Sample records for cell receptor profiles

  1. Mother and child T cell receptor repertoires: deep profiling study

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    Ekaterina V Putintseva


    Full Text Available The relationship between maternal and child immunity has been actively studied in the context of complications during pregnancy, autoimmune diseases, and haploidentical transplantation of hematopoietic stem cells (HSC and solid organs. Here, we have for the first time used high-throughput Illumina HiSeq sequencing to perform deep quantitative profiling of T-cell receptor (TCR repertoires for peripheral blood samples of three mothers and their six children. Advanced technology allowed accurate identification of 5х105–2х106 TCR beta clonotypes per individual. We performed comparative analysis of these TCR repertoires with the aim of revealing characteristic features that distinguish related mother-child pairs, such as relative TRBV segment usage frequency and relative overlap of TCR beta CDR3 repertoires. We show that thymic selection essentially and similarly shapes the initial output of the TCR recombination machinery in both related and unrelated pairs, with minor effect from inherited differences. The achieved depth of TCR profiling also allowed us to test the hypothesis that mature T cells transferred across the placenta during pregnancy can expand and persist as functional microchimeric clones in their new host, using characteristic TCR beta CDR3 variants as clonal identifiers.

  2. Activation profiles of opioid ligands in HEK cells expressing δ opioid receptors


    Clark J; Demirci Hasan; Gharagozlou Parham; Lameh Jelveh


    Abstract Background The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Results Naltrexone and nalorphine were classified as antagonists at δ opioid receptor....

  3. Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells. (United States)

    Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi


    GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca(2+) influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

  4. Progesterone Receptor Subcellular Localization and Gene Expression Profile in Human Astrocytoma Cells Are Modified by Progesterone

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    Aliesha González-Arenas


    Full Text Available Intracellular progesterone receptor (PR has been identified in human astrocytomas, the most common and aggressive primary brain tumors in humans. It has been reported that PR cell distribution affects their transcriptional activity and turnover. In this work we studied by immunofluorescence the effects of estradiol and progesterone on the subcellular localization of PR in a grade III human astrocytoma derived cell line (U373. We observed that total PR was mainly distributed in the cytoplasm without hormonal treatment. Estradiol (10 nM increased PR presence in the cytoplasm of U373 cells, whereas progesterone (10 nM and RU486 (PR antagonist, 1 μM blocked this effect. To investigate the role of PR activity in the regulation of gene expression pattern of U373 cells, we evaluated by microarray analysis the profile of genes regulated by progesterone, RU486, or both steroids. We found different genes regulated by steroid treatments that encode for proteins involved in metabolism, transport, cell cycle, proliferation, metastasis, apoptosis, processing of nucleic acids and proteins, adhesion, pathogenesis, immune response, cytoskeleton, and membrane receptors. We determined that 30 genes were regulated by progesterone, 41 genes by RU486 alone, and 13 genes by the cotreatment of progesterone+RU486, suggesting that there are many genes regulated by intracellular PR or through other signaling pathways modulated by progesterone. All these data suggest that PR distribution and activity should modify astrocytomas growth.

  5. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells. (United States)

    te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A


    Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

  6. Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous-cell carcinoma. (United States)

    Syed, Nazia; Barbhuiya, Mustafa A; Pinto, Sneha M; Nirujogi, Raja Sekhar; Renuse, Santosh; Datta, Keshava K; Khan, Aafaque Ahmad; Srikumar, Kotteazeth; Prasad, T S Keshava; Kumar, M Vijaya; Kumar, Rekha Vijay; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha


    Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early-stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non-neoplastic Het-1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry-based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA-based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.

  7. A Cell Model for Conditional Profiling of Androgen-Receptor-Interacting Proteins

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    K. A. Mooslehner


    Full Text Available Partial androgen insensitivity syndrome (PAIS is associated with impaired male genital development and can be transmitted through mutations in the androgen receptor (AR. The aim of this study is to develop a cell model suitable for studying the impact AR mutations might have on AR interacting proteins. For this purpose, male genital development relevant mouse cell lines were genetically modified to express a tagged version of wild-type AR, allowing copurification of multiprotein complexes under native conditions followed by mass spectrometry. We report 57 known wild-type AR-interacting proteins identified in cells grown under proliferating and 65 under nonproliferating conditions. Of those, 47 were common to both samples suggesting different AR protein complex components in proliferating and proliferation-inhibited cells from the mouse proximal caput epididymus. These preliminary results now allow future studies to focus on replacing wild-type AR with mutant AR to uncover differences in protein interactions caused by AR mutations involved in PAIS.

  8. Expression Profiles of the Nuclear Receptors and Their Transcriptional Coregulators During Differentiation of Neural Stem Cells (United States)

    Androutsellis-Theotokis, A.; Chrousos, G. P.; McKay, R. D.; DeCherney, A. H.; Kino, T.


    Neural stem cells (NSCs) are pluripotent precursors with the ability to proliferate and differentiate into 3 neural cell lineages, neurons, astrocytes and oligodendrocytes. Elucidation of the mechanisms underlying these biologic processes is essential for understanding both physiologic and pathologic neural development and regeneration after injury. Nuclear hormone receptors (NRs) and their transcriptional coregulators also play crucial roles in neural development, functions and fate. To identify key NRs and their transcriptional regulators in NSC differentiation, we examined mRNA expression of 49 NRs and many of their coregulators during differentiation (0–5 days) of mouse embryonic NSCs induced by withdrawal of fibroblast growth factor-2 (FGF2). 37 out of 49 NRs were expressed in NSCs before induction of differentiation, while receptors known to play major roles in neural development, such as THRα, RXRs, RORs, TRs, and COUPTFs, were highly expressed. CAR, which plays important roles in xenobiotic metabolism, was also highly expressed. FGF2 withdrawal induced mRNA expression of RORγ, RXRγ, and MR by over 20-fold. Most of the transcriptional coregulators examined were expressed basally and throughout differentiation without major changes, while FGF2 withdrawal strongly induced mRNA expression of several histone deacetylases (HDACs), including HDAC11. Dexamethasone and aldosterone, respectively a synthetic glucocorticoid and natural mineralocorticoid, increased NSC numbers and induced differentiation into neurons and astrocytes. These results indicate that the NRs and their coregulators are present and/or change their expression during NSC differentiation, suggesting that they may influence development of the central nervous system in the absence or presence of their ligands. PMID:22990992

  9. Isolation of the serotoninergic 5-HT4(e) receptor from human heart and comparative analysis of its pharmacological profile in C6-glial and CHO cell lines (United States)

    Mialet, Jeanne; Berque-Bestel, Isabelle; Eftekhari, Pierre; Gastineau, Monique; Giner, Mireille; Dahmoune, Yamina; Donzeau-Gouge, Patrick; Hoebeke, Johan; Langlois, Michel; Sicsic, Sames; Fischmeister, Rodolphe; Lezoualc'h, Frank


    RT–PCR technique was used to clone the human 5-HT4(e) receptor (h5-HT4(e)) from heart atrium. We showed that this h5-HT4(e) receptor splice variant is restricted to brain and heart atrium. Recombinant h5-HT4(e) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg−1 protein, respectively. Expression of h5-HT4(e) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [3H]-GR113808 of a number of 5-HT4 ligands, was consistent with that previously reported for other 5-HT4 receptor isoforms. Surprisingly, we found that the rank order of potencies (EC50) of 5-HT4 agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (Ki) obtained from binding assays. Furthermore, EC50 values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5-HT4(e) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L-type Ca2+ currents and myocyte contractility in human atrium. A constitutive activity of the h5-HT4(e) receptor was observed in CHO cells in the absence of any 5-HT4 ligand and two 5-HT4 antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5-HT4(e) receptor has a pharmacological profile which is close to the native h5-HT4 receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed. PMID:10683202

  10. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

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    Marion eDuriez


    Full Text Available Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis, where maternal and fetal cells are in close contact. Toll-like receptors (TLRs may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs and NK cells (dNKs, the major decidual immune cell populations.We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3 and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8 and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10 and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.


    NARCIS (Netherlands)



    A cDNA clone encoding the rat metabotropic glutamate receptor mGluR3 was stably transfected into human embryonic kidney 293 cells. Receptor-expressing cell lines were characterized by centrifugation binding assays using [H-3]glutamate as radioligand. The rank order of affinity was L-glutamate>(1S,3R

  12. Profiling gene expression induced by protease-activated receptor 2 (PAR2 activation in human kidney cells.

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    Jacky Y Suen

    Full Text Available Protease-Activated Receptor-2 (PAR2 has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD and key events in tumor progression (angiogenesis, metastasis, but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293, a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2 and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2. Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes, the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2 and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15. Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4 known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.

  13. Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling. (United States)

    Tamba, Shigero; Yodoi, Rieko; Morimoto, Kazushi; Inazumi, Tomoaki; Sukeno, Mamiko; Segi-Nishida, Eri; Okuno, Yasushi; Tsujimoto, Gozoh; Narumiya, Shuh; Sugimoto, Yukihiko


    To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2(-/-) cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix- (ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE(2)-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions.

  14. Cord blood Vα24-Vβ11 natural killer T cells display a Th2-chemokine receptor profile and cytokine responses.

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    Susanne Harner

    Full Text Available BACKGROUND: The fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24(+Vβ11(+ NKT cells expressing Th1/Th2-related chemokine receptors (CKR were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR(+ subsets. RESULTS: Cord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3(+ and CCR5(+ cord blood NKT cells (Th1-related were present at lower percentages. Within CD4(negCD8(neg (DN NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3(+, CCR4(+, CCR5(+, CCR6(+, CCR7(+, CCR8(+ and CXCR4(+ DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3(+, CCR6(+ and CCR8(+ cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells. CONCLUSIONS: Despite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens.

  15. Stress-Induced In Vivo Recruitment of Human Cytotoxic Natural Killer Cells Favors Subsets with Distinct Receptor Profiles and Associates with Increased Epinephrine Levels.

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    Marc B Bigler

    Full Text Available Acute stress drives a 'high-alert' response in the immune system. Psychoactive drugs induce distinct stress hormone profiles, offering a sought-after opportunity to dissect the in vivo immunological effects of acute stress in humans.3,4-methylenedioxymethamphetamine (MDMA, methylphenidate (MPH, or both, were administered to healthy volunteers in a randomized, double-blind, placebo-controlled crossover-study. Lymphocyte subset frequencies, natural killer (NK cell immune-phenotypes, and changes in effector function were assessed, and linked to stress hormone levels and expression of CD62L, CX3CR1, CD18, and stress hormone receptors on NK cells.MDMA/MPH > MDMA > MPH robustly induced an epinephrine-dominant stress response. Immunologically, rapid redistribution of peripheral blood lymphocyte-subsets towards phenotypically mature NK cells occurred. NK cytotoxicity was unaltered, but they expressed slightly reduced levels of the activating receptor NKG2D. Preferential circulation of mature NK cells was associated with high epinephrine receptor expression among this subset, as well as expression of integrin ligands previously linked to epinephrine-induced endothelial detachment.The acute epinephrine-induced stress response was characterized by rapid accumulation of mature and functional NK cells in the peripheral circulation. This is in line with studies using other acute stressors and supports the role of the acute stress response in rapidly mobilizing the innate immune system to counteract incoming threats.

  16. Profiling of glycan receptors for minute virus of mice in permissive cell lines towards understanding the mechanism of cell recognition.

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    Sujata Halder

    Full Text Available The recognition of sialic acids by two strains of minute virus of mice (MVM, MVMp (prototype and MVMi (immunosuppressive, is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM. Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3'SIA-LN and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X identified in a previous glycan microarray screen.

  17. Ex vivo generated natural killer cells acquire typical natural killer receptors and display a cytotoxic gene expression profile similar to peripheral blood natural killer cells

    NARCIS (Netherlands)

    Lehmann, D.; Spanholtz, J.; Osl, M.; Tordoir, M.; Lipnik, K.; Bilban, M.; Schlechta, B.; Dolstra, H.; Hofer, E.


    Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT

  18. Pomegranate extract demonstrate a selective estrogen receptor modulator profile in human tumor cell lines and in vivo models of estrogen deprivation. (United States)

    Sreeja, Sreekumar; Santhosh Kumar, Thankayyan R; Lakshmi, Baddireddi S; Sreeja, Sreeharshan


    Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands exhibiting tissue-specific agonistic or antagonistic biocharacter and are used in the hormonal therapy for estrogen-dependent breast cancers. Pomegranate fruit has been shown to exert antiproliferative effects on human breast cancer cells in vitro. In this study, we investigated the tissue-specific estrogenic/antiestrogenic activity of methanol extract of pericarp of pomegranate (PME). PME was evaluated for antiproliferative activity at 20-320 μg/ml on human breast (MCF-7, MDA MB-231) endometrial (HEC-1A), cervical (SiHa, HeLa), ovarian (SKOV3) carcinoma and normal breast fibroblast (MCF-10A) cells. Competitive radioactive binding studies were carried out to ascertain whether PME interacts with ER. The reporter gene assay measured the estrogenic/antiestrogenic activity of PME in MCF-7 and MDA MB-231 cells transiently transfected with plasmids coding estrogen response elements with a reporter gene (pG5-ERE-luc) and wild-type ERα (hEG0-ER). PME inhibited the binding of [³H] estradiol to ER and suppressed the growth and proliferation of ER-positive breast cancer cells. PME binds ER and down-regulated the transcription of estrogen-responsive reporter gene transfected into breast cancer cells. The expressions of selected estrogen-responsive genes were down-regulated by PME. Unlike 17β-estradiol [1 mg/kg body weight (BW)] and tamoxifen (10 mg/kg BW), PME (50 and 100 mg/kg BW) did not increase the uterine weight and proliferation in ovariectomized mice and its cardioprotective effects were comparable to that of 17β-estradiol. In conclusion, our findings suggest that PME displays a SERM profile and may have the potential for prevention of estrogen-dependent breast cancers with beneficial effects in other hormone-dependent tissues.

  19. Killer cell immunoglobulin-like receptor and human leukocyte antigen gene profiles in a cohort of HIV-infected Mexican Mestizos. (United States)

    Garrido-Rodríguez, Daniela; Ávila-Ríos, Santiago; García-Morales, Claudia; Valenzuela-Ponce, Humberto; Ormsby, Christopher; Reyes-Gopar, Helena; Fernandez-Lopez, Juan Carlos; Reyes-Terán, Gustavo


    Killer cell immunoglobulin-like receptors (KIRs) represent the most polymorphic genes responsible for natural killer cell function, while human leukocyte antigen (HLA) class I molecules define and restrict cytotoxic T lymphocyte responses. Specific KIR, HLA, or KIR-HLA combinations have been implicated in the outcome of human immunodeficiency virus (HIV) disease. The remarkable polymorphism of KIR and HLA genes warrants descriptive gene frequency studies in different populations, as well as their impact on HIV disease progression in different immunogenetic contexts. We report KIR and HLA class I gene profiles of 511 unrelated HIV-infected Mexican Mestizo individuals from 18 states for whom genetic ancestry proportions were assessed. KIR and HLA gene profiles were compared between individuals from the north and central-south regions of the country and between individuals with higher European (EUR) or Amerindian (AMI) genetic ancestry component. A total of 65 KIR genotypes were observed, 11 harboring novel KIR gene combinations. A total of 164 HLA alleles were observed: 43 HLA-A, 87 HLA-B, and 34 HLA-C. Differences in the distribution of 12 HLA alleles were observed between individuals with higher AMI or EUR ancestry components (p < 0.05, q < 0.2). After correcting for genetic ancestry, only individual HLA alleles were associated with HIV disease progression, including a novel association with A*02:06, an Amerindian HLA allele associated with lower CD4+ T cell counts. No KIR effects were significant. Our results highlight the advantages of considering a detailed genetic stratification within populations when studying genetic profiles that could be implicated in disease-association studies.

  20. Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line

    DEFF Research Database (Denmark)

    Pedersen, M.W.; Andersen, Thomas Thykjær; Ørntoft, Torben Falck;


    understanding of how the EGFRvIII contributes to the malignant phenotype is of major importance for future therapy. The GeneChip Hu6800Set developed by Affymetrix was used to identify changes in gene expression caused by the expression of EGFRvIII. The cell line selected for the study was an EGF receptor......Previous studies have shown a correlation between expression of the EGF receptor type III mutation (EGFRvIII) and a more malignant phenotype of various cancers including: non-small-cell lung cancer, glioblastoma multiforme, prostate cancer and breast cancer. Thus, a detailed molecular genetic...... negative small-cell-lung cancer cell line, GLC3, stably transfected with the EGFRvIII gene in a Tet-On system. By comparison of mRNA levels in EGFRvIII-GLC3 with those of Tet-On-GLC3, it was found that the levels of mRNAs encoding several transcription factors (ATF-3, JunD, and c-Myb), cell adhesion...

  1. Temporal gene expression profile of human precursor B leukemia cells induced by adhesion receptor: identification of pathways regulating B-cell survival. (United States)

    Astier, Anne Laurence; Xu, Ronghui; Svoboda, Marek; Hinds, Esther; Munoz, Olivier; de Beaumont, Rosalie; Crean, Colin Daniel; Gabig, Theodore; Freedman, Arnold Stephen


    The physical interactions between B cells and stromal cells from the lymphoid tissue microenvironment are critical to the survival of normal and malignant B cells. They are principally mediated by integrins expressed on B cells and counterreceptors on stromal cells. Specifically, alpha4beta1 integrin engagement rescues B cells from physiological or drug-induced apoptosis. Therefore, in order to understand the mechanisms by which integrins prevent apoptosis in leukemia B cells, we compared the temporal gene expression profiles induced by beta1-integrin ligation with fibronectin (Fn) or adhesion by poly-L-Lysine in serum-starved precursor B leukemia cells. Among the 38 selected differentially expressed genes, 6 genes involved in adhesion (VAV2, EPB41L1, CORO1A), proliferation (FRAP1, CCT4), and intercellular communication (GJB3) were validated by real-time quantitative polymerase chain reaction (RT-Q-PCR). Gene expression modulation could also be validated at the protein level for 5 other genes. We show that integrin stimulation up-regulated FBI-1 expression but inhibited CD79a, Requiem, c-Fos, and caspase 7 induction when the cells underwent apoptosis. We further demonstrate that Fn stimulation also inhibits caspase 3 activation but increases XIAP and survivin expression. Moreover, integrin stimulation also prevents caspase activation induced by doxorubicin. Therefore, we identified genes modulated by adhesion of human precursor B leukemia cells that regulate proliferation and apoptosis, highlighting new pathways that might provide insights into future therapy aiming at targeting apoptosis of leukemia cells.

  2. Frequencies of dendritic cells and Toll-like receptor 3 in neonates born to HBsAg-positive mothers with different HBV serological profiles. (United States)

    Guo, J; Gao, Y; Guo, Z; Zhang, L R; Wang, B; Wang, S P


    To investigate the frequencies of dendritic cells (DCs) and Toll-like receptor 3 (TLR3) in neonates of HBsAg-positive mothers with different HBV serological profiles, we conducted a study in Taiyuan, China. The study included 144 HBsAg-positive mothers and their neonates. The frequencies of DCs and TLR3 were determined using four-colour flow-cytometric analysis. DC and TLR3 frequencies were not related to HBV intrauterine transmission, maternal HBeAg positivity, maternal HBV DNA positivity and HBeAg/HBV DNA double-positivity. The plasmacytoid dendritic cell (pDC) frequencies in neonates whose maternal HBV DNA was >5 × 107 copies/ml decreased significantly compared to that in neonates whose maternal HBV DNA was ⩽5 × 107 copies/ml (Z = - 2·170, P = 0·03) or whose maternal HBV DNA was negative (Z = - 1·981 P = 0·048). This study suggests that neonatal pDC frequencies decrease when maternal HBV DNA loads are >5 × 107 copies/ml.

  3. Quantum dot multiplexing for the profiling of cellular receptors (United States)

    Lee-Montiel, Felipe T.; Li, Peter; Imoukhuede, P. I.


    The profiling of cellular heterogeneity has wide-reaching importance for our understanding of how cells function and react to their environments in healthy and diseased states. Our ability to interpret and model cell behavior has been limited by the difficulties of measuring cell differences, for example, comparing tumor and non-tumor cells, particularly at the individual cell level. This demonstrates a clear need for a generalizable approach to profile fluorophore sites on cells or molecular assemblies on beads. Here, a multiplex immunoassay for simultaneous detection of five different angiogenic markers was developed. We targeted angiogenic receptors in the vascular endothelial growth factor family (VEGFR1, VEGFR2 and VEGFR3) and Neuropilin (NRP) family (NRP1 and NRP2), using multicolor quantum dots (Qdots). Copper-free click based chemistry was used to conjugate the monoclonal antibodies with 525, 565, 605, 655 and 705 nm CdSe/ZnS Qdots. We tested and performed colocalization analysis of our nanoprobes using the Pearson correlation coefficient statistical analysis. Human umbilical vein endothelial cells (HUVEC) were tested. The ability to easily monitor the molecular indicators of angiogenesis that are a precursor to cancer in a fast and cost effective system is an important step towards personalized nanomedicine.The profiling of cellular heterogeneity has wide-reaching importance for our understanding of how cells function and react to their environments in healthy and diseased states. Our ability to interpret and model cell behavior has been limited by the difficulties of measuring cell differences, for example, comparing tumor and non-tumor cells, particularly at the individual cell level. This demonstrates a clear need for a generalizable approach to profile fluorophore sites on cells or molecular assemblies on beads. Here, a multiplex immunoassay for simultaneous detection of five different angiogenic markers was developed. We targeted angiogenic receptors

  4. The importance of molecular profiling in predicting response to epidermal growth factor receptor family inhibitors in non-small-cell lung cancer: focus on clinical trial results. (United States)

    Tsao, Anne S; Papadimitrakopoulou, Vassiliki


    In recent years, the epidermal growth factor receptor (EGFR) family has become a key focus of non-small-cell lung cancer biology and targeted therapies, such as the reversible EGFR tyrosine kinase inhibitors erlotinib and gefitinib. Initially, response to these agents was associated with certain demographic and clinical characteristics; subsequently, it was discovered that these subgroups were more likely to harbor specific mutations in the EGFR gene that enhanced tumor response. However, the presence of these mutations does not equate to therapeutic success. Other aspects of EGFR family signaling, including other types of EGFR mutations, EGFR protein expression, EGFR gene amplification, mediators of downstream signaling, and other receptors with similar downstream pathways may all play a role in response or resistance to treatment. The identification of these and other molecular determinants is driving the development of novel therapies designed to achieve improved clinical outcomes in patients.

  5. Cell cycle phase regulates glucocorticoid receptor function.

    Directory of Open Access Journals (Sweden)

    Laura Matthews

    Full Text Available The glucocorticoid receptor (GR is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1. Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G(1. This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase. The impact of cell cycle-driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle. In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.

  6. Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile (United States)

    Sánchez-Reyes, Karina; Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Jave-Suárez, Luis Felipe; Gómez-Lomelí, Paulina; de Celis, Ruth; Aguilar-Lemarroy, Adriana; Domínguez-Rodríguez, Jorge Ramiro; Ortiz-Lazareno, Pablo Cesar


    Cervical cancer (CC) is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV) is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated) and M2 (alternatively activated). Macrophage polarization exerts profound effects on the Toll-like receptor (TLR) profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages. PMID:25309919

  7. Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

    Directory of Open Access Journals (Sweden)

    Karina Sánchez-Reyes


    Full Text Available Cervical cancer (CC is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated and M2 (alternatively activated. Macrophage polarization exerts profound effects on the Toll-like receptor (TLR profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

  8. Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ. (United States)

    Słoniecka, Marta; Le Roux, Sandrine; Boman, Peter; Byström, Berit; Zhou, Qingjun; Danielson, Patrik


    Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides

  9. Comprehensive Profiling of GPCR Expression in Ghrelin-Producing Cells. (United States)

    Koyama, Hiroyuki; Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Hosoda, Hiroshi; Ariyasu, Hiroyuki; Kusakabe, Toru; Son, Choel; Hosoda, Kiminori; Akamizu, Takashi; Kangawa, Kenji; Nakao, Kazuwa


    To determine the comprehensive G protein-coupled receptor (GPCR) expression profile in ghrelin-producing cells and to elucidate the role of GPCR-mediated signaling in the regulation of ghrelin secretion, we determined GPCR expression profiles by RNA sequencing in the ghrelin-producing cell line MGN3-1 and analyzed the effects of ligands for highly expressed receptors on intracellular signaling and ghrelin secretion. Expression of selected GPCRs was confirmed in fluorescence-activated cell-sorted fluorescently tagged ghrelin-producing cells from ghrelin-promoter CreERT2/Rosa-CAG-LSL-ZsGreen1 mice. Expression levels of GPCRs previously suggested to regulate ghrelin secretion including adrenergic-β1 receptor, GPR81, oxytocin receptor, GPR120, and somatostatin receptor 2 were high in MGN3-1 cells. Consistent with previous reports, isoproterenol and oxytocin stimulated the Gs and Gq pathways, respectively, whereas lactate, palmitate, and somatostatin stimulated the Gi pathway, confirming the reliability of current assays. Among other highly expressed GPCRs, prostaglandin E receptor 4 agonist prostaglandin E2 significantly stimulated the Gs pathway and ghrelin secretion. Muscarine, the canonical agonist of cholinergic receptor muscarinic 4, stimulated both the Gq and Gi pathways. Although muscarine treatment alone did not affect ghrelin secretion, it did suppress forskolin-induced ghrelin secretion, suggesting that the cholinergic pathway may play a role in counterbalancing the stimulation of ghrelin by Gs (eg, by adrenaline). In addition, GPR142 ligand tryptophan stimulated ghrelin secretion. In conclusion, we determined the comprehensive expression profile of GPCRs in ghrelin-producing cells and identified two novel ghrelin regulators, prostaglandin E2 and tryptophan. These results will lead to a greater understanding of the physiology of ghrelin and facilitate the development of ghrelin-modulating drugs.

  10. Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain. (United States)

    Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Korn, Matthew J; Nakamura, Paul A; Shirkey, Nicole J; Wong, Jamie K; Miledi, Ricardo


    Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration.

  11. Gene expression profiling in Caco-2 human colon cells exposed to TCDD, benzo[a]pyrene, and natural Ah receptor agonists from cruciferous vegetables and citrus fruits

    NARCIS (Netherlands)

    Waard, de W.J.; Aarts, J.M.M.J.G.; Peijnenburg, A.A.C.M.; Baykus, H.; Talsma, E.F.; Punt, A.; Kok, de T.M.C.M.; Schooten, van F.J.; Hoogenboom, L.A.P.


    Cruciferous vegetables and citrus fruits are reported to possess health-beneficial properties, but also have been shown to contain natural aryl hydrocarbon receptor (AhR) agonists (NAhRAs). Binding to the AhR is widely assumed to activate the main pathway by which dioxins, like 2,3,7,8-tetrachlorodi

  12. Involvement of Ghrelin-Growth Hormone Secretagogue Receptor System in Pathoclinical Profiles of Digestive System Cancer

    Institute of Scientific and Technical Information of China (English)

    Zhigang WANG; Weigang WANG; Wencai QIU; Youben FAN; Jun ZHAO; Yu WANG; Qi ZHENG


    Ghrelin receptor has been shown to be expressed along the human gastrointestinal tract.Recent studies showed that ghrelin and a synthetic ghrelin receptor agonist improved weight gain and lean body mass retention in a rat model of cancer cachexia by acting on ghrelin receptor, that is, growth hormone secretagogue receptor (GHS-R). This study aims to explore the expression and the distribution of ghrelin receptor in human gastrointestinal tract cancers and to investigate the possible involvement of the ghrelin-GHS-R system in human digestive cancers. Surgical human digestive cancer specimens were obtained from various portions of the gastrointestinal tract from different patients. The expression of ghrelin receptor in these tissues was detected by tissue microarray technique. Our results showed that ghrelin receptor was expressed in cancers throughout the gastrointestinal tract, mainly in the cytoplasm of mucosal layer cells.Its expression level possibly correlated with organ type, histological grade, tumor-nodes-metastases stage,and nutrition status (weight loss) of the patients. For the first time, we identified the distribution of ghrelin receptor in digestive system cancers. Our results implied that the ghrelin-GHS-R system might be involved in the pathoclinical profiles of digestive cancers.

  13. Evolutionary vignettes of natural killer cell receptors. (United States)

    Sambrook, Jennifer G; Beck, Stephan


    The discovery of novel immune receptors has led to a recent renaissance of research into the innate immune system, following decades of intense research of the adaptive immune system. Of particular interest has been the discovery of the natural killer (NK) cell receptors which, depending on type, interact with classical or non-classical MHC class I antigens of the adaptive immune system, thus functioning at the interface of innate and adaptive immunity. Here, we review recent progress with respect to two such families of NK receptors, the killer immunoglobulin-like receptors (KIRs) and the killer cell lectin-like receptors (KLRs), and attempt to trace their evolution across vertebrates.

  14. Equilibrium and kinetic selectivity profiling on the human adenosine receptors. (United States)

    Guo, Dong; Dijksteel, Gabrielle S; van Duijl, Tirsa; Heezen, Maxime; Heitman, Laura H; IJzerman, Adriaan P


    Classical evaluation of target selectivity is usually undertaken by measuring the binding affinity of lead compounds against a number of potential targets under equilibrium conditions, without considering the kinetics of the ligand-receptor interaction. In the present study we propose a combined strategy including both equilibrium- and kinetics-based selectivity profiling. The adenosine receptor (AR) was chosen as a prototypical drug target. Six in-house AR antagonists were evaluated in a radioligand displacement assay for their affinity and in a competition association assay for their binding kinetics on three AR subtypes. One of the compounds with a promising kinetic selectivity profile was also examined in a [(35)S]-GTPγS binding assay for functional activity. We found that XAC and LUF5964 were kinetically more selective for the A1R and A3R, respectively, although they are non-selective in terms of their affinity. In comparison, LUF5967 displayed a strong equilibrium-based selectivity for the A1R over the A2AR, yet its kinetic selectivity thereon was less pronounced. In a GTPγS assay, LUF5964 exhibited insurmountable antagonism on the A3R while having a surmountable effect on the A1R, consistent with its kinetic selectivity profile. This study provides evidence that equilibrium and kinetic selectivity profiling can both be important in the early phases of the drug discovery process. Our proposed combinational strategy could be considered for future medicinal chemistry efforts and aid the design and discovery of different or even better leads for clinical applications.

  15. The evolution of natural killer cell receptors. (United States)

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


    Natural killer (NK) cells are 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 is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is "missing self" detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.

  16. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B


    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  17. Mast Cell and Immune Inhibitory Receptors

    Institute of Scientific and Technical Information of China (English)

    LixinLi; ZhengbinYao


    Modulation by balancing activating and inhibitory receptors constitutes an important mechanism for regulating immune responses. Cells that are activated following ligation of receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs) can be negatively regulated by other receptors bearing immunoreceptor tyrosine-based inhibition motifs (ITIMs). Human mast cells (MCs) are the major effector cells of type I hypersensitivity and important participants in a number of disease processes. Antigen-mediated aggregation of IgE bound to its high-affinity receptor on MCs initiates a complex series of biochemical events leading to MC activation. With great detailed description and analysis of several inhibitory receptors on human MCs, a central paradigm of negative regulation of human MC activation by these receptors has emerged. Cellular & Molecular Immunology. 2004;1(6):408-415.

  18. Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile. (United States)

    Hikichi, Yukiko; Yamaoka, Masuo; Kusaka, Masami; Hara, Takahito


    Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity.

  19. TRAIL Death Receptor-4, Decoy Receptor-1 and Decoy Receptor-2 Expression on CD8+ T Cells Correlate with the Disease Severity in Patients with Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Bisgin Atil


    Full Text Available Abstract Background Rheumatoid Arthritis (RA is a chronic autoimmune inflammatory disorder. Although the pathogenesis of disease is unclear, it is well known that T cells play a major role in both development and perpetuation of RA through activating macrophages and B cells. Since the lack of TNF-Related Apoptosis Inducing Ligand (TRAIL expression resulted in defective thymocyte apoptosis leading to an autoimmune disease, we explored evidence for alterations in TRAIL/TRAIL receptor expression on peripheral T lymphocytes in the molecular mechanism of RA development. Methods The expression of TRAIL/TRAIL receptors on T cells in 20 RA patients and 12 control individuals were analyzed using flow cytometry. The correlation of TRAIL and its receptor expression profile was compared with clinical RA parameters (RA activity scored as per DAS28 using Spearman Rho Analysis. Results While no change was detected in the ratio of CD4+ to CD8+ T cells between controls and RA patient groups, upregulation of TRAIL and its receptors (both death and decoy was detected on both CD4+ and CD8+ T cells in RA patients compared to control individuals. Death Receptor-4 (DR4 and the decoy receptors DcR1 and DcR2 on CD8+ T cells, but not on CD4+ T cells, were positively correlated with patients' DAS scores. Conclusions Our data suggest that TRAIL/TRAIL receptor expression profiles on T cells might be important in revelation of RA pathogenesis.

  20. Killer cell immunoglobulin receptor profile on CD4(+)  CD28(-) T cells and their pathogenic role in non-dialysis-dependent and dialysis-dependent chronic kidney disease patients. (United States)

    Zal, Behnam; Chitalia, Nihil; Ng, Yin Sing; Trieu, Verna; Javed, Sana; Warrington, Rachelle; Kaski, Juan Carlos; Banerjee, Debasish; Baboonian, Christina


    There is a progressive increase in cardiovascular disease with declining renal function, unexplained by traditional risk factors. A CD4(+) T-cell subpopulation (CD4(+)  CD28(-) ), activated by human heat-shock protein 60 (hHSP 60), expands in patients with acute coronary syndrome and is associated with vascular damage. These cells exhibit cytotoxicity via expression of activating killer cell-immunoglobulin-like receptor KIR2DS2, mainly in the absence of inhibitory KIR2DL3. We investigated expansion of these cells and the pathogenic role of the KIR in non-dialysis-dependent chronic kidney disease (NDD-CKD) and end-stage haemodialysis-dependent renal disease (HD-ESRD) patients. CD4(+)  CD28(-) cells were present in 27% of the NDD-CKD and HD-ESRD patients (8-11% and 10-11% of CD4(+) compartment, respectively). CD4(+)  CD28(-) cells were phenotyped for KIR and DAP12 expression. Cytotoxicity was assessed by perforin and pro-inflammatory function by interferon-γ expression on CD4(+)  CD28(-) clones (NDD-CKD n = 97, HD-ESRD n = 262). Thirty-four per cent of the CD4(+)  CD28(-) cells from NDD-CKD expressed KIR2DS2 compared with 56% in HD-ESRD patients (P = 0·03). However, 20% of clones expressed KIR2DL3 in NDD-CKD compared with 7% in HD-ESRD patients (P = 0·004). DAP12 expression in CD28(-)  2DS2(+) clones was more prevalent in HD-ESRD than NDD-CKD (92% versus 60%; P < 0·001). Only 2DS2(+)  2DL3(-)  DAP12(+) clones were cytotoxic in response to hHSP 60. CD4(+)  CD28(-) cells exhibited increased KIR2DS2, reduced KIR2DL3 and increased DAP12 expression in HD-ESRD compared with NDD-CKD patients. These findings suggest a gradual loss of expression, functionality and protective role of inhibitory KIR2DL3 as well as increased cytotoxic potential of CD4(+)  C28(-) cells with progressive renal impairment. Clonal expansion of these T cells may contribute to heightened cardiovascular events in HD-ESRD.

  1. Transcriptome profiling of LGR5 positive colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Daniela Hirsch


    Full Text Available The concept of cancer stem cells (CSCs claims that colorectal carcinomas (CRCs, like normal colorectal epithelium, are organized hierarchically and contain a subpopulation of qualitatively distinct cancer cells. The expression of distinctive surface markers or of certain enzymes is a prerequisite for the isolation and characterization of the CSC population. With respect to CRCs, putative CSCs can be identified by leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5, also known as G-protein-coupled receptor 49, Gpr49. However, the precise function of the intestinal stem cell marker Lgr5 in CRCs remains largely unknown. We silenced LGR5 expression in SW480 CRC cells via lentiviral shRNA constructs. This led to the depletion of a morphologically distinct subpopulation of SW480 CRC cells. Microarray gene expression profiling revealed a down-regulation of NOTCH signaling upon LGR5 silencing that could be confirmed by immunohistochemistry. Furthermore, we induced inflammation-driven colon tumors in Lgr5-EGFP-IRES-Cre-ERT2 mice via administration of azoxymethane and dextrane sodium sulfate. The induced tumors were flow-sorted into fractions of epithelial cells that expressed high or low levels of Lgr5 and were characterized using gene expression profiling. Lgr5 high tumor cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than Lgr5 low tumor cells and Lgr5 high normal stem cells. Here we provide a thorough description of our two gene expression datasets including quality control checks uploaded to Gene Expression Omnibus database (data accession number: GSE46200. The analysis and interpretation of our gene expression data and related results have been published recently by Hirsch and colleagues in Carcinogenesis in 2014.

