by CHPG-induced mGluR5 stimulation at the concentration, which increased tyrosine phosphorylation of Src and NR2A/2B but did not induce cell death. This effect was inhibited by selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). The results suggest that in hippocampal neurons, mGluR5 may regulate NMDA receptor activity, involving tyrosine phosphorylation of NR2A and NR2B and may be involved in NMDA receptor-mediated cell injury.

  14. Humanin Inhibits Neuronal Cell Death by Interacting with a Cytokine Receptor Complex or Complexes Involving CNTF Receptor α/WSX-1/gp130

    Science.gov (United States)

    Hashimoto, Yuichi; Kurita, Megumi; Aiso, Sadakazu; Nishimoto, Ikuo

    2009-01-01

    Humanin (HN) inhibits neuronal death induced by various Alzheimer's disease (AD)-related insults via an unknown receptor on cell membranes. Our earlier study indicated that the activation of STAT3 was essential for HN-induced neuroprotection, suggesting that the HN receptor may belong to the cytokine receptor family. In this study, a series of loss-of-function tests indicated that gp130, the common subunit of receptors belonging to the IL-6 receptor family, was essential for HN-induced neuroprotection. Overexpression of ciliary neurotrophic factor receptor α (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it. These results suggest that both CNTFR and WSX-1 may be also involved in HN binding to cells. Consistent with these results, loss-of-functions of CNTFR or WSX-1 in neuronal cells nullified their responsiveness to HN-mediated protection. In vitro–reconstituted binding assays showed that HN, but not the other control peptide, induced the hetero-oligomerization of CNTFR, WSX-1, and gp130. Together, these results indicate that HN protects neurons by binding to a complex or complexes involving CNTFR/WSX-1/gp130. PMID:19386761

  15. Importance of killer immunoglobulin-like receptors in allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Danilo Santana Alessio Franceschi

    2011-01-01

    Full Text Available Hematopoietic stem cell transplantation is the treatment of choice for many hematologic diseases, such as multiple myeloma, bone marrow aplasia and leukemia. Human leukocyte antigen (HLA compatibility is an important tool to prevent post-transplant complications such as graft rejection and graft-versus-host disease, but the high rates of relapse limit the survival of transplant patients. Natural Killer cells, a type of lymphocyte that is a key element in the defense against tumor cells, cells infected with viruses and intracellular microbes, have different receptors on their surfaces that regulate their cytotoxicity. Killer immunoglobulin-like receptors are the most important, interacting consistently with human leukocyte antigen class I molecules present in other cells and thus controlling the activation of natural killer cells. Several studies have shown that certain combinations of killer immunoglobulin-like receptors and human leukocyte antigens (in both donors and recipients can affect the chances of survival of transplant patients, particularly in relation to the graft-versusleukemia effect, which may be associated to decreased relapse rates in certain groups. This review aims to shed light on the mechanisms and effects of killer immunoglobulin-like receptors - human leukocyte antigen associations and their implications following hematopoietic stem cell transplantation, and to critically analyze the results obtained by the studies presented herein.

  16. Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

    Science.gov (United States)

    Minutti, Carlos M; Drube, Sebastian; Blair, Natalie; Schwartz, Christian; McCrae, Jame C; McKenzie, Andrew N; Kamradt, Thomas; Mokry, Michal; Coffer, Paul J; Sibilia, Maria; Sijts, Alice J; Fallon, Padraic G; Maizels, Rick M; Zaiss, Dietmar M

    2017-10-17

    Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

  18. Dysregulation of chemokine/chemokine receptor axes and NK cell tissue localization during diseases

    Directory of Open Access Journals (Sweden)

    Giovanni Bernardini

    2016-10-01

    Full Text Available ABSTRACTChemokines are small chemotactic molecules that play key roles in physiological and pathological conditions. Upon signaling via their specific receptors, chemokines regulate tissue mobilization and trafficking of a wide array of immune cells, including NK cells. Current research is focused in analyzing changes of chemokine/chemokine receptor expression during various diseases to interfere with pathological trafficking of cells, or to recruit selected cell types to specific tissues. NK cells are a heterogeneous lymphocyte population comprising several subsets endowed with distinct functional properties and mainly representing distinct stages of a linear development process. Because of their different functional potential, the type of subset that accumulates in a tissue drives the final outcome of NK cell-regulated immune response, leading to either protection or pathology. Correspondingly, chemokine receptors including CXCR4, CXCR3 and CX3CR1 are differentially expressed by NK cell subsets and their expression levels can be modulated during NK cell activation. This review will at first summarize the current knowledge on the contribution of chemokines to the localization and generation of NK cell subsets in homeostasis. How an inappropriate chemotactic response can lead to pathology and how chemokine targeting can therapeutically affect tissue recruitment/localization of distinct NK cell subsets will also be discussed.

  19. NF-κB activity marks cells engaged in receptor editing

    Science.gov (United States)

    Cadera, Emily J.; Wan, Fengyi; Amin, Rupesh H.; Nolla, Hector; Lenardo, Michael J.

    2009-01-01

    Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-κB–dependent IκBα gene was replaced with a lacZ (β-gal) reporter complementary DNA (cDNA; IκBα+/lacZ) suggests a potential role for NF-κB in receptor editing. Sorted β-gal+ pre–B cells showed increased levels of various markers of receptor editing. In IκBα+/lacZ reporter mice expressing either innocuous or self-specific knocked in BCRs, β-gal was preferentially expressed in pre–B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IκBα superrepressor in primary bone marrow cultures resulted in diminished germline κ and rearranged λ transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in β-gal+ pre–B cells. Because IRF4 is a target of NF-κB and is required for receptor editing, we suggest that NF-κB could be acting through IRF4 to regulate receptor editing. PMID:19581408

  20. NF-kappaB activity marks cells engaged in receptor editing.

    Science.gov (United States)

    Cadera, Emily J; Wan, Fengyi; Amin, Rupesh H; Nolla, Hector; Lenardo, Michael J; Schlissel, Mark S

    2009-08-03

    Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

  1. Scavenger receptors in human airway epithelial cells: role in response to double-stranded RNA.

    Directory of Open Access Journals (Sweden)

    Audrey Dieudonné

    Full Text Available Scavenger receptors and Toll-like receptors (TLRs cooperate in response to danger signals to adjust the host immune response. The TLR3 agonist double stranded (dsRNA is an efficient activator of innate signalling in bronchial epithelial cells. In this study, we aimed at defining the role played by scavenger receptors expressed by bronchial epithelial cells in the control of the innate response to dsRNA both in vitro and in vivo. Expression of several scavenger receptor involved in pathogen recognition was first evaluated in human bronchial epithelial cells in steady-state and inflammatory conditions. Their implication in the uptake of dsRNA and the subsequent cell activation was evaluated in vitro by competition with ligand of scavenger receptors including maleylated ovalbumin and by RNA silencing. The capacity of maleylated ovalbumin to modulate lung inflammation induced by dsRNA was also investigated in mice. Exposure to tumor necrosis factor-α increased expression of the scavenger receptors LOX-1 and CXCL16 and the capacity to internalize maleylated ovalbumin, whereas activation by TLR ligands did not. In contrast, the expression of SR-B1 was not modulated in these conditions. Interestingly, supplementation with maleylated ovalbumin limited dsRNA uptake and inhibited subsequent activation of bronchial epithelial cells. RNA silencing of LOX-1 and SR-B1 strongly blocked the dsRNA-induced cytokine production. Finally, administration of maleylated ovalbumin in mice inhibited the dsRNA-induced infiltration and activation of inflammatory cells in bronchoalveolar spaces and lung draining lymph nodes. Together, our data characterize the function of SR-B1 and LOX-1 in bronchial epithelial cells and their implication in dsRNA-induced responses, a finding that might be relevant during respiratory viral infections.

  2. TRIg: a robust alignment pipeline for non-regular T-cell receptor and immunoglobulin sequences.

    Science.gov (United States)

    Hung, Sheng-Jou; Chen, Yi-Lin; Chu, Chia-Hung; Lee, Chuan-Chun; Chen, Wan-Li; Lin, Ya-Lan; Lin, Ming-Ching; Ho, Chung-Liang; Liu, Tsunglin

    2016-10-26

    T cells and B cells are essential in the adaptive immunity via expressing T cell receptors and immunoglogulins respectively for recognizing antigens. To recognize a wide variety of antigens, a highly diverse repertoire of receptors is generated via complex recombination of the receptor genes. Reasonably, frequencies of the recombination events have been shown to predict immune diseases and provide insights into the development of immunity. The field is further boosted by high-throughput sequencing and several computational tools have been released to analyze the recombined sequences. However, all current tools assume regular recombination of the receptor genes, which is not always valid in data prepared using a RACE approach. Compared to the traditional multiplex PCR approach, RACE is free of primer bias, therefore can provide accurate estimation of recombination frequencies. To handle the non-regular recombination events, a new computational program is needed. We propose TRIg to handle non-regular T cell receptor and immunoglobulin sequences. Unlike all current programs, TRIg does alignments to the whole receptor gene instead of only to the coding regions. This brings new computational challenges, e.g., ambiguous alignments due to multiple hits to repetitive regions. To reduce ambiguity, TRIg applies a heuristic strategy and incorporates gene annotation to identify authentic alignments. On our own and public RACE datasets, TRIg correctly identified non-regularly recombined sequences, which could not be achieved by current programs. TRIg also works well for regularly recombined sequences. TRIg takes into account non-regular recombination of T cell receptor and immunoglobulin genes, therefore is suitable for analyzing RACE data. Such analysis will provide accurate estimation of recombination events, which will benefit various immune studies directly. In addition, TRIg is suitable for studying aberrant recombination in immune diseases. TRIg is freely available at

  3. Cyclic stretch induces human bladder smooth muscle cell proliferation in vitro through muscarinic receptors.

    Science.gov (United States)

    Dai, Yi; Tian, Ye; Luo, De-Yi; Wazir, Romel; Yue, Xuan; Li, Hong; Wang, Kun-Jie

    2015-03-01

    The present study aimed to investigate whether the cyclic stretch‑induced proliferation of human bladder smooth muscle cells (HBSMCs) is mediated by muscarinic (M) receptors, together with the signal transduction mechanisms involved in this process. HBSMCs seeded onto silicone membranes were subjected to different cyclic stretches (5, 10, 15 and 20%) for 6 and 12 h. As the effect of cyclic stretch on M2 and M3 mRNA expression levels was maximal at 6 h 10% stretch, all subsequent experiments were performed at this stretch. Western blot analysis was used to quantify M2, M3, protein kinase C (PKC) and phosphorylated (p)‑PKC protein expression levels, flow cytometry was employed to examine cell cycle distribution and a 5-bromo‑2-deoxyuridine (BrdU) incorporation assay was used to assess cell proliferation at this stretch. Subsequently, HBSMCs were exposed to different acetylcholine concentrations and/or cyclic stretch, M receptor antagonists [AF-DX16, an M2 receptor antagonist; 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), an M3 receptor antagonist and atropine, a non‑selective antagonist] and GF 109203X, a PKC antagonist, to assess the possible underlying signaling mechanisms. Cyclic stretch was found to increase the proliferation of HBSMCs and the expression levels of M2, M3, PKC and p‑PKC proteins. M receptor and PKC antagonists exerted no apparent effect on nonstretched cells, but reduced the incorporation of BrdU into stretched cells; the most pronounced effects were observed when non‑selective M receptor and PKC antagonists were applied. Notably, 4‑DAMP did not inhibit stretch‑induced PKC activation. These results indicate that the activation of the M3 receptor signaling pathway in stretch‑induced HBSMC proliferation occurs via PKC-independent mechanisms.

  4. YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells.

    Science.gov (United States)

    Poitelon, Yannick; Lopez-Anido, Camila; Catignas, Kathleen; Berti, Caterina; Palmisano, Marilena; Williamson, Courtney; Ameroso, Dominique; Abiko, Kansho; Hwang, Yoonchan; Gregorieff, Alex; Wrana, Jeffrey L; Asmani, Mohammadnabi; Zhao, Ruogang; Sim, Fraser James; Wrabetz, Lawrence; Svaren, John; Feltri, Maria Laura

    2016-07-01

    Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice, we show that Taz is required in Schwann cells for radial sorting and myelination and that Yap is redundant with Taz. Yap and Taz are activated in Schwann cells by mechanical stimuli and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells.

  5. Netrin-1 induces proliferation of Schwann cells through Unc5b receptor

    International Nuclear Information System (INIS)

    Lee, Hyun Kyoung; Seo, In Ae; Seo, Eunhui; Seo, Su-Yeong; Lee, Hye Jeong; Park, Hwan Tae

    2007-01-01

    Netrin and its receptors, DCC (Deleted in Colorectal Cancer) and Unc5, are proposed to be involved in the axon guidance and neuroglial migration during development. However, accumulating evidence implies that they may also participate in the cell survival and apoptosis. Here, we show that netrin-1 induces proliferation of Schwann cells. Unc5b is the sole receptor expressed in RT4 schwannoma cells and adult primary Schwann cells, and netrin-1 and Unc5b are found to be expressed in the injured sciatic nerve. It was also found that the netrin-1-induced Schwann cell proliferation was blocked by the specific inhibition of Unc5b expression with RNAi. These data suggest that netrin-1 could be an endogenous trophic factor for Schwann cells in the injured peripheral nerves

  6. Presence of dopamine D-2 receptors in human tumoral cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Sokoloff, P.; Riou, J.F.; Martres, M.P.; Schwartz, J.C. (Centre Paul Broca, Paris (France))

    1989-07-31

    ({sup 125}I) Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, ({sup 125}I) iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.

  7. Aberrant Expression of Functional BAFF-System Receptors by Malignant B-Cell Precursors Impacts Leukemia Cell Survival

    Science.gov (United States)

    Maia, Sara; Pelletier, Marc; Ding, Jixin; Hsu, Yen-Ming; Sallan, Stephen E.; Rao, Sambasiva P.; Nadler, Lee M.; Cardoso, Angelo A.

    2011-01-01

    Despite exhibiting oncogenic events, patient's leukemia cells are responsive and dependent on signals from their malignant bone marrow (BM) microenvironment, which modulate their survival, cell cycle progression, trafficking and resistance to chemotherapy. Identification of the signaling pathways mediating this leukemia/microenvironment interplay is critical for the development of novel molecular targeted therapies. We observed that primary leukemia B-cell precursors aberrantly express receptors of the BAFF-system, BAFF-R, BCMA, and TACI. These receptors are functional as their ligation triggers activation of NF-κB, MAPK/JNK, and Akt signaling. Leukemia cells express surface BAFF and APRIL ligands, and soluble BAFF is significantly higher in leukemia patients in comparison to age-matched controls. Interestingly, leukemia cells also express surface APRIL, which seems to be encoded by APRIL-δ, a novel isoform that lacks the furin convertase domain. Importantly, we observed BM microenvironmental cells express the ligands BAFF and APRIL, including surface and secreted BAFF by BM endothelial cells. Functional studies showed that signals through BAFF-system receptors impact the survival and basal proliferation of leukemia B-cell precursors, and support the involvement of both homotypic and heterotypic mechanisms. This study shows an unforeseen role for the BAFF-system in the biology of precursor B-cell leukemia, and suggests that the target disruption of BAFF signals may constitute a valid strategy for the treatment of this cancer. PMID:21687682

  8. Aberrant expression of functional BAFF-system receptors by malignant B-cell precursors impacts leukemia cell survival.

    Directory of Open Access Journals (Sweden)

    Sara Maia

    Full Text Available Despite exhibiting oncogenic events, patient's leukemia cells are responsive and dependent on signals from their malignant bone marrow (BM microenvironment, which modulate their survival, cell cycle progression, trafficking and resistance to chemotherapy. Identification of the signaling pathways mediating this leukemia/microenvironment interplay is critical for the development of novel molecular targeted therapies.We observed that primary leukemia B-cell precursors aberrantly express receptors of the BAFF-system, BAFF-R, BCMA, and TACI. These receptors are functional as their ligation triggers activation of NF-κB, MAPK/JNK, and Akt signaling. Leukemia cells express surface BAFF and APRIL ligands, and soluble BAFF is significantly higher in leukemia patients in comparison to age-matched controls. Interestingly, leukemia cells also express surface APRIL, which seems to be encoded by APRIL-δ, a novel isoform that lacks the furin convertase domain. Importantly, we observed BM microenvironmental cells express the ligands BAFF and APRIL, including surface and secreted BAFF by BM endothelial cells. Functional studies showed that signals through BAFF-system receptors impact the survival and basal proliferation of leukemia B-cell precursors, and support the involvement of both homotypic and heterotypic mechanisms.This study shows an unforeseen role for the BAFF-system in the biology of precursor B-cell leukemia, and suggests that the target disruption of BAFF signals may constitute a valid strategy for the treatment of this cancer.

  9. Segregation of receptor-ligand complexes in cell adhesion zones: phase diagrams and the role of thermal membrane roughness

    Science.gov (United States)

    Różycki, B.; Lipowsky, R.; Weikl, T. R.

    2010-09-01

    The adhesion zone of immune cells, the 'immunological synapse', exhibits characteristic domains of receptor-ligand complexes. The domain formation is probably caused by a length difference of the receptor-ligand complexes, and has been investigated in experiments in which T cells adhere to supported membranes with anchored ligands. For supported membranes with two types of anchored ligands, MHCp and ICAM1, which bind to the T-cell receptor (TCR) and the receptor LFA1 in the cell membrane, the coexistence of domains of the TCR-MHCp and LFA1-ICAM1 complexes in the cell adhesion zone has been observed for a wide range of ligand concentrations and affinities. For supported membranes with long and short ligands that bind to the same cell receptor CD2, in contrast, domain coexistence has been observed for a quite narrow ratio of ligand concentrations. In this paper, we determine detailed phase diagrams for cells adhering to supported membranes with a statistical-physical model of cell adhesion. We find a characteristic difference between the adhesion scenarios in which two types of ligands in a supported membrane bind (i) to the same cell receptor or (ii) to two different cell receptors, which helps us to explain the experimental observations. Our phase diagrams fully include thermal shape fluctuations of the cell membranes on nanometer scales, which lead to a critical point for the domain formation and to a cooperative binding of the receptors and ligands.

  10. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    International Nuclear Information System (INIS)

    Doi, Keiko; Fujimoto, Takahiro; Okamura, Tadashi; Ogawa, Masahiro; Tanaka, Yoko; Mototani, Yasumasa; Goto, Motohito; Ota, Takeharu; Matsuzaki, Hiroshi; Kuroki, Masahide; Tsunoda, Toshiyuki; Sasazuki, Takehiko; Shirasawa, Senji

    2012-01-01

    Highlights: ► We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. ► Zfat-deficiency leads to reduction in the number of the peripheral T cells. ► Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. ► Decreased expression of IL-7Rα, IL-2Rα and IL-2 in Zfat-deficient peripheral T cells. ► Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  11. Andrographolide inhibits growth of acute promyelocytic leukaemia cells by inducing retinoic acid receptor-independent cell differentiation and apoptosis.

    Science.gov (United States)

    Manikam, Shiamala D; Manikam, Shiamala T; Stanslas, Johnson

    2009-01-01

    The growth inhibiting potential of andrographolide was evaluated in three acute promyelocytic leukaemia cell line models (HL-60, NB4 and all-trans retinoic acid (ATRA)-resistant NB4-R2). In elucidating the mechanisms of growth inhibition, a special emphasis was placed on assessing the induction of differentiation and apoptosis by andrographolide in the primary acute promyelocytic leukaemia NB4 cells. The compound was 2- and 3-fold more active in inhibiting the growth of HL-60 and NB4-R2 cells compared with NB4 cells, respectively. At IC50 (concentration at which growth of 50% of the cells (compared with medium only treated control cells) is inhibited; 4.5 microM) the compound exhibited strong cell-differentiating activity in NB4 cells, similar to ATRA (IC50 1.5 microM). In the presence of a pure retinoic acid receptor antagonist AGN193109, the growth inhibition of NB4 cells by ATRA was reversed, whereas the activity of andrographolide was not affected. This clearly suggested that andrographolide's cell differentiating activity to induce growth inhibition of NB4 cells most likely occurred via a retinoic acid receptor-independent pathway. At higher concentration (2xIC50), andrographolide was an efficient inducer of apoptosis in NB4 cells. Taken together, these results suggest andrographolide and its derivatives, apparently with a novel cell differentiating mechanism and with ability to induce apoptosis, might be beneficial in the treatment of primary and ATRA-resistant acute promyelocytic leukaemia.

  12. The effects of black cohosh on the regulation of estrogen receptor (ERα and progesterone receptor (PR in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Szmyd M

    2018-01-01

    Full Text Available Monica Szmyd,1–3 Victoria Lloyd,1–3 Kelly Hallman,1–3 Katie Aleck,1–3 Viktoria Mladenovik,1–3 Christina McKee,1–3 Mia Morse,1–3 Tyler Bedgood,1–3 Sumi Dinda1–3 1Biomedical and Therapeutic Sciences, School of Health Sciences, 2Prevention Research Center, 3Center of Biomedical Sciences, Oakland University, Rochester, MI, USA Abstract: The North American plant Cimicifuga racemosa, also known as black cohosh (BC, is a herb that recently has gained attention for its hormonal effects. As the usage of hormone replacement therapy is declining due to its adverse effects in women with cancer, many are turning to herbal remedies like BC to treat menopausal symptoms. It is crucial to determine whether the effects of BC involve estrogen receptor-alpha (ERα. Previous studies from our laboratory have shown ERα to be a possible molecular target for BC. In this study, we examined the effects of BC (8% triterpene glycosides alone and in combination with hormones and antihormones on the cellular viability, expression of ERα and progesterone receptor (PR-A/B, and cytolocalization of ERα in ER (+ and PR-A/B (+ T-47D breast cancer cells. Cells were cultured and proteins were extracted and quantified. Western blot analysis revealed alterations in the expression of ERα and PR after treatment with BC (5–100 µM. BC induced a concentration-dependent decrease in ERα and PR protein levels when compared to the control. Image cytometric analysis with propidium iodide staining was used to enumerate changes in T-47D cell number and viability. A decrease in T-47D cell viability was observed upon treatment with 5–100 µM BC. The ideal concentration of BC (100 μM was used in combination with hormones and antihormones in an effort to further understand the possible similarities between this compound and other known effectors of ERα and PR. After a 24-hour concomitant treatment with and/or in combination of BC, estradiol, ICI 182, 780, and Tamoxifen

  13. Potentiation of NMDA receptor-dependent cell responses by extracellular high mobility group box 1 protein.

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    Marco Pedrazzi

    Full Text Available BACKGROUND: Extracellular high mobility group box 1 (HMGB1 protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca(2+ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. PRINCIPAL FINDINGS: Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca(2+-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130-139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1((130-139 peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. CONCLUSION: We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.

  14. Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells

    International Nuclear Information System (INIS)

    Bernier, M.; Chatelain, P.; Mather, J.P.; Saez, J.M.

    1986-01-01

    The author have investigated the effects of insulin and somatomedin-C/insulin like growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 μg/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 μg/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated [ 3 H]-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through the receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors

  15. Interleukin-21 induces proliferation and modulates receptor expression and effector function in canine natural killer cells.

    Science.gov (United States)

    Shin, Dong-Jun; Lee, Soo-Hyeon; Park, Ji-Yun; Kim, Ju-Sun; Lee, Je-Jung; Suh, Guk-Hyun; Lee, Youn-Kyung; Cho, Duck; Kim, Sang-Ki

    2015-05-15

    Interleukin (IL)-21 is an important modulator of natural killer (NK) cell function. However, little is known about IL-21 function in canine NK cells because the phenotype of these cells remains undefined. In this study, we selectively expanded non-B and non-T large granular NK lymphocytes (CD3(-)CD21(-)CD5(-)CD4(-)TCRαβ(-)TCRγδ(-)) ex vivo from the peripheral blood mononuclear cells (PBMCs) of healthy dogs using a combination of IL-2, IL-15, and IL-21 in the presence of 100 Gy-irradiated K562 cells. We investigated the effects of varying the duration and timing of IL-21 treatment on stimulation of proliferation, expression of NK-related receptors, anti-tumor activity and production of interferon (IFN)-γ. The expanded NK cells in each treatment group became enlarged and highly granular after 21 days in culture. NK cells proliferated rapidly in response to activation by IL-21 for 3 weeks, and IL-21 was able to induce changes in the mRNA expression of NK cell-related receptors and enhance the effector function of NK cells in perforin- and granzyme-B-dependent manners. The duration, frequency and timing of IL-21 stimulation during culture affected the rate of proliferation, patterns of receptor expression, cytokine production, and anti-tumor activity. The optimal conditions for maximizing the IL-21-induced proliferation and effector function of NK cells in the presence of IL-2 and IL-15 were seen in cells treated with IL-21 for the first 7 days of culture but without any further IL-21 stimulation other than an additional 2-day treatment prior to harvesting on day 21. The results of this study suggest that synergistic interactions of IL-21 with IL-2 and IL-15 play an important role in the proliferation, receptor expression, and effector function of canine NK cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

    OpenAIRE

    Van Schravendijk, C F; Hooghe-Peters, E L; De Meyts, P; Pipeleers, D G

    1984-01-01

    The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insu...