  2. Cell death sensitization of leukemia cells by opioid receptor activation (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich


    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  3. Expression profile of frizzled receptors in human medulloblastomas. (United States)

    Salsano, Ettore; Paterra, Rosina; Figus, Miriam; Menghi, Francesca; Maderna, Emanuela; Pollo, Bianca; Solero, Carlo Lazzaro; Massimi, Luca; Finocchiaro, Gaetano


    Secreted WNT proteins signal through ten receptors of the frizzled (FZD) family. Because of the relevance of the WNT/β-catenin (CTNNB1) signaling pathway in medulloblastomas (MBs), we investigated the expression of all ten members of the FZD gene family (FZD1-10) in 17 human MBs, four MB cell lines and in normal human cerebellum, using real-time PCR. We found that FZD2 transcript was over-expressed in all MBs and MB cell lines. Western blot analysis confirmed the expression of FZD2 at the protein level. Moreover, the levels of FZD2 transcript were found to correlate with those of ASPM transcript, a marker of mitosis essential for mitotic spindle function. Accordingly, ASPM mRNA was expressed at a very low level in the adult, post-mitotic, human cerebellum, at higher levels in fetal cerebellum and at highest levels in MB tissues and cell lines. Unlike FZD2, the other FZDs were overexpressed (e.g., FZD1, FZD3 and FZD8) or underexpressed (e.g., FZD7, FZD9 and FZD10) in a case-restricted manner. Interestingly, we did not find any nuclear immuno-reactivity to CTNNB1 in four MBs over-expressing both FZD2 and other FZD receptors, confirming the lack of nuclear CTNNB1 staining in the presence of increased FZD expression, as in other tumor types. Overall, our results indicate that altered expression of FZD2 might be associated with a proliferative status, thus playing a role in the biology of human MBs, and possibly of cerebellar progenitors from which these malignancies arise.

  4. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B


    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....

  5. A pharmacological profile of the high-affinity GluK5 kainate receptor. (United States)

    Møllerud, Stine; Kastrup, Jette Sandholm; Pickering, Darryl S


    Mouse GluK5 was expressed in Sf9 insect cells and radiolabelled with [(3)H]-kainate in receptor binding assays (Kd=6.9nM). Western immunoblotting indicated an Sf9 GluK5 band doublet corresponding to the glycosylated (128kDa) and deglycosylated (111kDa) protein, which was identical to the band pattern of native rat brain GluK5. A pharmacological profile of the high-affinity kainate receptor GluK5 is described which is distinct from the profiles of other kainate receptors (GluK1-3). The 27 tested ligands generally show a preferential affinity to GluK1 over GluK5, the exceptions being: dihydrokainate, (S)-5-fluorowillardiine, (S)-glutamate and quisqualate, where the affinity is similar at GluK1 and GluK5. In contrast, quisqualate shows 40-fold higher affinity at GluK5 over GluK3 whereas (S)-1-(2'-amino-2'-caboxyethyl)thienol[3,4-d]pyrimidin-2,4-dione (NF1231), (RS)-2-amino-3-(5-tert-butyl-3-hydroxyisoxazol-4-yl)propionate (ATPA), dihydrokainate and (2S,4R)-4-methyl-glutamate (SYM2081) have higher affinity at GluK3 compared to GluK5. Since some studies have indicated that GluK5 is associated with various diseases in the central nervous system (e.g. schizophrenia, temporal lobe epilepsy, bipolar disorder), selective GluK5 ligands could have therapeutic potential. The distinct pharmacological profile of GluK5 suggests that it would be possible to design ligands with selectivity towards GluK5.

  6. Pharmacological Profiles of Oligomerized μ-Opioid Receptors


    Ing-Kang Ho; Cynthia Wei-Sheng Lee


    Opioids are widely prescribed pain relievers with multiple side effects and potential complications. They produce analgesia via G-protein-protein coupled receptors: μ-, δ-, κ-opioid and opioid receptor-like 1 receptors. Bivalent ligands targeted to the oligomerized opioid receptors might be the key to developing analgesics without undesired side effects and obtaining effective treatment for opioid addicts. In this review we will update the biological effects of μ-opioids on homo- or hetero-ol...

  7. Pharmacological profile of lixisenatide: A new GLP-1 receptor agonist for the treatment of type 2 diabetes. (United States)

    Werner, Ulrich; Haschke, Guido; Herling, Andreas W; Kramer, Werner


    The glucagon-like peptide-1 (GLP-1) receptor represents an established therapeutic target in type 2 diabetes mellitus (T2DM). Agents that activate this receptor improve glucose tolerance alongside a low risk of hypoglycaemia, and have the potential to modify disease progression. Lixisenatide is a new potent and selective GLP-1 receptor agonist currently in development. The preclinical pharmacological profile of Lixisenatide suggests actions that are highly relevant to the long-term maintenance of glucose homeostasis. Lixisenatide protected Ins-1 cells (a rat-derived beta-cell line) from both lipid- and cytokine-induced apoptosis. More importantly, Lixisenatide also prevented lipotoxicity-induced insulin depletion in human islets and preserved insulin production, storage and pancreatic beta-cell function in vitro. Enhancement of insulin biosynthesis and pancreatic beta-cell volume could also be demonstrated in animal models of type 2 diabetes. The improvement of glucose-stimulated insulin secretion provided by Lixisenatide occurred in a strictly glucose-dependent manner. In animal models of diabetes, Lixisenatide improved basal blood glucose and HbA(1c) with a rapid onset and sustained duration of action, and prevented the deterioration of pancreatic responsiveness and glucose homeostasis. Lixisenatide also delayed gastric emptying and reduced food intake. The efficacy/safety profile of Lixisenatide is currently being studied further in an extensive ongoing Phase III clinical study programme. This article reviews the preclinical pharmacological profile of Lixisenatide.

  8. Chemokine receptor expression by inflammatory T cells in EAE. (United States)

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


    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 receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4(+) T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8(+) T cells. CD8(+) T cells expressed CXCR3, which was also expressed by CD4(+) T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6(+) and CXCR3(+) CD4(+) T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8(+) T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double 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 and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE.

  9. Striatal pre- and postsynaptic profile of adenosine A(2A receptor antagonists.

    Directory of Open Access Journals (Sweden)

    Marco Orru

    Full Text Available Striatal adenosine A(2A receptors (A(2ARs are highly expressed in medium spiny neurons (MSNs of the indirect efferent pathway, where they heteromerize with dopamine D(2 receptors (D(2Rs. A(2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1 receptors (A(1Rs. It has been hypothesized that postsynaptic A(2AR antagonists should be useful in Parkinson's disease, while presynaptic A(2AR antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A(2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261 showed no clear preference. Radioligand-binding experiments were performed in cells expressing A(2AR-D(2R and A(1R-A(2AR heteromers to determine possible differences in the affinity of these compounds for different A(2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2AR when co-expressed with D(2R than with A(1R. KW-6002 showed the best relative affinity for A(2AR co-expressed with D(2R than co-expressed with A(1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile

  10. ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor, Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

    Directory of Open Access Journals (Sweden)

    Rebecca Louise Harris


    Full Text Available Background. The asialoglycoprotein receptor (ASGPR is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2, encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR, expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver.

  11. Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions (United States)

    After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...

  12. A comparison of ovarian follicular and luteal cell gene expression profiles provides insight into cellular identities and functions (United States)

    After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...

  13. Regulation of Immune Cells by Eicosanoid Receptors

    Directory of Open Access Journals (Sweden)

    Nancy D. Kim


    Full Text Available Eicosanoids are potent, bioactive, lipid mediators that regulate important components of the immune response, including defense against infection, ischemia, and injury, as well as instigating and perpetuating autoimmune and inflammatory conditions. Although these lipids have numerous effects on diverse cell types and organs, a greater understanding of their specific effects on key players of the immune system has been gained in recent years through the characterization of individual eicosanoid receptors, the identification and development of specific receptor agonists and inhibitors, and the generation of mice genetically deficient in various eicosanoid receptors. In this review, we will focus on the receptors for prostaglandin D2, DP1 and DP2/CRTH2; the receptors for leukotriene B4, BLT1 and BLT2; and the receptors for the cysteinyl leukotrienes, CysLT1 and CysLT2, by examining their specific effects on leukocyte subpopulations, and how they may act in concert towards the development of immune and inflammatory responses.

  14. Expression profiling of constitutive mast cells reveals a unique identity within the immune system (United States)

    Dwyer, Daniel F.; Barrett, Nora A.; Austen, K. Frank


    Mast cells are evolutionarily ancient sentinel cells. Like basophils, mast cells express the high-affinity IgE receptor and are implicated in host defense and diverse immune-mediated diseases. To better characterize the function of these cells, we assessed the transcriptional profiles of mast cells isolated from peripheral connective tissues and basophils isolated from spleen and blood. We found that mast cells were transcriptionally distinct, clustering independently from all other profiled cells, and that mast cells demonstrated considerably greater heterogeneity across tissues than previously appreciated. We observed minimal homology between mast cells and basophils, which share more overlap with other circulating granulocytes than with mast cells. Derivation of mast cell and basophil transcriptional signatures underscores their differential capacity to detect environmental signals and influence the inflammatory milieu. PMID:27135604

  15. Molecular profiles of progesterone receptor loss in human breast tumors

    NARCIS (Netherlands)

    C.J. Creighton; C. Kent Osborne; M.J. van de Vijver; J.A. Foekens; J.G. Klijn; H.M. Horlings; D. Nuyten; Y. Wang; Y. Zhang; G.C. Chamness; S.G. Hilsenbeck; A.V. Lee; R. Schiff


    Background Patient prognosis and response to endocrine therapy in breast cancer correlate with protein expression of both estrogen receptor (ER) and progesterone receptor (PR), with poorer outcome in patients with ER+/PR- compared to ER+/PR+ tumors. Methods To better understand the underlying biolog

  16. Expression profiles and function of Toll-like receptors in human corneal epithelia

    Institute of Scientific and Technical Information of China (English)

    WU Xin-yi; GAO Jian-lu; REN Mei-yu


    Background Toll-like receptors play an important role in the human immune system. This study was conducted to investigate the expression profiles and function of Toll-like receptor (TLR)1-9 in human corneal epithelium.Methods The expression of TLR1-9 mRNA in 20 human donor corneal epithelia samples abraded during photorefractive keratotomy (PRK) and cultivated telomerase-immortalized human corneal epithelial cells (THCEs) was examined by semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Human peripheral blood mononuclear cells (PBMCs) were used as positive controls. The expression of the TLR2 and TLR4 proteins was detected by Western analysis. ELISA was used to detect IL-8 secretion from THCEs challenged with ligands for TLR3 and TLR4 with and without antibody blockade.Results The expression of TLR1-9 at the mRNA level was detected in the epithelia of 20 patients and in THCE.Significant differences among individuals were observed. One patient was found to lack of the expression of TLR3, 4, 6 and 8, whereas another did not express TLR5. The expression of TLR2 and TLR4 protein was detected in human corneal epithelial cells. As THCE cells express TLR1-9, cells were challenged with lipopolysaccharides (LPS) and poly I:C to determine whether TLR4 and TLR3 were functional. The results showed that secretion of IL-8 by cells stimulated with LPS and Poly I:C was 7 to 10 fold greater than secretion by unchallenged cells. Blocking TLR4 with an anti-TLR4 antibody significantly inhibited the LPS-induced IL-8 production by THCE (P<0.05).Conclusion Human corneal epithelial cells express multiple TLRs and are able to recognize LPS and poly I:C. Different expression profiles among individuals suggest that differences in the susceptibilities and sensitivities to bacterial and viral infection in human populations relate to different patterns of TLR expression.

  17. Soluble and cell surface receptors for tumor necrosis factor

    DEFF Research Database (Denmark)

    Wallach, D; Engelmann, H; Nophar, Y


    Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptor...... have recently been cloned. Antibodies to one of these receptor species (the p55, type I receptor) can trigger a variety of TNF like effects by cross-linking of the receptor molecules. Thus, it is not TNF itself but its receptors that provide the signal for the response to this cytokine...... in certain pathological situations. Release of the soluble receptors from the cells seems to occur by proteolytic cleavage of the cell surface forms and appears to be a way of down-regulating the cell response to TNF. Because of their ability to bind TNF, the soluble receptors exert an inhibitory effect...

  18. Pharmacological profile of DA-6886, a novel 5-HT4 receptor agonist to accelerate colonic motor activity in mice. (United States)

    Lee, Min Jung; Cho, Kang Hun; Park, Hyun Min; Sung, Hyun Jung; Choi, Sunghak; Im, Weonbin


    DA-6886, the gastrointestinal prokinetic benzamide derivative is a novel 5-HT4 receptor agonist being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C). The purpose of this study was to characterize in vitro and in vivo pharmacological profile of DA-6886. We used various receptor binding assay, cAMP accumulation assay, organ bath experiment and colonic transit assay in normal and chemically constipated mice. DA-6886 exhibited high affinity and selectivity to human 5-HT4 receptor splice variants, with mean pKi of 7.1, 7.5, 7.9 for the human 5-HT4a, 5-HT4b and 5-HT4d, respectively. By contrast, DA-6886 did not show significant affinity for several receptors including dopamine D2 receptor, other 5-HT receptors except for 5-HT2B receptor (pKi value of 6.2). The affinity for 5-HT4 receptor was translated into functional agonist activity in Cos-7 cells expressing 5-HT4 receptor splice variants. Furthermore, DA-6886 induced relaxation of the rat oesophagus preparation (pEC50 value of 7.4) in a 5-HT4 receptor antagonist-sensitive manner. The evaluation of DA-6886 in CHO cells expressing hERG channels revealed that it inhibited hERG channel current with an pIC50 value of 4.3, indicating that the compound was 1000-fold more selective for the 5-HT4 receptor over hERG channels. In the normal ICR mice, oral administration of DA-6886 (0.4 and 2mg/kg) resulted in marked stimulation of colonic transit. Furthermore, in the loperamide-induced constipation mouse model, 2mg/kg of DA-6886 significantly improved the delay of colonic transit, similar to 10mg/kg of tegaserod. Taken together, DA-6886 is a highly potent and selective 5-HT4 receptor agonist to accelerate colonic transit in mice, which might be therapeutic agent having a favorable safety profile in the treatment of gastrointestinal motor disorders such as IBS-C and chronic constipation.

  19. Effect of the FSH receptor single nucleotide polymorphisms (FSHR 307/680) on the follicular fluid hormone profile and the granulosa cell gene expression in human small antral follicles

    DEFF Research Database (Denmark)

    Borgbo, Tanni Kjær; Jeppesen, J V; Lindgren, I


    ), by evaluating the hormone and gene expression profiles of human small antral follicles collected under physiological conditions in connection with fertility preservation. In total 69 women at various time during the menstrual cycle were included in this study. The intrafollicular hormone content of 179......The most pronounced effects of FSH signalling are potentially displayed in the follicle fluid, which acts as a reservoir for FSH-induced granulosa cell (GC) secreted hormones. This study investigates the effects of two common polymorphisms of FSHR, FSHR 307 (rs6165) and FSHR 680 (rs6166...... small antral follicles collected under physiological FSH conditions....

  20. The Human Laminin Receptor is a Member of the Integrin Family of Cell Adhesion Receptors (United States)

    Gehlsen, Kurt R.; Dillner, Lena; Engvall, Eva; Ruoslahti, Erkki


    A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

  1. Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2.

    Directory of Open Access Journals (Sweden)

    D Malfacini

    Full Text Available Nociceptin/orphanin FQ (N/OFQ controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP. Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells. In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

  2. In vivo immunotoxicity of perfluorooctane sulfonate in BALB/c mice: Identification of T-cell receptor and calcium-mediated signaling pathway disruption through gene expression profiling of the spleen. (United States)

    Lv, Qi-Yan; Wan, Bin; Guo, Liang-Hong; Yang, Yu; Ren, Xiao-Min; Zhang, Hui


    Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that is used worldwide and is continuously being detected in biota and the environment, thus presenting potential threats to the ecosystem and human health. Although PFOS is highly immunotoxic, its underlying molecular mechanisms remain largely unknown. The present study examined PFOS-induced immunotoxicity in the mouse spleen and explored its underlying mechanisms by gene expression profiling. Oral exposure of male BALB/c mice for three weeks followed by one-week recovery showed that a 10 mg/kg/day PFOS exposure damaged the splenic architecture, inhibited T-cell proliferation in response to mitogen, and increased the percentages of T helper (CD3(+)CD4(+)) and cytotoxic T (CD3(+)CD8(+)) cells, despite the decrease in the absolute number of these cells. A delayed type of PFOS immunotoxicity was observed, which mainly occurred during the recovery period. Global gene expression profiling of mouse spleens and QRT-PCR analyses suggest that PFOS inhibited the expression of genes involved in cell cycle regulation and NRF2-mediated oxidative stress response, and upregulated those in TCR signaling, calcium signaling, and p38/MAPK signaling pathways. Western blot analysis confirmed that the expressions of CAMK4, THEMIS, and CD3G, which were involved in the upregulated pathways, were induced upon PFOS exposure. Acute PFOS exposure modulated calcium homoeostasis in splenocytes. These results indicate that PFOS exposure can activate TCR signaling and calcium ion influx, which provides a clue for the potential mechanism of PFOS immunotoxicity. The altered signaling pathways by PFOS treatment as revealed in the present study might facilitate in better understanding PFOS immunotoxicity and explain the association between immune disease and PFOS exposure.

  3. DNA methylation profiling of hematopoietic stem cells. (United States)

    Begtrup, Amber Hogart


    DNA methylation is a key epigenetic mark that is essential for properly functioning hematopoietic stem cells. Determining where functionally relevant DNA methylation marks exist in the genome is crucial to understanding the role that methylation plays in hematopoiesis. This chapter describes a method to profile DNA methylation by selectively enriching methylated DNA sequences that are bound in vitro by methyl-binding domain (MBD) proteins. The MBD-pulldown approach selects for DNA sequences that have the potential to be "read" by the endogenous machinery involved in epigenetic regulation. Furthermore, this approach is feasible with very small quantities of DNA, and is compatible with the use of any downstream high-throughput sequencing approach. This technique offers a reliable, simple, and powerful tool for exploration of the role of DNA methylation in hematopoietic stem cells.

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

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung


    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.

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

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    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.

  6. Monoamine transporter and receptor interaction profiles of novel psychoactive substances: para-halogenated amphetamines and pyrovalerone cathinones. (United States)

    Rickli, Anna; Hoener, Marius C; Liechti, Matthias E


    The pharmacology of novel psychoactive substances is mostly unknown. We evaluated the transporter and receptor interaction profiles of a series of para-(4)-substituted amphetamines and pyrovalerone cathinones. We tested the potency of these compounds to inhibit the norepinephrine (NE), dopamine (DA), and serotonin (5-HT) transporters (NET, DAT, and SERT, respectively) using human embryonic kidney 293 cells that express the respective human transporters. We also tested the substance-induced efflux of NE, DA, and 5-HT from monoamine-loaded cells, binding affinities to monoamine receptors, and 5-HT2B receptor activation. Para-(4)-substituted amphetamines, including 4-methylmethcathinone (mephedrone), 4-ethylmethcathinone, 4-fluoroamphetamine, 4-fluoromethamphetamine, 4-fluoromethcatinone (flephedrone), and 4-bromomethcathinone, were relatively more serotonergic (lower DAT:SERT ratio) compared with their analogs amphetamine, methamphetamine, and methcathinone. The 4-methyl, 4-ethyl, and 4-bromo groups resulted in enhanced serotonergic properties compared with the 4-fluoro group. The para-substituted amphetamines released NE and DA. 4-Fluoramphetamine, 4-flouromethamphetamine, 4-methylmethcathinone, and 4-ethylmethcathinone also released 5-HT similarly to 3,4-methylenedioxymethamphetamine. The pyrovalerone cathinones 3,4-methylenedioxypyrovalerone, pyrovalerone, α-pyrrolidinovalerophenone, 3,4-methylenedioxy-α-pyrrolidinopropiophenone, and 3,4-methylenedioxy-α-pyrrolidinobutiophenone potently inhibited the NET and DAT but not the SERT. Naphyrone was the only pyrovalerone that also inhibited the SERT. The pyrovalerone cathinones did not release monoamines. Most of the para-substituted amphetamines exhibited affinity for the 5-HT2A receptor but no relevant activation of the 5-HT2B receptor. All the cathinones exhibited reduced trace amine-associated receptor 1 binding compared with the non-β-keto-amphetamines. In conclusion, para-substituted amphetamines exhibited

  7. Gene expression profiles in irradiated cancer cells (United States)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.


    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  8. Gene expression profiles in irradiated cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C. [IBFM CNR - LATO, Cefalù, Segrate (Italy)


    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  9. Improving the developability profile of pyrrolidine progesterone receptor partial agonists

    Energy Technology Data Exchange (ETDEWEB)

    Kallander, Lara S.; Washburn, David G.; Hoang, Tram H.; Frazee, James S.; Stoy, Patrick; Johnson, Latisha; Lu, Qing; Hammond, Marlys; Barton, Linda S.; Patterson, Jaclyn R.; Azzarano, Leonard M.; Nagilla, Rakesh; Madauss, Kevin P.; Williams, Shawn P.; Stewart, Eugene L.; Duraiswami, Chaya; Grygielko, Eugene T.; Xu, Xiaoping; Laping, Nicholas J.; Bray, Jeffrey D.; Thompson, Scott K. (GSKPA)


    The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

  10. Gene expression profiling of gastric mucosa in mice lacking CCK and gastrin receptors

    DEFF Research Database (Denmark)

    Zhao, Chun-Mei; Kodama, Yosuke; Flatberg, Arnar


    acid secretion was impaired and the ECL cell signaling pathway was inhibited in CCK2 receptor KO mice but not in CCK1 receptor KO mice. However, in CCK1+2 receptor double KO mice the acid secretion in response to pylorus ligation-induced vagal stimulation and the ECL cell pathway were partially...... hydroxylase probably in trefoil peptide2-immunoreactive cells. In conclusion, mice lacking CCK receptors exhibited a functional shift from the gastrin-CCK pathways to the neuronal pathway in control of the ECL cells and eventually the acid secretion. Taking the present data together with previous findings, we......The stomach produces acid, which may play an important role in the regulation of bone homeostasis. The aim of this study was to reveal signaling pathways in the gastric mucosa that involve the acid secretion and possibly the bone metabolism in CCK1 and/or CCK2 receptor knockout (KO) mice. Gastric...

  11. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells. (United States)

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku


    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  12. Ginkgolide X is a potent antagonist of anionic Cys-loop receptors with a unique selectivity profile at glycine receptors

    DEFF Research Database (Denmark)

    Jensen, Anders Asbjørn; Bergmann, Marianne Lerbæk; Sander, Tommy


    identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6' M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABAAR. Conformational analysis and docking of low-energy conformations of the native ginkgolide...... arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor....

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

  14. Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells (United States)

    Moody, Terry W.; Berna, Marc J.; Mantey, Samuel; Sancho, Veronica; Ridnour, Lisa; Wink, David A.; Chan, Daniel; Giaccone, Giuseppe; Jensen, Robert T.


    Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr1068 of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)α antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species. PMID:20388507

  15. Molecular cell biology of androgen receptor signalling. (United States)

    Bennett, Nigel C; Gardiner, Robert A; Hooper, John D; Johnson, David W; Gobe, Glenda C


    The classical action of androgen receptor (AR) is to regulate gene transcriptional processes via AR nuclear translocation, response element binding and recruitment of, or crosstalk with, transcription factors. AR also utilises non-classical, non-genomic mechanisms of signal transduction. These precede gene transcription or protein synthesis, and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. Despite many decades of investigation, the role of AR in gene regulation of cells and tissues remains only partially characterised. AR exerts most of its effects in sex hormone-dependent tissues of the body, but the receptor is also expressed in many tissues not previously thought to be androgen sensitive. Thus it is likely that a complex, more over-arching, role for AR exists. Each AR domain co-ordinates a multitude of individual and vital roles via a diverse array of interacting partner molecules that are necessary for cellular and tissue development and maintenance. Aberrant AR activity, promoted by mutations or binding partner misregulation, can present as many clinical manifestations including androgen insensitivity syndrome and prostate cancer. In the case of malignant prostate cancer, treatment generally revolves around androgen deprivation therapies designed to interfere with AR action and the androgen signalling axis. Androgen therapies for prostate cancer often fail, highlighting a real need for increased research into AR function.

  16. Genetic engineering with T cell receptors. (United States)

    Zhang, Ling; Morgan, Richard A


    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.

  17. Freedom of expression: cell-type-specific gene profiling. (United States)

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H


    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  18. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.


    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  19. Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Joseph Ignatius Irudayam


    Full Text Available Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5, hepatoblast (day 15 and hepatocyte-like cells (day 21 were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21 had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.

  20. Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells. (United States)

    Ignatius Irudayam, Joseph; Contreras, Deisy; Spurka, Lindsay; Ren, Songyang; Kanagavel, Vidhya; Ramaiah, Arunachalam; Annamalai, Alagappan; French, Samuel W; Klein, Andrew S; Funari, Vincent; Arumugaswami, Vaithilingaraja


    Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs) to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5), hepatoblast (day 15) and hepatocyte-like cells (day 21) were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21) had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF) family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.

  1. Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli. (United States)

    Ilatovskaya, Daria V; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander


    Calcium flux in the podocytes is critical for normal and pathophysiological regulation of these types of cells, and excessive calcium signaling results in podocytes damage and improper glomeruli function. Purinergic activation of P2 receptors is a powerful and rapid signaling process; however, the exact physiological identity of P2 receptors subtypes in podocytes remains essentially unknown. The goal of this study was to determine the P2 receptor profile in podocytes of the intact Sprague-Dawley rat glomeruli using available pharmacological tools. Glomeruli were isolated by differential sieving and loaded with Fluo-4/Fura Red cell permeable calcium indicators, and the purinergic response in the podocytes was analyzed with ratiometric confocal fluorescence measurements. Various P2 receptors activators were tested and compared with the effect of ATP, specifically, UDP, MRS 2365, bzATP, αβ-methylene, 2-meSADP, MRS 4062, and MRS 2768, were analyzed. Antagonists (MRS 2500, 5-BDBD, A438079, and NF 449) were tested when 10 μM ATP was applied as the EC50 for ATP activation of the calcium influx in the podocytes was determined to be 10.7 ± 1.5 μM. Several agonists including MRS 2365 and 2-meSADP caused calcium flux. Importantly, only the P2Y1-specific antagonist MRS 2500 (1 nM) precluded the effects of ATP concentrations of the physiological range. Immunohistochemical analysis confirmed that P2Y1 receptors are highly expressed in the podocytes. We conclude that P2Y1 receptor signaling is the predominant P2Y purinergic pathway in the glomeruli podocytes and P2Y1 might be involved in the pathogenesis of glomerular injury and could be a target for treatment of kidney diseases.

  2. Receptor Expression in Rat Skeletal Muscle Cell Cultures (United States)

    Young, Ronald B.


    One on the most persistent problems with long-term space flight is atrophy of skeletal muscles. Skeletal muscle is unique as a tissue in the body in that its ability to undergo atrophy or hypertrophy is controlled exclusively by cues from the extracellular environment. The mechanism of communication between muscle cells and their environment is through a group of membrane-bound and soluble receptors, each of which carries out unique, but often interrelated, functions. The primary receptors include acetyl choline receptors, beta-adrenergic receptors, glucocorticoid receptors, insulin receptors, growth hormone (i.e., somatotropin) receptors, insulin-like growth factor receptors, and steroid receptors. This project has been initiated to develop an integrated approach toward muscle atrophy and hypertrophy that takes into account information on the populations of the entire group of receptors (and their respective hormone concentrations), and it is hypothesized that this information can form the basis for a predictive computer model for muscle atrophy and hypertrophy. The conceptual basis for this project is illustrated in the figure below. The individual receptors are shown as membrane-bound, with the exception of the glucocorticoid receptor which is a soluble intracellular receptor. Each of these receptors has an extracellular signalling component (e.g., innervation, glucocorticoids, epinephrine, etc.), and following the interaction of the extracellular component with the receptor itself, an intracellular signal is generated. Each of these intracellular signals is unique in its own way; however, they are often interrelated.

  3. Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

    Institute of Scientific and Technical Information of China (English)


    During the last decade,increasing evidence suggested that bone marrow stromal cells(MSCs) have the potential to differentiate into neural lineages.Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions.However,no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported.In this study,we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions.By using two-dimensional gel electrophoresis(2-DE),we compared the protein profiles of MSCs before and after induced differentiation.We obtained 792 protein spots in the protein profile by 2-DE,and found that 74 spots changed significantly before and after the differentiation using PDQuest software,with 43 up-regulated and 31 down-regulated.We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS) and by database searching,and found that they could be grouped into various classes,including cytoskeleton and structure proteins,growth factors,metabolic proteins,chaperone proteins,receptor proteins,cell cycle proteins,calcium binding proteins,and other proteins.These proteins also include neural and glial proteins,such as BDNF,CNTF and GFAP.The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells.

  4. Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

    Institute of Scientific and Technical Information of China (English)

    LI WenTing; SUN HuaLin; XU ZengLu; DING Fei; GU XiaoSong


    During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morpho-logical changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and in-duced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We ana-lyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells.

  5. 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;


    densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... 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...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  6. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye


    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.

  7. Expression profile of the entire family of Adhesion G protein-coupled receptors in mouse and rat

    Directory of Open Access Journals (Sweden)

    Ebendal Ted


    Full Text Available Abstract Background The Adhesion G protein-coupled receptors (GPCRs are membrane-bound receptors with long N termini. This family has 33 members in humans. Several Adhesion GPCRs are known to have important physiological functions in CNS development and immune system response mediated by large cell surface ligands. However, the majority of Adhesion GPCRs are still poorly studied orphans with unknown functions. Results In this study we performed the extensive tissue localization analysis of the entire Adhesion GPCR family in rat and mouse. By applying the quantitative real-time PCR technique we have produced comparable expression profile for each of the members in the Adhesion family. The results are compared with literature data and data from the Allen Brain Atlas project. Our results suggest that the majority of the Adhesion GPCRs are either expressed in the CNS or ubiquitously. In addition the Adhesion GPCRs from the same phylogenetic group have either predominant CNS or peripheral expression, although each of their expression profile is unique. Conclusion Our findings indicate that many of Adhesion GPCRs are expressed, and most probably, have function in CNS. The related Adhesion GPCRs are well conserved in their structure and interestingly have considerable overlap in their expression profiles, suggesting similarities among the physiological roles for members within many of the phylogenetically related clusters.

  8. Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality. (United States)

    Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W; Böttcher, Sebastian; van Dongen, Jacques J M; Orfao, Alberto; Almeida, Julia


    Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.

  9. Receptor interaction profiles of novel psychoactive tryptamines compared with classic hallucinogens. (United States)

    Rickli, Anna; Moning, Olivier D; Hoener, Marius C; Liechti, Matthias E


    The present study investigated interactions between the novel psychoactive tryptamines DiPT, 4-OH-DiPT, 4-OH-MET, 5-MeO-AMT, and 5-MeO-MiPT at monoamine receptors and transporters compared with the classic hallucinogens lysergic acid diethylamide (LSD), psilocin, N,N-dimethyltryptamine (DMT), and mescaline. We investigated binding affinities at human monoamine receptors and determined functional serotonin (5-hydroxytryptamine [5-HT]) 5-HT2A and 5-HT2B receptor activation. Binding at and the inhibition of human monoamine uptake transporters and transporter-mediated monoamine release were also determined. All of the novel tryptamines interacted with 5-HT2A receptors and were partial or full 5-HT2A agonists. Binding affinity to the 5-HT2A receptor was lower for all of the tryptamines, including psilocin and DMT, compared with LSD and correlated with the reported psychoactive doses in humans. Several tryptamines, including psilocin, DMT, DiPT, 4-OH-DiPT, and 4-OH-MET, interacted with the serotonin transporter and partially the norepinephrine transporter, similar to 3,4-methylenedioxymethamphetamine but in contrast to LSD and mescaline. LSD but not the tryptamines interacted with adrenergic and dopaminergic receptors. In conclusion, the receptor interaction profiles of the tryptamines predict hallucinogenic effects that are similar to classic serotonergic hallucinogens but also MDMA-like psychoactive properties.

  10. Role of laminin receptor in tumor cell migration

    DEFF Research Database (Denmark)

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


    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......). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor...... antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin...

  11. Profiling of olfactory receptor gene expression in whole human olfactory mucosa.

    Directory of Open Access Journals (Sweden)

    Christophe Verbeurgt

    Full Text Available Olfactory perception is mediated by a large array of olfactory receptor genes. The human genome contains 851 olfactory receptor gene loci. More than 50% of the loci are annotated as nonfunctional due to frame-disrupting mutations. Furthermore haplotypic missense alleles can be nonfunctional resulting from substitution of key amino acids governing protein folding or interactions with signal transduction components. Beyond their role in odor recognition, functional olfactory receptors are also required for a proper targeting of olfactory neuron axons to their corresponding glomeruli in the olfactory bulb. Therefore, we anticipate that profiling of olfactory receptor gene expression in whole human olfactory mucosa and analysis in the human population of their expression should provide an opportunity to select the frequently expressed and potentially functional olfactory receptors in view of a systematic deorphanization. To address this issue, we designed a TaqMan Low Density Array (Applied Biosystems, containing probes for 356 predicted human olfactory receptor loci to investigate their expression in whole human olfactory mucosa tissues from 26 individuals (13 women, 13 men; aged from 39 to 81 years, with an average of 67±11 years for women and 63±12 years for men. Total RNA isolation, DNase treatment, RNA integrity evaluation and reverse transcription were performed for these 26 samples. Then 384 targeted genes (including endogenous control genes and reference genes specifically expressed in olfactory epithelium for normalization purpose were analyzed using the same real-time reverse transcription PCR platform. On average, the expression of 273 human olfactory receptor genes was observed in the 26 selected whole human olfactory mucosa analyzed, of which 90 were expressed in all 26 individuals. Most of the olfactory receptors deorphanized to date on the basis of sensitivity to known odorant molecules, which are described in the literature, were

  12. Donor Killer Immunoglobulin-Like Receptor Profile Bx1 Imparts a Negative Effect and Centromeric B-Specific Gene Motifs Render a Positive Effect on Standard-Risk Acute Myeloid Leukemia/Myelodysplastic Syndrome Patient Survival after Unrelated Donor Hematopoietic Stem Cell Transplantation. (United States)

    Bao, Xiaojing; Wang, Miao; Zhou, Huifen; Zhang, Huanhuan; Wu, Xiaojin; Yuan, Xiaoni; Li, Yang; Wu, Depei; He, Jun


    Donor killer immunoglobulin-like receptor (KIR) group B profiles (Bx) and homozygous of centromeric motif B (Cen-B/B) are the most preferable KIR gene content motifs for hematopoietic stem cell transplantation (HSCT). The risk of transplant from Bx1 donors and the benefit of the presence of Cen-B (regardless of number) were observed for standard-risk acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) patients in this 4-year retrospective study. A total of 210 Chinese patients who underwent unrelated donor HSCT were investigated. Donor KIR profile Bx was associated with significantly improved overall survival (OS; P = .026) and relapse-free survival (RFS; P = .021) and reduced nonrelapse mortality (NRM; P = .017) in AML/MDS patients. A significantly lower survival rate was observed for transplants from Bx1 donors compared with Bx2, Bx3, and Bx4 donors for patients in first complete remission (n = 82; OS: P = .024; RFS: P = .021). Transplant from donors with Cen-B resulted in improved OS (HR = .256; 95% CI, .084 to .774; P = .016) and RFS (HR = .252; 95% CI, .084 to .758; P = .014) in AML/MDS patients at standard risk. However, this particular effect did not increase with a higher number of Cen-B motifs (cB/B versus cA/B; OS: P = .755; RFS: P = .768). No effect was observed on high-risk AML/MDS, acute lymphoblastic leukemia/non-Hodgkin lymphoma, and chronic myelogenous leukemia patients. Avoiding the selection of HSCT donors of KIR profile Bx1 is strongly advisable for standard-risk AML/MDS patients. The presence of the Cen-B motif rather than its number was more important in donor selection for the Chinese population.

  13. The potential of single-cell profiling in plants. (United States)

    Efroni, Idan; Birnbaum, Kenneth D


    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology.

  14. Identification and characterization of estrogen receptor-related receptor alpha and gamma in human glioma and astrocytoma cells


    Gandhari, Mukesh K; Frazier, Chester R.; Hartenstein, Julia S; Cloix, Jean-Francois; Bernier, Michel; Wainer, Irving W.


    The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRα and ERRγ proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC50 and IC50 value...

  15. Ubiquitylation of the chemokine receptor CCR7 enables efficient receptor recycling and cell migration


    Schäuble, Karin; Hauser, Mark A.; Rippl, Alexandra; Bruderer, Roland; Otero, Carolina; Gröttrup, Marcus; Legler, Daniel F.


    The chemokine receptor CCR7 is essential for lymphocyte and dendritic cell homing to secondary lymphoid organs. Owing to the ability to induce directional migration, CCR7 and its ligands CCL19 and CCL21 are pivotal for the regulation of the immune system. Here, we identify a novel function for receptor ubiquitylation in the regulation of the trafficking process of this G-protein-coupled seven transmembrane receptor. We discovered that CCR7 is ubiquitylated in a constitutive, ligand-independen...

  16. 5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

    DEFF Research Database (Denmark)

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


    in the CNS, none of the studies showed its expression and function in the endothelial cells. In the present study, we provide evidence for the first time that 5-HT4 receptor is expressed in the human umbilical vein endothelial cells (HUVECs). We demonstrate the transcription of 5-HT4 mRNA in the HUVECs using...... reverse transcription polimerase chain reaction. Additionally, we show 5- HT4 receptor expression in HUVECs by immunoblotting and immunofluorescent analysis with 5-HT4 specific antibody. Importantly, we determine that overexpression of 5-HT4 receptor leads to a pronounced cell rounding and intercellular...... 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...

  17. Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma (United States)

    Shahrabi-Farahani, Shokoufeh; Gallottini, Marina; Martins, Fabiana; Li, Erik; Mudge, Dayna R.; Nakayama, Hironao; Hida, Kyoko; Panigrahy, Dipak; D'Amore, Patricia A.; Bielenberg, Diane R.


    Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells. PMID:26877262

  18. Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma. (United States)

    Shahrabi-Farahani, Shokoufeh; Gallottini, Marina; Martins, Fabiana; Li, Erik; Mudge, Dayna R; Nakayama, Hironao; Hida, Kyoko; Panigrahy, Dipak; D'Amore, Patricia A; Bielenberg, Diane R


    Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells.

  19. Distribution of P2Y receptor subtypes on haematopoietic cells



    RT–PCR-southern hybridization analyses with radiolabelled P2Y receptor cDNAs as probes indicated that the peripheral blood leukocytes and the human umbilical vein endothelial cells express P2Y1, P2Y2, P2Y4 and P2Y6 receptors.Of the haematopoietic cell lines tested, promonocytic U937 cells express P2Y2 and P2Y6, but not P2Y1 or P2Y4; promyelocytic HL-60 cells express the P2Y1, P2Y2 and P2Y6 receptors but not the P2Y4 receptor; K562 cells express P2Y1 but not P2Y2, P2Y4 or P2Y6; and Dami cells ...

  20. 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)


    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 ce

  1. Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

    Directory of Open Access Journals (Sweden)

    Yiyan Fei


    Full Text Available A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA, with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

  2. Role of leptin receptors in granulosa cells during ovulation. (United States)

    Dupuis, Lisa; Schuermann, Yasmin; Cohen, Tamara; Siddappa, Dayananda; Kalaiselvanraja, Anitha; Pansera, Melissa; Bordignon, Vilceu; Duggavathi, Raj


    Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4 h after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein β (Cebpβ) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpβ to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpβ, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10 μg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpβ. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.

  3. Chemokine receptor expression by inflammatory T cells in EAE

    DEFF Research Database (Denmark)

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


    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...... receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed...... whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays...

  4. Challenges in imaging cell surface receptor clusters (United States)

    Medda, Rebecca; Giske, Arnold; Cavalcanti-Adam, Elisabetta Ada


    Super-resolution microscopy offers unique tools for visualizing and resolving cellular structures at the molecular level. STED microscopy is a purely optical method where neither complex sample preparation nor mathematical post-processing is required. Here we present the use of STED microscopy for imaging receptor cluster composition. We use two-color STED to further determine the distribution of two different receptor subunits of the family of receptor serine/threonine kinases in the presence or absence of their ligands. The implications of receptor clustering on the downstream signaling are discussed, and future challenges are also presented.

  5. Gravity and the cells of gravity receptors in mammals (United States)

    Ross, M. D.

    Two new findings, that crystals located in the inner ear gravity receptors of mammals have the internal organization requisite for the piezoelectric property, and that sensory hair cells of these same receptors possess contractile-appearing striated organelles, have prompted the author to model mammalian gravity receptors in the ear on the principles of piezoelectricity and bioenergetics. This model is presented and a brief discussion of its implications for the possible effects of weightlessness follows.

  6. Differential infection of receptor-modified host cells by receptor-specific influenza viruses. (United States)

    Carroll, S M; Paulson, J C


    Influenza viruses of contrasting receptor specificity have been examined for their ability to infect receptor-modified MDCK cells containing sialyloligosaccharide receptor determinants of defined sequence. Cells were treated with sialidase to remove sialic acid and render them resistant to infection and were then incubated with sialyltransferase and CMP-sialic acid to restore sialic acid in the SA alpha 2,6Gal or SA alpha 2,3Gal linkages. The viruses A/RI/5 + /57 and A/duck/Ukraine/1/63, previously shown to exhibit preferential binding of SA alpha 2,6Gal and SA alpha 2,3Gal linkages, respectively, were found to exhibit differential infection of the receptor-modified cells in accord with their receptor specificity. Coinfection of SA alpha 2,3Gal derivatized cells with a mixture of the two viruses resulted in selective propagation of the SA alpha 2,3Gal-specific A/duck/Ukraine/1/63 virus. The results demonstrate the potential for cell surface receptors to mediate selection of receptor-specific variants of influenza virus.

  7. Non-small cell lung cancer cell survival crucially depends on functional insulin receptors. (United States)

    Frisch, Carolin Maria; Zimmermann, Katrin; Zilleßen, Pia; Pfeifer, Alexander; Racké, Kurt; Mayer, Peter


    Insulin plays an important role as a growth factor and its contribution to tumor proliferation is intensely discussed. It acts via the cognate insulin receptor (IR) but can also activate the IGF1 receptor (IGF1R). Apart from increasing proliferation, insulin might have additional effects in lung cancer. Therefore, we investigated insulin action and effects of IR knockdown (KD) in three (NCI-H292, NCI-H226 and NCI-H460) independent non-small cell lung cancer (NSCLC) cell lines. All lung cancer lines studied were found to express IR, albeit with marked differences in the ratio of the two variants IR-A and IR-B. Insulin activated the classical signaling pathway with IR autophosphorylation and Akt phosphorylation. Moreover, activation of MAPK was observed in H292 cells, accompanied by enhanced proliferation. Lentiviral shRNA IR KD caused strong decrease in survival of all three lines, indicating that the effects of insulin in lung cancer go beyond enhancing proliferation. Unspecific effects were ruled out by employing further shRNAs and different insulin-responsive cells (human pre-adipocytes) for comparison. Caspase assays demonstrated that IR KD strongly induced apoptosis in these lung cancer cells, providing the physiological basis of the rapid cell loss. In search for the underlying mechanism, we analyzed alterations in the gene expression profile in response to IR KD. A strong induction of certain cytokines (e.g. IL20 and tumour necrosis factor) became obvious and it turned out that these cytokines trigger apoptosis in the NSCLC cells tested. This indicates a novel role of IR in tumor cell survival via suppression of pro-apoptotic cytokines.

  8. Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

    DEFF Research Database (Denmark)

    Corrêa, Natássia C R; Kuasne, Hellen; Faria, Jerusa A Q A


    Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1...... and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted....... This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines...

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

    Directory of Open Access Journals (Sweden)

    I. Hansenne


    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.

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

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


    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.

  11. HSP90 promotes Burkitt lymphoma cell survival by maintaining tonic B-cell receptor signaling. (United States)

    Walter, Roland; Pan, Kuan-Ting; Doebele, Carmen; Comoglio, Federico; Tomska, Katarzyna; Bohnenberger, Hanibal; Young, Ryan M; Jacobs, Laura; Keller, Ulrich; Bönig, Halvard; Engelke, Michael; Rosenwald, Andreas; Urlaub, Henning; Staudt, Louis M; Serve, Hubert; Zenz, Thorsten; Oellerich, Thomas


    Burkitt lymphoma (BL) is an aggressive B-cell neoplasm that is currently treated by intensive chemotherapy in combination with anti-CD20 antibodies. Because of their toxicity, current treatment regimens are often not suitable for elderly patients or for patients in developing countries where BL is endemic. Targeted therapies for BL are therefore needed. In this study, we performed a compound screen in 17 BL cell lines to identify small molecule inhibitors affecting cell survival. We found that inhibitors of heat shock protein 90 (HSP90) induced apoptosis in BL cells in vitro at concentrations that did not affect normal B cells. By global proteomic and phosphoproteomic profiling, we show that, in BL, HSP90 inhibition compromises the activity of the pivotal B-cell antigen receptor (BCR)-proximal effector spleen tyrosine kinase (SYK), which we identified as an HSP90 client protein. Consistently, expression of constitutively active TEL-SYK counteracted the apoptotic effect of HSP90 inhibition. Together, our results demonstrate that HSP90 inhibition impairs BL cell survival by interfering with tonic BCR signaling, thus providing a molecular rationale for the use of HSP90 inhibitors in the treatment of BL.

  12. Functional significance of erythropoietin receptor on tumor cells

    Institute of Scientific and Technical Information of China (English)

    Kodetthoor B Udupa


    Erythropoietin (Epo) is the regulator of red blood cell formation. Its receptor (EpoR) is now found in many cells and tissues of the body. EpoR is also shown to occur in tumor cells and Epo enhances the proliferation of these cells through cell signaling. EpoR antagonist can reduce the growth of the tumor in vivo. In view of our current knowledge of Epo, its recombinant forms and receptor,use of Epo in cancer patients to enhance the recovery of hematocrit after chemotherapy treatment has to be carefully evaluated.

  13. A comparison between protein profiles of B cell subpopulations and mantle cell lymphoma cells

    Directory of Open Access Journals (Sweden)

    Lehtiö Janne


    Full Text Available Abstract Background B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL is presumed to derive predominantly from naïve, pre-germinal centre (pre-GC B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods Subpopulations of B cells representing the germinal centre (GC, the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS. Results Quantitative MS data of significant protein peaks (p-value Conclusion AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.

  14. Lysophosphatidic Acid Receptor Is a Functional Marker of Adult Hippocampal Precursor Cells

    Directory of Open Access Journals (Sweden)

    Tara L. Walker


    Full Text Available Here, we show that the lysophosphatidic acid receptor 1 (LPA1 is expressed by a defined population of type 1 stem cells and type 2a precursor cells in the adult mouse dentate gyrus. LPA1, in contrast to Nestin, also marks the quiescent stem cell population. Combining LPA1-GFP with EGFR and prominin-1 expression, we have enabled the prospective separation of both proliferative and non-proliferative precursor cell populations. Transcriptional profiling of the isolated proliferative precursor cells suggested immune mechanisms and cytokine signaling as molecular regulators of adult hippocampal precursor cell proliferation. In addition to LPA1 being a marker of this important stem cell population, we also show that the corresponding ligand LPA is directly involved in the regulation of adult hippocampal precursor cell proliferation and neurogenesis, an effect that can be attributed to LPA signaling via the AKT and MAPK pathways.

  15. A pharmacological profile of the high-affinity GluK5 kainate receptor

    DEFF Research Database (Denmark)

    Møllerud, Stine; Kastrup, Jette Sandholm Jensen; Pickering, Darryl S


    Mouse GluK5 was expressed in Sf9 insect cells and radiolabelled with [3H]-kainate in receptor binding assays (Kd = 6.9 nM). Western immunoblotting indicated an Sf9 GluK5 band doublet corresponding to the glycosylated (128 kDa) and deglycosylated (111 kDa) protein, which was identical to the band...

  16. A response calculus for immobilized T cell receptor ligands

    DEFF Research Database (Denmark)

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


    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...

  17. Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells. (United States)

    Antoine, M; Wirz, W; Tag, C G; Mavituna, M; Emans, N; Korff, T; Stoldt, V; Gressner, A M; Kiefer, P


    Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.

  18. Myristoylation profiling in human cells and zebrafish

    Directory of Open Access Journals (Sweden)

    Malgorzata Broncel


    Full Text Available Human cells (HEK 293, HeLa, MCF-7 and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC. This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223–6 (PXD001863 and PXD001876 and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.

  19. TAM receptors support neural stem cell survival, proliferation and neuronal differentiation.

    Directory of Open Access Journals (Sweden)

    Rui Ji

    Full Text Available Tyro3, Axl and Mertk (TAM receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS. In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III+ and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF and brain-derived neurotrophic factor (BDNF, but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.

  20. Cell and molecular biology of epidermal growth factor receptor. (United States)

    Ceresa, Brian P; Peterson, Joanne L


    The epidermal growth factor receptor (EGFR) has been one of the most intensely studied cell surface receptors due to its well-established roles in developmental biology, tissue homeostasis, and cancer biology. The EGFR has been critical for creating paradigms for numerous aspects of cell biology, such as ligand binding, signal transduction, and membrane trafficking. Despite this history of discovery, there is a continual stream of evidence that only the surface has been scratched. New ways of receptor regulation continue to be identified, each of which is a potential molecular target for manipulating EGFR signaling and the resultant changes in cell and tissue biology. This chapter is an update on EGFR-mediated signaling, and describes some recent developments in the regulation of receptor biology.

  1. Cell Shape and Cardiosphere Differentiation: A Revelation by Proteomic Profiling

    Directory of Open Access Journals (Sweden)

    Nanako Kawaguchi


    Full Text Available Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions.

  2. Advances in human B cell phenotypic profiling

    Directory of Open Access Journals (Sweden)

    Denise A Kaminski


    Full Text Available To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation (Big Biology, necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.

  3. Massively parallel digital transcriptional profiling of single cells (United States)

    Zheng, Grace X. Y.; Terry, Jessica M.; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W.; Wilson, Ryan; Ziraldo, Solongo B.; Wheeler, Tobias D.; McDermott, Geoff P.; Zhu, Junjie; Gregory, Mark T.; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G.; Masquelier, Donald A.; Nishimura, Stefanie Y.; Schnall-Levin, Michael; Wyatt, Paul W.; Hindson, Christopher M.; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D.; Beppu, Lan W.; Deeg, H. Joachim; McFarland, Christopher; Loeb, Keith R.; Valente, William J.; Ericson, Nolan G.; Stevens, Emily A.; Radich, Jerald P.; Mikkelsen, Tarjei S.; Hindson, Benjamin J.; Bielas, Jason H.


    Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients. PMID:28091601

  4. P2 receptor-mediated signaling in mast cell biology. (United States)

    Bulanova, Elena; Bulfone-Paus, Silvia


    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.

  5. Chimeric Antigen Receptor T Cell Therapy in Hematology. (United States)

    Ataca, Pınar; Arslan, Önder


    It is well demonstrated that the 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. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. 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 1 July 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 benefits 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 and clinical studies, and the effectiveness and drawbacks of this strategy.

  6. Activation of intracellular angiotensin AT₂ receptors induces rapid cell death in human uterine leiomyosarcoma cells. (United States)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen; Zuhayra, Maaz; Schütze, Stefan; Steckelings, Ulrike M; Recanti, Chiara; Namsoleck, Pawel; Unger, Thomas; Culman, Juraj


    The presence of angiotensin type 2 (AT₂) receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT₂ receptors including their presence in mitochondria and their role in the induction 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 densities in mitochondria. Activation of the cell membrane AT₂ receptors by a concomitant treatment with angiotensin II and the AT₁ receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT₂ receptor agonist, Compound 21 (C21), penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT₂ receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped cell death, displayed activation of the mitochondrial apoptotic pathway, i.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 the elimination non-cycling uterine leiomyosarcoma cells providing that they over-express the AT₂ receptor.

  7. OVCAR-3 spheroid-derived cells display distinct metabolic profiles.

    Directory of Open Access Journals (Sweden)

    Kathleen A Vermeersch

    Full Text Available Recently, multicellular spheroids were isolated from a well-established epithelial ovarian cancer cell line, OVCAR-3, and were propagated in vitro. These spheroid-derived cells displayed numerous hallmarks of cancer stem cells, which are chemo- and radioresistant cells thought to be a significant cause of cancer recurrence and resultant mortality. Gene set enrichment analysis of expression data from the OVCAR-3 cells and the spheroid-derived putative cancer stem cells identified several metabolic pathways enriched in differentially expressed genes. Before this, there had been little previous knowledge or investigation of systems-scale metabolic differences between cancer cells and cancer stem cells, and no knowledge of such differences in ovarian cancer stem cells.To determine if there were substantial metabolic changes corresponding with these transcriptional differences, we used two-dimensional gas chromatography coupled to mass spectrometry to measure the metabolite profiles of the two cell lines.These two cell lines exhibited significant metabolic differences in both intracellular and extracellular metabolite measurements. Principal components analysis, an unsupervised dimensional reduction technique, showed complete separation between the two cell types based on their metabolite profiles. Pathway analysis of intracellular metabolomics data revealed close overlap with metabolic pathways identified from gene expression data, with four out of six pathways found enriched in gene-level analysis also enriched in metabolite-level analysis. Some of those pathways contained multiple metabolites that were individually statistically significantly different between the two cell lines, with one of the most broadly and consistently different pathways, arginine and proline metabolism, suggesting an interesting hypothesis about cancerous and stem-like metabolic phenotypes in this pair of cell lines.Overall, we demonstrate for the first time that metabolism

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

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    Fabio Cattaneo


    Full Text Available 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

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

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    Noam eRudich


    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.

  10. P2 receptor-mediated signaling in mast cell biology


    Bulanova, Elena; Bulfone-Paus, Silvia


    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of ...

  11. A balance between B cell receptor and inhibitory receptor signaling controls plasma cell differentiation by maintaining optimal Ets1 levels. (United States)

    Luo, Wei; Mayeux, Jessica; Gutierrez, Toni; Russell, Lisa; Getahun, Andrew; Müller, Jennifer; Tedder, Thomas; Parnes, Jane; Rickert, Robert; Nitschke, Lars; Cambier, John; Satterthwaite, Anne B; Garrett-Sinha, Lee Ann


    Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP1) or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling, thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn → CD22/SiglecG → SHP1 pathway in B cells.

  12. Genome-wide transcriptional profiling of human glioblastoma cells in response to ITE treatment. (United States)

    Kang, Bo; Zhou, Yanwen; Zheng, Min; Wang, Ying-Jie


    A ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is recently revealed to play a key role in embryogenesis and tumorigenesis (Feng et al. [1], Safe et al. [2]) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (Song et al. [3]) is an endogenous AhR ligand that possesses anti-tumor activity. In order to gain insights into how ITE acts via the AhR in embryogenesis and tumorigenesis, we analyzed the genome-wide transcriptional profiles of the following three groups of cells: the human glioblastoma U87 parental cells, U87 tumor sphere cells treated with vehicle (DMSO) and U87 tumor sphere cells treated with ITE. Here, we provide the details of the sample gathering strategy and show the quality controls and the analyses associated with our gene array data deposited into the Gene Expression Omnibus (GEO) under the accession code of GSE67986.

  13. Automated Detection of Binucleated Cell and Micronuclei using CellProfiler 2.0 Software

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    Dwi Ramadhani


    Full Text Available Micronucleus assay in human peripheral lymphocytes usually used to assess chromosomal damage. Manual scoring of micronuclei can be time consuming and large numbers of binucleated cells have to be analyzed to obtain statistically relevant data. Automation of the micronuclei analysis using image processing analysis software can provide a faster and more reliable analysis of micronucleus assay. Here the used of CellProfiler an open access cell image analysis software for automatic detection of binucleated cells and micronuclei were reported. We aimed to know whether there was a significant difference in the number of binucleated cells and micronuclei that obtained by manual and CellProfiler counting. Wilcoxon Rank test was used for statistical analysis to test H0 hypothesis that there was no significant difference in the number of binucleated cells and micronuclei that obtained by manual and CellProfiler counting. We analyzed 135 images for both manual and CellProfiler counting. Our results showed that there was no significant difference between manual and CellProfiler counting for binucleated cells (P = 0.851 and for micronuclei (P = 0.917. In conclusion, the binucleated cells and micronuclei counting using CellProfiler were comparable but not better than manual counting.

  14. Fluorescent ligand for human progesterone receptor imaging in live cells. (United States)

    Weinstain, Roy; Kanter, Joan; Friedman, Beth; Ellies, Lesley G; Baker, Michael E; Tsien, Roger Y


    We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core.

  15. Laminins and their receptors in Schwann cells and hereditary neuropathies. (United States)

    Feltri, Maria Laura; Wrabetz, Lawrence


    This review focuses on the influence of laminins, mediated through laminin receptors present on Schwann cells, on peripheral nerve development and pathology. Laminins influence multiple aspects of cell differentiation and tissue morphogenesis, including cell survival, proliferation, cytoskeletal rearrangements, and polarity. Peripheral nerves are no exception, as shown by the discovery that defective laminin signals contribute to the pathogenesis of diverse neuropathies such as merosin-deficient congenital muscular dystrophy and Charcot-Marie-Tooth 4F, neurofibromatosis, and leprosy. In the last 5 years, advanced molecular and cell biological techniques and conditional mutagenesis in mice began revealing the role of different laminins and receptors in developing nerves. In this way, we are starting to explain morphological and pathological observations beginning at the start of the last century. Here, we review these recent advances and show how the roles of laminins and their receptors are surprisingly varied in both time and place.


    Institute of Scientific and Technical Information of China (English)

    D-M,Chuang; M.Belchers; J.Barg; J.Rowinski; G.Clark; C.A.Gloeckner; A.Ho; X-M.Gao; C.J.Coscia


    The mechanisms underlying tolerance and dependence arising from chronic opioid exposure are poorly understood. However, the development of neuroblastoma and neurohybrid cell culturea, has provided a simplified model for the atudy of opioid receptor adaptation. Using neuroblastoma NG108-15 cells,

  17. Differential expression of functional Fc-receptors and additional immune complex receptors on mouse kidney cells. (United States)

    Suwanichkul, Adisak; Wenderfer, Scott E


    The precise mechanisms by which circulating immune complexes accumulate in the kidney to form deposits in glomerulonephritis are not well understood. In particular, the role of resident cells within glomeruli of the kidney has been widely debated. Immune complexes have been shown to bind one glomerular cell type (mesangial cells) leading to functional responses such as pro-inflammatory cytokine production. To further assess the presence of functional immunoreceptors on resident glomerular cells, cultured mouse renal epithelial, endothelial, and mesangial cells were treated with heat-aggregated mouse IgG or preformed murine immune complexes. Mesangial and renal endothelial cells were found to bind IgG complexes, whereas glomerular epithelial cell binding was minimal. A blocking antibody for Fc-gamma receptors reduced binding to mesangial cells but not renal endothelial cells, suggesting differential immunoreceptor utilization. RT-PCR and immunostaining based screening of cultured renal endothelial cells showed limited low-level expression of known Fc-receptors and Ig binding proteins. The interaction between mesangial cells and renal endothelial cells and immune complexes resulted in distinct, cell-specific patterns of chemokine and cytokine production. This novel pathway involving renal endothelial cells likely contributes to the predilection of circulating immune complex accumulation within the kidney and to the inflammatory responses that drive kidney injury.

  18. Mouse Leydig cells express multiple P2X receptor subunits



    ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X1–P2X7) and seven heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/6, P2X4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca2+]i and testosterone secretion, supporting the hypothesis that Ca2+ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Ley...

  19. The evolution of natural killer cell receptors

    NARCIS (Netherlands)

    Carrillo-Bustamante, Paola; Kesmir, C.; de Boer, Rob J


    Natural killer (NK) cells are 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 is tightly regulated by a sophisticated array of germline-encoded activating and inhibitin

  20. Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs. (United States)

    Stevens, James; Chen, Li-Mei; Carney, Paul J; Garten, Rebecca; Foust, Angie; Le, Jianhua; Pokorny, Barbara A; Manojkumar, Ramanunninair; Silverman, Jeanmarie; Devis, Rene; Rhea, Karen; Xu, Xiyan; Bucher, Doris J; Paulson, James C; Paulson, James; Cox, Nancy J; Klimov, Alexander; Donis, Ruben O


    Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of alpha2-6 and alpha2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to alpha2-6- and alpha2-3-type receptors but retained substantial binding to specific O- and N-linked alpha2-3 glycans, including alpha2-3GalNAc and fucosylated alpha2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.

  1. Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases. (United States)

    Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S


    3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting.

  2. Receptor-Dependent Coronavirus Infection of Dendritic Cells (United States)

    Turner, Brian C.; Hemmila, Erin M.; Beauchemin, Nicole; Holmes, Kathryn V.


    In several mammalian species, including humans, coronavirus infection can modulate the host immune response. We show a potential role of dendritic cells (DC) in murine coronavirus-induced immune modulation and pathogenesis by demonstrating that the JAW SII DC line and primary DC from BALB/c mice and p/p mice with reduced expression of the murine coronavirus receptor, murine CEACAM1a, are susceptible to murine coronavirus infection by a receptor-dependent pathway. PMID:15113927

  3. Gene expression profile changes in NB4 cells induced by realgar

    Institute of Scientific and Technical Information of China (English)

    王怀宇; 刘陕西; 吕晓虹; 赵晓艾; 陈思宇; 李信民


    Objectives To compare the gene expression profiles of acute promyelocytic leukemia cell line NB4 before and after 12 hours of realgar treatment using cDNA microarray.Methods Two cDNA probes were prepared through reverse transcription from mRNA of both untreated and realgar treated NB4 cells. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 1003 different human genes, and scanned for fluorescent intensity. The genes were screened through the analysis of the difference in two gene expression profiles. Results The analysis of gene expression profiles indicates that 9 genes were up-regulated and 37 genes were down-regulated. Among the 9 up-regulated genes, 2 genes were involved in a proteasome degradation pathway. Some genes related to protein synthesis, signal transduction and cell receptors were down-regulated. Conclusion PSMC2 and PSMD1 genes may play an important role in the apoptosis and partial differentiation of NB4 cells.

  4. Regulation of gene expression in ovarian cancer cells by luteinizing hormone receptor expression and activation

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    Dam Phuongan


    Full Text Available Abstract Background 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. Methods 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. Results 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

  5. Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells. (United States)

    Rodríguez-Hernández, A; Navarro-Villarán, E; González, R; Pereira, S; Soriano-De Castro, L B; Sarrias-Giménez, A; Barrera-Pulido, L; Álamo-Martínez, J M; Serrablo-Requejo, A; Blanco-Fernández, G; Nogales-Muñoz, A; Gila-Bohórquez, A; Pacheco, D; Torres-Nieto, M A; Serrano-Díaz-Canedo, J; Suárez-Artacho, G; Bernal-Bellido, C; Marín-Gómez, L M; Barcena, J A; Gómez-Bravo, M A; Padilla, C A; Padillo, F J; Muntané, J


    Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

  6. Activation of D4 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells. (United States)

    Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D; Jose, Pedro A; Zeng, Chunyu


    The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal angiotensin type 1 (AT1) receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto (WKY) rats, but the D4 receptor regulation of AT1 receptor is aberrant in renal proximal tubule cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time- and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na(+)-K(+) ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na(+)-K(+) ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na(+)-K(+) ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na(+)-K(+) ATPase activity in SHR cells. This was confirmed in vivo; pretreatment with PD128077 for 1 week augmented the antihypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension.

  7. Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic cells


    Basu, Sreyashi; Srivastava, Pramod


    Capsaicin (CP), the pungent component of chili pepper, acts on sensory neurons to convey the sensation of pain. The CP receptor, vanilloid receptor 1 (VR1), has been shown to be highly expressed by nociceptive neurons in dorsal root and trigeminal ganglia. We demonstrate here that the dendritic cell (DC), a key cell type of the vertebrate immune system, expresses VR1. Engagement of VR1 on immature DCs such as by treatment with CP leads to maturation of DCs as measured by up-regulation of anti...

  8. Opiate receptor blockade on human granulosa cells inhibits VEGF release. (United States)

    Lunger, Fabian; Vehmas, Anni P; Fürnrohr, Barbara G; Sopper, Sieghart; Wildt, Ludwig; Seeber, Beata


    The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

  9. Effect of the Cannabinoid Receptor-1 antagonist SR141716A on human adipocyte inflammatory profile and differentiation

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    Murumalla Ravi


    Full Text Available Abstract Background Obesity is characterized by inflammation, caused by increase in proinflammatory cytokines, a key factor for the development of insulin resistance. SR141716A, a cannabinoid receptor 1 (CB1 antagonist, shows significant improvement in clinical status of obese/diabetic patients. Therefore, we studied the effect of SR141716A on human adipocyte inflammatory profile and differentiation. Methods Adipocytes were obtained from liposuction. Stromal vascular cells were extracted and differentiated into adipocytes. Media and cells were collected for secretory (ELISA and expression analysis (qPCR. Triglyceride accumulation was observed using oil red-O staining. Cholesterol was assayed by a fluorometric method. 2-AG and anandamide were quantified using isotope dilution LC-MS. TLR-binding experiments have been conducted in HEK-Blue cells. Results In LPS-treated mature adipocytes, SR141716A was able to decrease the expression and secretion of TNF-a. This molecule has the same effect in LPS-induced IL-6 secretion, while IL-6 expression is not changed. Concerning MCP-1, the basal level is down-regulated by SR141716A, but not the LPS-induced level. This effect is not caused by a binding of the molecule to TLR4 (LPS receptor. Moreover, SR141716A restored adiponectin secretion to normal levels after LPS treatment. Lastly, no effect of SR141716A was detected on human pre-adipocyte differentiation, although the compound enhanced adiponectin gene expression, but not secretion, in differentiated pre-adipocytes. Conclusion We show for the first time that some clinical effects of SR141716A are probably directly related to its anti-inflammatory effect on mature adipocytes. This fact reinforces that adipose tissue is an important target in the development of tools to treat the metabolic syndrome.

  10. ATP induces P2X7 receptor-independent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells. (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Koch-Nolte, Friedrich; Haag, Friedrich; Bulfone-Paus, Silvia


    Extracellular ATP mediates a diverse array of biological responses in many cell types and tissues, including immune cells. We have demonstrated that ATP induces purinergic receptor P2X(7) mediated membrane permeabilization, apoptosis, and cytokine expression in murine mast cells (MCs). Here, we report that MCs deficient in the expression of the P2X(7) receptor are resistant to the ATP-induced membrane permeabilization and apoptosis. However, ATP affects the tyrosine phosphorylation pattern of P2X(7)knockout cells, leading to the activation of ERK1/2. Furthermore, ATP induces expression of several cytokines and chemokines in these cells, including IL-4, IL-6, IFN-gamma, TNF-alpha, RANTES, and MIP-2, at the mRNA level. In addition, the release of IL-6 and IL-13 to cell-conditioned medium was confirmed by ELISA. The ligand selectivity and pharmacological profile indicate the involvement of two P2X family receptors, P2X(1) and P2X(3). Thus, depending on genetic background, particular tissue microenvironment, and ATP concentration, MCs can presumably engage different P2X receptor subtypes, which may result in functionally distinct biological responses to extracellular nucleotides. This finding highlights a novel level of complexity in the sophisticated biology of MCs and may facilitate the development of new therapeutic approaches to modulate MC activities.

  11. Research Resource: Androgen Receptor Activity Is Regulated Through the Mobilization of Cell Surface Receptor Networks. (United States)

    Hsiao, Jordy J; Ng, Brandon H; Smits, Melinda M; Martinez, Harryl D; Jasavala, Rohini J; Hinkson, Izumi V; Fermin, Damian; Eng, Jimmy K; Nesvizhskii, Alexey I; Wright, Michael E


    The aberrant expression of androgen receptor (AR)-dependent transcriptional programs is a defining pathology of the development and progression of prostate cancers. Transcriptional cofactors that bind AR are critical determinants of prostate tumorigenesis. To gain a deeper understanding of the proteins linked to AR-dependent gene transcription, we performed a DNA-affinity chromatography-based proteomic screen designed to identify proteins involved in AR-mediated gene transcription in prostate tumor cells. Functional experiments validated the coregulator roles of known AR-binding proteins in AR-mediated transcription in prostate tumor cells. More importantly, novel coregulatory functions were detected in components of well-established cell surface receptor-dependent signal transduction pathways. Further experimentation demonstrated that components of the TNF, TGF-β, IL receptor, and epidermal growth factor signaling pathways modulated AR-dependent gene transcription and androgen-dependent proliferation in prostate tumor cells. Collectively, our proteomic dataset demonstrates that the cell surface receptor- and AR-dependent pathways are highly integrated, and provides a molecular framework for understanding how disparate signal-transduction pathways can influence AR-dependent transcriptional programs linked to the development and progression of human prostate cancers.

  12. Generation of antigen-specific T cell immunity through T cell receptor gene transfer

    NARCIS (Netherlands)

    Coccoris, Miriam


    Cancer cells often escape the attack of immune cells because they originate from self-tissue. Through T cell receptor gene transfer it is possible to equip peripheral T cells with a desired specificity, and this strategy may be useful to generate tumor-specific T cells for the treatment of cancer in

  13. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells (United States)

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.


    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  14. Sphingosine 1-Phosphate Receptor 1 Signaling in Mammalian Cells

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    Nigel J. Pyne


    Full Text Available The bioactive lipid, sphingosine 1-phosphate (S1P binds to a family of G protein-coupled receptors, termed S1P1-S1P5. These receptors function in, for example, the cardiovascular system to regulate vascular barrier integrity and tone, the nervous system to regulate neuronal differentiation, myelination and oligodendrocyte/glial cell survival and the immune system to regulate T- and B-cell subsets and trafficking. S1P receptors also participate in the pathophysiology of autoimmunity, inflammatory disease, cancer, neurodegeneration and others. In this review, we describe how S1P1 can form a complex with G-protein and β-arrestin, which function together to regulate effector pathways. We also discuss the role of the S1P1-Platelet derived growth factor receptor β functional complex (which deploys G-protein/β-arrestin and receptor tyrosine kinase signaling in regulating cell migration. Possible mechanisms by which different S1P-chaperones, such as Apolipoprotein M-High-Density Lipoprotein induce biological programmes in cells are also described. Finally, the role of S1P1 in health and disease and as a target for clinical intervention is appraised.

  15. Labeling of lectin receptors during the cell cycle. (United States)

    Garrido, J


    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.

  16. HER/ErbB receptor interactions and signaling patterns in human mammary epithelial cells

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    Chrisler William B


    constructed clone library we profiled the cell signaling patterns to establish the role of HER2 in the crosstalk between EGFR and HER3 receptors in HME cells. Our results for HME cells show that the weak linkage between EGFR and HER3 pathways can lead to distinct downstream cellular signaling patterns in response to the ligands of these two receptors.

  17. Tumor cell marker PVRL4 (nectin 4 is an epithelial cell receptor for measles virus.

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    Ryan S Noyce


    Full Text Available Vaccine and laboratory adapted strains of measles virus can use CD46 as a receptor to infect many human cell lines. However, wild type isolates of measles virus cannot use CD46, and they infect activated lymphocytes, dendritic cells, and macrophages via the receptor CD150/SLAM. Wild type virus can also infect epithelial cells of the respiratory tract through an unidentified receptor. We demonstrate that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon. Transfection of non-infectable adenocarcinoma cell lines with an expression vector encoding CD150/SLAM rendered them susceptible to measles virus, indicating that they were virus replication competent, but lacked a receptor for virus attachment and entry. Microarray analysis of susceptible versus non-susceptible cell lines was performed, and comparison of membrane protein gene transcripts produced a list of 11 candidate receptors. Of these, only the human tumor cell marker PVRL4 (Nectin 4 rendered cells amenable to measles virus infections. Flow cytometry confirmed that PVRL4 is highly expressed on the surfaces of susceptible lung, breast, and colon adenocarcinoma cell lines. Measles virus preferentially infected adenocarcinoma cell lines from the apical surface, although basolateral infection was observed with reduced kinetics. Confocal immune fluorescence microscopy and surface biotinylation experiments revealed that PVRL4 was expressed on both the apical and basolateral surfaces of these cell lines. Antibodies and siRNA directed against PVRL4 were able to block measles virus infections in MCF7 and NCI-H358 cancer cells. A virus binding assay indicated that PVRL4 was a bona fide receptor that supported virus attachment to the host cell. Several strains of measles virus were also shown to use PVRL4 as a receptor. Measles virus infection reduced PVRL4 surface expression in MCF7 cells, a

  18. Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells

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    Tseng, Y.L.; Burman, K.D.; Lahiri, S.; Abdelrahim, M.M.; D' Avis, J.C.; Wartofsky, L. (Walter Reed Army Medical Center, Washington, DC (USA))


    In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding (125I) ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound (125I)ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized (125I)ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity.

  19. Novel and highly potent histamine H3 receptor ligands. Part 3: an alcohol function to improve the pharmacokinetic profile. (United States)

    Labeeuw, Olivier; Levoin, Nicolas; Poupardin-Olivier, Olivia; Calmels, Thierry; Ligneau, Xavier; Berrebi-Bertrand, Isabelle; Robert, Philippe; Lecomte, Jeanne-Marie; Schwartz, Jean-Charles; Capet, Marc


    Synthesis and biological evaluation of potent histamine H3 receptor antagonists incorporating a hydroxyl function are described. Compounds in this series exhibited nanomolar binding affinities for human receptor, illustrating a new possible component for the H3 pharmacophore. As demonstrated with compound BP1.4160 (cyclohexanol 19), the introduction of an alcohol function counter-intuitively allowed to reach high in vivo efficiency and favorable pharmacokinetic profile with reduced half-life.

  20. Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors.

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    Lenka Plzakova

    Full Text Available Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell-pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ΔiglC and ΔftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria's internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

  1. Vaccination against Experimental Allergic Encephalomyelitis with T Cell Receptor Peptides (United States)

    Howell, Mark D.; Winters, Steven T.; Olee, Tsaiwei; Powell, Henry C.; Carlo, Dennis J.; Brostoff, Steven W.


    Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by CD4+ T cells reactive with myelin basic protein (MBP). Rats were rendered resistant to the induction of EAE by vaccination with synthetic peptides corresponding to idiotypic determinants of the β chain VDJ region and Jα regions of the T cell receptor (TCR) that are conserved among encephalitogenic T cells. These findings demonstrate the utility of TCR peptide vaccination for modulating the activity of autoreactive T cells and represent a general therapeutic approach for T cell--mediated pathogenesis.