  17. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  18. Quantitative mapping of RNA-mediated nuclear estrogen receptor β interactome in human breast cancer cells

    Science.gov (United States)

    Giurato, Giorgio; Nassa, Giovanni; Salvati, Annamaria; Alexandrova, Elena; Rizzo, Francesca; Nyman, Tuula A.; Weisz, Alessandro; Tarallo, Roberta

    2018-03-01

    The nuclear receptor estrogen receptor 2 (ESR2, ERβ) modulates cancer cell proliferation and tumor growth, exerting an oncosuppressive role in breast cancer (BC). Interaction proteomics by tandem affinity purification coupled to mass spectrometry was previously applied in BC cells to identify proteins acting in concert with ERβ to control key cellular functions, including gene transcription, RNA splicing and post-transcriptional mRNA regulation. These studies revealed an involvement of RNA in ERβ interactome assembly and functions. By applying native protein complex purification followed by nano LC-MS/MS before and after in vitro RNA removal, we generated a large dataset of newly identified nuclear ERβ interactors, including a subset associating with the receptor via RNA bridging. These datasets will be useful to investigate further the role of ERβ, nuclear RNAs and the other proteins identified here in BC and other cell types.

  19. Regulation of immune cell function and differentiation by the NKG2D receptor.

    Science.gov (United States)

    Zafirova, Biljana; Wensveen, Felix M; Gulin, Maja; Polić, Bojan

    2011-11-01

    NKG2D is one of the most intensively studied immune receptors of the past decade. Its unique binding and signaling properties, expression pattern, and functions have been attracting much interest within the field due to its potent antiviral and anti-tumor properties. As an activating receptor, NKG2D is expressed on cells of the innate and adaptive immune system. It recognizes stress-induced MHC class I-like ligands and acts as a molecular sensor for cells jeopardized by viral infections or DNA damage. Although the activating functions of NKG2D have been well documented, recent analysis of NKG2D-deficient mice suggests that this receptor may have a regulatory role during NK cell development. In this review, we will revisit known aspects of NKG2D functions and present new insights in the proposed influence of this molecule on hematopoietic differentiation.

  20. The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects

    Directory of Open Access Journals (Sweden)

    Farhad Dehkhoda

    2018-02-01

    Full Text Available The growth hormone receptor (GHR, although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK–signal transducer and activator of transcription (STAT signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK–STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling.

  1. A peptide antagonist of the ErbB1 receptor inhibits receptor activation, tumor cell growth and migration in vitro and xenograft tumor growth in vivo

    DEFF Research Database (Denmark)

    Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav

    2010-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization......B1 phosphorylation, cell growth, and migration in two human tumor cell lines, A549 and HN5, expressing moderate and high ErbB1 levels, respectively. Furthermore, we show that Inherbin3 inhibits tumor growth in vivo and induces apoptosis in a tumor xenograft model employing the human non-small cell...... lung cancer cell line A549. The Inherbin3 peptide may be a useful tool for investigating the mechanisms of ErbB receptor homo- and heterodimerization. Moreover, the here described biological effects of Inherbin3 suggest that peptide-based targeting of ErbB receptor dimerization is a promising anti...

  2. Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation

    DEFF Research Database (Denmark)

    Schmalzigaug, R; Ye, Q; Berchtold, M W

    2001-01-01

    stimulation of the B-cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII-/- cells. Despite higher Ca2+ resting levels, the CaMII-/- cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes....

  3. Complement Receptor Type 1 Suppresses Human B Cell Functions in SLE Patients

    Directory of Open Access Journals (Sweden)

    Mariann Kremlitzka

    2016-01-01

    Full Text Available Complement receptors (CRs play an integral role in innate immunity and also function to initiate and shape the adaptive immune response. Our earlier results showed that complement receptor type 1 (CR1, CD35 is a potent inhibitor of the B cell receptor- (BCR- induced functions of human B lymphocytes. Here we show that this inhibition occurs already at the initial steps of B cell activation since ligation of CR1 reduces the BCR-induced phosphorylation of key signaling molecules such as Syk and mitogen activated protein kinases (MAPKs. Furthermore, our data give evidence that although B lymphocytes of active systemic lupus erythematosus (SLE patients express lower level of CR1, the inhibitory capacity of this complement receptor is still maintained and its ligand-induced clustering results in significant inhibition of the main B cell functions, similar to that found in the case of healthy individuals. Since we have found that reduced CR1 expression of SLE patients does not affect the inhibitory capacity of the receptor, our results further support the therapeutical potential of CD35 targeting the decrease of B cell activation and autoantibody production in autoimmune patients.

  4. Complement Receptor Type 1 Suppresses Human B Cell Functions in SLE Patients.

    Science.gov (United States)

    Kremlitzka, Mariann; Mácsik-Valent, Bernadett; Polgár, Anna; Kiss, Emese; Poór, Gyula; Erdei, Anna

    2016-01-01

    Complement receptors (CRs) play an integral role in innate immunity and also function to initiate and shape the adaptive immune response. Our earlier results showed that complement receptor type 1 (CR1, CD35) is a potent inhibitor of the B cell receptor- (BCR-) induced functions of human B lymphocytes. Here we show that this inhibition occurs already at the initial steps of B cell activation since ligation of CR1 reduces the BCR-induced phosphorylation of key signaling molecules such as Syk and mitogen activated protein kinases (MAPKs). Furthermore, our data give evidence that although B lymphocytes of active systemic lupus erythematosus (SLE) patients express lower level of CR1, the inhibitory capacity of this complement receptor is still maintained and its ligand-induced clustering results in significant inhibition of the main B cell functions, similar to that found in the case of healthy individuals. Since we have found that reduced CR1 expression of SLE patients does not affect the inhibitory capacity of the receptor, our results further support the therapeutical potential of CD35 targeting the decrease of B cell activation and autoantibody production in autoimmune patients.

  5. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  6. An EGF receptor inhibitor induces RAR-β expression in breast and ovarian cancer cells

    International Nuclear Information System (INIS)

    Grunt, Thomas W.; Puckmair, Klaudia; Tomek, Katharina; Kainz, Birgit; Gaiger, Alexander

    2005-01-01

    Inhibition of the epidermal growth factor (EGF)-receptor (EGFR) has become a promising anticancer treatment strategy. In addition, application of retinoids yields encouraging results for cancer prevention and therapy. Many tumors express no or low amounts of retinoic acid receptor-β2 (RAR-β2) due to epigenetic silencing via DNA hypermethylation. RAR-β2 is the main mediator of the antiproliferative effect of retinoids. RAR-β2 re-expression causes reversal of transformation, cell cycle arrest, and restoration of retinoid sensitivity. RAR-β2 is thus a tumor suppressor. Western blotting, colorimetric in vitro cell proliferation assays, and reverse transcription-polymerase chain reaction demonstrated that the EGFR inhibitor PD153035 not only blocked activation of EGFR and inhibited cell growth, but also stimulated RAR-β expression in MDA-MB-468 breast and OVCAR-3 ovarian carcinoma cells. Upregulation of RAR-β by PD153035 was confirmed by real-time reverse transcription-polymerase chain reaction. In contrast, expression of other retinoid receptors and of estrogen receptor-α was not affected. PD153035-mediated re-induction of RAR-β was associated with demethylation of the RAR-β2 gene promoter P2 as demonstrated by methylation-specific polymerase chain reaction. These novel results on the ErbB/retinoid receptor cross-talk may be useful for designing future anticancer combination regimens

  7. Chimeric antigen receptor T cells: a novel therapy for solid tumors

    Directory of Open Access Journals (Sweden)

    Shengnan Yu

    2017-03-01

    Full Text Available Abstract The chimeric antigen receptor T (CAR-T cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2, and mesothelin (MSLN, as well as the challenges for CAR-T cell therapy.

  8. Characterization of a tachykinin peptide NK sub 2 receptor transfected into murine fibroblast B82 cells

    Energy Technology Data Exchange (ETDEWEB)

    Van Giersbergen, P.L.M. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States) Univ. of Cincinnati, OH (United States)); Shatzer, S.A.; Buck, S.H. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States)); Henderson, A.K.; Lai, J.; Yamamura, Henry, I. (Univ. of Arizona, Tucson (United States)); Nakanishi, Shigetada (Kyoto Univ. (Japan))

    1991-03-01

    Membranes isolated from a murine fibroblast B82 cell line (SKLKB82{number sign}3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of (His({sup 125}I){sup 1})neurokinin A ({sup 125}I-NKA) to a single population of sites with a B{sub max} of 147 fmol/mg of protein and a K{sub d} of 0.59 nM. The ligand binding in SKLKB82{number sign}3 cells was reversible. Thus, SKLKB82{number sign}3 cells have been transfected with NK{sub 2} receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK{sub 2} receptors, NK{sub 2} receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of {sup 125}I-NKA binding by nucleotide analogues was markedly different in SKLKB82{number sign}3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK{sub 2} receptors.

  9. Effect of outer hair cell piezoelectricity on high-frequency receptor potentials.

    Science.gov (United States)

    Spector, Alexander A; Brownell, William E; Popel, Aleksander S

    2003-01-01

    The low-pass voltage response of outer hair cells predicted by conventional equivalent circuit analysis would preclude the active force production at high frequencies. We have found that the band pass characteristics can be improved by introducing the piezoelectric properties of the cell wall. In contrast to the conventional analysis, the receptor potential does not tend to zero and at any frequency is greater than a limiting value. In addition, the phase shift between the transduction current and receptor potential tends to zero. The piezoelectric properties cause an additional, strain-dependent, displacement current in the cell wall. The wall strain is estimated on the basis of a model of the cell deformation in the organ of Corti. The limiting value of the receptor potential depends on the ratio of a parameter determined by the piezoelectric coefficients and the strain to the membrane capacitance. In short cells, we have found that for the low-frequency value of about 2-3 mV and the strain level of 0.1% the receptor potential can reach 0.4 mV throughout the whole frequency range. In long cells, we have found that the effect of the piezoelectric properties is much weaker. These results are consistent with major features of the cochlear amplifier.

  10. Nuclear receptors expression chart in peripheral blood mononuclear cells identifies patients with Metabolic Syndrome.

    Science.gov (United States)

    D'Amore, Simona; Vacca, Michele; Graziano, Giusi; D'Orazio, Andria; Cariello, Marica; Martelli, Nicola; Di Tullio, Giuseppe; Salvia, Roberto; Grandaliano, Giuseppe; Belfiore, Anna; Pellegrini, Fabio; Palasciano, Giuseppe; Moschetta, Antonio

    2013-12-01

    Nuclear receptors are a class of 48 ligand-activated transcription factors identified as key players of metabolic and developmental processes. Most of these receptors are potential targets for pharmacological strategies in the Metabolic Syndrome. In the present study, we analyzed changes in the mRNA expression of nuclear receptors in the peripheral blood mononuclear cells of patients with Metabolic Syndrome, in order to identify novel biomarkers of disease and candidate targets for putative therapeutical approaches. We enrolled thirty healthy controls (14 M:16 F) and thirty naïve patients (16 M: 14 F; >3 criteria for Metabolic Syndrome upon Adult Treatment Panel III) without organ damage. Using quantitative real-time PCR, we assessed the expression patterns of nuclear receptors in peripheral blood mononuclear cells. 33/48 nuclear receptors were expressed in peripheral blood mononuclear cells. In patients with Metabolic Syndrome, we found a significant down-regulation of the entire PPAR, NR4A and RAR families, together with a repression of RXRα, VDR, and Rev-Erbα. Furthermore, we performed a novel statistical analysis with classification trees, which allowed us to depict a predictive core of nuclear receptor expression patterns characterizing subjects with Metabolic Syndrome. Random Forest Analysis identified NOR1 and PPARδ, which were both reduced in peripheral blood mononuclear cells and specifically in CD14(+) cells (mostly monocytes), as classifiers of Metabolic Syndrome, with high specificity and sensitivity. Our results point to the use of PPAR and NR4A mRNA levels in the overall peripheral blood mononuclear cells as biomarkers of Metabolic Syndrome and bona fide putative targets of pharmacological therapy. © 2013.