  2. TLR expression profile of human gingival margin-derived stem progenitor cells (United States)

    Mekhemar, Mohamed; Adam-Klages, Sabine; Kabelitz, Dietrich; Dörfer, Christof


    Background Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile. Material and Methods Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs’ incubation in basic or inflammatory medium (IL-1β, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated. Results G-MSCs showed all stem/progenitor cells’ characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test). Conclusions The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions. Key words:Stem cells, TLR, gingiva, polymerase chain reaction, FACS. PMID:26615501

  3. B-cell receptor-driven MALT1 activity regulates MYC signaling in mantle cell lymphoma. (United States)

    Dai, Beiying; Grau, Michael; Juilland, Mélanie; Klener, Pavel; Höring, Elisabeth; Molinsky, Jan; Schimmack, Gisela; Aukema, Sietse M; Hoster, Eva; Vogt, Niklas; Staiger, Annette M; Erdmann, Tabea; Xu, Wendan; Erdmann, Kristian; Dzyuba, Nicole; Madle, Hannelore; Berdel, Wolfgang E; Trneny, Marek; Dreyling, Martin; Jöhrens, Korinna; Lenz, Peter; Rosenwald, Andreas; Siebert, Reiner; Tzankov, Alexandar; Klapper, Wolfram; Anagnostopoulos, Ioannis; Krappmann, Daniel; Ott, German; Thome, Margot; Lenz, Georg


    Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of B-cell receptor (BCR) signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 complex that links BCR signaling to the NF-κB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls an MYC-driven gene expression network predominantly through increasing MYC protein stability. Thus, our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1-induced MYC regulation is not restricted to MCL, but represents a common mechanism. MYC itself is pivotal for MCL survival because its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy of targeting the MALT1-MYC axis in MCL patients.

  4. Direct cell lysis for single-cell gene expression profiling

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    David eSvec


    Full Text Available The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA to be the best lysis agent, resulting in efficient cell lysis, high RNA stability and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single cells as well as samples composed of small numbers of cells.

  5. MicroRNA expression profiles in avian haemopoietic cells

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    Yongxiu eYao


    Full Text Available MicroRNAs (miRNAs are small, abundant, non-coding RNAs that modulate gene expression by interfering with translation or stability of mRNA transcripts in a sequence-specific manner. A total of 734 precursor and 996 mature miRNAs have so far been identified in the chicken genome. A number of these miRNAs are expressed in a cell type-specific manner, and understanding their function requires detailed examination of their expression in different cell types. We carried out deep sequencing of small RNA populations isolated from stimulated or transformed avian haemopoietic cell lines to determine the changes in the expression profiles of these important regulatory molecules during these biological events. There were significant changes in the expression of a number of miRNAs, including miR-155, in chicken B cells stimulated with CD40 ligand. Similarly, avian leukosis virus (ALV-transformed DT40 cells also showed changes in miRNA expression in relation to the naïve cells. Embryonic stem cell line BP25 demonstrated a distinct cluster of upregulated miRNAs, many of which were shown previously to be involved in embryonic stem cell development. Finally, chicken macrophage cell line HD11 showed changes in miRNA profiles, some of which are thought to be related to the transformation by v-myc transduced by the virus. This work represents the first publication of a catalog of microRNA expression in a range of important avian cells and provides insights into the potential roles of miRNAs in the hematopoietic lineages of cells in a model non-mammalian species.

  6. Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis. (United States)

    Zimmermann, Céline; Stévant, Isabelle; Borel, Christelle; Conne, Béatrice; Pitetti, Jean-Luc; Calvel, Pierre; Kaessmann, Henrik; Jégou, Bernard; Chalmel, Frédéric; Nef, Serge


    Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

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

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    Matsuda, Satoru, E-mail:; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)


    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.

  8. Toxicities of chimeric antigen receptor T cells: recognition and management. (United States)

    Brudno, Jennifer N; Kochenderfer, James N


    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy.

  9. Altered B cell receptor signaling in human systemic lupus erythematosus (United States)

    Jenks, Scott A.; Sanz, Iñaki


    Regulation of B cell receptor signaling is essential for the development of specific immunity while retaining tolerance to self. Systemic lupus erythematosus (SLE) is characterized by a loss of B cell tolerance and the production of anti-self antibodies. Accompanying this break down in tolerance are alterations in B cell receptor signal transduction including elevated induced calcium responses and increased protein phosphorylation. Specific pathways that negatively regulate B cell signaling have been shown to be impaired in some SLE patients. These patients have reduced levels of the kinase Lyn in lipid raft microdomains and this reduction is inversely correlated with increased CD45 in lipid rafts. Function and expression of the inhibitory immunoglobulin receptor FcγRIIB is also reduced in Lupus IgM- CD27+ memory cells. Because the relative contribution of different memory and transitional B cell subsets can be abnormal in SLE patients, we believe studies targeted to well defined B cell subsets will be necessary to further our understanding of signaling abnormalities in SLE. Intracellular flow cytometric analysis of signaling is a useful approach to accomplish this goal. PMID:18723129

  10. Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression.

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    Stéphanie Bertrand


    Full Text Available Nuclear receptors (NRs are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily.

  11. Antagonist profile of ibodutant at the tachykinin NK2 receptor in guinea pig isolated bronchi. (United States)

    Santicioli, Paolo; Meini, Stefania; Giuliani, Sandro; Lecci, Alessandro; Maggi, Carlo Alberto


    In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK2 receptor agonist [βAla(8)]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300nM) induced a concentration-dependent rightward shift of the [βAla(8)]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65-1.25), thus indicating a surmountable behaviour. The calculated apparent antagonist potency as pKB value was 8.31±0.05. Ibodutant (0.3-100nM), produced a concentration-dependent inhibition of the nonadrenergic-noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95±4% inhibition), the calculated IC50 value was 2.98nM (95% c.l. 1.73-5.16nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrate that ibodutant is a potent NK2 receptor antagonist in guinea pig airways.

  12. Expression profiling of pattern recognition receptors and selected cytokines in miniature dachshunds with inflammatory colorectal polyps. (United States)

    Igarashi, Hirotaka; Ohno, Koichi; Maeda, Shingo; Kanemoto, Hideyuki; Fukushima, Kenjiro; Uchida, Kazuyuki; Tsujimoto, Hajime


    Inflammatory colorectal polyps (ICRPs) are commonly seen in miniature dachshund (MD) dogs; typically, multiple polyps form with severe neutrophil infiltration. ICRP is thought to be a novel form of inflammatory bowel disease (IBD), but its etiology has not been investigated. The innate immune system is implicated in the pathogenesis of both human and canine IBD. Therefore, the aim of the current study was to evaluate the messenger RNA (mRNA) expression profiles of pattern recognition receptors (PRRs) and cytokines in ICRPs. Polyp tissues were collected by colonoscopic biopsies from 24 MDs with ICRPs. Non-polypoid colonic mucosa was collected from all MDs with ICRPs and 21 clinically healthy beagles (as the controls). The expression levels of the mRNAs encoding toll-like receptors (TLRs) 1-10; nucleotide-binding oligomerization domain (NOD)-like receptors NOD1 and NOD2; and cytokines IL-1β, IL-6, IL-8/CXCL8, and TNF-α were evaluated by quantitative real-time RT-PCR. Three of the 10 well-known candidate reference genes were selected as housekeeper genes based on analyses from the GeNorm, NormFinder, and BestKeeper programs. Levels of TLR1, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, NOD2, and all cytokines were significantly upregulated in the polyps relative to those in the controls. There was significant decrease in the expression levels of TLR3 and NOD1 in the polyp tissues compared to the non-polypoid colonic mucosa obtained from MDs with ICRPs. All upregulated PRR mRNAs were positively correlated with all proinflammatory cytokine mRNAs. This study demonstrated the dysregulation of PRRs and proinflammatory cytokines in ICRPs of MDs, which may play an important role in the pathogenesis of this disease.

  13. Transcriptional profiling of human brain endothelial cells reveals key properties crucial for predictive in vitro blood-brain barrier models.

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    Eduard Urich

    Full Text Available Brain microvascular endothelial cells (BEC constitute the blood-brain barrier (BBB which forms a dynamic interface between the blood and the central nervous system (CNS. This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local

  14. Functional bitter taste receptors are expressed in brain cells. (United States)

    Singh, Nisha; Vrontakis, Maria; Parkinson, Fiona; Chelikani, Prashen


    Humans are capable of sensing five basic tastes which are sweet, sour, salt, umami and bitter. Of these, bitter taste perception provides protection against ingestion of potentially toxic substances. Bitter taste is sensed by bitter taste receptors (T2Rs) that belong to the G-protein coupled receptors (GPCRs) superfamily. Humans have 25 T2Rs that are expressed in the oral cavity, gastrointestinal (GI) neuroendocrine cells and airway cells. Electrophysiological studies of the brain neurons show that the neurons are able to respond to different tastants. However, the presence of bitter taste receptors in brain cells has not been elucidated. In this report using RT-PCR, and immunohistochemistry analysis we show that T2Rs are expressed in multiple regions of the rat brain. RT-PCR analysis revealed the presence of T2R4, T2R107 and T2R38 transcripts in the brain stem, cerebellum, cortex and nucleus accumbens. The bitter receptor T2R4 was selected for further analysis at the transcript level by quantitative real time PCR and at the protein level by immunohistochemistry. To elucidate if the T2R4 expressed in these cells is functional, assays involving G-protein mediated calcium signaling were carried out. The functional assays showed an increase in intracellular calcium levels after the application of exogenous ligands for T2R4, denatonium benzoate and quinine to these cultured cells, suggesting that endogenous T2R4 expressed in these cells is functional. We discuss our results in terms of the physiological relevance of bitter receptor expression in the brain.

  15. Natural killer cells and cancer: regulation by the killer cell Ig-like receptors (KIR). (United States)

    Purdy, Amanda K; Campbell, Kerry S


    Natural killer (NK) cells are innate immune effector cells that make up approximately 10-15% of the peripheral blood lymphocytes in humans and are primarily involved in immunosurveillance to eliminate transformed and virally-infected cells. They were originally defined by their ability to spontaneously eliminate rare cells lacking expression of class I major histocompatibility complex (MHC-I) self molecules, which is commonly referred to as "missing self" recognition. The molecular basis for missing self recognition emerges from the expression of MHC-I-specific inhibitory receptors on the NK cell surface that tolerize NK cells toward normal MHC-I-expressing cells. By lacking inhibitory receptor ligands, tumor cells or virus-infected cells that have down-modulated surface MHC-I expression become susceptible to attack by NK cells. Killer cell Ig-like receptors (KIR; CD158) constitute a family of MHC-I binding receptors that plays a major role in regulating the activation thresholds of NK cells and some T cells in humans. Here, we review the multiple levels of KIR diversity that contribute to the generation of a highly varied NK cell repertoire and explain how this diversity can influence susceptibility to a variety of diseases, including cancer. We further describe strategies by which KIR can be manipulated therapeutically to treat cancer, through the exploitation of KIR/MHC-I ligand mismatch to potentiate hematopoietic stem cell transplantation and the use of KIR blockade to enhance tumor cell killing.

  16. Sex steroids and their receptors: molecular actions on brain cells. (United States)

    Mannella, Paolo; Simoncini, Tommaso


    Sex steroids exert actions of paramount importance on brain cells. They contribute to shape the central nervous system during embryo development. They modulate the formation and the turnover of the interconnections between neurons. They control the function of glial cells. And they do it through a signaling machinery that is apparently simple, but that hides a level of complexity that has been unveiled only in part. Different receptor isoforms, different interactions between receptors and co-regulators, chains of events originating at the cell membrane and leading to effects in the nucleus (or the other way around) all interact to determine selective modulations of brain cells. All these actions end up in phenomenal effects on brain function that change through adolescence, pregnancy, adulthood, up to menopause and ageing. Many of these actions are relevant for degenerative processes and research may offer soon new strategies to counteract these diseases.

  17. Localization of muscarinic acetylcholine receptor in plant guard cells

    Institute of Scientific and Technical Information of China (English)


    Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. In Vicia faba L. and Pisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane of Vicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.

  18. A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control. (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Thon, Lutz; Mamat, Uwe; Bellosta, Paola; Basilico, Claudio; Adam, Dieter; Paus, Ralf; Bulfone-Paus, Silvia


    Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a 'promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl-/- or IL-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha.

  19. FGFR4 profile as a prognostic marker in squamous cell carcinoma of the mouth and oropharynx.

    Directory of Open Access Journals (Sweden)

    Roberta Lelis Dutra

    Full Text Available BACKGROUND: Fibroblast growth factor receptor 4 (FGFR4 is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis. PATIENTS AND METHODS: DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC on paraffin-embedded tissue microarrays. RESULTS: Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death. CONCLUSION: Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.

  20. Functional pharmacological evidence for EP2 and EP4 prostanoid receptors in immortalized human trabecular meshwork and non-pigmented ciliary epithelial cells. (United States)

    Crider, J Y; Sharif, N A


    The aim of these studies was to characterize the molecular pharmacology of the prostanoid receptors positively coupled to stimulation of adenylyl cyclase activity in immortalized human trabecular meshwork (TM-3) cells and to compare these results with that of the receptors in immortalized human nonpigmented epithelial (NPE) cells. In general, the TM-3 and NPE cells showed a similar profile with respect to their responses to various prostaglandin (PG) receptor agonists. The rank order of potency (EC50; means +/- SEM) for these compounds in the TM-3 cells was: PGE2 (124 +/- 21 nM) > 13,14-dihydro-PGE1 (430 +/- 110 nM) = PGE1 (522 +/- 345 nM) > 11-deoxy-PGE1 (1063 +/- 118 nM) = 16,16-dimethyl-PGE2 (1776 +/- 460 nM) = butaprost (1920 +/- 527 nM) > PGD2 = PGI2 = PGF2alpha (n = 3 - 12). While the agonist profile indicated the presence of EP2 receptors, the effects of the EP4 receptor antagonists suggested the additional expression of EP4 receptors in both of these cells. Thus, the EP4 receptor antagonist, AH23848B, at a concentration of 30 microM, caused a dextral shift in the PGE2 concentration-response curves in both TM-3 and NPE cells coupled with a 20-28% decrease in the maximal response of PGE2, indicating apparent noncompetitive antagonism profiles. The antagonist potency of AH23848B in these cells was: Kb = 38.4 +/- 14.8 microM and 23.5 +/- 4.5 microM; -log Kb = 4.7. The other EP4 receptor antagonist, AH22921 (-log Kb = 4.1 - 4.7), was weaker than AH23848B. Taken together, these pharmacological studies have shown than TM-3 and NPE cells apparently contain functional EP2 and EP4 prostanoid receptors positively coupled to adenylyl cyclase.

  1. 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;


    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...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...... 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...

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

    DEFF Research Database (Denmark)

    Kornblit, Brian; Enevold, Christian; Wang, Tao;


    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...

  3. Estrogen receptors and cell proliferation in breast cancer. (United States)

    Ciocca, D R; Fanelli, M A


    Most of the actions of estrogens on the normal and abnormal mammary cells are mediated via estrogen receptors (ERs), including control of cell proliferation; however, there are also alternative pathways of estrogen action not involving ERs. Estrogens control several genes and proteins that induce the cells to enter the cell cycle (protooncogenes, growth factors); estrogens also act on proteins directly involved in the control of the cell cycle (cyclins), and moreover, estrogens stimulate the response of negative cell cycle regulators (p53, BRCA1). The next challenge for researchers is elucidating the integration of the interrelationships of the complex pathways involved in the control of cell proliferation. This brief review focuses on the mechanisms of estrogen action to control cell proliferation and the clinical implications in breast cancer. (Trends Endocrinol Metab 1997;8:313-321). (c) 1997, Elsevier Science Inc.

  4. Transcriptome Profiling and Analysis during Cotton Fiber Cell Development

    Institute of Scientific and Technical Information of China (English)

    ZHU Yu-xian


    @@ In this project,we aim to elucidate the molecular mechanism controlling initiation and elongation of tetraploid Gossypium hirsutum fiber cells by setting up a high throughput custom-designed cDNA microarray and a systematic gene expression profiling during cotton fiber development.We first constructed a microarray consisting of more than 28,000 cotton UniESTs that we obtained by deep-sequencing of several cotton ovule cDNA libraries.

  5. Global renal gene expression profiling analysis in B2-kinin receptor null mice: impact of diabetes.

    Directory of Open Access Journals (Sweden)

    Miran A Jaffa

    Full Text Available Diabetic nephropathy (DN, the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The risk factors and mechanisms that contribute to the development and progression of DN are still incompletely defined. To address the involvement of bradykinin B(2-receptors (B(2R in DN, we used a genome wide approach to study the effects of diabetes on differential renal gene expression profile in wild type and B(2R knockout (B(2R(-/- mice. Diabetes was induced with streptozotocin and plasma glucose levels and albumin excretion rate (AER were measured at predetermined times throughout the 23 week study period. Longitudinal analysis of AER indicated that diabetic B(2R(-/-D null mice had a significantly decreased AER levels compared to wild type B(2R(+/+D mice (P = 0.0005. Results from the global microarray study comparing gene expression profiles among four groups of mice respectively: (B(2R(+/+C, B(2R(+/+D, B(2R(-/-C and B(2R(-/-D highlighted the role of several altered pathological pathways in response to disruption of B(2R and to the diabetic state that included: endothelial injury, oxidative stress, insulin and lipid metabolism and inflammatory process with a marked alteration in the pro-apoptotic genes. The findings of the present study provide a global genomics view of biomarkers that highlight the mechanisms and putative pathways involved in DN.

  6. Direct Cell Lysis for Single-Cell Gene Expression Profiling


    David eSvec; Daniel eAndersson; Milos ePekny; Robert eSjöback; Mikael eKubista; Anders eStåhlberg


    The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously express...

  7. The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells (United States)

    Leisi, Remo; Von Nordheim, Marcus; Ros, Carlos; Kempf, Christoph


    Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells. PMID:27690083

  8. Limitations in plasticity of the T-cell receptor repertoire.


    Nanda, N K; Apple, R; Sercarz, E.


    How constrained is T-cell recognition? Is a truncated T-cell receptor (TCR) repertoire, missing half of its V beta components (where V indicates variable), still broad enough to produce an antigen-specific T-cell response to all determinants? These questions can be answered for certain T-cell antigenic determinants whose response in the wild type is limited to specific gene segments. Our results show that mice with such a deletion in their TCR V beta genes (V beta truncated haplotype, Va beta...

  9. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors. (United States)

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


    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.

  10. Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

    Directory of Open Access Journals (Sweden)

    Monica Marzagalli

    Full Text Available Cutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor β (ERβ is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its tumor suppressive function. These experiments were performed to investigate the effects of ERβ activation on melanoma cell growth.Protein expression was analyzed by Western blot and immunofluorescence assays. Cell proliferation was assessed by counting the cells by hemocytometer. ERβ transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERβ isoforms were identified by qRT-PCR. We demonstrated that ERβ is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552. In BLM (NRAS-mutant cells, ERβ agonists significantly and specifically inhibited cell proliferation. ERβ activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins. In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation. ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant cells. Finally, we could observe that ERβ isoforms are expressed at different levels in the various cell lines. Specific oncogenic mutations or differential expression of receptor isoforms might be responsible for the different responses of cell lines to ERβ agonists.Our results demonstrate that ERβ is expressed in melanoma cell lines and that ERβ agonists differentially regulate the proliferation of these cells. These data confirm the notion that melanoma is a

  11. Nanometer-Scale Electrical Potential Profiling Across Perovskite Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Chuanxiao; Jiang, Chun-Sheng; Ke, Weijun; Wang, Changlei; Gorman, Brian; Yan, Yanfa; Al-Jassim, Mowafak


    We used Kelvin probe force microscopy to study the potential distribution on cross-section of perovskite solar cells with different types of electron-transporting layers (ETLs). Our results explain the low open-circuit voltage and fill factor in ETL-free cells, and support the fact that intrinsic SnO2 as an alternative ETL material can make high-performance devices. Furthermore, the potential-profiling results indicate a reduction in junction-interface recombination by the optimized SnO2 layer and adding a fullerene layer, which is consistent with the improved device performance and current-voltage hysteresis.

  12. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J


    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  13. Profiling helper T cell subset gene expression in deer mice

    Directory of Open Access Journals (Sweden)

    Hjelle Brian


    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

  14. 4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes

    DEFF Research Database (Denmark)

    Madsen, Ulf; Pickering, Darryl S; Nielsen, Birgitte;


    4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies...

  15. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer. (United States)

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R; Tang, Dean G


    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

  16. Integrated expression profiling and ChIP-seq analyses of the growth inhibition response program of the androgen receptor.

    Directory of Open Access Journals (Sweden)

    Biaoyang Lin

    Full Text Available BACKGROUND: The androgen receptor (AR plays important roles in the development of male phenotype and in different human diseases including prostate cancers. The AR can act either as a promoter or a tumor suppressor depending on cell types. The AR proliferative response program has been well studied, but its prohibitive response program has not yet been thoroughly studied. METHODOLOGY/PRINCIPAL FINDINGS: Previous studies found that PC3 cells expressing the wild-type AR inhibit growth and suppress invasion. We applied expression profiling to identify the response program of PC3 cells expressing the AR (PC3-AR under different growth conditions (i.e. with or without androgens and at different concentration of androgens and then applied the newly developed ChIP-seq technology to identify the AR binding regions in the PC3 cancer genome. A surprising finding was that the comparison of MOCK-transfected PC3 cells with AR-transfected cells identified 3,452 differentially expressed genes (two fold cutoff even without the addition of androgens (i.e. in ethanol control, suggesting that a ligand independent activation or extremely low-level androgen activation of the AR. ChIP-Seq analysis revealed 6,629 AR binding regions in the cancer genome of PC3 cells with an FDR (false discovery rate cut off of 0.05. About 22.4% (638 of 2,849 can be mapped to within 2 kb of the transcription start site (TSS. Three novel AR binding motifs were identified in the AR binding regions of PC3-AR cells, and two of them share a core consensus sequence CGAGCTCTTC, which together mapped to 27.3% of AR binding regions (1,808/6,629. In contrast, only about 2.9% (190/6,629 of AR binding sites contains the canonical AR matrix M00481, M00447 and M00962 (from the Transfac database, which is derived mostly from AR proliferative responsive genes in androgen dependent cells. In addition, we identified four top ranking co-occupancy transcription factors in the AR binding regions, which

  17. A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures

    DEFF Research Database (Denmark)

    Bundgaard Christiansen, Gitte; Harbak, Barbara; E. Hede, Susanne;


    -mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two...... information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing...

  18. Calcium Activation Profile In Electrically Stimulated Intact Rat Heart Cells (United States)

    Geerts, Hugo; Nuydens, Rony; Ver Donck, Luc; Nuyens, Roger; De Brabander, Marc; Borgers, Marcel


    Recent advances in fluorescent probe technology and image processing equipment have made available the measurement of calcium in living systems on a real-time basis. We present the use of the calcium indicator Fura-2 in intact normally stimulated rat heart cells for the spatial and dynamic measurement of the calcium excitation profile. After electric stimulation (1 Hz), the activation proceeds from the center of the myocyte toward the periphery. Within two frame times (80 ms), the whole cell is activated. The activation is slightly faster in the center of the cell than in the periphery. The mean recovery time is 200-400 ms. There is no difference along the cell's long axis. The effect of a beta-agonist and of a calcium antagonist is described.

  19. Killer Immunoglobulin-Like Receptor Profiles are not Associated with risk of Amoxicillin-Clavulanate-Induced Liver Injury in Spanish Patients

    Directory of Open Access Journals (Sweden)

    Camilla Stephens


    Full Text Available Natural killer cells are an integral part of the immune system and represent a large proportion of the lymphocyte population in the liver. The activity of these cells is regulated by various cell surface receptors, such as killer Ig-like receptors (KIR that bind to human leukocyte antigen (HLA class I ligands on the target cell. The composition of KIR receptors has been suggested to influence the development of specific diseases, in particularly autoimmune diseases, cancer and reproductive diseases. The role played in idiosyncratic drug-induced liver injury (DILI is currently unknown. In this study we examined KIR gene profiles and HLA class I polymorphisms in amoxicillin-clavulanate (AC DILI patients in search for potential risk associations. 102 AC DILI patients and 226 controls were genotyped for the presence or absence of 16 KIR loci, including the two pseudogenes 2DP1 and 3DP1. No significant differences were found in the distribution of individual KIRs between patients and controls, which were comparable to previously reported KIR data from ethnically similar cohorts. 21.6% and 21.2% of the patients and controls, respectively, were homozygous haplotype A carriers, while 78.4% and 78.8%, respectively, contained at least one B haplotype (Bx. The genotypes translated into 27 (AC DILI and 46 (controls different gene profiles, with 19 being present in both groups. The most frequent Bx gene profile containing 2DS2, 2DL2, 2DL3, 2DP1, 2DL1, 3DL1, 2DS4, 3DL2, 3DL3, 2DL4 and 3PD1 was present in 16% of the DILI patients and 14% of the controls. The distribution of HLA class I epitopes did not differ significantly between AC DILI patients and controls. The most frequent receptor-ligand combinations in the DILI patients were 2DL3 + epitope C1 (67% and 3DL1 + Bw4 motif (67%, while 2DL1 + epitope C2 (69% and 3DL1 + Bw4 motif (69% predominated in the controls. This is to our knowledge the first analysis of KIR receptor-HLA ligand associations in DILI

  20. Killer Immunoglobulin-Like Receptor Profiles Are not Associated with Risk of Amoxicillin-Clavulanate–Induced Liver Injury in Spanish Patients (United States)

    Stephens, Camilla; Moreno-Casares, Antonia; López-Nevot, Miguel-Ángel; García-Cortés, Miren; Medina-Cáliz, Inmaculada; Hallal, Hacibe; Soriano, German; Roman, Eva; Ruiz-Cabello, Francisco; Romero-Gomez, Manuel; Lucena, M. Isabel; Andrade, Raúl J.


    Natural killer cells are an integral part of the immune system and represent a large proportion of the lymphocyte population in the liver. The activity of these cells is regulated by various cell surface receptors, such as killer Ig-like receptors (KIR) that bind to human leukocyte antigen (HLA) class I ligands on the target cell. The composition of KIR receptors has been suggested to influence the development of specific diseases, in particularly autoimmune diseases, cancer and reproductive diseases. The role played in idiosyncratic drug-induced liver injury (DILI) is currently unknown. In this study, we examined KIR gene profiles and HLA class I polymorphisms in amoxicillin-clavulanate (AC) DILI patients in search for potential risk associations. One hundred and two AC DILI patients and 226 controls were genotyped for the presence or absence of 16 KIR loci, including the two pseudogenes 2DP1 and 3DP1. No significant differences were found in the distribution of individual KIRs between patients and controls, which were comparable to previously reported KIR data from ethnically similar cohorts. The 21.6 and 21.2% of the patients and controls, respectively, were homozygous haplotype A carriers, while 78.4 and 78.8%, respectively, contained at least one B haplotype (Bx). The genotypes translated into 27 (AC DILI) and 46 (controls) different gene profiles, with 19 being present in both groups. The most frequent Bx gene profile containing KIRs 2DS2, 2DL2, 2DL3, 2DP1, 2DL1, 3DL1, 2DS4, 3DL2, 3DL3, 2DL4, and 3PD1 was present in 16% of the DILI patients and 14% of the controls. The distribution of HLA class I epitopes did not differ significantly between AC DILI patients and controls. The most frequent receptor-ligand combinations in the DILI patients were 2DL3 + epitope C1 (67%) and 3DL1 + Bw4 motif (67%), while 2DL1 + epitope C2 (69%) and 3DL1 + Bw4 motif (69%) predominated in the controls. This is to our knowledge the first analysis of KIR receptor-HLA ligand

  1. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation


    Au-Yeung, Byron B.; Zikherman, Julie; James L. Mueller; Ashouri, Judith F.; Matloubian, Mehrdad; Cheng, Debra A.; Chen, Yiling; Shokat, Kevan M; Weiss, Arthur


    Biochemical signals triggered by the T-cell receptor (TCR) are required for stimulating T cells and can be initiated within seconds. However, a hallmark of T-cell activation, cell division, occurs hours after TCR signaling has begun, implying that T cells require a minimum duration and/or accumulate TCR signaling events to drive proliferation. To visualize the accumulated signaling experienced by T cells, we used a fluorescent reporter gene that is activated by TCR stimulation. This technique...

  2. Toxicological profiles of selected synthetic cannabinoids showing high binding affinities to the cannabinoid receptor subtype CB₁. (United States)

    Koller, Verena J; Zlabinger, Gerhard J; Auwärter, Volker; Fuchs, Sabine; Knasmueller, Siegfried


    Products containing synthetic cannabinoids are consumed as a surrogate for marihuana due to their non-detectability with commonly used drug tests and their strong cannabimimetic effects. Because data concerning their toxicological properties are scarce, the cytotoxic, genotoxic, immunomodulatory, and hormonal activities of four naphthoylindole compounds (JWH-018, JWH-073, JWH-122 and JWH-210) and of one benzoylindole (AM-694) were studied in human cell lines and primary cells; tetrahydrocannabinol was included as the classical non-endogenous cannabinoid receptor ligand. All compounds induced damage to the cell membranes of buccal (TR146) and breast (MCF-7) derived cells at concentrations of ≥75-100 μM. No cytotoxic responses were seen in other assays which reflect mitochondrial damage, protein synthesis, and lysosomal activities. JWH-073 and JWH-122 induced DNA migration in buccal and liver cells (HepG2) in single cell gel electrophoresis assays, while JWH-210 was only in the latter cell line active. No estrogenic activities were detected in bone marrow cells (U2-OS), but all compounds caused anti-estrogenic effects at levels between 2.1 and 23.0 μM. Furthermore, no impact on cytokine release (i.e., on IL-10, IL-6, IL-12/23p40 and TNFα levels) was seen in LPS-stimulated human PBMCs, except with JWH-210 and JWH-122 which caused a decrease of TNFα and IL-12/23p40. All toxic effects were observed with concentrations higher than those expected in body fluids of users. Since genotoxic effects are in general linear over a wide concentration range and the exposure levels may be higher in epithelial cells than [corrected] in serum, further experimental work is required to find out if DNA damage takes place in drug users.

  3. Salivary Protein Profiles among HER2/neu-Receptor-Positive and -Negative Breast Cancer Patients: Support for Using Salivary Protein Profiles for Modeling Breast Cancer Progression

    Directory of Open Access Journals (Sweden)

    Charles F. Streckfus


    Full Text Available Purpose. The objective of this study was to compare the salivary protein profiles from individuals diagnosed with breast cancer that were either HER2/neu receptor positive or negative. Methods. Two pooled saliva specimens underwent proteomic analysis. One pooled specimen was from women diagnosed with stage IIa HER2/neu-receptor-positive breast cancer patients (n=10 and the other was from women diagnosed with stage IIa HER2/neu-receptor-negative cancer patients (n=10. The pooled samples were trypsinized and the peptides labeled with iTRAQ reagent. Specimens were analyzed using an LC-MS/MS mass spectrometer. Results. The results yielded approximately 71 differentially expressed proteins in the saliva specimens. There were 34 upregulated proteins and 37 downregulated proteins.

  4. Identification and characterization of estrogen receptor-related receptor alpha and gamma in human glioma and astrocytoma cells. (United States)

    Gandhari, Mukesh K; Frazier, Chester R; Hartenstein, Julia S; Cloix, Jean-Francois; Bernier, Michel; Wainer, Irving W


    The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRalpha and ERRgamma proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC(50) and IC(50) values were consistent with previous reported values determined in other types of cancer cell lines. Induction of luciferase expression under the control of ERR isoform-specific promoters was also observed in these cells. These results indicate that ERRalpha and ERRgamma are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity.

  5. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. (United States)

    Etheridge, S Leah; Spencer, Gary J; Heath, Deborah J; Genever, Paul G


    Through their broad differentiation potential, mesenchymal stem cells (MSCs) are candidates for a range of therapeutic applications, but the precise signaling pathways that determine their differentiated fate are not fully understood. Evidence is emerging that developmental signaling cues may be important in regulating stem cell self-renewal and differentiation programs. Here we have identified a consistent expression profile of Wnt signaling molecules in MSCs and provide evidence that an endogenous canonical Wnt pathway functions in these cells. Wnts bind to Frizzled (Fz) receptors and subsequent canonical signaling inhibits glycogen synthase kinase-3beta (GSK-3beta), causing beta-catenin translocation into the nucleus to induce target gene expression. In human MSCs isolated from bone marrow of different donors, we appear to have identified a common Wnt/Fz expression profile using reverse transcriptase polymerase chain reaction (RT-PCR). Associated Wnt signaling components, including low-density lipoprotein receptor-related protein-5 (LRP-5), kremen-1, dickkopf-1 (Dkk-1), secreted Frizzled-related peptide (sFRP)-2, sFRP3, sFRP4, Disheveled (Dvl), GSK-3beta, adenomatous polyposis coli (APC), beta-catenin,T-cell factor (TCF)-1, and TCF-4, were also identified. Nuclear beta-catenin was observed in 30%-40% of MSCs, indicative of endogenous Wnt signaling. Exposure to both Wnt3a and Li+ ions, which promotes canonical Wnt signaling by inhibiting GSK-3beta, reduced phosphorylation of beta-catenin in MSCs and increased beta-catenin nuclear translocation approximately threefold over that of the controls. Our findings indicate that autocrine Wnt signaling operates in primitive MSC populations and supports previous evidence that Wnt signaling regulates mesenchymal lineage specification. The identification of a putative common Wnt/Fz molecular signature in MSCs will contribute to our understanding of the molecular mechanisms that regulate self-renewal and lineage

  6. Gravity and the cells of gravity receptors in mammals (United States)

    Ross, M. D.


    A model of the mammalian gravity receptor system is presented, with attention given to the effects of weightlessness. Two receptors are on each side of the head, with end organs in the saccule and utricle of the vestibular membranous labyrinth of the inner ear, embedded in the temporal bone. Each end organ has a macula, containing hair cells and supporting cells, and an otoconial complex, an otoconial membrane and mineral masses called otoconia. X ray powder diffraction examinations have revealed that the otoconia can behave like crystals, i.e., with piezoelectric properties, due to the mineral deposits. Bending of the hair cells because of acceleration can put pressure on the otoconial mineral, producing an electrical signal in the absence of a gravitational field. The possibility that pyroelectricity, as well as piezoelectricity, is present in the otoconial complexes, is discussed.

  7. Gene expression profiles in peripheral blood mononuclear cells of SARS patients

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Yu; Yun-Wen Hu; Xiao-Ying Liu; Wei Xiong; Zhi-Tong Zhou; Zheng-Hong Yuan


    AIM: To investigate the role of inflammatory and anti-viral genes in the pathogenesis of SARS.METHODS: cDNA microarrays were used to screen the gene expression profiles of peripheral blood mononuclear cells (PBMCs) in two SARS patients (one in the acute severe phase and the other in the convalescent phase)and a healthy donor. In addition, real-time qualitative PCR was also performed to verify the reproducibility of the microarray results. The data were further analyzed.RESULTS: Many inflammatory and anti-viral genes were differentially expressed in SARS patients. Compared to the healthy control or the convalescent case, plenty of pro-inflammatory cytokines such as IL-1, TNF-α, IL-8, and MAPK signaling pathway were significantly upregulated in the acute severe case. However, anti-inflammatory agents such as IL-4 receptor, IL-13 receptor, IL-1Ra,and TNF-α-induced proteins 3 and 6 also increased dramatically in the acute severe case. On the contrary, a lot of IFN-stimulated genes like PKR, GBP-1 and 2, CXCL-10and 11, and JAK/STAT signal pathway were downregulated in the acute severe case compared to the convalescent case.CONCLUSION: Gene expression in SARS patients mirrors a host state of inflammation and anti-viral immunity at the transcription level, and understanding of gene expression profiles may make contribution to further studies of the SARS pathogenesis.

  8. SHP-1 phosphatase activity counteracts increased T cell receptor affinity. (United States)

    Hebeisen, Michael; Baitsch, Lukas; Presotto, Danilo; Baumgaertner, Petra; Romero, Pedro; Michielin, Olivier; Speiser, Daniel E; Rufer, Nathalie


    Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 μM), those with supraphysiological affinity (K(d) = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity.

  9. Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants. (United States)

    Markwell, M A; Paulson, J C


    Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC in conjunction with neuraminidase (acylneuraminyl hydrolase, EC were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphological characteristics. By either criterion, treatment of the cells with Vibrio cholerae neuraminidase to remove cell surface sialic acids rendered them resistant to infection by Sendai virus. Endogenous replacement of receptors by the cell occurred slowly but supported maximal levels of infection within 6 hr. In contrast, sialylation during a 20-min incubation with CMP-sialic acid and beta-galactoside alpha 2,3-sialytransferase restored full susceptibility to infection. This enzyme elaborates the NeuAc alpha 2,3Gal beta 1,3GalNAc (NeuAc, N-acetylneuraminic acid) sequence on glycoproteins and glycolipids. No restoration of infectivity was observed when neuraminidase-treated cells were sialylated by using beta-galactoside alpha 2,6-sialytransferase, which elaborates the NeuAc-alpha 2,6Gal beta 1,4GlcNAc sequence. These results suggest that sialyloligosaccharide receptor determinants of defined sequence are required for Sendai virus infection of host cells.

  10. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)


    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  11. A Comprehensive Nuclear Receptor Network for Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ralf Kittler


    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.

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

    Lifescience Database Archive (English)

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

  13. P2X and P2Y receptor signaling in red blood cells. (United States)

    Sluyter, Ronald


    Purinergic signaling involves the activation of cell surface P1 and P2 receptors by extracellular nucleosides and nucleotides such as adenosine and adenosine triphosphate (ATP), respectively. P2 receptors comprise P2X and P2Y receptors, and have well-established roles in leukocyte and platelet biology. Emerging evidence indicates important roles for these receptors in red blood cells. P2 receptor activation stimulates a number of signaling pathways in progenitor red blood cells resulting in microparticle release, reactive oxygen species formation, and apoptosis. Likewise, activation of P2 receptors in mature red blood cells stimulates signaling pathways mediating volume regulation, eicosanoid release, phosphatidylserine exposure, hemolysis, impaired ATP release, and susceptibility or resistance to infection. This review summarizes the distribution of P2 receptors in red blood cells, and outlines the functions of P2 receptor signaling in these cells and its implications in red blood cell biology.

  14. Comparative transcriptional profiling of human Merkel cells and Merkel cell carcinoma. (United States)

    Mouchet, Nicolas; Coquart, Nolwenn; Lebonvallet, Nicolas; Le Gall-Ianotto, Christelle; Mogha, Ariane; Fautrel, Alain; Boulais, Nicholas; Dréno, Brigitte; Martin, Ludovic; Hu, Weiguo; Galibert, Marie-Dominique; Misery, Laurent


    Merkel cell carcinoma is believed to be derived from Merkel cells after infection by Merkel cell polyomavirus (MCPyV) and other poorly understood events. Transcriptional profiling using cDNA microarrays was performed on cells from MCPy-negative and MCPy-positive Merkel cell carcinomas and isolated normal Merkel cells. This microarray revealed numerous significantly upregulated genes and some downregulated genes. The extensive list of genes that were identified in these experiments provides a large body of potentially valuable information of Merkel cell carcinoma carcinogenesis and could represent a source of potential targets for cancer therapy.

  15. Altered Immune Profiles of Natural Killer Cells in Chronic Hepatitis B Patients: A Systematic Review and Meta-Analysis (United States)

    Zhang, Qiong-Fang; Shao, Jian-Ying; Yin, Wen-Wei; Xia, Yang; Chen, Ling; Wang, Xing; Hu, Huai-Dong; Hu, Peng; Ren, Hong; Zhang, Da-Zhi


    Background Natural killer (NK) cells are the main effective component of the innate immune system that responds to chronic hepatitis B (CHB) infection. Although numerous studies have reported the immune profiles of NK cells in CHB patients, they are limited by inconsistent results. Thus, we performed a meta-analysis to characterize reliably the immune profiles of NK cells after CHB infection, specifically frequency, phenotype, and function. Methods A literature search of the computer databases MEDLINE, PUBMED, EMBASE, and Cochrane Center Register of Controlled Trails was performed and 19 studies were selected. The standard mean difference (SMD) and 95% confidence interval (CI) of each continuous variable was estimated with a fixed effects model when I2 NUCs) showed no statistical difference in NK frequency. The activating receptors were upregulated, whereas inhibitory receptors were comparable in the peripheral NK cells of CHB individuals and healthy controls. NK cells of CHB patients displayed higher cytotoxic potency as evidenced by CD107a protein levels and conserved potency to produce interferon-gamma (IFNγ), compared with their healthy counterparts. Conclusion Our results revealed that CHB patients had a lower frequency of NK cells compared with healthy individuals not treatable with antiviral NUC therapy. With an activating phenotype, NK cells in CHB patients showed better cytotoxic potency and conserved IFNγ production. PMID:27513564

  16. Comparative Metabolic Flux Profiling of Melanoma Cell Lines (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.


    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  17. Simultaneous transcriptional profiling of bacteria and their host cells.

    Directory of Open Access Journals (Sweden)

    Michael S Humphrys

    Full Text Available We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness. Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

  18. CD44 is the principal cell surface receptor for hyaluronate. (United States)

    Aruffo, A; Stamenkovic, I; Melnick, M; Underhill, C B; Seed, B


    CD44 is a broadly distributed cell surface protein thought to mediate cell attachment to extracelular matrix components or specific cell surface ligands. We have created soluble CD44-immunoglobulin fusion proteins and characterized their reactivity with tissue sections and lymph node high endothelial cells in primary culture. The CD44 target on high endothelial cells is sensitive to enzymes that degrade hyaluronate, and binding of soluble CD44 is blocked by low concentrations of hyaluronate or high concentrations of chondroitin 4- and 6-sulfates. A mouse anti-hamster hyaluonate receptor antibody reacts with COS cells expressing hamster CD44 cDNA. In sections of all tissues examined, including lymph nodes and Peyer's patches, predigestion with hyaluronidase eliminated CD44 binding.

  19. Gene expression profile of renal cell carcinoma clear cell type

    Directory of Open Access Journals (Sweden)

    Marcos F. Dall’Oglio


    Full Text Available PURPOSE: The determination of prognosis in patients with renal cell carcinoma (RCC is based, classically, on stage and histopathological aspects. The metastatic disease develops in one third of patients after surgery, even in localized tumors. There are few options for treating those patients, and even the new target designed drugs have shown low rates of success in controlling disease progression. Few studies used high throughput genomic analysis in renal cell carcinoma for determination of prognosis. This study is focused on the identification of gene expression signatures in tissues of low-risk, high-risk and metastatic RCC clear cell type (RCC-CCT. MATERIALS AND METHODS: We analyzed the expression of approximately 55,000 distinct transcripts using the Whole Genome microarray platform hybridized with RNA extracted from 19 patients submitted to surgery to treat RCC-CCT with different clinical outcomes. They were divided into three groups (1 low risk, characterized by pT1, Fuhrman grade 1 or 2, no microvascular invasion RCC; (2 high risk, pT2-3, Fuhrman grade 3 or 4 with, necrosis and microvascular invasion present and (3 metastatic RCC-CCT. Normal renal tissue was used as control. RESULTS: After comparison of differentially expressed genes among low-risk, high-risk and metastatic groups, we identified a group of common genes characterizing metastatic disease. Among them Interleukin-8 and Heat shock protein 70 were over-expressed in metastasis and validated by real-time polymerase chain reaction. CONCLUSION: These findings can be used as a starting point to generate molecular markers of RCC-CCT as well as a target for the development of innovative therapies.

  20. Role of Prokineticin Receptor-1 in Epicardial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Canan G. Nebigil


    Full Text Available G protein-coupled receptors (GPCRs form a large class of seven transmembrane (TM domain receptors. The use of endogenous GPCR ligands to activate the stem cell maintenance or to direct cell differentiation would overcome many of the problems currently encountered in the use of stem cells, such as rapid in vitro differentiation and expansion or rejection in clinical applications. This review focuses on the definition of a new GPCR signaling pathway activated by peptide hormones, called “prokineticins”, in epicardium-derived cells (EPDCs. Signaling via prokineticin-2 and its receptor, PKR1, is required for cardiomyocyte survival during hypoxic stress. The binding of prokineticin-2 to PKR1 induces proliferation, migration and angiogenesis in endothelial cells. The expression of prokineticin and PKR1 increases during cardiac remodeling after myocardial infarction. Gain of function of PKR1 in the adult mouse heart revealed that cardiomyocyte-PKR1 signaling activates EPDCs in a paracrine fashion, thereby promoting de novo vasculogenesis. Transient PKR1 gene therapy after myocardial infarction in mice decreases mortality and improves heart function by promoting neovascularization, protecting cardiomyocytes and mobilizing WT1+ cells. Furthermore, PKR1 signaling promotes adult EPDC proliferation and differentiation to adopt endothelial and smooth muscle cell fate, for the induction of de novo vasculogenesis. PKR1 is expressed in the proepicardium and epicardial cells derived from mice kidneys. Loss of PKR1 causes deficits in EPDCs in the neonatal mice hearts and kidneys and impairs vascularization and heart and kidney function. Taken together, these data indicate a novel role for PKR1 in heart-kidney complex via EPDCs.

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

    Directory of Open Access Journals (Sweden)

    Tilo Beyer


    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.

  2. Glycone-rich Soy Isoflavone Extracts Promote Estrogen Receptor Positive Breast Cancer Cell Growth. (United States)

    Johnson, Kailee A; Vemuri, Sravan; Alsahafi, Sameerh; Castillo, Rudy; Cheriyath, Venugopalan


    Due to the association of hormone replacement therapy (HRT) with breast cancer risk, estrogenically active soy isoflavones are considered as an HRT alternative to alleviate menopausal symptoms. However, several recent reports challenged the health benefits of soy isoflavones and associated them with breast cancer promotion. While glyconic isoflavones are the major constituents of soybean seeds, due to their low cell permeability, they are considered to be biologically inactive. The glyconic isoflavones may exert their effects on membrane-bound estrogen receptors or could be converted to aglycones by extracellular β-glucosidases. Therefore, we hypothesized that despite their low cell permeability, soybean cultivars with high glyconic isoflavones may promote breast cancer cell growth. To test this, composition and estrogenic activity of isoflavones from 54 commercial soybean cultivars were determined. Soybean seeds produced in identical climate and growth conditions were used to minimize the effects of extraneous factors on isoflavone profile and concentrations. The glyconic daidzin concentration negatively correlated with genistin and with other aglycones. Relative to control, isoflavone extracts from 51 cultivars were estrogenic and promoted the growth of estrogen receptor positive (ER+) breast cancer cell line MCF-7 from 1.14 to 4.59 folds and other three cultivars slightly reduced the growth. Among these, extracts from three cultivars were highly estrogenic and promoted MCF-7 cell growth by 2.59-4.64 folds (Psoy isoflavone extracts may exert estrogenic effects and promote ER+ breast cancer growth.

  3. Effects of 3-styrylchromones on metabolic profiles and cell death in oral squamous cell carcinoma cells

    Directory of Open Access Journals (Sweden)

    Hiroshi Sakagami


    Full Text Available 4H-1-benzopyran-4-ones (chromones are important naturally-distributing compounds. As compared with flavones, isoflavones and 2-styrylchromones, there are only few papers of 3-styrylchromones that have been published. We have previously reported that among fifteen 3-styrylchromone derivatives, three new synthetic compounds that have OCH3 group at the C-6 position of chromone ring, (E-3-(4-hydroxystyryl-6-methoxy-4H-chromen-4-one (compound 11, (E-6-methoxy-3-(4-methoxystyryl-4H-chromen-4-one (compound 4, (E-6-methoxy-3-(3,4,5-trimethoxystyryl-4H-chromen-4-one (compound 6 showed much higher cytotoxicities against four epithelial human oral squamous cell carcinoma (OSCC lines than human normal oral mesenchymal cells. In order to further confirm the tumor specificities of these compounds, we compared their cytotoxicities against both human epithelial malignant and non-malignant cells, and then investigated their effects on fine cell structures and metabolic profiles and cell death in human OSCC cell line HSC-2. Cytotoxicities of compounds 4, 6, 11 were assayed with MTT method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to CE-TOFMS analysis. Compounds 4, 6, 11 showed much weaker cytotoxicity against human oral keratinocyte and primary human gingival epithelial cells, as compared with HSC-2, confirming their tumor-specificity, whereas doxorubicin and 5-FU were highly cytotoxic to these normal epithelial cells, giving unexpectedly lower tumor-specificity. The most cytotoxic compound 11, induced the mitochondrial vacuolization, autophagy suppression followed by apoptosis induction, and changes in the metabolites involved in amino acid and glycerophospholipid metabolisms. Chemical modification of lead compound 11 may be a potential choice for designing new type of anticancer drugs.

  4. Bovine mammary stem cells: Transcriptome profiling and the stem cell niche (United States)

    Identification and transcriptome analysis of mammary stem cells (MaSC) are important steps toward understanding the molecular basis of mammary epithelial growth, homeostasis and tissue repair. Our objective was to evaluate the molecular profiles of four categories of cells within the bovine mammary ...

  5. Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

    DEFF Research Database (Denmark)

    Boergesen, Michael; Pedersen, Thomas Åskov; Gross, Barbara;


    The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs...

  6. Determination of the Antibiotic Resistance Profile of Student Cell Phones

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    Lisa Ann Blankinship


    Full Text Available Sampling of common use items (e.g., student cell phones for bacterial presence, identification, and antibiotic resistance profiling helps students to recognize the need for routine cleaning of personal items and encourages thoughtful use of currently available medications. This multilab period project can be used to teach or reinforce several methods from general microbiology including aseptic technique, isolation streak, serial dilution, spread plating, Kirby Bauer testing, unknown identification, and media production. The data generated can be saved and added to each semester, thus providing a data set that reflects a local trend of antibiotic resistance.      

  7. Intraocular caspofungin: in vitro safety profile for human ocular cells. (United States)

    Kernt, M; Kampik, A


    Endogenous Candida endophthalmitis is sight-threatening, difficult to treat and sometimes leads to loss of the eye. Only a few therapeutic agents are available for its treatment. Caspofungin is the first of a new class of antifungal drugs (echinocandins) with a high activity against Candida species, the most common pathogens found in endogenous endophthalmitis. This study investigates the safety profile of caspofungin for intraocular application in a cell-culture model. Endothelial toxicity of caspofungin was evaluated in cultured human corneas. Possible toxic effects of caspofungin (5-300 μg ml(-1)) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC) and primary human retinal pigment epithelium (RPE) cells were evaluated after 24 h and under conditions of inflammatory stress by treatment with tumour necrosis factor-alpha (TNF-α), lipopolysaccharides (LPS) or interleukin-6 (IL-6) and hydrogen peroxide (H(2)O(2)). Toxicity was evaluated by tetrazolium dye-reduction assay; cell viability was quantified by a microscopic live-dead assay. No corneal endothelial toxicity could be detected after 30 days of treatment with 75 μg ml(-1) of caspofungin. Concentrations up to 75 μg ml(-1) had no influence on CEC, TMC or RPE cell proliferation, or on cell viability when administered for 24 h. Exposure to H(2)O(2) did not increase cellular toxicity of caspofungin at concentrations of 5-50 μg ml(-1). After preincubation with TNF-α, LPS or IL-6 for 24 h followed by treatment with caspofungin for 24 h, no significant decrease in cell proliferation or viability was observed. This study showed no significant toxicity for caspofungin on CEC, TMC or RPE cells, or human corneal endothelium when administered in therapeutic concentrations up to 50 μg ml(-1).

  8. Comparative proteomic and phosphoproteomic profiling of pancreatic adenocarcinoma cells treated with CB1 or CB2 agonists. (United States)

    Brandi, Jessica; Dando, Ilaria; Palmieri, Marta; Donadelli, Massimo; Cecconi, Daniela


    The pancreatic adenocarcinoma cell line Panc1 was treated with cannabinoid receptor ligands (arachidonylcyclopropylamide or GW405833) in order to elucidate the molecular mechanism of their anticancer effect. A proteomic approach was used to analyze the protein and phosphoprotein profiles. Western blot and functional data mining were also employed in order to validate results, classify proteins, and explore their potential relationships. We demonstrated that the two cannabinoids act through a widely common mechanism involving up- and down-regulation of proteins related to energetic metabolism and cell growth regulation. Overall, the results reported might contribute to the development of a therapy based on cannabinoids for pancreatic adenocarcinoma.

  9. Microarray gene expression profiling and analysis in renal cell carcinoma

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    Sadhukhan Provash


    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  10. Erythropoietin regulates Treg cells in asthma through TGFβ receptor signaling. (United States)

    Wan, Guoshi; Wei, Bing


    Asthma is a chronic inflammatory disorder of the airways, the development of which is suppressed by regulatory T cells (Treg). Erythropoietin (EPO) is originally defined as a hematopoietic growth factor. Recently, the anti-inflammatory effects of EPO in asthma have been acknowledged. However, the underlying mechanisms remain ill-defined. Here, we showed that EPO treatment significantly reduced the severity of an ovalbumin (OVA)-induced asthma in mice, seemingly through promoting Foxp3-mediated activation of Treg cells in OVA-treated mouse lung. The activation of Treg cells resulted from increases in transforming growth factor β1 (TGFβ1), which were mainly produced by M2 macrophages (M2M). In vitro, Co-culture with M2M increased Foxp3 levels in Treg cells and the Treg cell number, in a TGFβ receptor signaling dependent manner. Moreover, elimination of macrophages abolished the therapeutic effects of EPO in vivo. Together, our data suggest that EPO may increase M2M, which activate Treg cells through TGFβ receptor signaling to mitigate the severity of asthma.

  11. Mineralocorticoid Receptors in Immune Cells; Emerging Role in Cardiovascular Disease


    Bene, Nicholas C.; Alcaide, Pilar; Wortis, Henry H.; Jaffe, Iris Z.


    Mineralocorticoid receptors (MR) contribute to the pathophysiology of hypertension and cardiovascular disease in humans. As such, MR antagonists improve cardiovascular outcomes but the molecular mechanisms remain unclear. The actions of the MR in the kidney to increase blood pressure are well known, but the recent identification of MRs in immune cells has led to novel discoveries in the pathogenesis of cardiovascular disease that are reviewed here. MR regulates macrophage activation to the pr...

  12. Expression profile of cholecystokinin type-A receptor in gallbladder cancer and gallstone disease

    Institute of Scientific and Technical Information of China (English)

    Rajani Rai; Mallika Tewari; Mohan Kumar; Tej Bali Singh; Hari S Shukla


    BACKGROUND: Regulatorypeptidereceptorshaveattractedthe interest of oncologists as a new promising approach for cancer pathology, imaging and therapy. Although cholecystokinin (CCK) is a potent modulator of gallbladder contractility and plays a potential role in pancreatic carcinogenesis through CCK type-A receptor (CCKAR), its role in gallbladder cancer (GBC) is still unknown and immunohistochemical detection of CCKAR in the gallbladder has not yet been reported. This novel case-control study aimed to investigate the expression profile of CCKAR in GBC and gallstone disease (GSD). METHODS: This study included 162 samples of gallbladder: 94 fromGBCand68fromGSD.ExpressionofCCKARwasanalyzed by immunohistochemistry and immunoblotting. The results were statistically correlated with disease history including age, sex, presenceofgallstone,stageanddifferentiation. RESULTS: CCKAR was positive in 30/68 (44.1%) of GSD and 72/94 (76.6%) of GBC samples. Fifty-one of the 72 (70.8%) CCKAR-positive GBC samples showed over-expression. Interestingly, consistent results also appeared in the immuno-blotting study. CONCLUSIONS: CCKARexpressionwassignificantlyincreased in GBC compared to GSD. Moreover, CCKAR expression was associated with the degree of tumor differentiation, i.e., less expression in poorly-differentiated tumors. Thus, it has future prognostic and therapeutic implications in the management of GBC.

  13. Analysis of Chemokines and Receptors Expression Profile in the Myelin Mutant Taiep Rat

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    Guadalupe Soto-Rodriguez


    Full Text Available Taiep rat has a failure in myelination and remyelination processes leading to a state of hypomyelination throughout its life. Chemokines, which are known to play a role in inflammation, are also involved in the remyelination process. We aimed to demonstrate that remyelination-stimulating factors are altered in the brainstem of 1- and 6-month-old taiep rats. We used a Rat RT2 Profiler PCR Array to assess mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors. We also evaluated protein levels of CCL2, CCR1, CCR2, CCL5, CCR5, CCR8, CXCL1, CXCR2, CXCR4, FGF2, and VEGFA by ELISA. Sprague-Dawley rats were used as a control. PCR Array procedure showed that proinflammatory cytokines were not upregulated in the taiep rat. In contrast, some mRNA levels of beta and alpha chemokines were upregulated in 1-month-old rats, but CXCR4 was downregulated at their 6 months of age. ELISA results showed that CXCL1, CCL2, CCR2, CCR5, CCR8, and CXCR4 protein levels were decreased in brainstem at the age of 6 months. These results suggest the presence of a chronic neuroinflammation process with deficiency of remyelination-stimulating factors (CXCL1, CXCR2, and CXCR4, which might account for the demyelination in the taiep rat.

  14. Analysis of Chemokines and Receptors Expression Profile in the Myelin Mutant Taiep Rat (United States)

    Soto-Rodriguez, Guadalupe; Gonzalez-Barrios, Juan-Antonio; Martinez-Fong, Daniel; Blanco-Alvarez, Victor-Manuel; Eguibar, Jose R.; Ugarte, Araceli; Martinez-Perez, Francisco; Millán-Perez Peña, Lourdes; Pazos-Salazar, Nidia-Gary; Torres-Soto, Maricela; Garcia-Robles, Guadalupe; Tomas-Sanchez, Constantino


    Taiep rat has a failure in myelination and remyelination processes leading to a state of hypomyelination throughout its life. Chemokines, which are known to play a role in inflammation, are also involved in the remyelination process. We aimed to demonstrate that remyelination-stimulating factors are altered in the brainstem of 1- and 6-month-old taiep rats. We used a Rat RT2 Profiler PCR Array to assess mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors. We also evaluated protein levels of CCL2, CCR1, CCR2, CCL5, CCR5, CCR8, CXCL1, CXCR2, CXCR4, FGF2, and VEGFA by ELISA. Sprague-Dawley rats were used as a control. PCR Array procedure showed that proinflammatory cytokines were not upregulated in the taiep rat. In contrast, some mRNA levels of beta and alpha chemokines were upregulated in 1-month-old rats, but CXCR4 was downregulated at their 6 months of age. ELISA results showed that CXCL1, CCL2, CCR2, CCR5, CCR8, and CXCR4 protein levels were decreased in brainstem at the age of 6 months. These results suggest the presence of a chronic neuroinflammation process with deficiency of remyelination-stimulating factors (CXCL1, CXCR2, and CXCR4), which might account for the demyelination in the taiep rat. PMID:25883747

  15. Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells. (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Hein, Martina; Petersen, Frank; Thon, Lutz; Adam, Dieter; Bulfone-Paus, Silvia


    Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

  16. Sf9 cells: a versatile model system to investigate the pharmacological properties of G protein-coupled receptors. (United States)

    Schneider, Erich H; Seifert, Roland


    The Sf9 cell/baculovirus expression system is widely used for high-level protein expression, often with the purpose of purification. However, proteins may also be functionally expressed in the defined Sf9 cell environment. According to the literature, the pharmacology of G-protein-coupled receptors (GPCRs) functionally reconstituted in Sf9 cells is similar to the receptor properties in mammalian cells. Sf9 cells express both recombinant GPCRs and G-proteins at much higher levels than mammalian cells. Sf9 cells can be grown in suspension culture, providing an inexpensive way of obtaining large protein amounts. Co-infection with various baculoviruses allows free combination of GPCRs with different G-proteins. The absence of constitutively active receptors in Sf9 cells provides an excellent signal-to background ratio in functional assays, allowing the detection of agonist-independent receptor activity and of small ligand-induced signals including partial agonistic and inverse agonistic effects. Insect cell Gα(i)-like proteins mostly do not couple productively to mammalian GPCRs. Thus, unlike in mammalian cells, Sf9 cells do not require pertussis toxin treatment to obtain a Gα(i)-free environment. Co-expression of GPCRs with Gα(i1), Gα(i2), Gα(i3) or Gα(o) in Sf9 cells allows the generation of a selectivity profile for these Gα(i/o)-isoforms. Additionally, GPCR-G-protein combinations can be compared with defined 1:1 stoichiometry by expressing GPCR-Gα fusion proteins. Sf9 cells can also be employed for ligand screening in medicinal chemistry programs, using radioligand binding assays or functional assays, like the steady-state GTPase- or [(35)S]GTPγS binding assay. This review shows that Sf9 cells are a versatile model system to investigate the pharmacological properties of GPCRs.

  17. Defining an olfactory receptor function in airway smooth muscle cells (United States)

    Aisenberg, William H.; Huang, Jessie; Zhu, Wanqu; Rajkumar, Premraj; Cruz, Randy; Santhanam, Lakshmi; Natarajan, Niranjana; Yong, Hwan Mee; De Santiago, Breann; Oh, Jung Jin; Yoon, A-Rum; Panettieri, Reynold A.; Homann, Oliver; Sullivan, John K.; Liggett, Stephen B.; Pluznick, Jennifer L.; An, Steven S.


    Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma. PMID:27905542

  18. RNA cell typing and DNA profiling of mixed samples: can cell types and donors be associated? (United States)

    Harteveld, Joyce; Lindenbergh, Alexander; Sijen, Titia


    Forensic samples regularly involve mixtures, which are readily recognised in forensic analyses. Combined DNA and mRNA profiling is an upcoming forensic practice to examine donors and cell types from the exact same sample. From DNA profiles individual genotypes may be deconvoluted, but to date no studies have established whether the cell types identified in corresponding RNA profiles can be associated with individual donors. Although RNA expression levels hold many variables from which an association may not be expected, proof of concept is important to forensic experts who may be cross examined about this possible correlation in court settings. Clearly, the gender-specificity of certain body fluids (semen, vaginal mucosa, menstrual secretion) can be instructive. However, when donors of the same gender or gender-neutral cell types are involved, alternatives are needed. Here we analyse basic two-component mixtures (two cell types provided by different donors) composed of six different cell types, and assess whether the heights of DNA and RNA peaks may guide association of donor and cell type. Divergent results were obtained; for some mixtures RNA peak heights followed the DNA results, but for others the major DNA component did not present higher RNA peaks. Also, variation in mixture ratios was observed for RNA profiling replicates and when different donor couples gave the same two body fluids. As sample degradation may affect the two nucleic acids and/or distinct cell types differently (and thus influence donor and cell type association), mixtures were subjected to elevated temperature or UV-light. Variation in DNA and RNA stability was observed both between and within cell types and depended on the method inducing degradation. Taken together, we discourage to associate cell types and donors from peak heights when performing RNA and DNA profiling.

  19. Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells. (United States)

    Zheng, Xiangjun; Cheung, Luthur Siu-Lun; Schroeder, Joyce A; Jiang, Linan; Zohar, Yitshak


    Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

  20. Distribution, Arrangement and Interconnectedness of Cell Surface Receptor sites in the body of an Organism

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    Full Text Available Cell surface receptors have been identified as the sites of disease infectivity in living organisms in a previous study. Drugs used for the treatment or cure of infections have to eliminate infections through attacking infective organisms at the cell surface receptors to which the infective organisms are attached. Problem statement: The present study examines a wide sample of living things to get more information on the relationship of one cell surface receptor to other cell surface receptors in the body of an organism. Approach: The arrangement of cell surface receptors on the external covering of a few samples of fruits, leaves, stems, dry wood of a plant; wall gecko and some parts of the human body, were examined and photographed. Transverse and/or Longitudinal sections of soursop fruit and sycamore fruit were also examined and photographed. The five different coverings of the fleshy part of a coconut were also photographed. The photographs were studied to note the relationship of disease infection attached to cell surface receptors on the external surface of an organ to disease infection on the innermost covering of the same organ. Results: The results of the study showed that all living things had ubiquitous distribution of cell surface receptors which are usually observable with the unaided eye as dots or spots on the external covering of an organ, tissue or cell. The dots or receptor sites of cell surface receptors in the study are arranged in lines which were perpendicular, oblique, transverse or arranged in any other lineal geometrical form. The lineally arranged cell surface receptors were noted to be connected by grooves, channels or pipes which joined other receptor channels or intersected with them. Smaller cell surface receptor channels emptied into bigger channels or continued as small sized channels that ran side by side in a connective tissue bundle. These connective tissue bundles that carried many independent small-sized cell

  1. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations. (United States)

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H


    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors.

  2. Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation



    Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire coul...

  3. Gene expression profiling of chicken primordial germ cell ESTs

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    Lim Dajeong


    Full Text Available Abstract Background Germ cells are the only cell type that can penetrate from one generation to next generation. At the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. This study was conducted to investigate a large-scale expressed sequence tag (EST analysis in chicken PGCs and compare the expression of the PGC ESTs with that of embryonic gonad. Results We constructed 10,851 ESTs from a chicken cDNA library of a collection of highly separated embryonic PGCs. After chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were 5,093 resulting unique sequences consisting of 156 contigs and 4,937 singlets. Pearson chi-square tests of gene ontology terms in the 2nd level between PGC and embryonic gonad set showed no significance. However, digital gene expression profiling using the Audic's test showed that there were 2 genes expressed significantly with higher number of transcripts in PGCs compared with the embryonic gonads set. On the other hand, 17 genes in embryonic gonads were up-regulated higher than those in the PGC set. Conclusion Our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages.

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

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    Pinar Ataca


    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.

  5. Transcriptional Profile of HIV-induced Nuclear Translocation of Amyloid β in Brain Endothelial Cells (United States)

    András, Ibolya E.; Rampersaud, Evadnie; Eum, Sung Yong; Toborek, Michal


    Background and Aims Increased amyloid deposition in HIV-infected brains may contribute to the pathogenesis of neurocognitive dysfunction in infected patients. We have previously shown that exposure to HIV results in enhanced amyloid β (Aβ) levels in human brain microvascular endothelial cells, suggesting that brain endothelial cells contribute to accumulation of Aβ in HIV-infected brains. Importantly, Aβ not only accumulates in the cytoplasm of HIV-exposed cells but also enters the nuclei of brain endothelial cells. Methods cDNA microarray analysis was performed in order to examine changes in the transcriptional profile associated with Aβ nuclear entry in the presence of HIV-1. Results Gene network analysis indicated that inhibition of nuclear entry of Aβ resulted in enrichment in gene sets involved in apoptosis and survival, endoplasmic reticulum stress response, immune response, cell cycle, DNA damage, oxidative stress, cytoskeleton remodeling and transforming growth factor b (TGFβ) receptor signaling. Conclusions The obtained data indicate that HIV-induced Aβ nuclear uptake affects several cellular stress-related pathways relevant for HIV-induced Aβ pathology. PMID:25446617

  6. Fingolimod alters the transcriptome profile of circulating CD4+ cells in multiple sclerosis (United States)

    Friess, Jörg; Hecker, Michael; Roch, Luisa; Koczan, Dirk; Fitzner, Brit; Angerer, Ines Charlotte; Schröder, Ina; Flechtner, Kristin; Thiesen, Hans-Jürgen; Winkelmann, Alexander; Zettl, Uwe Klaus


    Multiple sclerosis is a demyelinating disease affecting the central nervous system. T cells are known to contribute to this immune-mediated condition. Fingolimod modulates sphingosine-1-phosphate receptors, thereby preventing the egress of lymphocytes, especially CCR7-expressing CD8+ and CD4+ T cells, from lymphoid tissues. Using Affymetrix Human Transcriptome Arrays (HTA 2.0), we performed a transcriptome profiling analysis of CD4+ cells obtained from the peripheral blood of patients with highly active relapsing-remitting multiple sclerosis. The samples were drawn before the first administration of fingolimod as well as 24 hours and 3 months after the start of therapy. Three months after treatment initiation, 890 genes were found to be differentially expressed with fold-change >2.0 and t-test p-value CCR7 as expected, while 618 genes showed an increase in expression, e.g., CCR2, CX3CR1, CD39, CD58 as well as LYN, PAK1 and TLR2. To conclude, we studied the gene expression of CD4+ cells to evaluate the effects of fingolimod treatment, and we identified 890 genes to be altered in expression after continuous drug administration. T helper cells circulating in the blood during fingolimod therapy present a distinct gene expression signature. PMID:28155899

  7. Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells

    Institute of Scientific and Technical Information of China (English)

    胡庆柳; 朴英杰; 邹飞


    Objective To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.Methods Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with α-32P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by AtlasImage analysis software Version 1.01a.Results Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells.Conclusion The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

  8. Tumor-derived death receptor 6 modulates dendritic cell development. (United States)

    DeRosa, David C; Ryan, Paul J; Okragly, Angela; Witcher, Derrick R; Benschop, Robert J


    Studies in murine models of cancer as well as in cancer patients have demonstrated that the immune response to cancer is often compromised. This paradigm is viewed as one of the major mechanisms of tumor escape. Many therapies focus on employing the professional antigen presenting dendritic cells (DC) as a strategy to overcome immune inhibition in cancer patients. Death receptor 6 (DR6) is an orphan member of the tumor necrosis factor receptor superfamily (TNFRSF21). It is overexpressed on many tumor cells and DR6(-/-) mice display altered immunity. We investigated whether DR6 plays a role in tumorigenesis by negatively affecting the generation of anti-tumor activity. We show that DR6 is uniquely cleaved from the cell surface of tumor cell lines by the membrane-associated matrix metalloproteinase (MMP)-14, which is often overexpressed on tumor cells and is associated with malignancy. We also demonstrate that >50% of monocytes differentiating into DC die when the extracellular domain of DR6 is present. In addition, DR6 affects the cell surface phenotype of the resulting immature DC and changes their cytokine production upon stimulation with LPS/IFN-gamma. The effects of DR6 are mostly amended when these immature DC are matured with IL-1beta/TNF-alpha, as measured by cell surface phenotype and their ability to present antigen. These results implicate MMP-14 and DR6 as a mechanism tumor cells can employ to actively escape detection by the immune system by affecting the generation of antigen presenting cells.

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

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    Francesco Marras


    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.

  10. Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis. (United States)

    Riggio, Marcello P; Lappin, David F; Bennett, David


    The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (2 CD, 10 SA) and detected in 4 samples (3 CD, 1 SA) using culture-independent methods. Statistically significant increases in TLR2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p=0.043) and IL-12 (p=0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p=0.033) in dogs up to 4 years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

  11. Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

    Directory of Open Access Journals (Sweden)

    Helle Jensen

    Full Text Available NKG2D is a stimulatory receptor expressed by natural killer (NK cells, CD8(+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+ T-cells, however recently a subset of NKG2D(+ CD4(+ T-cells has been found, which is specific for human cytomegalovirus (HCMV. This particular subset of HCMV-specific NKG2D(+ CD4(+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+ CD4(+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA to investigate the gene expression profile of NKG2D(+ CD4(+ T-cells, generated from HCMV-primed CD4(+ T-cells. We show that the HCMV-primed NKG2D(+ CD4(+ T-cells possess a higher differentiated phenotype than the NKG2D(- CD4(+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+ T-cells, whereas it is produced de novo in resting CD4(+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+ CD4(+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression.

  12. A novel gene delivery system targeting cells expressing VEGF receptors

    Institute of Scientific and Technical Information of China (English)



    Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.

  13. Analysis of tanshinone IIA induced cellular apoptosis in leukemia cells by genome-wide expression profiling

    Directory of Open Access Journals (Sweden)

    Liu Chang


    Full Text Available Abstract Background Tanshinone IIA (Tan IIA is a diterpene quinone extracted from the root of Salvia miltiorrhiza, a Chinese traditional herb. Although previous studies have reported the anti-tumor effects of Tan IIA on various human cancer cells, the underlying mechanisms are not clear. The current study was undertaken to investigate the molecular mechanisms of Tan IIA's apoptotic effects on leukemia cells in vitro. Methods The cytotoxicity of Tan IIA on different types of leukemia cell lines was evaluated by the 3-[4,5-dimethylthiazol-2,5]-diphenyl tetrazolium bromide (MTT assay on cells treated without or with Tan IIA at different concentrations for different time periods. Cellular apoptosis progression with and without Tan IIA treatment was analyzed by Annexin V and Caspase 3 assays. Gene expression profiling was used to identify the genes regulated after Tan IIA treatment and those differentially expressed among the five cell lines. Confirmation of these expression regulations was carried out using real-time quantitative PCR and ELISA. The antagonizing effect of a PXR inhibitor L-SFN on Tan IIA treatment was tested using Colony Forming Unit Assay. Results Our results revealed that Tan IIA had different cytotoxic activities on five types of leukemia cells, with the highest toxicity on U-937 cells. Tan IIA inhibited the growth of U-937 cells in a time- and dose-dependent manner. Annexin V and Caspase-3 assays showed that Tan IIA induced apoptosis in U-937 cells. Using gene expression profiling, 366 genes were found to be significantly regulated after Tan IIA treatment and differentially expressed among the five cell lines. Among these genes, CCL2 was highly expressed in untreated U-937 cells and down-regulated significantly after Tan IIA treatment in a dose-dependent manner. RT-qPCR analyses validated the expression regulation of 80% of genes. Addition of L- sulforaphane (L-SFN, an inhibitor of Pregnane × receptor (PXR significantly

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

    Directory of Open Access Journals (Sweden)

    Marie-Pier Lecours

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

  15. 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: [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)


    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.