  11. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Directory of Open Access Journals (Sweden)

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  12. The receptors for gibbon ape leukemia virus and amphotropic murine leukemia virus are not downregulated in productively infected cells

    Directory of Open Access Journals (Sweden)

    Eiden Maribeth V

    2011-07-01

    Full Text Available Abstract Background Over the last several decades it has been noted, using a variety of different methods, that cells infected by a specific gammaretrovirus are resistant to infection by other retroviruses that employ the same receptor; a phenomenon termed receptor interference. Receptor masking is thought to provide an earlier means of blocking superinfection, whereas receptor down regulation is generally considered to occur in chronically infected cells. Results We used replication-competent GFP-expressing viruses containing either an amphotropic murine leukemia virus (A-MLV or the gibbon ape leukemia virus (GALV envelope. We also constructed similar viruses containing fluorescence-labeled Gag proteins for the detection of viral particles. Using this repertoire of reagents together with a wide range of antibodies, we were able to determine the presence and availability of viral receptors, and detect viral envelope proteins and particles presence on the cell surface of chronically infected cells. Conclusions A-MLV or GALV receptors remain on the surface of chronically infected cells and are detectable by respective antibodies, indicating that these receptors are not downregulated in these infected cells as previously proposed. We were also able to detect viral envelope proteins on the infected cell surface and infected cells are unable to bind soluble A-MLV or GALV envelopes indicating that receptor binding sites are masked by endogenously expressed A-MLV or GALV viral envelope. However, receptor masking does not completely prevent A-MLV or GALV superinfection.

  13. Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells.

    Directory of Open Access Journals (Sweden)

    Claudio eElgueta

    2015-02-01

    Full Text Available Abstract Acetylcholine (ACh is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline, suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca2+ accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs and intracellular Ca2+ stores. Inhibition of acetyl-cholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram, suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

  14. Ruthenium complexes with phenylterpyridine derivatives target cell membrane and trigger death receptors-mediated apoptosis in cancer cells.

    Science.gov (United States)

    Deng, Zhiqin; Gao, Pan; Yu, Lianling; Ma, Bin; You, Yuanyuan; Chan, Leung; Mei, Chaoming; Chen, Tianfeng

    2017-06-01

    Elucidation of the communication between metal complexes and cell membrane may provide useful information for rational design of metal-based anticancer drugs. Herein we synthesized a novel class of ruthenium (Ru) complexes containing phtpy derivatives (phtpy = phenylterpyridine), analyzed their structure-activity relationship and revealed their action mechanisms. The result showed that, the increase in the planarity of hydrophobic Ru complexes significantly enhanced their lipophilicity and cellular uptake. Meanwhile, the introduction of nitro group effectively improved their anticancer efficacy. Further mechanism studies revealed that, complex (2c), firstly accumulated on cell membrane and interacted with death receptors to activate extrinsic apoptosis signaling pathway. The complex was then transported into cell cytoplasm through transferrin receptor-mediated endocytosis. Most of the intracellular 2c accumulated in cell plasma, decreasing the level of cellular ROS, inducing the activation of caspase-9 and thus intensifying the apoptosis. At the same time, the residual 2c can translocate into cell nucleus to interact with DNA, induce DNA damage, activate p53 pathway and enhance apoptosis. Comparing with cisplatin, 2c possesses prolonged circulation time in blood, comparable antitumor ability and importantly, much lower toxicity in vivo. Taken together, this study uncovers the role of membrane receptors in the anticancer actions of Ru complexes, and provides fundamental information for rational design of membrane receptor targeting anticancer drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Anandamide and Δ9-Tetrahydrocannabinol Directly Inhibit Cells of the Immune System via CB2 Receptors

    Science.gov (United States)

    Eisenstein, Toby K.; Meissler, Joseph J.; Wilson, Qiana; Gaughan, John P.; Adler, Martin W.

    2007-01-01

    This study shows that two cannabinoids, Δ9-tetrahydrocannabinol (THC) and anandamide, induce dose related immunosuppression in both the primary and secondary in vitro plaque-forming cell assays of antibody formation. The immunosuppression induced by both compounds could be blocked by SR144528, an antagonist specific for the CB2 receptor, but not by SR141716, a CB1 antagonist. These studies are novel in that they show that both anadamide and THC are active in the nanomolar to picomolar (for anandamide) range in these assays of immune function, and that both mediate their effects directly on cells of the immune system through the CB2 receptor. PMID:17640739

  16. Potentiating action of propofol at GABAA receptors of retinal bipolar cells

    DEFF Research Database (Denmark)

    Yue, Lan; Xie, An; Bruzik, Karol S

    2011-01-01

    Purpose. Propofol (2,6-diisopropyl phenol), a widely used systemic anesthetic, is known to potentiate GABA(A) receptor activity in a number of CNS neurons and to produce changes in electroretinographically recorded responses of the retina. However, little is known about propofol's effects...... on specific retinal neurons. The authors investigated the action of propofol on GABA-elicited membrane current responses of retinal bipolar cells, which have both GABA(A) and GABA(C) receptors. Methods. Single, enzymatically dissociated bipolar cells obtained from rat retina were treated with propofol...

  17. 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

  18. Regulation of gene expression in ovarian cancer cells by luteinizing hormone receptor expression and activation

    International Nuclear Information System (INIS)

    Cui, Juan; Miner, Brooke M; Eldredge, Joanna B; Warrenfeltz, Susanne W; Dam, Phuongan; Xu, Ying; Puett, David

    2011-01-01

    Since a substantial percentage of ovarian cancers express gonadotropin receptors and are responsive to the relatively high concentrations of pituitary gonadotropins during the postmenopausal years, it has been suggested that receptor activation may contribute to the etiology and/or progression of the neoplasm. The goal of the present study was to develop a cell model to determine the impact of luteinizing hormone (LH) receptor (LHR) expression and LH-mediated LHR activation on gene expression and thus obtain insights into the mechanism of gonadotropin action on ovarian surface epithelial (OSE) carcinoma cells. The human ovarian cancer cell line, SKOV-3, was stably transfected to express functional LHR and incubated with LH for various periods of time (0-20 hours). Transcriptomic profiling was performed on these cells to identify LHR expression/activation-dependent changes in gene expression levels and pathways by microarray and qRT-PCR analyses. Through comparative analysis on the LHR-transfected SKOV-3 cells exposed to LH, we observed the differential expression of 1,783 genes in response to LH treatment, among which five significant families were enriched, including those of growth factors, translation regulators, transporters, G-protein coupled receptors, and ligand-dependent nuclear receptors. The most highly induced early and intermediate responses were found to occupy a network impacting transcriptional regulation, cell growth, apoptosis, and multiple signaling transductions, giving indications of LH-induced apoptosis and cell growth inhibition through the significant changes in, for example, tumor necrosis factor, Jun and many others, supportive of the observed cell growth reduction in in vitro assays. However, other observations, e.g. the substantial up-regulation of the genes encoding the endothelin-1 subtype A receptor, stromal cell-derived factor 1, and insulin-like growth factor II, all of which are potential therapeutic targets, may reflect a positive

  19. Investigation of T cell receptors in the peripheral blood of patients with active pulmonary tuberculosis.

    Science.gov (United States)

    Akbulut, H H; Deveci, F; Celik, I; Ilhan, F; Turgut, T

    2009-01-01

    T cells have the capability of recognizing target cells through their T cell receptors (TCRs). Thus, the percentages of CD3+/gamma-delta (gammadelta) TCR+ and CD3+/alpha-beta (alphabeta) TCR+ T lymphocytes were investigated in active and inactive pulmonary tuberculosis (PT) patients and in healthy individuals. CD3+ and CD3+/alphabeta TCR+ cell percentages were significantly lower in all PT patients than in healthy subjects. Percentages of CD3+/gammadelta and CD3+/alphabeta TCR+ were not statistically different between active and inactive PT patients. It was concluded that alphabeta TCR+ T cells might have a protective role in tuberculosis infection.

  20. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

    OpenAIRE

    Kontny, U; Kurane, I; Ennis, F A

    1988-01-01

    It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

  1. The vitamin d receptor and T cell function

    DEFF Research Database (Denmark)

    Kongsbak, Martin; Levring, Trine B; Geisler, Carsten

    2013-01-01

    The vitamin D receptor (VDR) is a nuclear, ligand-dependent transcription factor that in complex with hormonally active vitamin D, 1,25(OH)2D3, regulates the expression of more than 900 genes involved in a wide array of physiological functions. The impact of 1,25(OH)2D3-VDR signaling on immune...... function has been the focus of many recent studies as a link between 1,25(OH)2D3 and susceptibility to various infections and to development of a variety of inflammatory diseases has been suggested. It is also becoming increasingly clear that microbes slow down immune reactivity by dysregulating the VDR...

  2. Soluble urokinase plasminogen activator receptor during allogeneic stem cell transplantation

    DEFF Research Database (Denmark)

    Haastrup, E; Andersen, J; Ostrowski, S R

    2011-01-01

    Previous studies have found that soluble urokinase plasminogen activation receptor (suPAR) increases during inflammatory and malignant illness and elevated suPAR levels may be associated with poor clinical outcome. The purpose of this study was to investigate plasma levels of suPAR during...... to pretreatment levels. High suPAR levels at day 0 were associated with increased mortality (P = 0.011). The present study found increased suPAR levels during the conditioning in SCT patients. Further, the data indicated that increased suPAR levels may be associated with increased mortality, suggesting su...

  3. Photoreceptor cells display a daily rhythm in the orphan receptor Esrrβ.

    Science.gov (United States)

    Kunst, Stefanie; Wolloscheck, Tanja; Grether, Markus; Trunsch, Patricia; Wolfrum, Uwe; Spessert, Rainer

    2015-01-01

    Nuclear orphan receptors are critical for the development and long-term survival of photoreceptor cells. In the present study, the expression of the nuclear orphan receptor Esrrβ--a transcriptional regulator of energy metabolism that protects rod photoreceptors from dystrophy--was tested under daily regulation in the retina and photoreceptor cells. The daily transcript and protein amount profiles were recorded in preparations of the whole retina and microdissected photoreceptor cells using quantitative PCR (qPCR) and western blot analysis. Esrrβ displayed a daily rhythm with elevated values at night in the whole retina and enriched photoreceptor cells. Daily regulation of Esrrβ mRNA depended on light input but not on melatonin, and evoked a corresponding rhythm in the Esrrβ protein. The data presented in this study indicate that daily regulation of Esrrβ in photoreceptor cells may contribute to their adaptation to 24-h changes in metabolic demands.

  4. Strong Expression of Chemokine Receptor CXCR4 by Renal Cell Carcinoma Correlates with Advanced Disease

    Directory of Open Access Journals (Sweden)

    Thomas C. Wehler

    2008-01-01

    Full Text Available Diverse chemokines and their receptors have been associated with tumor growth, tumor dissemination, and local immune escape. In different tumor entities, the level of chemokine receptor CXCR4 expression has been linked with tumor progression and decreased survival. The aim of this study was to evaluate the influence of CXCR4 expression on the progression of human renal cell carcinoma. CXCR4 expression of renal cell carcinoma was assessed by immunohistochemistry in 113 patients. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human renal cell carcinoma revealed variable intensities of CXCR4 expression. Strong CXCR4 expression of renal cell carcinoma was significantly associated with advanced T-status (P=.039, tumor dedifferentiation (P = .0005, and low hemoglobin (P = .039. In summary, strong CXCR4 expression was significantly associated with advanced dedifferentiated renal cell carcinoma.

  5. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Ploug, M; Behrendt, N

    1997-01-01

    relative to the reaction in solution. The time course of uPA-catalyzed cleavage of cell-bound uPAR was studied using U937 cells stimulated with phorbol 12-myristate 13-acetate. Only 30 min was required for 10 nM uPA to cleave 50% of the cell-bound uPAR. This uPA-catalyzed cleavage reaction was inhibited...

  6. A systematic approach for peptide characterization of B-cell receptor in chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Díez, Paula; Ibarrola, Nieves; Dégano, Rosa M; Lécrevisse, Quentin; Rodriguez-Caballero, Arancha; Criado, Ignacio; Nieto, Wendy G; Góngora, Rafael; González, Marcos; Almeida, Julia; Orfao, Alberto; Fuentes, Manuel

    2017-06-27

    A wide variety of immunoglobulins (Ig) is produced by the immune system thanks to different mechanisms (V(D)J recombination, somatic hypermutation, and antigen selection). The profiling of Ig sequences (at both DNA and peptide levels) are of great relevance to developing targeted vaccines or treatments for specific diseases or infections. Thus, genomics and proteomics techniques (such as Next-Generation Sequencing (NGS) and mass spectrometry (MS)) have notably increased the knowledge in Ig sequencing and serum Ig peptide profiling in a high-throughput manner. However, the peptide characterization of membrane-bound Ig (e.g., B-cell receptors, BCR) is still a challenge mainly due to the poor recovery of mentioned Ig.Herein, we have evaluated three different sample processing methods for peptide sequencing of BCR belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/MS data are available via ProteomeXchange with identifier PXD004466). Moreover, as a consequence of the results here obtained, recommended guidelines have been described for BCR-sequencing of B-CLL samples by MS approaches.For this purpose, an in-house algorithm has been designed and developed to compare the MS/MS results with those obtained by molecular biology in order to integrate both proteomics and genomics results and establish the steps to follow when sequencing membrane-bound Ig by MS/MS.