  16. Transcriptional network profile on synovial fluid T cells in psoriatic arthritis. (United States)

    Fiocco, Ugo; Martini, Veronica; Accordi, Benedetta; Caso, Francesco; Costa, Luisa; Oliviero, Francesca; Scanu, Anna; Facco, Monica; Boso, Daniele; Gatto, Mariele; Felicetti, Mara; Frallonardo, Paola; Ramonda, Roberta; Piva, Lucia; Zambello, Renato; Agostini, Carlo; Scarpa, Raffaele; Basso, Giuseppe; Semenzato, Gianpietro; Dayer, Jean-Michel; Punzi, Leonardo; Doria, Andrea


    The objective of the study was to quantify the transcriptional profile, as the main T cell lineage-transcription factors on synovial fluid (SF) T cells, in relation to SF cytokines and T cell frequencies (%) of psoriatic arthritis (PsA) patients. Reverse phase protein array was employed to identify interleukin (IL)-23Rp19-, FOXP3- and related orphan receptor gamma T (RORγt)- protein and Janus associated tyrosine kinases 1 (JAK1), signal transducer and activator and transcription 1 (STAT1), STAT3 and STAT5 phosphoproteins in total T cell lysates from SF of PsA patients. IL-1β, IL-2, IL-6, IL-21 and interferon (INF)-γ were measured using a multiplex bead immunoassay in SF from PsA patients and peripheral blood (PB) from healthy controls (HC). Frequencies of CD4(+)CD25(-), CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg, and either mean fluorescence intensity (MFI) of FOXP3(+) on CD4(+) Treg or MFI of classic IL-6 receptor (IL-6R) α expression on CD4(+)CD25(-) helper/effector T cells (Th/eff) and Treg cells, were quantified in SF of PsA patients and in PB from HC by flow cytometry (FC). In PsA SF samples, IL-2, IL-21 and IFN-γ were not detectable, whereas IL-6 and IL-1β levels were higher than in SF of non-inflammatory osteoarthritis patients. Higher levels of IL-23R-, FOXP3- and RORγt proteins and JAK1, STAT1, STAT3 and STAT5 were found in total T cells from SF of PsA patients compared with PB from HC. Direct correlations between JAK1 Y1022/Y1023 and STAT5 Y694, and STAT3 Y705 and IL6, were found in SF of PsA patients. Increased proportion of CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg cells and brighter MFI of IL-6Rα were observed both on CD4(+)CD25(high)- and CD4(+)CD25(-) T cells in PsA SF. The study showed a distinctive JAK1/STAT3/STAT5 transcriptional network on T cells in the joint microenvironment, outlining the interplay of IL-6, IL-23, IL-1β and γC cytokines in the polarization and plasticity of Th17 and Treg cells

  17. 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


    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...

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

    NARCIS (Netherlands)

    Haastert, Peter J.M. van


    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

  19. Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes

    NARCIS (Netherlands)

    Watts, A. O.; van Lipzig, M. M. H.; Jaeger, W. C.; Seeber, R. M.; van Zwam, M.; Vinet, J.; van der Lee, M. M. C.; Siderius, M.; Zaman, G. J. R.; Boddeke, H. W. G. M.; Smit, M. J.; Pfleger, K. D. G.; Leurs, R.; Vischer, H. F.


    Background and Purpose The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heter

  20. Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells. (United States)

    Daud, Hasbullah; Browne, Susan; Al-Majmaie, Rasoul; Murphy, William; Al-Rubeai, Mohamed


    An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71(-)/GpA(+) surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells.


    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...

  2. Studies of phosphorus Gaussian profile emitter silicon solar cells

    Directory of Open Access Journals (Sweden)

    N. Stem


    Full Text Available Considering recent modifications on n-type highly doped silicon parameters, an emitter optimization was made based on one-dimensional models with analytical solutions. In order to get good accuracy, a fifth order approximation has been considered. Two kinds of emitters, homogeneous and non-homogeneous, with phosphorus Gaussian profile emitter solar cells were optimized. According to our results: homogeneous emitter solar cells show their maximum efficiencies (h @ 21.60-21.74%with doping levelsnus = 1x10(19 - 5x10(18 (cm-3 and (1.2-2.0 mum emitter thickness range. Non-homogeneous emitter solar cells provide a slightly higher efficiency (eta = 21.82-21.92%, with Ns = 1x10(20 (cm-3 with 2.0 mum thickness under metal-contacted surface and Ns = 1x10(19 - 5x10(18 (cm-3 with (1.2-2.0 mum thickness range, (sheet resistance range 90-100 W/ under passivated surface. Although non-homogeneous emitter solar cells have a higher efficiency than homogeneous emitter ones, the required technology is more complex and their overall interest for practical applications is questionable.

  3. Melanocortin receptor accessory protein 2 (MRAP2) interplays with the zebrafish melanocortin 1 receptor (MC1R) but has no effect on its pharmacological profile. (United States)

    Cortés, Raúl; Agulleiro, Maria Josep; Navarro, Sandra; Guillot, Raúl; Sánchez, Elisa; Cerdá-Reverter, José Miguel


    The melanocortin system is probably one of the most complex hormonal systems since it integrates agonist, encoded in the proopiomelanocortin precursor, endogenous antagonist, agouti signaling protein and agouti-related protein, five different G-protein coupled receptors and two accessory proteins. These accessory proteins interact with melanocortin receptors to allow traffic to the plasma membrane or to regulate the pharmacological profile. The MC1R fill the extension locus, which is primarily responsible for the regulation of pigmentation. In zebrafish, both MC1R and MRAP2 system are expressed in the skin. We demonstrate that zebrafish MC1R physically, or closely, interacts with the MRAP2 system, although this interaction did not result in modification of the studied pharmacological profile. However, progressive fasting induced skin darkening but also an upregulation of the MRAP2 expression in the skin, suggesting an unknown role for MRAP2a that could involve receptor desensitization processes. We also demonstrate that crowding stress induces skin darkening and a downregulation of MC1R expression in the skin.

  4. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines. (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H


    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  5. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells. (United States)

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike


    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  6. Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression. (United States)

    Saby, Charles; Buache, Emilie; Brassart-Pasco, Sylvie; El Btaouri, Hassan; Courageot, Marie-Pierre; Van Gulick, Laurence; Garnotel, Roselyne; Jeannesson, Pierre; Morjani, Hamid


    Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

  7. Five layers of receptor signalling in γδ T cell differentiation and activation

    Directory of Open Access Journals (Sweden)

    Sérgio T. Ribeiro


    Full Text Available The contributions of gamma-delta T cells to immunity to infection or tumours critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate gamma-delta T cell physiology. The understanding of how environmental signals are integrated by gamma-delta T cells is critical for their manipulation in clinical settings. Here we discuss how different classes of surface receptors impact on human and murine gamma-delta T cell differentiation, activation and expansion. In particular, we review the role of five receptor types: the T cell receptor (TCR, costimulatory receptors, cytokine receptors, NK receptors and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of gamma-delta T cells; the cytokine receptors IL-2R, IL-7R and IL-15R, which drive functional differentiation and expansion of gamma-delta T cells; the NK receptor NKG2D and its contribution to gamma-delta T cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control gamma-delta T cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signalling in gamma-delta T cell differentiation and activation, and discuss its implications for the manipulation of gamma-delta T cells in immunotherapy.

  8. Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder;


    will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...... their Ph-chromosome status. Interestingly, we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+), whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph(+) and Ph(-) candidate CML...

  9. 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


    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.

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

    DEFF Research Database (Denmark)

    de Turco, Elena B; Diemer, Nils Henrik; Bazan, Nicolas G


    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....

  11. Activin Receptor Signaling Regulates Prostatic Epithelial Cell Adhesion and Viability

    Directory of Open Access Journals (Sweden)

    Derek P. Simon


    Full Text Available Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s in cell proliferation. Using prostatic cancer cell (PCC lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII, as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS. Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.

  12. 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


    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

  13. Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutantsin the Sf9 Cell Expression System. (United States)

    Schneider, Erich H; Seifert, Roland


    A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [(35)S]GTPγS assays). The human H1R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH1R and insect cell Gαq. By contrast, functional expression of the hH2R required the generation of an hH2R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH2R to the long (GsαL) or short (GsαS) splice variant of Gαs resulted in comparable constitutive hH2R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH1R/hH2R and their guinea pig orthologues gpH1R/gpH2R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH3R with Gαi1, Gαi2, Gαi3, and Gαi/o in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li(+), Na(+), K(+)) and anions (Cl(-), Br(-), I(-)) revealed that anions with large radii most efficiently stabilize the inactive hH3R state. Potential sodium binding sites in the hH3R protein were analyzed by expressing specific hH3R mutants in Sf9 cells. In contrast to the hH3R, the hH4R preferentially couples to co-expressed Gαi2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH4R shows structural instability and adopts a G protein-independent high

  14. Activation of Estrogen Receptor Transfected into a Receptor-Negative Brest Cancer Cell Line Decreases the Metastatic and Invasive Potential of the Cells (United States)

    Garcia, Marcel; Derocq, Danielle; Freiss, Gilles; Rochefort, Henri


    Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas the antiestrogens 4-hydroxytamoxifen and ICI 164,384 reversed these effects. These results show that estradiol inhibits the metastatic ability of estrogen receptornegative breast cancer cells following transfection with the estrogen receptor, whereas estrogen receptor-positive breast cancers are stimulated by estrogen, indicating that factors other than the estrogen receptor are involved in progression toward hormone independence. Reactivation or transfer of the estrogen receptor gene can therefore be considered as therapeutic approaches to hormone-independent cancers

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

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil


    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.

  16. Transcriptional profiling of rhesus monkey embryonic stem cells. (United States)

    Byrne, James A; Mitalipov, Shoukhrat M; Clepper, Lisa; Wolf, Don P


    Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

  17. Neurophysiologic and antipsychotic profiles of TASP0433864, a novel positive allosteric modulator of metabotropic glutamate 2 receptor. (United States)

    Hiyoshi, Tetsuaki; Marumo, Toshiyuki; Hikichi, Hirohiko; Tomishima, Yasumitsu; Urabe, Hiroki; Tamita, Tomoko; Iida, Izumi; Yasuhara, Akito; Karasawa, Jun-ichi; Chaki, Shigeyuki


    Excess glutamatergic neurotransmission has been implicated in the pathophysiology of schizophrenia, and the activation of metabotropic glutamate 2 (mGlu2) receptor may exert antipsychotic effects by normalizing glutamate transmission. In the present study, we investigated the neurophysiologic and antipsychotic profiles of TASP0433864 [(2S)-2-[(4-tert-butylphenoxy)methyl]-5-methyl-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide], a newly synthesized positive allosteric modulator (PAM) of mGlu2 receptor. TASP0433864 exhibited PAM activity at human and rat mGlu2 receptors with EC50 values of 199 and 206 nM, respectively, without exerting agonist activity at rat mGlu2 receptor. TASP0433864 produced a leftward and upward shift in the concentration-response curve of glutamate-increased guanosine 5'-O-(3-[(35)S]thio)triphosphate binding to mGlu2 receptor. In contrast, TASP0433864 had negligible activities for other mGlu receptors, including mGlu3 receptor, and did not have any affinity for other receptors or transporters. In hippocampal slices, TASP0433864 potentiated an inhibitory effect of DCG-IV [(2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine], a mGlu2/3 receptor agonist, on the field excitatory postsynaptic potentials in the dentate gyrus, indicating that TASP0433864 potentiates the mGlu2 receptor-mediated presynaptic inhibition of glutamate release. Moreover, TASP0433864 inhibited both MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate]- and ketamine-increased cortical γ band oscillation in the rat cortical electroencephalogram, which have been considered to reflect the excess activation of cortical pyramidal neurons. The inhibitory effect of TASP0433864 on cortical activation was also observed in the mouse 2-deoxy-glucose uptake study. In a behavioral study, TASP0433864 significantly inhibited both ketamine- and methamphetamine-increased locomotor activities in mice and rats, respectively. Collectively, these

  18. Glycine receptors contribute to cytoprotection of glycine in myocardial cells

    Institute of Scientific and Technical Information of China (English)

    QI Ren-bin; ZHANG Jun-yan; LU Da-xiang; WANG Hua-dong; WANG Hai-hua; LI Chu-jie


    Background The classic glycine receptor (GlyR) in the central nervous system is a ligand-gated membrane-spanning ion channel. Recent studies have provided evidence for the existence of GlyR in endothelial cells, renal proximal tubular cells and most leukocytes. In contrast, no evidence for GlyR in myocardial cells has been found so far. Our recent researches have showed that glycine could protect myocardial cells from the damage induced by lipopolysaccharide (LPS). Further studies suggest that myocardial cells could contain GlyR or binding site of glycine.Methods In isolated rat heart damaged by LPS, the myocardial monophasic action potential (MAP), the heart rate (HR),the myocardial tension and the activities of lactate dehydrogenase (LDH) from the coronary effluent were determined.The concentration of intracellular free calcium ([Ca2+]i) was measured in cardiomyocytes injured by LPS and by hypoxia/reoxygenation (H/R), which excludes the possibility that reduced calcium influx because of LPS neutralized by glycine. Immunohistochemistry was used to detect the GlyR in myocardial tissue. GlyR and its subunit in the purified cultured cardiomyocytes were identified by Western blotting.Results Although significant improvement in the MAP/MAPD20, HR, and reduction in LDH release were observed in glycine + LPS hearts, myocardial tension did not recover. Further studies demonstrated that glycine could prevent rat mycordial cells from LPS and hypoxia/reoxygenation injury (no endotoxin) by attenuating calcium influx.Immunohistochemistry exhibited a positive green-fluorescence signaling along the cardiac muscle fibers. Western blotting shows that the purified cultured cardiomyocytes express GlyR β subunit, but GlyR α1 subunit could not be detected.Conclusions The results suggest that glycine receptor is expressed in cardiomyocytes and participates in cytoprotection from LPS and hypoxia/reoxygenation injury. Glycine could directly activate GlyR on the cardiomyocytes and

  19. Extracellular calcium sensing receptor in human pancreatic cells (United States)

    Rácz, G Z; Kittel, Á; Riccardi, D; Case, R M; Elliott, A C; Varga, G


    Background and aims: The extracellular calcium sensing receptor (CaR) plays a key role in the calcium homeostatic system and is therefore widely expressed in tissues involved in calcium metabolism. However, the CaR has also been identified in other tissues where its role is less clear. We have investigated the presence of the CaR in the human pancreas. Methods: Messenger RNA for the CaR was detected by reverse transcription-polymerase chain reaction and the protein was localised by immunostaining. CaR function was assayed in Capan-1 cells by measuring intracellular calcium and [3H] thymidine incorporation. Results: The receptor was highly expressed in human pancreatic ducts. It was also expressed in exocrine acinar cells, in islets of Langerhans, and in intrapancreatic nerves and blood vessels. The CaR was expressed in both normal and neoplastic human tissue samples but was detected in only one of five ductal adenocarcinoma cells lines examined. Experiments on the CaR expressing adenocarcinoma cell line Capan-1 showed that the CaR was functional and was linked to mobilisation of intracellular calcium. Stimulation of the CaR reduced Capan-1 cell proliferation. Conclusions: We propose that the CaR may play multiple functional roles in the human pancreas. In particular, the CaR on the duct luminal membrane may monitor and regulate the Ca2+ concentration in pancreatic juice by triggering ductal electrolyte and fluid secretion. This could help to prevent precipitation of calcium salts in the duct lumen. The CaR may also help to regulate the proliferation of pancreatic ductal cells. PMID:12377811

  20. Proteomic profile of human monocytic cells infected with dengue virus

    Institute of Scientific and Technical Information of China (English)

    Viviana Martnez-Betancur; Marlen Martnez-Gutierrez


    Objective: To identify the changes in the proteome of U937 cells infected with dengue virus (DENV). Methods: In this study, differentiated U937 cultures were infected with two DENV-2 strains, one of which was associated with dengue (DENV-2/NG) and the other one with severe dengue (DENV-2/16681), with the aim of determining the cellular proteomic profiles under different infection conditions. Cellular proteins were extracted and sepa-rated by two-dimensional electrophoresis, and those proteins with differential expression profiles were identified by mass spectrometry. The obtained results were correlated with cellular viability, the number of infectious viral particles, and the viral DNA/protein quantity. Results: In comparison with non-infected cultures, in the cells infected with the DENV-2/NG strain, nine proteins were expressed differentially (five were upregulated and four were downregulated); in those cultures infected with the DENV-2/16681 strain, six proteins were differentially expressed (two were downregulated and four were upregu-lated). The downregulated proteins included fatty acid-binding protein, heterogeneous nuclear ribonucleoprotein 1, protein disulfide isomerase, enolase 1, heat shock 70 kDa protein 9, phosphotyrosyl phosphatase, and annexin IV. The upregulated proteins included heat shock 90 kDa protein AA1, tubulin beta, enolase 1, pyruvate kinase, transaldolase and phospholipase C-alpha. Conclusions: Because the monocyte/macrophage lineage is critical for disease patho-genicity, additional studies on these proteins could provide a better understanding of the cellular response to DENV infection and could help identify new therapeutic targets against infection.

  1. Role of nuclear receptors in breast cancer stem cells

    Institute of Scientific and Technical Information of China (English)

    Alessio; Papi; Marina; Orlandi


    The recapitulation of primary tumour heterogenity and the existence of a minor sub-population of cancer cells,capable of initiating tumour growth in xenografts on serial passages, led to the hypothesis that cancer stem cells(CSCs) exist. CSCs are present in many tumours, among which is breast cancer. Breast CSCs(BCSCs) are likely to sustain the growth of the primary tumour mass, as wellas to be responsible for disease relapse and metastatic spreading. Consequently, BCSCs represent the most significant target for new drugs in breast cancer therapy. Both the hypoxic condition in BCSCs biology and proinflammatory cytokine network has gained increasing importance in the recent past. Breast stromal cells are crucial components of the tumours milieu and are a major source of inflammatory mediators. Recently, the antiinflammatory role of some nuclear receptors ligands has emerged in several diseases, including breast cancer. Therefore, the use of nuclear receptors ligands may be a valid strategy to inhibit BCSCs viability and consequently breast cancer growth and disease relapse.

  2. From drug response profiling to target addiction scoring in cancer cell models

    Directory of Open Access Journals (Sweden)

    Bhagwan Yadav


    Full Text Available Deconvoluting the molecular target signals behind observed drug response phenotypes is an important part of phenotype-based drug discovery and repurposing efforts. We demonstrate here how our network-based deconvolution approach, named target addiction score (TAS, provides insights into the functional importance of druggable protein targets in cell-based drug sensitivity testing experiments. Using cancer cell line profiling data sets, we constructed a functional classification across 107 cancer cell models, based on their common and unique target addiction signatures. The pan-cancer addiction correlations could not be explained by the tissue of origin, and only correlated in part with molecular and genomic signatures of the heterogeneous cancer cells. The TAS-based cancer cell classification was also shown to be robust to drug response data resampling, as well as predictive of the transcriptomic patterns in an independent set of cancer cells that shared similar addiction signatures with the 107 cancers. The critical protein targets identified by the integrated approach were also shown to have clinically relevant mutation frequencies in patients with various cancer subtypes, including not only well-established pan-cancer genes, such as PTEN tumor suppressor, but also a number of targets that are less frequently mutated in specific cancer types, including ABL1 oncoprotein in acute myeloid leukemia. An application to leukemia patient primary cell models demonstrated how the target deconvolution approach offers functional insights into patient-specific addiction patterns, such as those indicative of their receptor-type tyrosine-protein kinase FLT3 internal tandem duplication (FLT3-ITD status and co-addiction partners, which may lead to clinically actionable, personalized drug treatment developments. To promote its application to the future drug testing studies, we have made available an open-source implementation of the TAS calculation in the form

  3. Expression of EPO Receptor in Pancreatic Cells and Its Effect on Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hongxia SHUAI; Ji ZHANG; Yikai YU; Muxun ZHANG


    In order to explore the expression of erythropoietin receptor (EPOR) in pancreatic cell ine NIT-1 and its effect on cell apoptosis after binding with erythropoietin (EPO), NIT-1 cells were cultured and expanded. The expression of EPOR was detected using electrophoresis. NIT-1 apoptosis was induced by cytokines and their effects on cell apoptosis and cell insulin secretion were assayed after binding of EPO to EPOR. The results showed that EPOR was expressed in NIT-1 cells. Recom- binant human EPO (rHuEPO) had no effect on cell apoptosis but significantly inhibited apoptosis in- duced by cytokines, rHuEPO had no effect on cell insulin secretion but significantly improved insulin secretion inhibited by cytokines. From these findings, it was concluded that EPOR was expressed in NIT-1 cells and EPO could protect N1T-1 cells from apoptosis induced by cytokines.

  4. Radio-sensitization of Prostate Cancer Cells by Monensin Treatment and its associated Gene Expression Profiling Changes (United States)

    Zhang Ye; Rohde, Larry H.; Wu, Honglu


    Radio-resistant or recurrent prostate cancer represents a serious health risk for approximately 20%-30% of patients treated with primary radiation therapy for clinically localized prostate cancer. Here, we investigated the effect of monensin on sensitizing radiation mediated cell killing of two radio-resistant prostate cell lines Lncap (P53+ and AR+) and PC3 (P53- and AR-). Treatment with monensin alone (5 micromoles-20 micromoles) showed a significant direct cell killing of Lncap (10-30%), but not PC3 cells. Monensin was also shown to successfully sensitize Lncap cells to X-ray radiation (2Gy-10Gy) mediated cell death, up to 50% of killing with the combined treatment. To better understand the mechanisms of radio-resistance of these two cell lines and their different response to monensin, the apoptosis related gene expression profiles in both cell lines were analyzed using cDNA PCR array. Without any treatment, PC3 showed a much higher expression level of antiapoptosis genes than Lncap in the BCL2 family, the caspase/card family and the TNF ligand/receptor family. At 2 hr after 20 micormolar monensin treatment alone, only the TRAF and CIDE family showed a greater induction in Lncap cells than in PC3. Exposures to 10 Gy X-rays alone of Lncap cells significantly induced gene expression levels in the death and death receptor domain family, the TNF ligand and receptor family, and apoptotic group of BCL2 family; whereas exposures of PC3 induced only the expression of genes in the anti-apoptosis group of CASP and CARD family. Furthermore, we selectively suppressed the expression of several anti-apoptosis genes (BCL-xl, Bcl2A1, BIRC2, BIRC3 and CASP2) in PC3 cells by using the siRNA treatment. Exposure to 10Gy X-rays alone showed an enhanced cell killing (about 15%) in BCL-x1 silenced cells, but not in cells with siRNA treatment targeting other anti-apoptosis genes. We also exposed PC3 cells to protons in the Bragg peak region to compare the effectiveness of cell killing

  5. Similar chemokine receptor profiles in lymphomas with central nervous system involvement - possible biomarkers for patient selection for central nervous system prophylaxis, a retrospective study. (United States)

    Lemma, Siria A; Pasanen, Anna Kaisa; Haapasaari, Kirsi-Maria; Sippola, Antti; Sormunen, Raija; Soini, Ylermi; Jantunen, Esa; Koivunen, Petri; Salokorpi, Niina; Bloigu, Risto; Turpeenniemi-Hujanen, Taina; Kuittinen, Outi


    Central nervous system (CNS) relapse occurs in around 5% of diffuse large B-cell lymphoma (DLBCL) cases. No biomarkers to identify high-risk patients have been discovered. We evaluated the expression of lymphocyte-guiding chemokine receptors in systemic and CNS lymphomas. Immunohistochemical staining for CXCR4, CXCR5, CCR7, CXCL12, and CXCL13 was performed on 89 tissue samples, including cases of primary central nervous system lymphoma (PCNSL), secondary CNS lymphoma (sCNSL), and systemic DLBCL. Also, 10 reactive lymph node samples were included. Immunoelectron microscopy was performed on two PCNSLs, one sCNSL, one systemic DLBCL, and one reactive lymph node samples, and staining was performed for CXCR4, CXCR5, CXCL12, and CXCL13. Chi-square test was used to determine correlations between clinical parameters, diagnostic groups, and chemokine receptor expression. Strong nuclear CXCR4 positivity correlated with systemic DLBCL, whereas strong cytoplasmic CXCR5 positivity correlated with CNS involvement (P = 0.003 and P = 0.039). Immunoelectron microscopy revealed a nuclear CXCR4 staining in reactive lymph node, compared with cytoplasmic and membranous localization seen in CNS lymphomas. We found that CNS lymphoma presented a chemokine receptor profile different from systemic disease. Our findings give new information on the CNS tropism of DLBCL and, if confirmed, may contribute to more effective targeting of CNS prophylaxis among patients with DLBCL.

  6. Differential gene expression profiling of human epidermal growth factor receptor 2-overexpressing mammary tumor

    Institute of Scientific and Technical Information of China (English)

    Yan Wang; Haining Peng; Yingli Zhong; Daiqiang Li; Mi Tang; Xiaofeng Ding; Jian Zhang


    Human epidermal growth factor receptor 2 (HER2) is highly expressed in approximately 30% of breast cancer patients,and substantial evidence supports the relationship between HER2 overexpression and poor overall survival. However,the biological function of HER2 signaltransduction pathways is not entirely clear. To investigate gene activation within the pathways, we screened differentially expressed genes in HER2-positive mouse mammary tumor using two-directional suppression subtractive hybridization combined with reverse dot-blotting analysis. Forty genes and expressed sequence tags related to transduction, cell proliferation/growth/apoptosis and secreted/extracellular matrix proteins were differentially expressed in HER2-positive mammary tumor tissue. Among these, 19 were already reported to be differentially expressed in mammary tumor, 11 were first identified to be differentially expressed in mammary tumor in this study but were already reported in other tumors, and 10 correlated with other cancers. These genes can facilitate the understanding of the role of HER2 signaling in breast cancer.

  7. Muscarinic receptors stimulate cell proliferation in the human urothelium-derived cell line UROtsa. (United States)

    Arrighi, Nicola; Bodei, Serena; Lucente, Alessandra; Michel, Martin C; Zani, Danilo; Simeone, Claudio; Cunico, Sergio Cosciani; Spano, PierFranco; Sigala, Sandra


    The widespread non-neuronal synthesis of acetylcholine (ACh) has changed the paradigm of ACh acting solely as a neurotransmitter. Indeed, the presence of ACh in many types of proliferating cells suggests a role for this neurotransmitter in the control of cell division. The parasympathetic system is a major pathway regulating micturition, but ACh-mediated control plays a more complex role than previously described, acting not only in the detrusor muscle, but also influencing detrusor function through the activity of urothelial muscarinic receptors. Here we investigated the role of muscarinic receptors in mediating cell proliferation in the human UROtsa cell line, which is a widely used experimental model to study urothelium physiology and pathophysiology. Our results demonstrate that UROtsa cells express the machinery for ACh synthesis and that muscarinic receptors, with the rank order of M3>M2>M5>M1=M4, are present and functionally linked to their known second messengers. Indeed, the cholinergic receptor agonist carbachol (CCh) (1-100 μM) concentration-dependently raised IP(3) levels, reaching 66±5% over basal. The forskolin-mediated adenylyl cyclase activation was reduced by CCh exposure (forskolin: 1.4±0.14 pmol/ml; forskolin+100 μM CCh: 0.84±0.12 pmol/ml). CCh (1-100 μM) concentration-dependently increased UROtsa cell proliferation and this effect was inhibited by the non-selective antagonist atropine and the M(3)-selective antagonists darifenacin and J104129. Finally, CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation.

  8. Identification of testosterone-/androgen receptor-regulated genes in mouse Sertoli cells

    Institute of Scientific and Technical Information of China (English)

    Qiao-Xia Zhang; Xiao-Yan Zhang; Zhen-Ming Zhang; Wei Lu; Ling Liu; Gang Li; Zhi-Ming Cai; Yao-Ting Gui; Chawnshang Chang


    Androgen and androgen receptor (AR) play important roles in male spermatogenesis and fertility,yet detailed androgenlAR signals in Sertoli cells remain unclear.To identify AR target genes in Sertoli cells,we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells (S-AR-/y) and their littermate wild-type (WT) mice.Digital gene expression analysis identified 2276 genes downregulated and 2865 genes upregulated in the S-AR-/y mice testis compared to WT ones.To further nail down the difference within Sertoli cells,we first constructed Sertoli cell line TM4 with stably transfected AR (named as TM4/AR) and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells.Interestingly,additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing 10 times more androgen sensitivity than TM4 cells.In the condition where maximal androgen response was demonstrated,we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone.Among these genes,603 androgen-/ AR-regulated genes,including 164 upregulated and 439 downregulated,were found in both S-AR-/y mice testis and TM4/AR cells.Using informatics analysis,the gene ontology was applied to analyze these androgen-/AR-regulated genes to predict the potential roles of androgen/AR in the process of spermatogenesis.Together,using gene analysis in both S-AR-/y mice testis and TM4/AR cells may help us to better understand the androgen/AR signals in Sertoli cells and their influences in spermatogenesis.

  9. Regulatory Role of a Receptor-Like Kinase in Specifying Anther Cell Identity1[OPEN (United States)

    Yang, Li; Qian, Xiaoling; Chen, Mingjiao


    In flowering plants, sequential formation of anther cell types is a highly ordered process that is essential for successful meiosis and sexual reproduction. Differentiation of meristematic cells and cell-cell communication are proposed to coordinate anther development. Among the proposed mechanisms of cell fate specification are cell surface-localized Leu-rich repeat receptor-like kinases (LRR-RLKs) and their putative ligands. Here, we present the genetic and biochemical evidence that a rice (Oryza sativa) LRR-RLK, MSP1 (MULTIPLE SPOROCYTE1), interacts with its ligand OsTDL1A (TPD1-like 1A), specifying the cell identity of anther wall layers and microsporocytes. An in vitro assay indicates that the 21-amino acid peptide of OsTDL1A has a physical interaction with the LRR domain of MSP1. The ostdl1a msp1 double mutant showed the defect in lacking middle layers and tapetal cells and having an increased number of microsporocytes similar to the ostdl1a or msp1 single mutant, indicating the same pathway of OsTDL1A-MSP1 in regulating anther development. Genome-wide expression profiles showed the altered expression of genes encoding transcription factors, particularly basic helix-loop-helix and basic leucine zipper domain transcription factors in ostdl1a and msp1. Among these reduced expressed genes, one putatively encodes a TGA (TGACGTCA cis-element-binding protein) factor OsTGA10, and another one encodes a plant-specific CC-type glutaredoxin OsGrx_I1. OsTGA10 was shown to interact with OsGrx_I1, suggesting that OsTDL1A-MSP1 signaling specifies anther cell fate directly or indirectly affecting redox status. Collectively, these data point to a central role of the OsTDL1A-MSP1 signaling pathway in specifying somatic cell identity and suppressing overproliferation of archesporial cells in rice. PMID:27208278

  10. Gene Expression Profiling of Dendritic Cells in Different Physiological Stages under Cordyceps sinensis Treatment (United States)

    Li, Chia-Yang; Chiang, Chi-Shiun; Cheng, Wei-Chung; Wang, Shu-Chi; Cheng, Hung-Tsu; Chen, Chaang-Ray; Shu, Wun-Yi; Tsai, Min-Lung; Hseu, Ruey-Shyang; Chang, Cheng-Wei; Huang, Chao-Ying; Fang, Shih-Hua; Hsu, Ian C.


    Cordyceps sinensis (CS) has been commonly used as herbal medicine and a health supplement in China for over two thousand years. Although previous studies have demonstrated that CS has benefits in immunoregulation and anti-inflammation, the precise mechanism by which CS affects immunomodulation is still unclear. In this study, we exploited duplicate sets of loop-design microarray experiments to examine two different batches of CS and analyze the effects of CS on dendritic cells (DCs), in different physiology stages: naïve stage and inflammatory stage. Immature DCs were treated with CS, lipopolysaccharide (LPS), or LPS plus CS (LPS/CS) for two days, and the gene expression profiles were examined using cDNA microarrays. The results of two loop-design microarray experiments showed good intersection rates. The expression level of common genes found in both loop-design microarray experiments was consistent, and the correlation coefficients (Rs), were higher than 0.96. Through intersection analysis of microarray results, we identified 295 intersecting significantly differentially expressed (SDE) genes of the three different treatments (CS, LPS, and LPS/CS), which participated mainly in the adjustment of immune response and the regulation of cell proliferation and death. Genes regulated uniquely by CS treatment were significantly involved in the regulation of focal adhesion pathway, ECM-receptor interaction pathway, and hematopoietic cell lineage pathway. Unique LPS regulated genes were significantly involved in the regulation of Toll-like receptor signaling pathway, systemic lupus erythematosus pathway, and complement and coagulation cascades pathway. Unique LPS/CS regulated genes were significantly involved in the regulation of oxidative phosphorylation pathway. These results could provide useful information in further study of the pharmacological mechanisms of CS. This study also demonstrates that with a rigorous experimental design, the biological effects of a complex

  11. Gene expression profiling of dendritic cells in different physiological stages under Cordyceps sinensis treatment.

    Directory of Open Access Journals (Sweden)

    Chia-Yang Li

    Full Text Available Cordyceps sinensis (CS has been commonly used as herbal medicine and a health supplement in China for over two thousand years. Although previous studies have demonstrated that CS has benefits in immunoregulation and anti-inflammation, the precise mechanism by which CS affects immunomodulation is still unclear. In this study, we exploited duplicate sets of loop-design microarray experiments to examine two different batches of CS and analyze the effects of CS on dendritic cells (DCs, in different physiology stages: naïve stage and inflammatory stage. Immature DCs were treated with CS, lipopolysaccharide (LPS, or LPS plus CS (LPS/CS for two days, and the gene expression profiles were examined using cDNA microarrays. The results of two loop-design microarray experiments showed good intersection rates. The expression level of common genes found in both loop-design microarray experiments was consistent, and the correlation coefficients (Rs, were higher than 0.96. Through intersection analysis of microarray results, we identified 295 intersecting significantly differentially expressed (SDE genes of the three different treatments (CS, LPS, and LPS/CS, which participated mainly in the adjustment of immune response and the regulation of cell proliferation and death. Genes regulated uniquely by CS treatment were significantly involved in the regulation of focal adhesion pathway, ECM-receptor interaction pathway, and hematopoietic cell lineage pathway. Unique LPS regulated genes were significantly involved in the regulation of Toll-like receptor signaling pathway, systemic lupus erythematosus pathway, and complement and coagulation cascades pathway. Unique LPS/CS regulated genes were significantly involved in the regulation of oxidative phosphorylation pathway. These results could provide useful information in further study of the pharmacological mechanisms of CS. This study also demonstrates that with a rigorous experimental design, the biological effects

  12. CD84 is a survival receptor for CLL cells. (United States)

    Binsky-Ehrenreich, I; Marom, A; Sobotta, M C; Shvidel, L; Berrebi, A; Hazan-Halevy, I; Kay, S; Aloshin, A; Sagi, I; Goldenberg, D M; Leng, L; Bucala, R; Herishanu, Y; Haran, M; Shachar, I


    Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B lymphocytes in peripheral blood, lymphoid organs and bone marrow. The main feature of the disease is accumulation of the malignant cells due to decreased apoptosis. CD84 belongs to the signaling lymphocyte activating molecule family of immunoreceptors, and has an unknown function in CLL cells. Here, we show that the expression of CD84 is significantly elevated from the early stages of the disease, and is regulated by macrophage migration inhibitory factor and its receptor, CD74. Activation of cell surface CD84 initiates a signaling cascade that enhances CLL cell survival. Both downmodulation of CD84 expression and its immune-mediated blockade induce cell death in vitro and in vivo. In addition, analysis of samples derived from an on-going clinical trial, in which human subjects were treated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84 messenger RNA and protein levels in milatuzumab-treated cells. This downregulation was correlated with reduction of Bcl-2 and Mcl-1 expression. Thus, our data show that overexpression of CD84 in CLL is an important survival mechanism that appears to be an early event in the pathogenesis of the disease. These findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

  13. Proteome Profiling in Lung Injury after Hematopoietic Stem Cell Transplantation. (United States)

    Bhargava, Maneesh; Viken, Kevin J; Dey, Sanjoy; Steinbach, Michael S; Wu, Baolin; Jagtap, Pratik D; Higgins, LeeAnn; Panoskaltsis-Mortari, Angela; Weisdorf, Daniel J; Kumar, Vipin; Arora, Mukta; Bitterman, Peter B; Ingbar, David H; Wendt, Chris H


    infectious lung injury, 96 proteins were differentially expressed. Gene ontology enrichment analysis showed that these proteins participate in biological processes involved in the development of lung injury after HSCT. These include acute phase response signaling, complement system, coagulation system, liver X receptor (LXR)/retinoid X receptor (RXR), and farsenoid X receptor (FXR)/RXR modulation. We identified 2 canonical pathways modulated by TNF-α, FXR/RXR activation, and IL2 signaling in macrophages. The proteins also mapped to blood coagulation, fibrinolysis, and wound healing-processes that participate in organ repair. Cell movement was identified as significantly over-represented by proteins with differential expression between IPS and infection. In conclusion, the BALF protein expression in IPS differed significantly from infectious lung injury in HSCT recipients. These differences provide insights into mechanisms that are activated in lung injury in HSCT recipients and suggest potential therapeutic targets to augment lung repair.

  14. Growth and by-product profiles of Kluyveromyces marxianus cells immobilized in foamed alginate. (United States)

    Wilkowska, Agnieszka; Kregiel, Dorota; Guneser, Onur; Karagul Yuceer, Yonca


    The aim of this research was to study how the yeast cell immobilization technique influences the growth and fermentation profiles of Kluyveromyces marxianus cultivated on apple/chokeberry and apple/cranberry pomaces. Encapsulation of the cells was performed by droplet formation from a foamed alginate solution. The growth and metabolic profiles were evaluated for both free and immobilized cells. Culture media with fruit waste produced good growth of free as well as immobilized yeast cells. The fermentation profiles of K. marxianus were different with each waste material. The most varied aroma profiles were noted for immobilized yeast cultivated on apple/chokeberry pomace.

  15. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor


    Harrison, Hannah; Farnie, Gillian; Howell, Sacha J.; Rock, Rebecca E; Stylianou, Spyros; Brennan, Keith R.; Bundred, Nigel J; Clarke, Robert B.


    Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA+/CD44+/CD24low. Using these breast cancer stem cell populations, we compared the activation status of Notch...

  16. 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


    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....

  17. Modeling and simulation of ion channels and action potentials in taste receptor cells

    Institute of Scientific and Technical Information of China (English)

    CHEN PeiHua; LIU Xiaodong; ZHANG Wei; ZHOU Jun; WANG Ping; YANG Wei; LUO JianHong


    Based on patch clamp data on the ionic currents of rat taste receptor cells,a mathematical model of mammalian taste receptor cells was constructed to simulate the action potentials of taste receptor cells and their corresponding ionic components,including voltage-gated Na~+ currents and outward delayed rectifier K~+ currents.Our simulations reproduced the action potentials of taste receptor cells in response to electrical stimuli or sour tastants.The kinetics of ion channels and their roles in action potentials of taste receptor cells were also analyzed.Our prototype model of single taste receptor cell and simulation results presented in this paper provide the basis for the further study of taste information processing in the gustatory system.

  18. Modeling and simulation of ion channels and action potentials in taste receptor cells

    Institute of Scientific and Technical Information of China (English)


    Based on patch clamp data on the ionic currents of rat taste receptor cells, a mathematical model of mammalian taste receptor cells was constructed to simulate the action potentials of taste receptor cells and their corresponding ionic components, including voltage-gated Na+ currents and outward delayed rectifier K+ currents. Our simulations reproduced the action potentials of taste receptor cells in response to electrical stimuli or sour tastants. The kinetics of ion channels and their roles in action potentials of taste receptor cells were also analyzed. Our prototype model of single taste receptor cell and simulation results presented in this paper provide the basis for the further study of taste information processing in the gustatory system.

  19. Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells (United States)

    He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.


    Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

  20. Neonatal diethylstilbestrol exposure alters the metabolic profile of uterine epithelial cells

    Directory of Open Access Journals (Sweden)

    Yan Yin


    Developmental exposure to diethylstilbestrol (DES causes reproductive tract malformations, affects fertility and increases the risk of clear cell carcinoma of the vagina and cervix in humans. Previous studies on a well-established mouse DES model demonstrated that it recapitulates many features of the human syndrome, yet the underlying molecular mechanism is far from clear. Using the neonatal DES mouse model, the present study uses global transcript profiling to systematically explore early gene expression changes in individual epithelial and mesenchymal compartments of the neonatal uterus. Over 900 genes show differential expression upon DES treatment in either one or both tissue layers. Interestingly, multiple components of peroxisome proliferator-activated receptor-γ (PPARγ-mediated adipogenesis and lipid metabolism, including PPARγ itself, are targets of DES in the neonatal uterus. Transmission electron microscopy and Oil-Red O staining further demonstrate a dramatic increase in lipid deposition in uterine epithelial cells upon DES exposure. Neonatal DES exposure also perturbs glucose homeostasis in the uterine epithelium. Some of these neonatal DES-induced metabolic changes appear to last into adulthood, suggesting a permanent effect of DES on energy metabolism in uterine epithelial cells. This study extends the list of biological processes that can be regulated by estrogen or DES, and provides a novel perspective for endocrine disruptor-induced reproductive abnormalities.

  1. Troglitazone inhibits cell proliferation by attenuation of epidermal growth factor receptor signaling independent of peroxisome proliferator-activated receptor γ

    Institute of Scientific and Technical Information of China (English)

    Xiaoqi Li; Xuanming Yang; Youli Xu; Xuejun Jiang; Xin Li; Fajun Nan; Hong Tang


    Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. Recent results have shown that agonists of PPARy, such as troglitazone (TGZ), can inhibit cell proliferation and promote cell differentiation independent of PPARγ. In the present study, we provide evidence that TGZ may bind directly to EGFR and trigger its signaling and internalization independent of PPARγ. Detailed studies revealed that prolonged incubation with TGZ effectively attenuated EGFR signaling by target-ing the receptor to the endo-lysosomal degradation machinery. Although the extracellular signal-regulated kinase-signaling pathway was transiently activated by TGZ in EGFR overexpressing cancer cells, inhibition of EGF-induced Akt phosphorylation most likely accounted for the growth arrest of tumor cells caused by TGZ at pharmacologically achievable concentrations. Therefore, we have provided a new line of evidence indicating that TGZ inhibits cell pro-liferation by promoting EGFR degradation and attenuating Akt phosphorylation.

  2. Ryanodine Receptors Selectively Interact with L Type Calcium Channels in Mouse Taste Cells.

    Directory of Open Access Journals (Sweden)

    Michelle R Rebello

    Full Text Available WE REPORTED THAT RYANODINE RECEPTORS ARE EXPRESSED IN TWO DIFFERENT TYPES OF MAMMALIAN PERIPHERAL TASTE RECEPTOR CELLS: Type II and Type III cells. Type II cells lack voltage-gated calcium channels (VGCCs and chemical synapses. In these cells, ryanodine receptors contribute to the taste-evoked calcium signals that are initiated by opening inositol trisphosphate receptors located on internal calcium stores. In Type III cells that do have VGCCs and chemical synapses, ryanodine receptors contribute to the depolarization-dependent calcium influx.The goal of this study was to establish if there was selectivity in the type of VGCC that is associated with the ryanodine receptor in the Type III taste cells or if the ryanodine receptor opens irrespective of the calcium channels involved. We also wished to determine if the ryanodine receptors and VGCCs require a physical linkage to interact or are simply functionally associated with each other. Using calcium imaging and pharmacological inhibitors, we found that ryanodine receptors are selectively associated with L type VGCCs but likely not through a physical linkage.Taste cells are able to undergo calcium induced calcium release through ryanodine receptors to increase the initial calcium influx signal and provide a larger calcium response than would otherwise occur when L type channels are activated in Type III taste cells.

  3. The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells. (United States)

    Wenner, Maria I; Maker, Garth L; Dawson, Linda F; Drummond, Peter D; Mullaney, Ian


    Several studies of neuropathic pain have linked abnormal adrenergic signalling to the development and maintenance of pain, although the mechanisms underlying this are not yet fully understood. Metabolomic analysis is a technique that can be used to give a snapshot of biochemical status, and can aid in the identification of the mechanisms behind pathological changes identified in cells, tissues and biological fluids. This study aimed to use gas chromatography-mass spectrometry-based metabolomic profiling in combination with reverse transcriptase-polymerase chain reaction and immunocytochemistry to identify functional α1-adrenergic receptors on cultured N1E-115 mouse neuroblastoma cells. The study was able to confirm the presence of mRNA for the α1D subtype, as well as protein expression of the α1-adrenergic receptor. Furthermore, metabolomic data revealed changes to the metabolite profile of cells when exposed to adrenergic pharmacological intervention. Agonist treatment with phenylephrine hydrochloride (10 µM) resulted in altered levels of several metabolites including myo-inositol, glucose, fructose, alanine, leucine, phenylalanine, valine, and n-acetylglutamic acid. Many of the changes observed in N1E-115 cells by agonist treatment were modulated by additional antagonist treatment (prazosin hydrochloride, 100 µM). A number of these changes reflected what is known about the biochemistry of α1-adrenergic receptor activation. This preliminary study therefore demonstrates the potential of metabolomic profiling to confirm the presence of functional receptors on cultured cells.

  4. Generalized paired-agent kinetic model for in vivo quantification of cancer cell-surface receptors under receptor saturation conditions (United States)

    Sadeghipour, N.; Davis, S. C.; Tichauer, K. M.


    New precision medicine drugs oftentimes act through binding to specific cell-surface cancer receptors, and thus their efficacy is highly dependent on the availability of those receptors and the receptor concentration per cell. Paired-agent molecular imaging can provide quantitative information on receptor status in vivo, especially in tumor tissue; however, to date, published approaches to paired-agent quantitative imaging require that only ‘trace’ levels of imaging agent exist compared to receptor concentration. This strict requirement may limit applicability, particularly in drug binding studies, which seek to report on a biological effect in response to saturating receptors with a drug moiety. To extend the regime over which paired-agent imaging may be used, this work presents a generalized simplified reference tissue model (GSRTM) for paired-agent imaging developed to approximate receptor concentration in both non-receptor-saturated and receptor-saturated conditions. Extensive simulation studies show that tumor receptor concentration estimates recovered using the GSRTM are more accurate in receptor-saturation conditions than the standard simple reference tissue model (SRTM) (% error (mean  ±  sd): GSRTM 0  ±  1 and SRTM 50  ±  1) and match the SRTM accuracy in non-saturated conditions (% error (mean  ±  sd): GSRTM 5  ±  5 and SRTM 0  ±  5). To further test the approach, GSRTM-estimated receptor concentration was compared to SRTM-estimated values extracted from tumor xenograft in vivo mouse model data. The GSRTM estimates were observed to deviate from the SRTM in tumors with low receptor saturation (which are likely in a saturated regime). Finally, a general ‘rule-of-thumb’ algorithm is presented to estimate the expected level of receptor saturation that would be achieved in a given tissue provided dose and pharmacokinetic information about the drug or imaging agent being used, and physiological

  5. Transcriptome profile of human neuroblastoma cells in the hypomagnetic field. (United States)

    Mo, WeiChuan; Liu, Ying; Bartlett, Perry F; He, RongQiao


    Research has shown that the hypomagnetic field (HMF) can affect embryo development, cell proliferation, learning and memory, and in vitro tubulin assembly. In the present study, we aimed to elucidate the molecular mechanism by which the HMF exerts its effect, by comparing the transcriptome profiles of human neuroblastoma cells exposed to either the HMF or the geomagnetic field. A total of 2464 differentially expressed genes (DEGs) were identified, 216 of which were up-regulated and 2248 of which were down-regulated after exposure to the HMF. These DEGs were found to be significantly clustered into several key processes, namely macromolecule localization, protein transport, RNA processing, and brain function. Seventeen DEGs were verified by real-time quantitative PCR, and the expression levels of nine of these DEGs were measured every 6 h. Most notably, MAPK1 and CRY2, showed significant up- and down-regulation, respectively, during the first 6 h of HMF exposure, which suggests involvement of the MAPK pathway and cryptochrome in the early bio-HMF response. Our results provide insights into the molecular mechanisms underlying the observed biological effects of the HMF.

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

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


    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.

  7. 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;


    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...

  8. In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures. (United States)

    Ng, Jia-Hui; Kumar, Vibhor; Muratani, Masafumi; Kraus, Petra; Yeo, Jia-Chi; Yaw, Lai-Ping; Xue, Kun; Lufkin, Thomas; Prabhakar, Shyam; Ng, Huck-Hui


    The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale chromatin immunoprecipitation sequencing (ChIP-seq) method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac, and H2BK20ac). Comparison of active chromatin state between somatic, embryonic stem, and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. We found the nuclear receptor Nr5a2 motif to be enriched at a subset of germ cell cis-regulatory regions, and our results implicate Nr5a2 in germ cell biology. Interestingly, in germ cells, the H3K27me3 histone modification occurs more frequently at regions that are enriched for retrotransposons and MHC genes, indicating that these loci are specifically silenced in germ cells. Together, our study provides genome-wide histone modification maps of in vivo germ cells and reveals the molecular chromatin signatures of germ cells.

  9. Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells

    DEFF Research Database (Denmark)

    Jørgensen, Claus; Sherman, Andrew; Chen, Ginny I;


    Cells have self-organizing properties that control their behavior in complex tissues. Contact between cells expressing either B-type Eph receptors or their transmembrane ephrin ligands initiates bidirectional signals that regulate cell positioning. However, simultaneously investigating how...... information is processed in two interacting cell types remains a challenge. We implemented a proteomic strategy to systematically determine cell-specific signaling networks underlying EphB2- and ephrin-B1-controlled cell sorting. Quantitative mass spectrometric analysis of mixed populations of EphB2......- and ephrin-B1-expressing cells that were labeled with different isotopes revealed cell-specific tyrosine phosphorylation events. Functional associations between these phosphotyrosine signaling networks and cell sorting were established with small interfering RNA screening. Data-driven network modeling...

  10. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells. (United States)

    Marquardt, D L; Walker, L L; Heinemann, S


    Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

  11. Targeting beta- and alpha-adrenergic receptors differentially shifts Th1, Th2, and inflammatory cytokine profiles in immune organs to attenuate adjuvant arthritis

    Directory of Open Access Journals (Sweden)

    Dianne eLorton


    Full Text Available The sympathetic nervous system (SNS regulates host defense responses and restores homeostasis. SNS-immune regulation is altered in rheumatoid arthritis (RA and rodent models of RA, characterized by nerve remodeling in immune organs and defective adrenergic receptor (AR signaling to immune cell targets that typically promotes or suppresses inflammation via α- and β2-AR activation, respectively, and indirectly drives humoral immunity by blocking Th1 cytokine secretion. Here, we investigate how β2-AR stimulation and/or α-AR blockade at disease onset affects disease pathology and cytokine profiles in relevant immune organs from male Lewis rats with adjuvant-induced arthritis (AA. Rats challenged to induce AA were treated with terbutaline (TERB, a β2-AR agonist (600 μg/kg/day and/or phentolamine (PHEN, an α-AR antagonist (5.0 mg/kg/day or vehicle from disease onset through severe disease. We report that in spleen, mesenteric (MLN and draining lymph node (DLN cells, TERB reduces proliferation, an effect independent of IL-2. TERB also fails to shift Th cytokines from a Th1 to Th2 profile in spleen and MLN (no effect on IFN-γ and DLN (greater IFN-γ cells. In splenocytes, TERB, PHEN and co-treatment (PT promotes an anti-inflammatory profile (greater IL-10 and lowers TNF-α (PT only. In DLN cells, drug treatments do not affect inflammatory profiles, except PT, which raised IL-10. In MLN cells, TERB or PHEN lowers MLN cell secretion of TNF-α or IL-10, respectively. Collectively, our findings indicate disrupted β2-AR, but not α-AR signaling in AA. Aberrant β2-AR signaling consequently derails the sympathetic regulation of lymphocyte expansion, Th cell differentiation, and inflammation in the spleen, DLNs and MLNs that is required for immune system homeostasis. Importantly, this study provides potential mechanisms through which reestablished balance between α- and β2-AR function in the immune system ameliorates inflammation and joint

  12. Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Simon A., E-mail: [Molecular Pharmacology Laboratory, School of Pharmacy, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA (Australia); Richards, Alex K.; Kusumah, Ivonne; Perumal, Vanathi [Molecular Pharmacology Laboratory, School of Pharmacy, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA (Australia); Bolitho, Erin M. [Western Australian Institute for Medical Research, The University of Western Australia Centre for Medical Research, Perth, WA (Australia); Mutsaers, Steven E. [Lung Institute of Western Australia, Centre for Asthma Allergy and Respiratory Research, University of Western Australia, Nedlands (Australia); Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, University of Western Australia and Western Australian Institute for Medical Research, Nedlands (Australia); Dharmarajan, Arun M. [School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA (Australia)


    Highlights: •Expression profile of Wnt pathway related genes in mesothelioma cells. •Differential expression of key Wnt pathway molecules and regulators. •Wnt3a stimulated mesothelioma growth whereas sFRP4 was inhibitory. •Targeting β-Catenin can sensitise mesothelioma cells to cytotoxic drugs. -- Abstract: Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.

  13. Estrogen receptors regulate innate immune cells and signaling pathways. (United States)

    Kovats, Susan


    Humans show strong sex differences in immunity to infection and autoimmunity, suggesting sex hormones modulate immune responses. Indeed, receptors for estrogens (ERs) regulate cells and pathways in the innate and adaptive immune system, as well as immune cell development. ERs are ligand-dependent transcription factors that mediate long-range chromatin interactions and form complexes at gene regulatory elements, thus promoting epigenetic changes and transcription. ERs also participate in membrane-initiated steroid signaling to generate rapid responses. Estradiol and ER activity show profound dose- and context-dependent effects on innate immune signaling pathways and myeloid cell development. While estradiol most often promotes the production of type I interferon, innate pathways leading to pro-inflammatory cytokine production may be enhanced or dampened by ER activity. Regulation of innate immune cells and signaling by ERs may contribute to the reported sex differences in innate immune pathways. Here we review the recent literature and highlight several molecular mechanisms by which ERs regulate the development or functional responses of innate immune cells.

  14. Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate. (United States)

    Montiel, F; Ortiz-Caro, J; Villa, A; Pascual, A; Aranda, A


    The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells. PMID:2930502

  15. Profile of Steroid Receptors and Increased Aromatase Immunoexpression in Canine Inflammatory Mammary Cancer as a Potential Therapeutic Target. (United States)

    De Andrés, P J; Cáceres, S; Clemente, M; Pérez-Alenza, M D; Illera, J C; Peña, L


    Canine inflammatory mammary cancer (IMC) has been proposed as a model for the study of human inflammatory breast cancer (IBC). The aims of this study were to compare the immunohistochemical expression of aromatase (Arom) and several hormone receptors [estrogen receptor α (ERα), estrogen receptor β (ERβ), progesterone receptor (PR) and androgen receptor (AR)], in 21 IMC cases vs 19 non-IMC; and to study the possible effect of letrozole on canine IMC and human inflammatory breast cancer (IBC) in vitro using IPC-366 and SUM-149 cell lines. Significant elevations of the means of Arom Total Score (TS), ERβ TS and PR TS were found in the IMC group (p = 0.025, p = 0.038 and p = 0.037, respectively). Secondary IMC tumours expressed higher levels of Arom than primary IMC (p = 0.029). Non-IMC PR- tumours contained higher levels of Arom than non-IMC PR+ tumours (p = 0.007). After the addition of letrozole, the number of IMC and IBC cells dropped drastically. The overexpression of Arom found and the results obtained in vitro further support canine IMC as a model for the study of IBC and future approaches to the treatment of dogs with mammary cancer, and especially IMC, using Arom inhibitors.

  16. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing, E-mail: [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Yang, Qifeng, E-mail: [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Haffty, Bruce G., E-mail: [Department of Radiation Oncology, UMDNJ-Robert Wood Johnson School of Medicine, Cancer Institute of New Jersey, NB (United States); Li, Xiaoyan, E-mail: [Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Moran, Meena S., E-mail: [Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT (United States)


    Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT

  17. Gender differences in the T-cell profiles of the airways in COPD patients associated with clinical phenotypes (United States)

    Forsslund, Helena; Yang, Mingxing; Mikko, Mikael; Karimi, Reza; Nyrén, Sven; Engvall, Benita; Grunewald, Johan; Merikallio, Heta; Kaarteenaho, Riitta; Wahlström, Jan; Wheelock, Åsa M; Sköld, C Magnus


    T lymphocytes are believed to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). How T cells are recruited to the lungs and contribute to the inflammatory process is largely unknown. COPD is a heterogeneous disease, and discriminating disease phenotypes based on distinct molecular and cellular pathways may provide new approaches for individualized diagnosis and therapies. Bronchoalveolar lavage (BAL) and blood samples were obtained from 40 never-smokers, 40 smokers with normal lung function, and 38 COPD patients. T-cell chemokine receptor expression was analyzed with flow cytometry, and soluble BAL cytokines and chemokines were measured using a cytokine multiplex assay. Correlations with gender and clinical characteristics including lung imaging were investigated using multivariate modeling. Th1/Tc1- and Th2/Tc2-associated soluble analytes and T-cell chemokine receptors were analyzed as cumulative Th1/Tc1 and Th2/Tc2 immune responses. A higher expression of chemokine receptor CCR5 on CD8+ T cells in BAL and higher percentage of CXCR3+CD8+ T cells in blood was found in female smokers with COPD compared to those without COPD. CCR5 expression on CD4+ and CD8+ T cells was lower in BAL from male smokers with COPD compared to those without COPD. Among female smokers with COPD, Th1/Tc1 immune response was linked to BAL macrophage numbers and goblet cell density, and Th2/Tc2 response was associated with the measures of emphysema on high-resolution computed tomography. The highly gender-dependent T-cell profile in COPD indicates different links between cellular events and clinical manifestations in females compared to males. Our findings may reveal mechanisms of importance for the difference in clinical course in female COPD patients compared to males. PMID:28053515

  18. Differential S1P Receptor Profiles on M1- and M2-Polarized Macrophages Affect Macrophage Cytokine Production and Migration (United States)

    Müller, Jan; von Bernstorff, Wolfram; Heidecke, Claus-Dieter


    Introduction. Macrophages are key players in complex biological processes. In response to environmental signals, macrophages undergo polarization towards a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that acts via 5 G-protein coupled receptors (S1P1–5) in order to influence a broad spectrum of biological processes. This study assesses S1P receptor expression on macrophages before and after M1 and M2 polarization and performs a comparative analysis of S1P signalling in the two activational states of macrophages. Methods. Bone marrow derived macrophages (BMDM) from C57 BL/6 mice were cultured under either M1- or M2-polarizing conditions. S1P-receptor expression was determined by quantitative RT-PCR. Influence of S1P on macrophage activation, migration, phagocytosis, and cytokine secretion was assessed in vitro. Results. All 5 S1P receptor subclasses were expressed in macrophages. Culture under both M1- and M2-polarizing conditions led to significant downregulation of S1P1. In contrast, M1-polarized macrophages significantly downregulated S1P4. The expression of the remaining three S1P receptors did not change. S1P increased expression of iNOS under M2-polarizing conditions. Furthermore, S1P induced chemotaxis in M1 macrophages and changed cytokine production in M2 macrophages. Phagocytosis was not affected by S1P-signalling. Discussion. The expression of different specific S1P receptor profiles may provide a possibility to selectively influence M1- or M2-polarized macrophages.

  19. p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma (United States)

    Sánchez-Prieto, Ricardo; Saada, Sofiane; Naves, Thomas; Guillaudeau, Angélique; Perraud, Aurélie; Sindou, Philippe; Lacroix, Aurélie; Descazeaud, Aurélien; Lalloué, Fabrice; Jauberteau, Marie-Odile


    p75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCC lines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking anti-p75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together, these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC. PMID:27120782

  20. Gene expression profile correlates with T cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy (United States)

    Prins, Robert M.; Soto, Horacio; Konkankit, Vera; Odesa, Sylvia K.; Eskin, Ascia; Yong, William H.; Nelson, Stanley F.; Liau, Linda M.


    Purpose To assess the feasibility, safety, and toxicity of autologous tumor lysate-pulsed dendritic cell (DC) vaccination and toll-like receptor (TLR) agonists in patients with newly diagnosed and recurrent glioblastoma. Clinical and immune responses were monitored and correlated with tumor gene expression profiles. Experimental Design Twenty-three patients with glioblastoma (WHO grade IV) were enrolled in this dose-escalation study and received three biweekly injections of glioma lysate-pulsed DCs followed by booster vaccinations with either imiquimod or poly-ICLC adjuvant every three months until tumor progression. Gene expression profiling, IHC, FACS, and cytokine bead arrays were performed on patient tumors and PBMC. Results DC vaccinations are safe and not associated with any dose-limiting toxicity. The median overall survival from the time of initial surgical diagnosis of glioblastoma was 31.4 months, with a one-, two-, and three-year survival rate of 91%, 55% and 47%, respectively. Patients whose tumors had mesenchymal gene expression signatures exhibited increased survival following DC vaccination compared to historical controls of the same genetic subtype. Tumor samples with a mesenchymal gene expression signature had a higher number of CD3+ and CD8+ tumor infiltrating lymphocytes (TILs) compared with glioblastomas of other gene expression signatures (p = 0.006). Conclusion Autologous tumor lysate-pulsed DC vaccination in conjunction with TLR agonists is safe as adjuvant therapy in newly diagnosed and recurrent glioblastoma patients. Our results suggest that the mesenchymal gene expression profile may identify an immunogenic subgroup of glioblastoma that may be more responsive to immune-based therapies. PMID:21135147

  1. 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


    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...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  2. Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

    DEFF Research Database (Denmark)

    Jacobsen, Stine Engesgaard; Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie;


    receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort...... of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A...

  3. 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


    MII gene causes the intracellular CaM level to decrease by 60%. CaMII-/- cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon......Calmodulin (CaM) is the main intracellular Ca2+ sensor protein responsible for mediating Ca2+ triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the Ca...... 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....

  4. [Research Progress on Expression and Function of P2 Purinergic Receptor in Blood Cells]. (United States)

    Feng, Wen-Li; Wang, Li-Na; Zheng, Guo-Guang


    Nucleotides have unambiguously emerged as a family of mediators of intercellular communication, which bind a class of plasma membrane receptors, P2 purinergic receptors, to trigger intercellular signaling. P2 receptors can be further divided into two structurally and functionally different sub-famlies, the P2X and P2Y receptors. Different blood cells express diverse spectrum of P2 receptors at different levels. Extracellular adenosine triphosphate (ATP) exerts different effects on blood cells, regulating cell proliferation, differentiation, migration, chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species. The relationship between abnormal P2 receptors and human diseases attracts more and more attention. This review briefly discusses the expression and function of P2 receptors in hematopoietic system.

  5. Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles (United States)

    Agrawal, Rahul; Dale, Tina P.; Al-Zubaidi, Mohammed A.; Benny Malgulwar, Prit; Forsyth, Nicholas R.; Kulshreshtha, Ritu


    MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O2) in hESCs and hMSCs, respectively, with a negligible overlap of only three miRNAs. We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology. PMID:27783707

  6. Targeting Gallium to Cancer Cells through the Folate Receptor

    Directory of Open Access Journals (Sweden)

    Nerissa Viola-Villegas


    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.

  7. The mu1, mu2, delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine

    DEFF Research Database (Denmark)

    Kristensen, K; Christensen, C B; Christrup, Lona Louring


    The binding affinities of racemic methadone and its optical isomers R-methadone and S-methadone were evaluated for the opioid receptors mu1, mu2, delta and kappa, in comparison with that of morphine. The analgesic R-methadone had a 10-fold higher affinity for mu1 receptors than S-methadone (IC50 3.......0 nM and 26.4 nM, respectively). At the mu2 receptor, the IC50 value of R-methadone was 6.9 nM and 88 nM for S-methadone, respectively. As expected, R-methadone had twice the affinity for mu1 and mu2 receptors than the racemate. All of the compounds tested had low affinity for the delta and kappa...

  8. Distinct chemokine receptor and cytokine expression profile in secondary progressive MS

    DEFF Research Database (Denmark)

    Sørensen, Torben Lykke; Sellebjerg, F


    Chemokines, small chemotactic cytokines, have been implicated in active relapsing-remitting MS (RRMS). However, the role of chemokines and chemokine receptors has not been specifically studied in secondary progressive MS (SPMS)....

  9. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Akiyoshi Uezumi


    Full Text Available Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.

  10. 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


    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

  11. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes


    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A; Sutter, Thomas R.


    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell diff...

  12. 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.

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

    Deniger, Drew C.; Yu, Jianqiang; Huls, M. Helen; Figliola, Matthew J.; Mi, Tiejuan; Maiti, Sourindra N.; Widhopf, George F.; Hurton, Lenka V.; Thokala, Radhika; Singh, Harjeet; Olivares, Simon; Champlin, Richard E.; Wierda, William G.; Kipps, Thomas J.; Cooper, Laurence J. N.


    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. PMID:26030772

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

    Directory of Open Access Journals (Sweden)

    Ahmed Nessar


    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.

  15. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

    Institute of Scientific and Technical Information of China (English)

    ZOU Hong-yun; MA Li; MENG Min-jie; YAO Xin-sheng; LIN Ying; WU Zhen-qiang; HE Xiao-wei; WANG Ju-fang; WANG Xiao-ning


    Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor (TCR) gene recombination.Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVβ chain was examined by the TCR GeneScan technique.Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ25' and Dβ 23' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation.Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire. However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  16. MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Shuqiang Li

    Full Text Available Chronic lymphocytic leukemia (CLL is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+ and IgV(H unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

  17. Activation by SLAM Family Receptors Contributes to NK Cell Mediated "Missing-Self" Recognition. (United States)

    Alari-Pahissa, Elisenda; Grandclément, Camille; Jeevan-Raj, Beena; Leclercq, Georges; Veillette, André; Held, Werner


    Natural Killer (NK) cells attack normal hematopoietic cells that do not express inhibitory MHC class I (MHC-I) molecules, but the ligands that activate NK cells remain incompletely defined. Here we show that the expression of the Signaling Lymphocyte Activation Molecule (SLAM) family members CD48 and Ly9 (CD229) by MHC-I-deficient tumor cells significantly contributes to NK cell activation. When NK cells develop in the presence of T cells or B cells that lack inhibitory MHC-I but express activating CD48 and Ly9 ligands, the NK cells' ability to respond to MHC-I-deficient tumor cells is severely compromised. In this situation, NK cells express normal levels of the corresponding activation receptors 2B4 (CD244) and Ly9 but these receptors are non-functional. This provides a partial explanation for the tolerance of NK cells to MHC-I-deficient cells in vivo. Activating signaling via 2B4 is restored when MHC-I-deficient T cells are removed, indicating that interactions with MHC-I-deficient T cells dominantly, but not permanently, impair the function of the 2B4 NK cell activation receptor. These data identify an important role of SLAM family receptors for NK cell mediated "missing-self" reactivity and suggest that NK cell tolerance in MHC-I mosaic mice is in part explained by an acquired dysfunction of SLAM family receptors.

  18. Atypical nuclear localization of VIP receptors in glioma cell lines and patients

    Energy Technology Data Exchange (ETDEWEB)

    Barbarin, Alice; Séité, Paule [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Godet, Julie [Laboratoire d’anatomie et de cytologie pathologiques, CHU de Poitiers, 2 rue de la Milétrie, 86000 Poitiers (France); Bensalma, Souheyla; Muller, Jean-Marc [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Chadéneau, Corinne, E-mail: [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France)


    Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

  19. Effects of peripheral-type benzodiazepine receptor ligands on Ehrlich tumor cell proliferation. (United States)

    Sakai, Mônica; Fonseca, Evelise Souza Monteiro; Oloris, Silvia Catarina Salgado; Matsuzaki, Patrícia; Otake, Andréia Hanada; Leite, Kátia Ramos Moura; Massoco, Cristina Oliveira; Dagli, Maria Lúcia Zaidan; Palermo-Neto, João


    Peripheral-type benzodiazepine receptors have been found throughout the body, and particularly, in high numbers, in neoplastic tissues such as the ovary, liver, colon, breast, prostate and brain cancer. Peripheral-type benzodiazepine receptor expression has been associated with tumor malignity, and its subcellular localization is important to define its function in tumor cells. We investigated the presence of peripheral-type benzodiazepine receptors in Ehrlich tumor cells, and the in vitro effects of peripheral-type benzodiazepine receptors ligands on tumor cell proliferation. Our results demonstrate the presence of peripheral-type benzodiazepine receptor in the nucleus of Ehrlich tumor cells (85.53+/-12.60%). They also show that diazepam and Ro5-4864 (peripheral-type benzodiazepine receptor agonists) but not clonazepam (a molecule with low affinity for the peripheral-type benzodiazepine receptor) decreased the percentage of tumor cells in G0-G1 phases and increased that of cells in S-G2-M phases. The effects of those agonists were prevented by PK11195 (a peripheral-type benzodiazepine receptor antagonist) that did not produce effects by itself. Altogether, these data suggest that the presence of peripheral-type benzodiazepine receptor within the nucleus of Ehrlich tumor cells is associated with tumor malignity and proliferation capacity.

  20. Epigenetic control of the basal-like gene expression profile via Interleukin-6 in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Mitrugno Valentina


    Full Text Available Abstract Background Basal-like carcinoma are aggressive breast cancers that frequently carry p53 inactivating mutations, lack estrogen receptor-α (ERα and express the cancer stem cell markers CD133 and CD44. These tumors also over-express Interleukin 6 (IL-6, a pro-inflammatory cytokine that stimulates the growth of breast cancer stem/progenitor cells. Results Here we show that p53 deficiency in breast cancer cells induces a loss of methylation at IL-6 proximal promoter region, which is maintained by an IL-6 autocrine loop. IL-6 also elicits the loss of methylation at the CD133 promoter region 1 and of CD44 proximal promoter, enhancing CD133 and CD44 gene transcription. In parallel, IL-6 induces the methylation of estrogen receptor (ERα promoter and the loss of ERα mRNA expression. Finally, IL-6 induces the methylation of IL-6 distal promoter and of CD133 promoter region 2, which harbour putative repressor regions. Conclusion We conclude that IL-6, whose methylation-dependent autocrine loop is triggered by the inactivation of p53, induces an epigenetic reprogramming that drives breast carcinoma cells towards a basal-like/stem cell-like gene expression profile.

  1. Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling. (United States)

    Börner, Christine; Smida, Michal; Höllt, Volker; Schraven, Burkhart; Kraus, Jürgen


    The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

  2. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire (United States)

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan


    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  3. A Computational Study of the Effects of Syk Activity on B Cell Receptor Signaling Dynamics

    Directory of Open Access Journals (Sweden)

    Reginald L. McGee


    Full Text Available The kinase Syk is intricately involved in early signaling events in B cells and isrequired for proper response when antigens bind to B cell receptors (BCRs. Experimentsusing an analog-sensitive version of Syk (Syk-AQL have better elucidated its role, buthave not completely characterized its behavior. We present a computational model for BCRsignaling, using dynamical systems, which incorporates both wild-type Syk and Syk-AQL.Following the use of sensitivity analysis to identify significant reaction parameters, we screenfor parameter vectors that produced graded responses to BCR stimulation as is observedexperimentally. We demonstrate qualitative agreement between the model and dose responsedata for both mutant and wild-type kinases. Analysis of our model suggests that the level of NF-KB activation, which is reduced in Syk-AQL cells relative to wild-type, is more sensitiveto small reductions in kinase activity than Erkp activation, which is essentially unchanged.Since this profile of high Erkp and reduced NF-KB is consistent with anergy, this implies thatanergy is particularly sensitive to small changes in catalytic activity. Also, under a range offorward and reverse ligand binding rates, our model of Erkp and NF-KB activation displaysa dependence on a power law affinity: the ratio of the forward rate to a non-unit power of thereverse rate. This dependence implies that B cells may respond to certain details of bindingand unbinding rates for ligands rather than simple affinity alone.

  4. The association of killer cell immunoglobulin like receptor gene polylmorphism with cytomegalovirus infection after hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)



    Objective To explore the influence of the killer cell immunoglobulin like receptor(KIR)gene polymorphism on cytomegalovirus(CMV)infection and pathogenesis after hematopoietic stem cell transplantation(HSCT)

  5. NKG2D functions as an activating receptor on natural killer cells in the common marmoset (Callithrix jacchus). (United States)

    Watanabe, Masamichi; Kudo, Yohei; Kawano, Mitsuko; Nakayama, Masafumi; Nakamura, Kyohei; Kameda, Mai; Ebara, Masamune; Sato, Takeki; Nakamura, Marina; Omine, Kaito; Kametani, Yoshie; Suzuki, Ryuji; Ogasawara, Kouetsu


    The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.

  6. Mas receptor deficiency is associated with worsening of lipid profile and severe hepatic steatosis in ApoE-knockout mice. (United States)

    Silva, Analina R; Aguilar, Edenil C; Alvarez-Leite, Jacqueline I; da Silva, Rafaela F; Arantes, Rosa M E; Bader, Michael; Alenina, Natalia; Pelli, Graziano; Lenglet, Sébastien; Galan, Katia; Montecucco, Fabrizio; Mach, François; Santos, Sérgio H S; Santos, Robson A S


    The classical renin-angiotensin system pathway has been recently updated with the identification of additional molecules [such as angiotensin converting enzyme 2, ANG-(1-7), and Mas receptor] that might improve some pathophysiological processes in chronic inflammatory diseases. In the present study, we focused on the potential protective role of Mas receptor activation on mouse lipid profile, liver steatosis, and atherogenesis. Mas/apolipoprotein E (ApoE)-double-knockout (DKO) mice (based on C57BL/6 strain of 20 wk of age) were fed under normal diet and compared with aged-matched Mas and ApoE-single-knockout (KO), as well as wild-type mice. Mas/ApoE double deficiency was associated with increased serum levels of atherogenic fractions of cholesterol, triglycerides, and fasting glucose compared with wild-type or single KO. Serum levels of HDL or leptin in DKO were lower than in other groups. Hepatic lipid content as well as alanine aminotransferase serum levels were increased in DKO compared with wild-type or single-KO animals. Accordingly, the hepatic protein content of mediators related to atherosclerotic inflammation, such as peroxisome proliferator-activated receptor-α and liver X receptor, was altered in an adverse way in DKO compared with ApoE-KO. On the other hand, DKO mice did not display increased atherogenesis and intraplaque inflammation compared with ApoE-KO group. In conclusion, Mas deletion in ApoE-KO mice was associated with development of severe liver steatosis and dyslipidemia without affecting concomitant atherosclerosis. Mas receptor activation might represent promising strategies for future treatments targeting both hepatic and metabolic alterations in chronic conditions clustering these disorders.