  7. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    Directory of Open Access Journals (Sweden)

    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  8. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2012-02-01

    BACKGROUND: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. RESULTS: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. CONCLUSION: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  9. Optogenetic control of chemokine receptor signal and T-cell migration

    Science.gov (United States)

    Xu, Yuexin; Hyun, Young-Min; Lim, Kihong; Lee, Hyunwook; Cummings, Ryan J.; Gerber, Scott A.; Bae, Seyeon; Cho, Thomas Yoonsang; Lord, Edith M.; Kim, Minsoo

    2014-01-01

    Adoptive cell transfer of ex vivo-generated immune-promoting or tolerogenic T cells to either enhance immunity or promote tolerance in patients has been used with some success. However, effective trafficking of the transferred cells to the target tissue sites is the main barrier to achieving successful clinical outcomes. Here we developed a strategy for optically controlling T-cell trafficking using a photoactivatable (PA) chemokine receptor. Photoactivatable-chemokine C-X-C motif receptor 4 (PA-CXCR4) transmitted intracellular CXCR4 signals in response to 505-nm light. Localized activation of PA-CXCR4 induced T-cell polarization and directional migration (phototaxis) both in vitro and in vivo. Directing light onto the melanoma was sufficient to recruit PA-CXCR4–expressing tumor-targeting cytotoxic T cells and improved the efficacy of adoptive T-cell transfer immunotherapy, with a significant reduction in tumor growth in mice. These findings suggest that the use of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with outstanding spatial resolution in tissues and may be feasible in other cell transfer therapies. PMID:24733886

  10. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Hatt Hanns

    2011-08-01

    Full Text Available Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  11. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2011-08-22

    Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  12. HUMAN NK CELLS: FROM SURFACE RECEPTORS TO THE THERAPY OF LEUKEMIAS AND SOLID TUMORS

    Directory of Open Access Journals (Sweden)

    LORENZO eMORETTA

    2014-03-01

    Full Text Available Natural Killer (NK cells are major effector cells of the innate immunity. The discovery, over two decades ago, of MHC-class I specific NK receptors and subsequently of activating receptors, recognizing ligands expressed by tumor or virus-infected cells, paved the way to our understanding of the mechanisms of selective recognition and killing of tumor cells. Although NK cells can efficiently kill tumor cells of different histotypes in vitro, their activity may be limited in vivo by their inefficient trafficking to tumor lesions and by the inhibition of their function induced by tumor cells themselves and by the tumor microenvironment. On the other hand, the important role of NK cells has been clearly demonstrated in the therapy of high risk leukemias in the haploidentical hematopoietic cell (HSC transplantation setting. NK cells derived from donor HSC kill leukemic cells residual after the conditioning regimen, thus preventing leukemia relapses. In addition, they also kill residual dendritic cells and T lymphocytes, thus preventing both GvHD and graft rejection.

  13. Increased angiotensin II type 1 receptor expression in temporal arteries from patients with giant cell arteritis

    DEFF Research Database (Denmark)

    Dimitrijevic, Ivan; Malmsjö, Malin; Andersson, Christina

    2009-01-01

    PURPOSE: Currently, giant cell arteritis (GCA) is primarily treated with corticosteroids or immunomodulating agents, but there is interest in identifying other noncorticosteroid alternatives. Similarities exist in the injury pathways between GCA and atherosclerosis. Angiotensin II is a vasoactive...... peptide involved in vessel inflammation during atherosclerosis, and angiotensin II receptor inhibitors are effective in preventing atherosclerosis. The present study was performed to elucidate the role of angiotensin type 1 (AT(1)) and type 2 (AT(2)) receptors in GCA. DESIGN: Experimental retrospective...... with antibodies for the AT(2) receptor was similar in the patients with GCA and in controls. CONCLUSIONS: These results suggest that AT(1) receptors play a role in the development of GCA. Inhibition of the angiotensin system may thus provide a noncorticosteroid alternative for the treatment of GCA. FINANCIAL...

  14. The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

    International Nuclear Information System (INIS)

    Mei, Lin.

    1989-01-01

    The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 1 type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

  15. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  16. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Onuma, Hirohisa; Inukai, Kouichi, E-mail: kinukai@ks.kyorin-u.ac.jp; Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  17. Targeting non-small cell lung cancer cells by dual inhibition of the insulin receptor and the insulin-like growth factor-1 receptor.

    Directory of Open Access Journals (Sweden)

    Emma E Vincent

    Full Text Available Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R antibody figitumumab in non-small cell lung cancer (NSCLC patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R and IR47-9 (IR, and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.

  18. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  19. Mast cell degranulation following adenosine A3 receptor activation in rats.

    Science.gov (United States)

    Fozard, J R; Pfannkuche, H J; Schuurman, H J

    1996-03-18

    The present studies were carried out to provide further evidence for the hypothesis that the hypotensive response to adenosine A3 receptor activation in the anaesthetized rat involves mediator release from mast cells. Male Sprague-Dawley rats were anaesthetized and given just supramaximal hypotensive doses of either the non-selective A3 receptor agonist, N6(-2)-(4-aminophenyl)ethyladenosine (APNEA: 100 micrograms/kg, preceded by the A1/A2 receptor antagonist, 8-p-(sulphophenyl)theophylline, to "isolate' the A3 receptor-mediated component of the response), the mast cell degranulating agent, compound 48/80 (300 micrograms/kg) or the vehicle for APNEA, intravenously. Blood was withdrawn from a carotid artery between 2 and 10 min after the injection and plasma and serum histamine concentrations measured. Samples of connective tissue (surrounding the abdominal musculature), thymus, mesenteric lymph node, kidney, skin and diaphragm were removed for histological analysis. The plasma and serum histamine concentrations were markedly and significantly higher in the APNEA- or compound 48/80-treated animals compared to vehicle-treated controls. Consistent with this, a substantially greater proportion of mast cells was seen to be undergoing degranulation in all tissues removed from animals treated with APNEA or compound 48/80 compared to those from rats treated with vehicle. Thus, adenosine A3 receptor activation results in rapid mast cell degranulation in the anaesthetized rat. The data provide further evidence of a key role for the mast cell in adenosine A3 receptor-mediated hypotension in this species.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-02-15

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

  1. Non-Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases.

    Science.gov (United States)

    Isozaki, Hideko; Ichihara, Eiki; Takigawa, Nagio; Ohashi, Kadoaki; Ochi, Nobuaki; Yasugi, Masayuki; Ninomiya, Takashi; Yamane, Hiromichi; Hotta, Katsuyuki; Sakai, Katsuya; Matsumoto, Kunio; Hosokawa, Shinobu; Bessho, Akihiro; Sendo, Toshiaki; Tanimoto, Mitsune; Kiura, Katsuyuki

    2016-03-15

    Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult. ©2015 American Association for Cancer Research.

  2. Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay.

    Science.gov (United States)

    Zhu, Zhengrong; Puglisi, Jaime; Connors, David; Stewart, Jeremy; Herbst, John; Marino, Anthony; Sinz, Michael; O'Connell, Jonathan; Banks, Martyn; Dickinson, Kenneth; Cacace, Angela

    2007-03-01

    Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

  3. Receptors for short-chain fatty acids in brush cells at the gastric groove

    Directory of Open Access Journals (Sweden)

    Julia Anna-Maria Eberle

    2014-04-01

    Full Text Available In the stomach of rodents clusters of brush cells are arranged at the gastric groove, immediately at the transition zone from the non-glandular reservoir compartment to the glandular digestive compartment. Based on their taste cell-like molecular phenotype it has been speculated that the cells may be capable to sense constituents of the ingested food, however, searches for nutrient receptors have not been successful. In this study, it was hypothesized that the cells may express receptors for short-chain fatty acids, metabolites generated by microorganisms during the storage of ingested food in the murine forestomach, which lacks the acidic milieu of more posterior regions of the stomach and is colonized with numerous microbiota. Experimental approaches, including RT-PCR analysis and immunohistochemical studies, revealed that the majority of these brush cells express the G-protein coupled receptor types GPR41 (FFAR3 and GPR43 (FFAR2, which are activated by short-chain fatty acids. Both, the GPR41 receptor proteins as well as an appropriate G-protein, α-gustducin, were found to be segregated at the apical brush border of the cells, indicating a direct contact with the luminal content of this gastric region. The exposure of microvillar processes with appropriate receptors and signaling elements to the gastric lumen suggests that the brush cells may in fact be capable to sense the short-chain fatty acids which originate from fermentation processes during the retention of ingested food in the anterior part of the stomach.

  4. GABA agonist promoted formation of low affinity GABA receptors on cerebellar granule cells is restricted to early development

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, Gert Helge; Schousboe, A

    1988-01-01

    The ability of the GABA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) to promote formation of low affinity GABA receptors on cerebellar granule cells was tested using primary cultures of these neurons. Granule cells were exposed to THIP (150 microM) for 6 hr after......, respectively, 4, 7, 10 and 14 days in culture. It was found that THIP treatment of 4- and 7-day-old cultures led to formation of low affinity GABA receptors, whereas such receptors could not be detected after THIP treatment in the older cultures (10 and 14 days) in spite of the fact that these cultured granule...... cells expressed a high density of high affinity GABA receptors. It is concluded that the ability of THIP to promote formation of low affinity GABA receptors on cerebellar granule cells is restricted to an early developmental period....

  5. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    This study aims to investigate EPCR expression in renal tubular epithelial cells and related influencing factors. EPCR expression was assessed by flow cytometry in renal tubular epithelial cells. The effects of some reagents (high glucose, tumor necrosis factor–α and interleukin-1β) were measured by RT-PCR. The results ...

  6. Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate

    DEFF Research Database (Denmark)

    Picone, R; Kajtaniak, E L; Nielsen, Lars Søegaard

    1989-01-01

    A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribu......A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization...... and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits...... in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis....

  7. Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells

    Directory of Open Access Journals (Sweden)

    Sergiy V. Korol

    2018-04-01

    Full Text Available In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100–1000 nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1 but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D. Keywords: GABA, GABAA receptor, Pancreatic islet, Type 2 diabetes

  8. Copy number and nucleotide variation of the LILR family of myelomonocytic cell activating and inhibitory receptors.

    Science.gov (United States)

    López-Álvarez, María R; Jones, Des C; Jiang, Wei; Traherne, James A; Trowsdale, John

    2014-02-01

    Leukocyte immunoglobulin-like receptors (LILR) are cell surface molecules that regulate the activities of myelomonocytic cells through the balance of inhibitory and activation signals. LILR genes are located within the leukocyte receptor complex (LRC) on chromosome 19q13.4 adjacent to KIR genes, which are subject to allelic and copy number variation (CNV). LILRB3 (ILT5) and LILRA6 (ILT8) are highly polymorphic receptors with similar extracellular domains. LILRB3 contains inhibitory ITIM motifs and LILRA6 is coupled to an adaptor with activating ITAM motifs. We analysed the sequences of the extracellular immunoglobulin domain-encoding regions of LILRB3 and LILRA6 in 20 individuals, and determined the copy number of these receptors, in addition to those of other members of the LILR family. We found 41 polymorphic sites within the extracellular domains of LILRB3 and LILRA6. Twenty-four of these sites were common to both receptors. LILRA6, but not LILRB3, exhibited CNV. In 20 out of 48 human cell lines from the International Histocompatibility Working Group, LILRA6 was deleted or duplicated. The only other LILR gene exhibiting genomic aberration was LILRA3, in this case due to a partial deletion.

  9. 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.

  10. 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

  11. ErbB receptors and cell polarity: New pathways and paradigms for understanding cell migration and invasion

    International Nuclear Information System (INIS)

    Feigin, Michael E.; Muthuswamy, Senthil K.

    2009-01-01

    The ErbB family of receptor tyrosine kinases is involved in initiation and progression of a number of human cancers, and receptor activation or overexpression correlates with poor patient survival. Research over the past two decades has elucidated the molecular mechanisms underlying ErbB-induced tumorigenesis, which has resulted in the development of effective targeted therapies. ErbB-induced signal transduction cascades regulate a wide variety of cell processes, including cell proliferation, apoptosis, cell polarity, migration and invasion. Within tumors, disruption of these core processes, through cooperative oncogenic lesions, results in aggressive, metastatic disease. This review will focus on the ErbB signaling networks that regulate migration and invasion and identify a potential role for cell polarity pathways during cancer progression

  12. Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro.

    Science.gov (United States)

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Schaarschmidt, Grit; Härtig, Wolfgang; Schwarz, Sigrid C; Schwarz, Johannes

    2009-10-01

    Human midbrain-derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson's disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole-cell patch-clamp recordings, l-glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration-response plots of differentiated NPCs yielded an EC(50) of 2.2 microM for glutamate and an EC(50) of 36 microM for NMDA. Glutamate-induced currents were markedly inhibited by memantine in contrast to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggesting a higher density of functional NMDA than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. NMDA-evoked currents and calcium signals were blocked by the NR2B-subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage-gated calcium channels by verapamil abolished AMPA-induced calcium responses but only partially reduced NMDA-evoked transients suggesting the expression of calcium-impermeable, GluR2-containing AMPA receptors. Quantitative real-time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 microM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase-immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.

  13. 5-HT2A receptor antagonists inhibit hepatic stellate cell activation and facilitate apoptosis.

    Science.gov (United States)

    Kim, Dong Chan; Jun, Dae Won; Kwon, Young Il; Lee, Kang Nyeong; Lee, Hang Lak; Lee, Oh Young; Yoon, Byung Chul; Choi, Ho Soon; Kim, Eun Kyung

    2013-04-01

    5-hydroxytryptamine (5-HT) receptors are upregulated in activated hepatic stellate cells (HSCs), and are therefore thought to play an important role in their activation. The aim of this study was to determine whether 5-HT2A receptor antagonists affect the activation or apoptosis of HSCs in vitro and/or in vivo. For the in vitro experiments, the viability, apoptosis and wound healing ability of LX-2 cells were examined after treatment with various 5-HT2A receptor antagonists. Levels of HSC activation markers (procollagen type I, α-SMA, TGF-β and Smad 2/3) were measured. For in vivo experiments, rats were divided into three groups: (i) a control group, (ii) a disease group, in which cirrhosis was induced by thioacetamide (iii) a treatment group, in which cirrhosis was induced and a 5-HT2A receptor antagonist (sarpogrelate, 30 mg/kg) was administered. 5-HT2A , but not 5-HT2B receptor mRNA increased with time upon HSC activation. 5-HT2A receptor antagonists (ketanserin and sarpogrelate) inhibited viability and wound healing in LX-2 cells and induced apoptosis. Expression of α-SMA and procollagen type I was also inhibited. In the in vivo study, lobular inflammation was reduced in the sarpogrelate-treated group, but there was only slight and statistically insignificant attenuation of periportal fibrosis. Expression of α-SMA, TGF-β and Smad 2/3 was also reduced in the treatment group. 5-HT2A receptor antagonists can reduce inflammation and the activation of HSCs in this cirrhotic model. © 2013 John Wiley & Sons A/S.

  14. Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Jun Ishii

    Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

  15. GABAB receptor subtypes differentially modulate synaptic inhibition in the dentate gyrus to enhance granule cell output.

    Science.gov (United States)

    Foster, Joshua D; Kitchen, Ian; Bettler, Bernhard; Chen, Ying

    2013-04-01

    Activation of GABAB receptors in the dentate gyrus (DG) enhances granule cell (GC) activity by reducing synaptic inhibition imposed by hilar interneurons. This disinhibitory action facilitates signal transfer from the perforant path to the hippocampus. However, as the two main molecular subtypes, GABA(B(1a,2)) and GABA(B(1b,2)) receptors, prefer axonal terminal and dendritic compartments, respectively, they may modulate the hilar pathways at different synaptic localizations. We examined their relative expression and functions in the DG. The localization of GABAB subtypes was revealed immunohistochemically using subunit-selective antibodies in GABA(B1a)(-/-) and GABA(B1b)(-/-) mice. Effects of subtype activation by the GABAB receptor agonist, baclofen, were examined on the perforant path-stimulated GC population activities in brain slices. GABA(B(1a,2)) receptors were concentrated in the inner molecular layer, the neuropil of the hilus and hilar neurons at the border zone; while GABA(B(1b,2)) receptors dominated the outer molecular layer and hilar neurons in the deep layer, showing their differential localization on GC dendrite and in the hilus. Baclofen enhanced the GC population spike to a larger extent in the GABA(B1b)(-/-) mice, demonstrating exclusively disinhibitory roles of the GABA(B(1a,2)) receptors. Conversely, in the GABA(B1a)(-/-) mice baclofen not only enhanced but also inhibited the population spike during GABAA blockade, revealing both disinhibitory and inhibitory effects of GABA(B(1b,2)) receptors. The GABA(B(1a,2)) and GABA(B(1b,2)) receptor subtypes differentially modulate GC outputs via selective axonal terminal and dendritic locations in the hilar pathways. The GABA(B(1a,2)) receptors exclusively mediate disinhibition, thereby playing a greater role in gating signal transfer for hippocampal spatial and pattern learning. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  16. Involvement of mTOR in Type 2 CRF Receptor Inhibition of Insulin Signaling in Muscle Cells

    OpenAIRE

    Chao, Hongxia; Li, Haochen; Grande, Rebecca; Lira, Vitor; Yan, Zhen; Harris, Thurl E.; Li, Chien

    2015-01-01

    Type 2 corticotropin-releasing factor receptor (CRFR2) is expressed in skeletal muscle and stimulation of the receptor has been shown to inhibit the effect of insulin on glucose uptake in muscle cells. Currently, little is known about the mechanisms underlying this process. In this study, we first showed that both in vivo and in vitro CRFR2 expression in muscle was closely correlated with insulin sensitivity, with elevated receptor levels observed in insulin resistant muscle cells. Stimulatio...

  17. Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro

    International Nuclear Information System (INIS)

    Rubin, L.A.; Kurman, C.C.; Fritz, M.E.; Biddison, W.E.; Boutin, B.; Yarchoan, R.; Nelson, D.L.

    1985-01-01

    With the use of an enzyme-linked immunoabsorbent assay to measure soluble human interleukin 2 receptors (IL 2R), certain human T cell leukemia virus I (HTLV I)-positive T cell lines were found to spontaneously release large quantities of IL 2R into culture supernatants. This was not found with HTLV I-negative and IL 2 independent T cell lines, and only one of seven B cell-derived lines examined produced small amounts of IL 2R. In addition to this constitutive production of soluble IL 2R by certain cell lines, normal human peripheral blood mononuclear cells (PBMC) could be induced to release soluble IL 2R by plant lectins, the murine monoclonal antibody OKT3, tetanus toxoid, and allogeneic cells. Such activated cells also expressed cellular IL 2R measurable in detergent solubilized cell extracts. The generation of cellular and supernatant IL 2R was: dependent on cellular activation, rapid, radioresistant (3000 rad), and inhibited by cycloheximide treatment. NaDodSO 4 -polyacrylamide gel electrophoresis analysis of soluble IL 2R demonstrated molecules of apparent Mr = 35,000 to 40,000, and 45,000 to 50,000, respectively, somewhat smaller than the mature surface receptor on these cells. The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation

  18. Proteinase-Activated Receptor 1 (PAR1) regulates leukemic stem cell functions.

    Science.gov (United States)

    Bäumer, Nicole; Krause, Annika; Köhler, Gabriele; Lettermann, Stephanie; Evers, Georg; Hascher, Antje; Bäumer, Sebastian; Berdel, Wolfgang E; Müller-Tidow, Carsten; Tickenbrock, Lara

    2014-01-01

    External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.

  19. Proteinase-Activated Receptor 1 (PAR1 regulates leukemic stem cell functions.

    Directory of Open Access Journals (Sweden)

    Nicole Bäumer

    Full Text Available External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.

  20. CD3 receptor modulation in Jurkat leukemic cell line.

    Directory of Open Access Journals (Sweden)

    Jacek M Witkowski

    2004-03-01

    Full Text Available CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low (217.6, CD3+(217.9 or CD3(low (217.7. The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.

  1. Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.

    Science.gov (United States)

    Feyaerts, D; Kuret, T; van Cranenbroek, B; van der Zeeuw-Hingrez, S; van der Heijden, O W H; van der Meer, A; Joosten, I; van der Molen, R G

    2018-02-13

    Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR

  2. Placental Kisspeptins Differentially Modulate Vital Parameters of Estrogen Receptor-Positive and -Negative Breast Cancer Cells

    Science.gov (United States)

    Rasoulzadeh, Zahra; Ghods, Roya; Kazemi, Tohid; Mirzadegan, Ebrahim; Ghaffari-Tabrizi-Wizsy, Nassim; Rezania, Simin; Kazemnejad, Somaieh; Arefi, Soheila; Ghasemi, Jamileh; Vafaei, Sedigheh; Mahmoudi, Ahmad-Reza; Zarnani, Amir-Hassan

    2016-01-01

    Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2–30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production. PMID:27101408

  3. Mediation of autophagic cell death by type 3 ryanodine receptor (RyR3 in adult hippocampal neural stem cells

    Directory of Open Access Journals (Sweden)

    Kyung Min eChung

    2016-05-01

    Full Text Available Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs and IP3 receptors (IP3Rs, the main Ca2+ release channels located in endoplasmic reticulum (ER membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN cells utilizing an insulin withdrawal model of autophagic cell death. Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs — especially RyR3 — were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished autophagic cell death of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology.

  4. Effect of glucocorticoids on melatonin receptor expression under T-cell activated immune response

    International Nuclear Information System (INIS)

    Tauschanova, P.; Georgiev, G.; Manchev, S.; Konakchieva, R.

    2007-01-01

    The present study was aimed to explore the stress response in rats under conditions of T-cell antigen-activated immune function and to investigate the specific melatonin (MEL) receptor binding in primary and secondary immune tissue of rats employing 2-( 125 I)-iodo melatonin autoradiography and in vitro ligand binding assay. The study revealed that melatonin receptor binding was specifically expressed in discrete areas of the lymphoid sheath of the spleen and in a network of interdigitating cells of the experimental rats. Demonstration of the modulation of MEL receptor binding in the course of a primary immune response under hypercorticalemic conditions indicate that the pineal hormone might interfere in the processes of glucocorticoid-dependent immune competency. (authors)

  5. Immunolocalization of progesterone receptors in binucleate trophoblast cells of the buffalo placenta (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Carlos Eduardo Ambrósio

    2007-06-01

    Full Text Available The binucleate trophoblast cells (CTBs of the water buffalo placenta (Bubalus bubalis were studied with emphasis on the presence of progesterone receptor. Placentomal tissues from 27 buffalos (2-10 months of pregnancy were processed and embedded in paraplast (Paraplast Embedding Media – Paraplast Plus to locate the progesterone receptors using the immunohistochemistry technique. The immunohistochemical reaction for progesterone receptor through monoclonal antibody PgR Ab2 showed staining of CTBs, caruncular epithelial and estromal cells and blood vessel estromal pericitos present in the placentome throughout the entire gestational period analyzed. These results indicate the production of progesterone with autocrine and paracrine action in the placentome growth, differentiation and functional regulation.

  6. Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes

    International Nuclear Information System (INIS)

    Hagopian, W.A.; Tager, H.S.

    1984-01-01

    A reverse-phase HPLC method has been developed to purify 125 I-labeled products resulting from the chloramine-T-based iodination of glucagon. In addition the products [( 125 I)iodoTyr 10 13 ]glucagon, [( 125 I)iodoTyr 13 ]glucagon, and [( 125 I)iodoTyr 10 ]glucagon) have been used to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. It was concluded that (a) not withstanding apparent differences in affinities exhibited by the three peptides, the interactions with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH 2 -terminal region of the hormone has been modified by limited proteolytic cleavage

  7. Neuroprotective Effect of σ1-Receptors on the Cell Model of Huntington's Disease.

    Science.gov (United States)

    Bol'shakova, A V; Kraskovskaya, N A; Gainullina, A N; Kukanova, E O; Vlasova, O L; Bezprozvanny, I B

    2017-12-01

    Huntington's disease is a hereditary neurodegenerative disease that primarily affects striatal neurons. Recent studies demonstrated abnormalities in calcium regulation in striatal neurons in Huntington's disease, which leads to elimination of synaptic connections between cortical and striatal neurons. In the present study, we focused on the neuroprotective properties of σ1-receptor, because one of its main functions is associated with modulation of calcium homeostasis in cells. The application of selective σ1-receptor agonists to the corticostriatal cell culture restores synaptic connections between the cortical and striatal neurons. Based on the obtained data, we assume that σ1-receptor is a promising target for the development of drugs for the therapy of Huntington's disease.

  8. Impact of blood processing variations on Natural Killer cell frequency, activation, chemokine receptor expression and function

    Science.gov (United States)

    Naranbhai, Vivek; Bartman, Pat; Ndlovu, Dudu; Ramkalawon, Pamela; Ndung’u, Thumbi; Wilson, Douglas; Altfeld, Marcus; Carr, William H

    2011-01-01

    Understanding the role of natural killer (NK) cells in human disease pathogenesis is crucial and necessitates study of patient samples directly ex vivo. Manipulation of whole blood by density gradient centrifugation or delays in sample processing due to shipping, however, may lead to artifactual changes in immune response measures. Here, we assessed the impact of density gradient centrifugation and delayed processing of both whole blood and peripheral blood mononuclear cells (PBMC) at multiple timepoints (2–24 hrs) on flow cytometric measures of NK cell frequency, activation status, chemokine receptor expression, and effector functions. We found that density gradient centrifugation activated NK cells and modified chemokine receptor expression. Delays in processing beyond 8 hours activated NK cells in PBMC but not in whole blood. Likewise, processing delays decreased chemokine receptor (CCR4 and CCR7) expression in both PBMC and whole blood. Finally, delays in processing PBMC were associated with a decreased ability of NK cells to degranulate (as measured by CD107a expression) or secrete cytokines (IFN-γ and TNF-α). In summary, our findings suggest that density gradient centrifugation and delayed processing of PBMC can alter measures of clinically relevant NK cell characteristics including effector functions; and therefore should be taken into account in designing clinical research studies. PMID:21255578

  9. Gastric Cancer Cell Glycosylation as a Modulator of the ErbB2 Oncogenic Receptor

    Directory of Open Access Journals (Sweden)

    Henrique O. Duarte

    2017-10-01

    Full Text Available Aberrant expression and hyperactivation of the human epidermal growth factor receptor 2 (ErbB2 constitute crucial molecular events underpinning gastric neoplastic transformation. Despite ErbB2 extracellular domain being a well-known target for glycosylation, its glycosylation profile and the molecular mechanisms through which it actively tunes tumorigenesis in gastric cancer (GC cells remain elusive. We aimed at disclosing relevant ErbB2 glycan signatures and their functional impact on receptor’s biology in GC cells. The transcriptomic profile of cancer-relevant glycosylation enzymes, and the expression and activation of the ErbB receptors were characterized in four GC cell lines. Cellular- and receptor-specific glycan profiling of ErbB2-overexpressing NCI-N87 cells unveiled a heterogeneous glycosylation pattern harboring the tumor-associated sialyl Lewis a (SLea antigen. The expression of SLea and key enzymes integrating its biosynthetic pathway were strongly upregulated in this GC cell line. An association between the expression of ERBB2 and FUT3, a central gene in SLea biosynthesis, was disclosed in GC patients, further highlighting the crosstalk between ErbB2 and SLea expression. Moreover, cellular deglycosylation and CA 19.9 antibody-mediated blocking of SLea drastically altered ErbB2 expression and activation in NCI-N87 cells. Altogether, NCI-N87 cell line constitutes an appealing in vitro model to address glycan-mediated regulation of ErbB2 in GC.

  10. A new insight in chimeric antigen receptor-engineered T cells for cancer immunotherapy

    Directory of Open Access Journals (Sweden)

    Erhao Zhang

    2017-01-01

    Full Text Available Abstract Adoptive cell therapy using chimeric antigen receptor (CAR-engineered T cells has emerged as a very promising approach to combating cancer. Despite its ability to eliminate tumors shown in some clinical trials, CAR-T cell therapy involves some significant safety challenges, such as cytokine release syndrome (CRS and “on-target, off-tumor” toxicity, which is related to poor control of the dose, location, and timing of T cell activity. In the past few years, some strategies to avoid the side effects of CAR-T cell therapy have been reported, including suicide gene, inhibitory CAR, dual-antigen receptor, and the use of exogenous molecules as switches to control the CAR-T cell functions. Because of the advances of the CAR paradigm and other forms of cancer immunotherapy, the most effective means of defeating the cancer has become the integration therapy with the combinatorial control system of switchable dual-receptor CAR-T cell and immune checkpoint blockade.

  11. The regulation of steroid receptors by epigallocatechin-3-gallate in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Hallman K

    2017-05-01

    Full Text Available Kelly Hallman,* Katie Aleck,* Meghan Quigley, Brigitte Dwyer, Victoria Lloyd, Monica Szmyd, Sumi Dinda Biomedical Diagnostic and Therapeutic Sciences, School of Health Sciences, Center for Biomedical Research, Oakland University, Rochester, MI, USA *These authors contributed equally to this work Abstract: It has been reported that phytoestrogen epigallocatechin gallate (EGCG suppresses cancer cell proliferation and may have antitumor properties. In this study, we analyzed the effects of EGCG on estrogen receptor α (ERα and progesterone receptor in hormone-dependent T-47D breast cancer cells. Western blot analysis revealed EGCG induced a concentration-dependent decrease in ERα protein levels, with a 56% reduction occurring with 60 µM EGCG when compared to controls. Downregulation of ERα protein levels was observed after 24-hour co-treatment of T-47D cells with 60 µM EGCG and 10 nM 17β-estradiol (E2. The proliferative effect of E2 on cell viability was reversed when treated in combination with EGCG. In contrast, the combination of EGCG with the pure ER antagonist, ICI 182, 780, showed no further reduction in cell number as only 5% of the cells were viable after 6 days of treatment. These studies may provide further understanding of the interactions among flavonoids and steroid receptors in breast cancer cells. Keywords: phytoestrogen, ER, PR, T-47D, antiestrogens

  12. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

    Science.gov (United States)

    Parrish-Novak, J; Dillon, S R; Nelson, A; Hammond, A; Sprecher, C; Gross, J A; Johnston, J; Madden, K; Xu, W; West, J; Schrader, S; Burkhead, S; Heipel, M; Brandt, C; Kuijper, J L; Kramer, J; Conklin, D; Presnell, S R; Berry, J; Shiota, F; Bort, S; Hambly, K; Mudri, S; Clegg, C; Moore, M; Grant, F J; Lofton-Day, C; Gilbert, T; Rayond, F; Ching, A; Yao, L; Smith, D; Webster, P; Whitmore, T; Maurer, M; Kaushansky, K; Holly, R D; Foster, D

    2000-11-02

    Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

  13. Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

    Directory of Open Access Journals (Sweden)

    Fu-Nan Cho

    Full Text Available Natural killer (NK cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs, which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

  14. Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

    Science.gov (United States)

    Cho, Fu-Nan; Chang, Tsung-Hsien; Shu, Chih-Wen; Ko, Ming-Chin; Liao, Shuen-Kuei; Wu, Kang-Hsi; Yu, Ming-Sun; Lin, Shyh-Jer; Hong, Ying-Chung; Chen, Chien-Hsun; Hung, Chien-Hui; Chang, Yu-Hsiang

    2014-01-01

    Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

  15. Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow.

    Science.gov (United States)

    Jung, Younghun; Decker, Ann M; Wang, Jingcheng; Lee, Eunsohl; Kana, Lulia A; Yumoto, Kenji; Cackowski, Frank C; Rhee, James; Carmeliet, Peter; Buttitta, Laura; Morgan, Todd M; Taichman, Russell S

    2016-05-03

    GAS6 and its receptors (Tryo 3, Axl, Mer or "TAM") are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment.We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133-/CD44-) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G1) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo.Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

  16. The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

    Science.gov (United States)

    Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

    2004-09-01

    Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

  17. Extracellular Matrix Regulation of Estrogen Receptors in Mouse Mammary Cells

    National Research Council Canada - National Science Library

    Novaro, Virginia

    2002-01-01

    .... We present evidence that cell adhesion to the BM components collagen-IV, through alpha 2 and beta 1 integrin subunits and laminin-l, through alpha 2, alpha 6 and beta 1 subunits are the relevant...

  18. Effects of melatonin and its receptor antagonist on retinal pigment epithelial cells against hydrogen peroxide damage

    Science.gov (United States)

    Rosen, Richard B.; Hu, Dan-Ning; Chen, Min; McCormick, Steven A.; Walsh, Joseph

    2012-01-01

    Purpose Recently, we reported finding that circulating melatonin levels in age-related macular degeneration patients were significantly lower than those in age-matched controls. The purpose of this study was to investigate the hypothesis that melatonin deficiency may play a role in the oxidative damage of the retinal pigment epithelium (RPE) by testing the protective effect of melatonin and its receptor antagonist on RPE cells exposed to H2O2 damage. Methods Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H2O2. Cell viability was measured using the microculture tetrazoline test (MTT) assay. Cells were pretreated with or without melatonin for 24 h. Luzindole (50 μM), a melatonin membrane-receptor antagonist, was added to the culture 1 h before melatonin to distinguish direct antioxidant effects from indirect receptor-dependent effects. All tests were performed in triplicate. Results H2O2 at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H2O2. Cell viability of RPE cells pretreated with 10−10, 10−8, 10−6, and 10−4 M melatonin for 24 h was 130%, 160%, 187%, and 230% of cells treated with H2O2 alone (all p<0.05). Using cells cultured without H2O2 as the control, cell viability of cells treated with H2O2 after pretreatment with 10−10-10−4 M melatonin was still significantly lower than that of the controls, suggesting that melatonin significantly decreased but did not completely abolish the in vitro cytotoxic effects of H2O2. Luzindole completely blocked melatonin’s protective effects at low concentrations of melatonin (10−10-10−8 M) but not at high concentrations (10−6-10−4 M). Conclusions Melatonin has a partial protective effect on RPE cells against H2O2 damage across a wide range of concentrations (10−10-10−4 M). This protective effect occurs through the activation of melatonin membrane receptors at low concentrations (10−10

  19. An equine infectious anemia virus variant superinfects cells through novel receptor interactions.

    Science.gov (United States)

    Brindley, Melinda A; Zhang, Baoshan; Montelaro, Ronald C; Maury, Wendy

    2008-10-01

    Wild-type strains of equine infectious anemia virus (EIAV) prevent superinfection of previously infected cells. A variant strain of virus that spontaneously arose during passage, EIAV(vMA-1c), can circumvent this mechanism in some cells, such as equine dermis (ED) cells, but not in others, such as equine endothelial cells. EIAV(vMA-1c) superinfection of ED cells results in a buildup of unintegrated viral DNA and rapid killing of the cell monolayer. Here, we examined the mechanism of resistance that is used by EIAV to prevent superinfection and explored the means by which EIAV(vMA-1c) overcomes this restriction. We found that the cellular receptor used by EIAV, equine lentivirus receptor 1 (ELR1), remains on the surface of cells chronically infected with EIAV, suggesting that wild-type EIAV interferes with superinfection by masking ELR1. The addition of soluble wild-type SU protein to the medium during infection blocked infection by wild-type strains of virus, implicating SU as the viral protein responsible for interfering with virion entry into previously infected cells. Additionally, interference of wild-type EIAV binding to ELR1 by the addition of either anti-ELR1 antibodies or the ELR1 ectodomain prevented entry of the wild-type strains of EIAV into two permissive cell populations. Many of these same interference treatments prevented EIAV(vMA-1c) infection of endothelial cells but only modestly affected the ability of EIAV(vMA-1c) to enter and kill previously infected ED cells. These findings indicate that EIAV(vMA-1c) retains the ability to use ELR1 for entry and suggest that this virus can interact with an additional, unidentified receptor to superinfect ED cells.

  20. NF-κB Protects NKT Cells from Tumor Necrosis Factor Receptor 1-induced Death.

    Science.gov (United States)

    Kumar, Amrendra; Gordy, Laura E; Bezbradica, Jelena S; Stanic, Aleksandar K; Hill, Timothy M; Boothby, Mark R; Van Kaer, Luc; Joyce, Sebastian

    2017-11-15

    Semi-invariant natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. NKT cell survival during development requires signal processing by activated RelA/NF-κB. Nonetheless, the upstream signal(s) integrated by NF-κB in developing NKT cells remains incompletely defined. We show that the introgression of Bcl-x L -coding Bcl2l1 transgene into NF-κB signalling-deficient IκBΔN transgenic mouse rescues NKT cell development and differentiation in this mouse model. We reasoned that NF-κB activation was protecting developing NKT cells from death signals emanating either from high affinity agonist recognition by the T cell receptor (TCR) or from a death receptor, such as tumor necrosis factor receptor 1 (TNFR1) or Fas. Surprisingly, the single and combined deficiency in PKC-θ or CARMA-1-the two signal transducers at the NKT TCR proximal signalling node-only partially recapitulated the NKT cell deficiency observed in IκBΔN tg mouse. Accordingly, introgression of the Bcl2l1 transgene into PKC-θ null mouse failed to rescue NKT cell development. Instead, TNFR1-deficiency, but not the Fas-deficiency, rescued NKT cell development in IκBΔN tg mice. Consistent with this finding, treatment of thymocytes with an antagonist of the inhibitor of κB kinase -which blocks downstream NF-κB activation- sensitized NKT cells to TNF-α-induced cell death in vitro. Hence, we conclude that signal integration by NF-κB protects developing NKT cells from death signals emanating from TNFR1, but not from the NKT TCR or Fas.

  1. Cooperation of tyrosine kinase receptor TrkB and epidermal growth factor receptor signaling enhances migration and dispersal of lung tumor cells.

    Directory of Open Access Journals (Sweden)

    Rudolf Götz

    Full Text Available TrkB mediates the effects of brain-derived neurotrophic factor (BDNF in neuronal and nonnneuronal cells. Based on recent reports that TrkB can also be transactivated through epidermal growth-factor receptor (EGFR signaling and thus regulates migration of early neurons, we investigated the role of TrkB in migration of lung tumor cells. Early metastasis remains a major challenge in the clinical management of non-small cell lung cancer (NSCLC. TrkB receptor signaling is associated with metastasis and poor patient prognosis in NSCLC. Expression of this receptor in A549 cells and in another adenocarcinoma cell line, NCI-H441, promoted enhanced migratory capacity in wound healing assays in the presence of the TrkB ligand BDNF. Furthermore, TrkB expression in A549 cells potentiated the stimulatory effect of EGF in wound healing and in Boyden chamber migration experiments. Consistent with a potential loss of cell polarity upon TrkB expression, cell dispersal and de-clustering was induced in A549 cells independently of exogeneous BDNF. Morphological transformation involved extensive cytoskeletal changes, reduced E-cadherin expression and suppression of E-cadherin expression on the cell surface in TrkB expressing tumor cells. This function depended on MEK and Akt kinase activity but was independent of Src. These data indicate that TrkB expression in lung adenoma cells is an early step in tumor cell dissemination, and thus could represent a target for therapy development.

  2. Viability of D283 medulloblastoma cells treated with a histone deacetylase inhibitor combined with bombesin receptor antagonists.

    Science.gov (United States)

    Jaeger, Mariane; Ghisleni, Eduarda C; Fratini, Lívia; Brunetto, Algemir L; Gregianin, Lauro José; Brunetto, André T; Schwartsmann, Gilberto; de Farias, Caroline B; Roesler, Rafael

    2016-01-01

    Medulloblastoma (MB) comprises four distinct molecular subgroups, and survival remains particularly poor in patients with Group 3 tumors. Mutations and copy number variations result in altered epigenetic regulation of gene expression in Group 3 MB. Histone deacetylase inhibitors (HDACi) reduce proliferation, promote cell death and neuronal differentiation, and increase sensitivity to radiation and chemotherapy in experimental MB. Bombesin receptor antagonists potentiate the antiproliferative effects of HDACi in lung cancer cells and show promise as experimental therapies for several human cancers. Here, we examined the viability of D283 cells, which belong to Group 3 MB, treated with an HDACi alone or combined with bombesin receptor antagonists. D283 MB cells were treated with different doses of the HDACi sodium butyrate (NaB), the neuromedin B receptor (NMBR) antagonist BIM-23127, the gastrin releasing peptide receptor (GRPR) antagonist RC-3095, or combinations of NaB with each receptor antagonist. Cell viability was examined by cell counting. NaB alone or combined with receptor antagonists reduced cell viability at all doses tested. BIM-23127 alone did not affect cell viability, whereas RC-3095 at an intermediate dose significantly increased cell number. Although HDACi are promising agents to inhibit MB growth, the present results provide preliminary evidence that combining HDACi with bombesin receptor antagonists is not an effective strategy to improve the effects of HDACi against MB cells.

  3. Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells.

    Science.gov (United States)

    Rasmussen, Andreas H; Strøbæk, Dorte; Dyhring, Tino; Jensen, Marianne L; Peters, Dan; Grunnet, Morten; Timmermann, Daniel B; Ahring, Philip K

    2014-01-13

    Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6 receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6 receptor expression. Combinations of chimeric and mutated α6, β2 and β3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca(2+)-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3(V9'S) gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3(V9'S):β2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type β3, significant functional expression of α6/α3β2β3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of α6/α3β2β3(V9'S) to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca(2+)-imaging experiments showed preservation of basic α6 receptor pharmacology. Our results establish that α6/α3β2β3(V9'S) replicate several basic features of native α6 receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts. © 2013 Published by Elsevier B.V.

  4. Purinergic Receptors in Quiescence and Localization of Leukemic Stem Cells

    Science.gov (United States)

    2012-05-01

    samples, five human acute leukemia samples (AML) (M0 or M1) were chosen based on FAB classification , cytogenetic abnormalities, CD34 and CD14...treatment) to xenotransplanted human leukemia cells. 9 Patient Age FAB Cytogenetic abnormalities Blast CD34+ CD14+ 104 66 M0/M1 Negative PML-RARA...Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT How leukemia stem cells gained resistance to

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

    DEFF Research Database (Denmark)

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

    2018-01-01

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

  6. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-08-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  7. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  8. Nuclear thyroid hormone receptor binding in human mononuclear blood cells after goitre resection

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E; Blichert-Toft, M

    1989-01-01

    Nuclear thyroxine and triiodothyronine receptor-binding in human mononuclear blood cells were examined in 14 euthyroid persons prior to and 1, 6, 24 and 53 weeks after goitre resection. One week after resection decreased serum T3 from 1.47 nmol/l to 1.14 nmol/l (P less than 0.05), FT4I from 103 a...

  9. Novel primary thymic defect with T lymphocytes expressing gamma delta T cell receptor

    DEFF Research Database (Denmark)

    Geisler, C; Pallesen, G; Platz, P

    1989-01-01

    . Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that most of the gamma delta T cell receptors existed as disulphide-linked heterodimers. Proliferative responses to mitogens were severely reduced, but specific antibody responses after vaccination could be detected...

  10. Endothelin receptors stimulate both phospholipase C and phospholipase D activities in different cell lines.

    Science.gov (United States)

    Ambar, I; Sokolovsky, M

    1993-03-15

    Endothelin (ET) receptor-binding assays using [125I]ET-1 in C6-glioma cells and in Rat-1 and Swiss 3T3 fibroblasts indicated the presence of two binding sites, one of which binds agonists at the pM range and the other at the nM range. All three cell lines exhibited the same pharmacological profile for agonist binding (ET-1 congruent to sarafotoxin-b > ET-3), which suggests that the receptor is of the ETA type. Binding of ET-1 to the receptor resulted in activation of two phospholipases, phospholipase C (PLC) and phospholipase D (PLD). The activation of PLC or PLD by endothelin in the three cell lines was mediated by the high affinity binding site (nM range) and was not significantly affected by either extracellular or intracellular Ca2+. Measurement of PLD activation by ET-1 and/or phorbol 12-myristate 13-acetate (PMA), in the presence and absence of two potent inhibitors of protein kinase C (PKC), strongly suggests that activation of PLD by ET receptor in C6 glioma cells as well as in Rat-1 and Swiss 3T3 fibroblasts involves both PKC-dependent and PKC-independent mechanisms.

  11. Functional pharmacology of cloned heterodimeric GABA-B receptors expressed in mammalian cells

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Krogsgaard-Larsen, P

    1999-01-01

    1. In this study we report a new assay of heterodimeric gamma-amino-butanoic acid subtype B (GABAB) receptors where either GABABR1a or GABABR1b are co-expressed with GABABR2 and the chimeric G-protein Galphaq-z5 in tsA cells. In this manner we obtained a robust response to GABAB agonists measured...

  12. Lenalidomide enhances antitumor functions of chimeric antigen receptor modified T cells

    Czech Academy of Sciences Publication Activity Database

    Otáhal, Pavel; Průková, D.; Král, Vlastimil; Fábry, Milan; Vockova, P.; Lateckova, L.; Trněný, M.; Klener, P.

    2016-01-01

    Roč. 5, č. 4 (2016), č. článku e1115940. ISSN 2162-402X R&D Projects: GA MZd(CZ) NT13201 Institutional support: RVO:68378050 Keywords : Chimeric anti genic receptor * lenalidomide * lymphoma * tumor immunotherapy * T cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.719, year: 2016

  13. Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP

    NARCIS (Netherlands)

    L.J. Blok (Leen); J.E. Perry; J.K. Lindzey; D.J. Tindall; Y. Gong (Yuewen)

    1995-01-01

    textabstractElevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of

  14. MicroRNAs regulate B-cell receptor signaling-induced apoptosis

    NARCIS (Netherlands)

    Kluiver, J. L.; Chen, C-Z

    Apoptosis induced by B-cell receptor (BCR) signaling is critical for antigen-driven selection, a process critical to tolerance and immunity. Here, we examined the roles of microRNAs (miRNAs) in BCR signaling-induced apoptosis using the widely applied WEHI-231 model. Comparison of miRNA levels in

  15. The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

    DEFF Research Database (Denmark)

    Christensen, Claus; Lauridsen, Jes B; Berezin, Vladimir

    2006-01-01

    The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface...

  16. Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice

    NARCIS (Netherlands)

    Ellenbroek, J.H.; Tons, H.A.; Westerouen van Meeteren, M.J.; de Graaf, N.; Hanegraaf, M.A.; Rabelink, T.J.; Carlotti, F.; de Koning, E.J.

    2013-01-01

    AIMS/HYPOTHESIS: Incretin-based therapies improve glycaemic control in patients with type 2 diabetes. In animal models of diabetes, glucagon-like peptide-1 receptor agonists (GLP-1RAs) increase beta cell mass. GLP-1RAs are also evaluated in non-diabetic individuals with obesity and cardiovascular

  17. Studying NK cell lectin receptors and their interactions using HEK293T eukaryotic expression system

    Czech Academy of Sciences Publication Activity Database

    Vaněk, O.; Celadová, P.; Kolenko, Petr; Dohnálek, Jan; Bezouška, Karel

    2009-01-01

    Roč. 276, Suppl. 1 (2009), s. 170 ISSN 1742-464X. [FEBS Congress "Life´s Molecular Interactions /34./. 04.07.2009-09.07.2009, Praha] Institutional research plan: CEZ:AV0Z40500505 Keywords : NK cell lectin receptors * HEK293T * eukaryotic expression system Subject RIV: CD - Macromolecular Chemistry

  18. Amplification of epidermal growth factor receptor gene in renal cell carcinoma

    DEFF Research Database (Denmark)

    El-Hariry, Iman; Powles, Thomas; Lau, Mike R

    2010-01-01

    Expression of epidermal growth factor receptor (EGFR) may be of prognostic value in renal cell cancer (RCC). Gene amplification of EGFR was investigated in a cohort of 315 patients with advanced RCC from a previously reported randomised study. Using fluorescent in situ hybridisation, only 2 patie...

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

    NARCIS (Netherlands)

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

    1988-01-01

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

  20. Impact of NMDA Receptor Overexpression on Cerebellar Purkinje Cell Activity and Motor Learning

    NARCIS (Netherlands)

    Galliano, Elisa; Schonewille, Martijn; Peter, Saša; Rutteman, Mandy; Houtman, Simone; Jaarsma, Dick; Hoebeek, Freek E; De Zeeuw, Chris I

    2018-01-01

    In many brain regions involved in learning NMDA receptors (NMDARs) act as coincidence detectors of pre- and postsynaptic activity, mediating Hebbian plasticity. Intriguingly, the parallel fiber (PF) to Purkinje cell (PC) input in the cerebellar cortex, which is critical for procedural learning,

  1. Bacillus thuringiensis toxins trigger receptor shedding from gypsy moth midgut cells

    Science.gov (United States)

    Algimantas P. Valaitis

    2007-01-01

    The mechanism of action of the Cry1 insecticidal proteins produced by Bacillus thuringiensis (Bt) begins with the processing of these proteins in the larval gut. After proteolytic activation, the Bt toxins bind to specific midgut receptors and insert into the membrane of the gut epithelial cells, causing insect death.

  2. LH-Receptor Gene Expression in Human Granulosa and Cumulus Cells from Antral and Preovulatory Follicles

    DEFF Research Database (Denmark)

    Jeppesen, Janni Vikkelsø; Kristensen, Stine Gry; Nielsen, Maria Eilsø

    2012-01-01

    Context: Human granulosa cells (GC) acquire LH receptor (LHR) expression during the follicular phase of the menstrual cycle. Currently, the precise follicular stage is unknown, and specific roles of LH in the follicular development are not fully understood. Objective: Our objective was to measure...

  3. Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis

    NARCIS (Netherlands)

    Determann, Rogier M.; Weisfelt, Martijn; de Gans, Jan; van der Ende, Arie; Schultz, Marcus J.; van de Beek, Diederik

    2006-01-01

    OBJECTIVE: To evaluate whether soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in CSF can serve as a biomarker for the presence of bacterial meningitis and outcome in patients with this disease. DESIGN: Retrospective study of diagnostic accuracy. SETTING AND PATIENTS: CSF was

  4. Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor

    International Nuclear Information System (INIS)

    Schols, D.; Baba, M.; Pauwels, R.; Desmyter, J.; De Clercq, E.

    1989-01-01

    The triphenylmethane derivative aurintricarboxylic acid (ATA), but not aurin, selectively prevented the binding of OKT4A/Leu-3a mo