  7. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns. (United States)

    Bengsch, Bertram; Ohtani, Takuya; Herati, Ramin Sedaghat; Bovenschen, Niels; Chang, Kyong-Mi; Wherry, E John


    The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56(hi) versus CD56(dim) defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation

  8. Variant B Cell Receptor Isotype Functions Differ in Hairy Cell Leukemia with Mutated BRAF and IGHV Genes

    NARCIS (Netherlands)

    Weston-Bell, Nicola J.; Forconi, Francesco; Kluin-Nelemans, Hanneke C.; Sahota, Surinder S.


    A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes

  9. 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.

  10. Glyphosate induces human breast cancer cells growth via estrogen receptors. (United States)

    Thongprakaisang, Siriporn; Thiantanawat, Apinya; Rangkadilok, Nuchanart; Suriyo, Tawit; Satayavivad, Jutamaad


    Glyphosate is an active ingredient of the most widely used herbicide and it is believed to be less toxic than other pesticides. However, several recent studies showed its potential adverse health effects to humans as it may be an endocrine disruptor. This study focuses on the effects of pure glyphosate on estrogen receptors (ERs) mediated transcriptional activity and their expressions. Glyphosate exerted proliferative effects only in human hormone-dependent breast cancer, T47D cells, but not in hormone-independent breast cancer, MDA-MB231 cells, at 10⁻¹² to 10⁻⁶M in estrogen withdrawal condition. The proliferative concentrations of glyphosate that induced the activation of estrogen response element (ERE) transcription activity were 5-13 fold of control in T47D-KBluc cells and this activation was inhibited by an estrogen antagonist, ICI 182780, indicating that the estrogenic activity of glyphosate was mediated via ERs. Furthermore, glyphosate also altered both ERα and β expression. These results indicated that low and environmentally relevant concentrations of glyphosate possessed estrogenic activity. Glyphosate-based herbicides are widely used for soybean cultivation, and our results also found that there was an additive estrogenic effect between glyphosate and genistein, a phytoestrogen in soybeans. However, these additive effects of glyphosate contamination in soybeans need further animal study.

  11. Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling. (United States)

    Ahsan, Nagib; Salomon, Arthur R


    TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.

  12. CTLA4 blockade broadens the peripheral T cell receptor repertoire (United States)

    Robert, Lidia; Tsoi, Jennifer; Wang, Xiaoyan; Emerson, Ryan; Homet, Blanca; Chodon, Thinle; Mok, Stephen; Huang, Rong Rong; Cochran, Alistair J.; Comin-Anduix, Begonya; Koya, Richard C.; Graeber, Thomas G.; Robins, Harlan; Ribas, Antoni


    Purpose To evaluate the immunomodulatory effects of CTLA-4 blockade with tremelimumab in peripheral blood mononuclear cells (PBMC). Experimental Design We used next generation sequencing to study the complementarity determining region 3 (CDR3) from the rearranged T cell receptor (TCR) variable beta (V-beta) in PBMC of 21 patients, at baseline and 30–60 days after receiving tremelimumab. Results After receiving tremelimumab there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (p=0.01) and for Shannon index diversity (p=0.04). In comparison, serially collected PBMC from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over one year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared to patients without toxicity (p=0.05). No relevant differences were noted between clinical responders and non-responders. Conclusions CTLA4 blockade with tremelimumab diversifies the peripheral T cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system. PMID:24583799

  13. Gene expression profiling analysis of bisphenol A-induced perturbation in biological processes in ER-negative HEK293 cells. (United States)

    Yin, Rong; Gu, Liang; Li, Min; Jiang, Cizhong; Cao, Tongcheng; Zhang, Xiaobai


    Bisphenol A (BPA) is an environmental endocrine disruptor which has been detected in human bodies. Many studies have implied that BPA exposure is harmful to human health. Previous studies mainly focused on BPA effects on estrogen receptor (ER)-positive cells. Genome-wide impacts of BPA on gene expression in ER-negative cells is unclear. In this study, we performed RNA-seq to characterize BPA-induced cellular and molecular impacts on ER-negative HEK293 cells. The microscopic observation showed that low-dose BPA exposure did not affect cell viability and morphology. Gene expression profiling analysis identified a list of differentially expressed genes in response to BPA exposure in HEK293 cells. These genes were involved in variable important biological processes including ion transport, cysteine metabolic process, apoptosis, DNA damage repair, etc. Notably, BPA up-regulated the expression of ERCC5 encoding a DNA endonuclease for nucleotide-excision repair. Further electrochemical experiment showed that BPA induced significant DNA damage in ER-positive MCF-7 cells but not in ER-negative HEK293 cells. Collectively, our study revealed that ER-negative HEK293 cells employed mechanisms in response to BPA exposure different from ER-positive cells.

  14. Gene expression profiling analysis of bisphenol A-induced perturbation in biological processes in ER-negative HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Rong Yin

    Full Text Available Bisphenol A (BPA is an environmental endocrine disruptor which has been detected in human bodies. Many studies have implied that BPA exposure is harmful to human health. Previous studies mainly focused on BPA effects on estrogen receptor (ER-positive cells. Genome-wide impacts of BPA on gene expression in ER-negative cells is unclear. In this study, we performed RNA-seq to characterize BPA-induced cellular and molecular impacts on ER-negative HEK293 cells. The microscopic observation showed that low-dose BPA exposure did not affect cell viability and morphology. Gene expression profiling analysis identified a list of differentially expressed genes in response to BPA exposure in HEK293 cells. These genes were involved in variable important biological processes including ion transport, cysteine metabolic process, apoptosis, DNA damage repair, etc. Notably, BPA up-regulated the expression of ERCC5 encoding a DNA endonuclease for nucleotide-excision repair. Further electrochemical experiment showed that BPA induced significant DNA damage in ER-positive MCF-7 cells but not in ER-negative HEK293 cells. Collectively, our study revealed that ER-negative HEK293 cells employed mechanisms in response to BPA exposure different from ER-positive cells.

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

    DEFF Research Database (Denmark)

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


    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...

  16. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG. (United States)

    Kyakumoto, S; Kurokawa, R; Ota, M


    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  17. Omeprazole and lansoprazole enantiomers induce CYP3A4 in human hepatocytes and cell lines via glucocorticoid receptor and pregnane X receptor axis. (United States)

    Novotna, Aneta; Dvorak, Zdenek


    Benzimidazole drugs lansoprazole and omeprazole are used for treatment of various gastrointestinal pathologies. Both compounds cause drug-drug interactions because they activate aryl hydrocarbon receptor and induce CYP1A genes. In the current paper, we examined the effects of lansoprazole and omeprazole enantiomers on the expression of key drug-metabolizing enzyme CYP3A4 in human hepatocytes and human cancer cell lines. Lansoprazole enantiomers, but not omeprazole, were equipotent inducers of CYP3A4 mRNA in HepG2 cells. All forms (S-, R-, rac-) of lansoprazole and omeprazole induced CYP3A4 mRNA and protein in human hepatocytes. The quantitative profiles of CYP3A4 induction by individual forms of lansoprazole and omeprazole exerted enantiospecific patterns. Lansoprazole dose-dependently activated pregnane X receptor PXR in gene reporter assays, and slightly modulated rifampicin-inducible PXR activity, with similar potency for each enantiomer. Omeprazole dose-dependently activated PXR and inhibited rifampicin-inducible PXR activity. The effects of S-omeprazole were much stronger as compared to those of R-omeprazole. All forms of lansoprazole, but not omeprazole, slightly activated glucocorticoid receptor and augmented dexamethasone-induced GR transcriptional activity. Omeprazole and lansoprazole influenced basal and ligand inducible expression of tyrosine aminotransferase, a GR-target gene, in HepG2 cells and human hepatocytes. Overall, we demonstrate here that omeprazole and lansoprazole enantiomers induce CYP3A4 in HepG2 cells and human hepatocytes. The induction comprises differential interactions of omeprazole and lansoprazole with transcriptional regulators PXR and GR, and some of the effects were enantiospecific. The data presented here might be of toxicological and clinical importance, since the effects occurred in therapeutically relevant concentrations.

  18. HIV-1 infection is associated with changes in nuclear receptor transcriptome, pro-inflammatory and lipid profile of monocytes

    Directory of Open Access Journals (Sweden)

    Renga Barbara


    Full Text Available Abstract Background Persistent residual immune activation and lipid dysmetabolism are characteristics of HIV positive patients receiving an highly active antiretroviral therapy (HAART. Nuclear Receptors are transcription factors involved in the regulation of immune and metabolic functions through the modulation of gene transcription. The objective of the present study was to investigate for the relative abundance of members of the nuclear receptor family in monocytic cells isolated from HIV positive patients treated or not treated with HAART. Methods Monocytes isolated from peripheral blood mononuclear cells (PBMC were used for analysis of the relative mRNA expressions of FXR, PXR, LXR, VDR, RARα, RXR, PPARα, PPARβ, PPARγ and GR by Real-Time polymerase chain reaction (PCR. The expression of a selected subset of inflammatory and metabolic genes MCP-1, ICAM-1, CD36 and ABCA1 was also measured. Results Monocytes isolated from HIV infected patients expressed an altered pattern of nuclear receptors characterized by a profound reduction in the expressions of FXR, PXR, PPARα, GR, RARα and RXR. Of interest, the deregulated expression of nuclear receptors was not restored under HAART and was linked to an altered expression of genes which supports both an immune activation and altered lipid metabolism in monocytes. Conclusions Altered expression of genes mediating reciprocal regulation of lipid metabolism and immune function in monocytes occurs in HIV. The present findings provide a mechanistic explanation for immune activation and lipid dysmetabolism occurring in HIV infected patients and could lead to the identification of novel potential therapeutic targets.

  19. NJK14013, a novel synthetic estrogen receptor-α agonist, exhibits estrogen receptor-independent, tumor cell-specific cytotoxicity. (United States)

    Kim, Hye-In; Kim, Taelim; Kim, Ji-Eun; Lee, Jun; Heo, Jinyuk; Lee, Na-Rae; Kim, Nam-Jung; Inn, Kyung-Soo


    Estrogens act through interactions with estrogen receptors (ERs) to play diverse roles in various pathophysiological conditions. A number of synthetic selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, have been developed and used to treat ER-related diseases, including breast cancer and osteoporosis. Here, we identified a novel compound, bis(4-hydroxyphenyl)methanone-O-isopentyl oxime, designated NJK14013, as an ER agonist. NJK14013 activated ER-dependent transcription in a concentration-dependent manner, while suppressing androgen receptor-dependent transcriptional activity. It induced the activation-related phosphorylation of ER and enhanced the transcription of growth regulation by estrogen in breast cancer 1 (GREB1), further supporting its ER-stimulating activity. NJK14013 exerted anti-proliferative effects on various cancer cell lines, including an ER-negative breast cancer cell line, suggesting that it is capable of suppressing the growth of cancer cells independent of its ER-modulating activity. In addition, NJK14013 treatment resulted in significant apoptotic death of MCF7 and Ishikawa cancer cells, but did not induce apoptosis in non-cancer human umbilical vein endothelial cells. Collectively, our findings demonstrate that NJK14013 is a novel SERM that can activate ER-mediated transcription in MCF7 cells and suppress the proliferation of various cancer cells, including breast cancer cells and endometrial cancer cells. These results suggest that NJK14013 has potential as a novel SERM for anticancer or hormone-replacement therapy with reduced risk of carcinogenesis.

  20. A Coevolutionary Arms Race between Hosts and Viruses Drives Polymorphism and Polygenicity of NK Cell Receptors

    NARCIS (Netherlands)

    Carrillo-Bustamante, Paola; Kesmir, C.; de Boer, Rob J


    Natural killer cell receptors (NKRs) monitor the expression of major histocompatibility class I (MHC-I) and stress molecules to detect unhealthy tissue, such as infected or tumor cells. The NKR gene family shows a remarkable genetic diversity, containing several genes encoding receptors with activat

  1. 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;


    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...

  2. 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;


    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....

  3. Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays (Communities of Practice) (United States)

    Understanding potential health risks is a significant challenge for large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study analyzes chemical-target activity profiles of 976 chemicals (including failed pharmaceuticals...

  4. The A3 adenosine receptor attenuates the calcium rise triggered by NMDA receptors in retinal ganglion cells. (United States)

    Zhang, Mei; Hu, Huiling; Zhang, Xiulan; Lu, Wennan; Lim, Jason; Eysteinsson, Thor; Jacobson, Kenneth A; Laties, Alan M; Mitchell, Claire H


    The A(3) adenosine receptor is emerging as an important regulator of neuronal signaling, and in some situations receptor stimulation can limit excitability. As the NMDA receptor frequently contributes to neuronal excitability, this study examined whether A(3) receptor activation could alter the calcium rise accompanying NMDA receptor stimulation. Calcium levels were determined from fura-2 imaging of isolated rat retinal ganglion cells as these neurons possess both receptor types. Brief application of glutamate or NMDA led to repeatable and reversible elevations of intracellular calcium. The A(3) agonist Cl-IB-MECA reduced the response to both glutamate and NMDA. While adenosine mimicked the effect of Cl-IB-MECA, the A(3) receptor antagonist MRS 1191 impeded the block by adenosine, implicating a role for the A(3) receptor in response to the natural agonist. The A(1) receptor antagonist DPCPX provided additional inhibition, implying a contribution from both A(1) and A(3) adenosine receptors. The novel A(3) agonist MRS 3558 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide and mixed A(1)/A(3) agonist MRS 3630 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide also inhibited the calcium rise induced by NMDA. Low levels of MRS 3558 were particularly effective, with an IC(50) of 400 pM. In all cases, A(3) receptor stimulation inhibited only 30-50% of the calcium rise. In summary, stimulation of the A(3) adenosine receptor by either endogenous or synthesized agonists can limit the calcium rise accompanying NMDA receptor activation. It remains to be determined if partial block of the calcium rise by A(3) agonists can modify downstream responses to NMDA receptor stimulation.

  5. The Split Virus Influenza Vaccine rapidly activates immune cells through Fcγ receptors. (United States)

    O'Gorman, William E; Huang, Huang; Wei, Yu-Ling; Davis, Kara L; Leipold, Michael D; Bendall, Sean C; Kidd, Brian A; Dekker, Cornelia L; Maecker, Holden T; Chien, Yueh-Hsiu; Davis, Mark M


    Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.

  6. Unique Responses are Observed in Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and TRPV1 Co-Expressing Cells

    Directory of Open Access Journals (Sweden)

    Laura R. Sadofsky


    Full Text Available Transient receptor potential (TRP ankyrin 1 (TRPA1 and vanilloid 1 (TRPV1 receptors are implicated in modulation of cough and nociception. In vivo, TRPA1 and TRPV1 are often co-expressed in neurons and TRPA1V1 hetero-tetramer formation is noted in cells co-transfected with the respective expression plasmids. In order to understand the impact of TRP receptor interaction on activity, we created stable cell lines expressing the TRPA1, TRPV1 and co-expressing the TRPA1 and TRPV1 (TRPA1V1 receptors. Among the 600 compounds screened against these receptors, we observed a number of compounds that activated the TRPA1, TRPV1 and TRPA1V1 receptors; compounds that activated TRPA1 and TRPA1V1; compounds that activated TRPV1 and TRPA1V1; compounds in which TRPA1V1 response was modulated by either TRPA1 or TRPV1; and compounds that activated only TRPV1 or TRPA1 or TRPA1V1; and one compound that activated TRPA1 and TRPV1, but not TRPA1V1. These results suggest that co-expression of TRPA1 and TRPV1 receptors imparts unique activation profiles different from that of cells expressing only TRPA1 or TRPV1.

  7. Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily.



    During morphogenesis, cell-cell association patterns are dynamically altered. We are interested in how cell adhesion molecules can regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells are switched from one subtype to a...

  8. Infection Profiles of Selected Aquabirnavirus Isolates in CHSE Cells.

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    Amr A A Gamil

    Full Text Available The wide host range and antigenic diversity of aquabirnaviruses are reflected by the presence of a collection of isolates with different sero- and genotypic properties that have previously been classified as such. Differences in cytopathogenic mechanisms and host responses induced by these isolates have not been previously examined. In the present study, we investigated infection profiles induced by genetically and serologically closely related as well as distant isolates in-vitro. CHSE-214 cells were infected with either E1S (serotype A3, genogroup 3, VR-299 (serotype A1, genogroup 1, highly virulent Sp (TA or avirulent Sp (PT (serotype A2, genogroup 5. The experiments were performed at temperatures most optimum for each of the isolates namely 15°C for VR-299, TA and PT strains and 20°C for E1S. Differences in virus loads and ability to induce cytopathic effect, inhibition of protein synthesis, apoptosis, and induction of IFNa, Mx1, PKR or TNFα gene expression at different times post infection were examined. The results showed on one hand, E1S with the highest ability to replicate, induce apoptosis and IFNa gene expression while VR-299 inhibited protein synthesis and induced Mx1 and PKR gene expression the most. The two Sp isolates induced the highest TNFα gene expression but differed in their ability to replicate, inhibit protein synthesis, and induce gene expression, with TA being more superior. Collectively, these findings point towards the adaptation by different virus isolates to suit environments and hosts that they patronize. Furthermore, the results also suggest that genetic identity is not prerequisite to functional similarities thus results of one aquabirnavirus isolate cannot necessarily be extrapolated to another.

  9. Global evaluation of Eph receptors and ephrins in lung adenocarcinomas identifies EphA4 as an inhibitor of cell migration and invasion. (United States)

    Saintigny, Pierre; Peng, Shaohua; Zhang, Li; Sen, Banibrata; Wistuba, Ignacio I; Lippman, Scott M; Girard, Luc; Minna, John D; Heymach, John V; Johnson, Faye M


    The Eph family of receptors is the largest family of receptor tyrosine kinases, but it remains poorly studied in lung cancer. We aimed to systematically explore the human Eph receptors and their ligands, the ephrins, in lung adenocarcinoma. The prognostic impact of Eph receptor and ephrin gene expression was analyzed using 2 independent cohorts of lung adenocarcinoma. Gene expression profiles in lung adenocarcinoma compared with normal adjacent lung were studied in 3 independent cohorts and in cell lines. Gene expression profiles were validated with quantitative polymerase chain reaction (qPCR) and Western blotting in cell lines. Functional studies to assess the role of Eph receptor A4 (EphA4) were carried out in vitro. The biological effects of EphA4 in lung cancer cell lines were assayed following overexpression and knockdown. Of the 11 Eph receptors and 8 ephrins analyzed, only EphA4 and ephrin A1 gene expression were consistently associated with an improved outcome in patients with lung adenocarcinoma. Expression levels of EphA4 by microarray correlated well with expression levels measured by qPCR and Western blotting. EphA4 overexpression reduced cell migration and invasion but did not affect cell cycle, apoptosis, or drug sensitivity. Surprisingly, EphA4 was expressed at higher levels in cancer compared with non-cancer tissues and cell lines. EphA4 gene expression is associated with an improved outcome in patients with resected lung adenocarcinoma, possibly by affecting cancer cell migration and invasion.

  10. Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro (United States)

    Svegliati, Silvia; Amico, Donatella; Spadoni, Tatiana; Fischetti, Colomba; Finke, Doreen; Moroncini, Gianluca; Paolini, Chiara; Tonnini, Cecilia; Grieco, Antonella; Rovinelli, Marina; Gabrielli, Armando


    One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia. PMID:28228756

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

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    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

  12. Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis. (United States)

    Xu, Gang; Gu, Gui-Xiang; Teng, Zi-Wen; Wu, Shun-Fan; Huang, Jia; Song, Qi-Sheng; Ye, Gong-Yin; Fang, Qi


    In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors.

  13. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface. (United States)

    Becker, Björn; Shaebani, M Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J


    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTA(H/KDEL)), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

  14. Diverse activation states of RhoA in human lung cancer cells: contribution of G protein coupled receptors. (United States)

    Touge, Hirokazu; Chikumi, Hiroki; Igishi, Tadashi; Kurai, Jun; Makino, Haruhiko; Tamura, Yoshisato; Takata, Miyako; Yoneda, Kazuhiko; Nakamoto, Masaki; Suyama, Hisashi; Gutkind, J Silvio; Shimizu, Eiji


    Rho GTPases play an essential role in the control of various cellular functions. Accumulating evidence suggests that RhoA overexpression contributes to human cancer development. However, the activation states of RhoA are poorly defined in cancer cells. In this study, we examined both the expression levels and the activation states of RhoA in various lung cancer cells by quantitative real-time reverse transcriptase-polymerase chain reaction and in vivo Rho guanine nucleotide exchange assay, respectively. Moreover, we dissected the signaling pathway from the cell surface receptors to RhoA using a broad-spectrum G protein coupled receptor (GPCR) antagonist, [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP), and a recently reported Galphaq/11-selective inhibitor, YM-254890. We found that RhoA was expressed highly in large cell carcinoma cells but only weakly in adenocarcinoma cells. The activation states of RhoA are considerably different from its expression profiles. We found that four of six small cell lung carcinoma (SCLC) cell lines exhibited a moderate to high activation rate of RhoA. The addition of [D-Arg1,D-Trp5,7,9,Leu11]SP reduced RhoA activity by almost 60% in H69 SCLC cells. The addition of YM-254890 had no effect on RhoA activity in H69 cells. Our results suggest that RhoA is activated in various lung cancer cells independent of its expression levels, and the high activation state of RhoA in SCLC cells mainly depends on a neuroendocrine peptide autocrine system which signals through Galpha12 coupled GPCR to RhoA. This study provides new insights into RhoA signaling in lung cancer cells and may help in developing novel therapeutic strategies against lung cancer.

  15. Expression and function of P2 receptors in hematopoietic stem and progenitor cells. (United States)

    Feng, Wenli; Wang, Lina; Zheng, Guoguang


    Nucleotides have unambiguously emerged as a family of mediators of intercellular communication, which bind to a class of plasma membrane receptors, P2 receptors, to trigger intercellular signaling. P2 receptors can be further divided into P2X and P2Y subfamilies based on structure and function. Different hematopoietic cells express diverse spectrums of P2 receptors at different levels, including hematopoietic stem and progenitor cells (HSPCs). Extracellular adenosine triphosphate (ATP) exerts different effects on HSPCs, regulating cell proliferation, differentiation, migration, and chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species. The relationship between abnormal P2 receptor function and human diseases attracts more and more attention. This review summarizes the expression and function of P2 receptors in HSPCs and the relationship to hematopoietic diseases.

  16. Octreotide scintigraphy localizes somatostatin receptor-positive islet cell carcinomas

    NARCIS (Netherlands)

    W. Becker (W.); J. Marienhagen (J.); R. Scheubel (R.); A. Saptogino (A.); W.H. Bakker (Willem); W.A.P. Breeman (Wouter); F. Wolf (F.)


    textabstractTyr-3-Octreotide is a synthetic derivative of somatostatin and a somatostatin-receptor analogue. The iodine-123-labelled compound localizes somatostatin-receptor-positive tumours. In this paper two patients are reported in whom somatostatin receptors were demonstrated in vitro. In a 60-y

  17. Using expression profiling to understand the effects of chronic cadmium exposure on MCF-7 breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Zelmina Lubovac-Pilav

    Full Text Available Cadmium is a metalloestrogen known to activate the estrogen receptor and promote breast cancer cell growth. Previous studies have implicated cadmium in the development of more malignant tumors; however the molecular mechanisms behind this cadmium-induced malignancy remain elusive. Using clonal cell lines derived from exposing breast cancer cells to cadmium for over 6 months (MCF-7-Cd4, -Cd6, -Cd7, -Cd8 and -Cd12, this study aims to identify gene expression signatures associated with chronic cadmium exposure. Our results demonstrate that prolonged cadmium exposure does not merely result in the deregulation of genes but actually leads to a distinctive expression profile. The genes deregulated in cadmium-exposed cells are involved in multiple biological processes (i.e. cell growth, apoptosis, etc. and molecular functions (i.e. cadmium/metal ion binding, transcription factor activity, etc.. Hierarchical clustering demonstrates that the five clonal cadmium cell lines share a common gene expression signature of breast cancer associated genes, clearly differentiating control cells from cadmium exposed cells. The results presented in this study offer insights into the cellular and molecular impacts of cadmium on breast cancer and emphasize the importance of studying chronic cadmium exposure as one possible mechanism of promoting breast cancer progression.

  18. Using expression profiling to understand the effects of chronic cadmium exposure on MCF-7 breast cancer cells. (United States)

    Lubovac-Pilav, Zelmina; Borràs, Daniel M; Ponce, Esmeralda; Louie, Maggie C


    Cadmium is a metalloestrogen known to activate the estrogen receptor and promote breast cancer cell growth. Previous studies have implicated cadmium in the development of more malignant tumors; however the molecular mechanisms behind this cadmium-induced malignancy remain elusive. Using clonal cell lines derived from exposing breast cancer cells to cadmium for over 6 months (MCF-7-Cd4, -Cd6, -Cd7, -Cd8 and -Cd12), this study aims to identify gene expression signatures associated with chronic cadmium exposure. Our results demonstrate that prolonged cadmium exposure does not merely result in the deregulation of genes but actually leads to a distinctive expression profile. The genes deregulated in cadmium-exposed cells are involved in multiple biological processes (i.e. cell growth, apoptosis, etc.) and molecular functions (i.e. cadmium/metal ion binding, transcription factor activity, etc.). Hierarchical clustering demonstrates that the five clonal cadmium cell lines share a common gene expression signature of breast cancer associated genes, clearly differentiating control cells from cadmium exposed cells. The results presented in this study offer insights into the cellular and molecular impacts of cadmium on breast cancer and emphasize the importance of studying chronic cadmium exposure as one possible mechanism of promoting breast cancer progression.

  19. Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability. (United States)

    Fairfax, Benjamin P; Pitcher, Julie A; Scott, Mark G H; Calver, Andrew R; Pangalos, Menelas N; Moss, Stephen J; Couve, Andrés


    GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

  20. Generalized stochastic profiling of transcriptional regulatory heterogeneities in tissues, tumors, and cultured cells



    Single-cell variations in gene and protein expression are important during development and disease. Cell-to-cell heterogeneities can be directly inspected one cell at a time, but global methods are usually not sensitive enough to work with such a small amount of starting material. Here, we provide a detailed protocol for stochastic profiling, a method that infers single-cell regulatory heterogeneities by repeatedly sampling small collections of cells at random. Repeated stochastic sampling is...

  1. 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


    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

  2. Expression profiles of genes involved in xenobiotic metabolism and disposition in human renal tissues and renal cell models

    Energy Technology Data Exchange (ETDEWEB)

    Van der Hauwaert, Cynthia; Savary, Grégoire [EA4483, Université de Lille 2, Faculté de Médecine de Lille, Pôle Recherche, 59045 Lille (France); Buob, David [Institut de Pathologie, Centre de Biologie Pathologie Génétique, Centre Hospitalier Régional Universitaire de Lille, 59037 Lille (France); Leroy, Xavier; Aubert, Sébastien [Institut de Pathologie, Centre de Biologie Pathologie Génétique, Centre Hospitalier Régional Universitaire de Lille, 59037 Lille (France); Institut National de la Santé et de la Recherche Médicale, UMR837, Centre de Recherche Jean-Pierre Aubert, Equipe 5, 59045 Lille (France); Flamand, Vincent [Service d' Urologie, Hôpital Huriez, Centre Hospitalier Régional Universitaire de Lille, 59037 Lille (France); Hennino, Marie-Flore [EA4483, Université de Lille 2, Faculté de Médecine de Lille, Pôle Recherche, 59045 Lille (France); Service de Néphrologie, Hôpital Huriez, Centre Hospitalier Régional Universitaire de Lille, 59037 Lille (France); Perrais, Michaël [Institut National de la Santé et de la Recherche Médicale, UMR837, Centre de Recherche Jean-Pierre Aubert, Equipe 5, 59045 Lille (France); and others


    Numerous xenobiotics have been shown to be harmful for the kidney. Thus, to improve our knowledge of the cellular processing of these nephrotoxic compounds, we evaluated, by real-time PCR, the mRNA expression level of 377 genes encoding xenobiotic-metabolizing enzymes (XMEs), transporters, as well as nuclear receptors and transcription factors that coordinate their expression in eight normal human renal cortical tissues. Additionally, since several renal in vitro models are commonly used in pharmacological and toxicological studies, we investigated their metabolic capacities and compared them with those of renal tissues. The same set of genes was thus investigated in HEK293 and HK2 immortalized cell lines in commercial primary cultures of epithelial renal cells and in proximal tubular cell primary cultures. Altogether, our data offers a comprehensive description of kidney ability to process xenobiotics. Moreover, by hierarchical clustering, we observed large variations in gene expression profiles between renal cell lines and renal tissues. Primary cultures of proximal tubular epithelial cells exhibited the highest similarities with renal tissue in terms of transcript profiling. Moreover, compared to other renal cell models, Tacrolimus dose dependent toxic effects were lower in proximal tubular cell primary cultures that display the highest metabolism and disposition capacity. Therefore, primary cultures appear to be the most relevant in vitro model for investigating the metabolism and bioactivation of nephrotoxic compounds and for toxicological and pharmacological studies. - Highlights: • Renal proximal tubular (PT) cells are highly sensitive to xenobiotics. • Expression of genes involved in xenobiotic disposition was measured. • PT cells exhibited the highest similarities with renal tissue.

  3. Interleukin-1 receptors are differentially expressed in normal and psoriatic T cells. (United States)

    Bebes, Attila; Kovács-Sólyom, Ferenc; Prihoda, Judit; Kui, Róbert; Kemény, Lajos; Gyulai, Rolland


    This study was carried out to examine the possible role of interleukin-1 (IL-1) in the functional insufficiency of regulatory T cells in psoriasis, by comparing the expression of IL-1 receptors on healthy control and psoriatic T cells. Patients with moderate-to-severe chronic plaque psoriasis and healthy volunteers, matched in age and sex, were selected for all experiments. CD4(+)CD25(-) effector and CD4(+)CD25(+)CD127(low) regulatory T cells were separated and used for the experiments. Expression of the mRNA of IL-1 receptors (IL-1R1, IL-1R2, and sIL-1R2) was determined by quantitative real-time RT-PCR. Cell surface IL-1 receptor expression was assessed by flow cytometry. Relative expression of the signal transmitting IL-1 receptor type 1 (IL-1R1) mRNA is higher in resting psoriatic effector and regulatory T cells, and activation induces higher IL-1R1 protein expression in psoriatic T cells than in healthy cells. Psoriatic regulatory and effector T cells express increased mRNA levels of the decoy IL-1 receptors (IL-1R2 and sIL-1R2) upon activation compared to healthy counterparts. Psoriatic T cells release slightly more sIL-1R2 into their surrounding than healthy T cells. In conclusion, changes in the expression of IL-1 receptors in psoriatic regulatory and effector T cells could contribute to the pathogenesis of psoriasis.

  4. Categorizing Cells on the Basis of their Chemical Profiles: Progress in Single-Cell Mass Spectrometry (United States)


    The chemical differences between individual cells within large cellular populations provide unique information on organisms’ homeostasis and the development of diseased states. Even genetically identical cell lineages diverge due to local microenvironments and stochastic processes. The minute sample volumes and low abundance of some constituents in cells hinder our understanding of cellular heterogeneity. Although amplification methods facilitate single-cell genomics and transcriptomics, the characterization of metabolites and proteins remains challenging both because of the lack of effective amplification approaches and the wide diversity in cellular constituents. Mass spectrometry has become an enabling technology for the investigation of individual cellular metabolite profiles with its exquisite sensitivity, large dynamic range, and ability to characterize hundreds to thousands of compounds. While advances in instrumentation have improved figures of merit, acquiring measurements at high throughput and sampling from large populations of cells are still not routine. In this Perspective, we highlight the current trends and progress in mass-spectrometry-based analysis of single cells, with a focus on the technologies that will enable the next generation of single-cell measurements. PMID:28135079

  5. 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: [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)


    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.

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

    Directory of Open Access Journals (Sweden)

    Muellhaupt Beat


    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.

  7. Metabolic profiling-based data-mining for an effective chemical combination to induce apoptosis of cancer cells. (United States)

    Kumazoe, Motofumi; Fujimura, Yoshinori; Hidaka, Shiori; Kim, Yoonhee; Murayama, Kanako; Takai, Mika; Huang, Yuhui; Yamashita, Shuya; Murata, Motoki; Miura, Daisuke; Wariishi, Hiroyuki; Maeda-Yamamoto, Mari; Tachibana, Hirofumi


    Green tea extract (GTE) induces apoptosis of cancer cells without adversely affecting normal cells. Several clinical trials reported that GTE was well tolerated and had potential anti-cancer efficacy. Epigallocatechin-3-O-gallate (EGCG) is the primary compound responsible for the anti-cancer effect of GTE; however, the effect of EGCG alone is limited. To identify GTE compounds capable of potentiating EGCG bioactivity, we performed metabolic profiling of 43 green tea cultivar panels by liquid chromatography-mass spectrometry (LC-MS). Here, we revealed the polyphenol eriodictyol significantly potentiated apoptosis induction by EGCG in vitro and in a mouse tumour model by amplifying EGCG-induced activation of the 67-kDa laminin receptor (67LR)/protein kinase B/endothelial nitric oxide synthase/protein kinase C delta/acid sphingomyelinase signalling pathway. Our results show that metabolic profiling is an effective chemical-mining approach for identifying botanical drugs with therapeutic potential against multiple myeloma. Metabolic profiling-based data mining could be an efficient strategy for screening additional bioactive compounds and identifying effective chemical combinations.

  8. SLAM family receptors and the SLAM-associated protein (SAP) modulate T cell functions. (United States)

    Detre, Cynthia; Keszei, Marton; Romero, Xavier; Tsokos, George C; Terhorst, Cox


    One or more of the signaling lymphocytic activation molecule (SLAM) family (SLAMF) of cell surface receptors, which consists of nine transmembrane proteins, i.e., SLAMF1-9, are expressed on most hematopoietic cells. While most SLAMF receptors serve as self-ligands, SLAMF2 and SLAMF4 use each other as counter structures. Six of the receptors carry one or more copies of a unique intracellular tyrosine-based switch motif, which has high affinity for the single SH2-domain signaling molecules SLAM-associated protein and EAT-2. Whereas SLAMF receptors are costimulatory molecules on the surface of CD4+, CD8+, and natural killer (NK) T cells, they also involved in early phases of lineage commitment during hematopoiesis. SLAMF receptors regulate T lymphocyte development and function and modulate lytic activity, cytokine production, and major histocompatibility complex-independent cell inhibition of NK cells. Furthermore, they modulate B cell activation and memory generation, neutrophil, dendritic cell, macrophage and eosinophil function, and platelet aggregation. In this review, we will discuss the role of SLAM receptors and their adapters in T cell function, and we will examine the role of these receptors and their adapters in X-linked lymphoproliferative disease and their contribution to disease susceptibility in systemic lupus erythematosus.

  9. An Essential Role of the Avidity of T-Cell Receptor in Differentiation of Self-Antigen-reactive CD8+ T Cells. (United States)

    Kondo, Kenta; Fujiki, Fumihiro; Nakajima, Hiroko; Yatsukawa, Erika; Morimoto, Soyoko; Tatsumi, Naoya; Nishida, Sumiyuki; Nakata, Jun; Oka, Yoshihiro; Tsuboi, Akihiro; Hosen, Naoki; Oji, Yusuke; Sugiyama, Haruo


    Many studies demonstrated crucial roles of avidity of T-cell receptor (TCR) in T-cell fate. However, majority of these findings resulted from analysis of non-self-antigen-specific CD8 T cells, and little is known about roles of TCR avidity in the fate of self-antigen-specific CD8 T cells. Wilms tumor gene 1 (WT1) protein is a self-antigen most suitable for addressing this issue because WT1 protein is a highly immunogenic, typical self-antigen. Here, we isolated 2 distinct and functional TCRs, TCR1 and TCR2, from murine WT1 peptide (RMFPNAPYL)-specific cytotoxic T lymphocytes (WT1-CTLs) and generated TCR1-retrogenic (Rg) and TCR2-Rg mice under T and B-cell-deficient and -reconstituted conditions. TCR1-transduced CD8 T (TCR1-T) cells had approximately 2-fold higher avidity to WT1 peptide than TCR2-transduced CD8 T (TCR2-T) cells. Cytokine production profiles and cell surface phenotypes showed that TCR1-T cells were more differentiated than TCR2-T cells under both conditions. Therefore, TCR1-T cells with TCR avidity higher than that of TCR2-T cells are more differentiated compared with TCR2-T cells. Furthermore, TCR1-T cells that developed under T and B-cell-reconstituted conditions displayed cytotoxicity against endogenously WT1-expressing tumor cells, whereas TCR2 T cells that developed under the same conditions did not. Thus, it was demonstrated, for the first time, that TCR avidity played an essential role in differentiation of self-antigen-reactive T cells, through the success of establishment of two distinct WT1-CTLs with a difference in only TCR avidity under the identical genetic background. Present results should provide us with an insight for elucidation of the differentiation mechanisms of self-antigen-reactive T cells, including tumor antigen-reactive T cells.

  10. 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.

  11. The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alphaT3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size. (United States)

    Forrest-Owen, W; Willars, G B; Nahorski, S R; Assefa, D; Davidson, J S; Hislop, J; McArdle, C A


    The phospholipase C (PLC)-activating gonadotrophin-releasing hormone (GnRH) receptor is thought not to rapidly desensitise in alphaT3-1 cells. This extremely unusual characteristic raises the concern that it might be a feature of the cell type, rather than the receptor per se. Here we have used video imaging to establish whether the effects of endogenous PLC-activating G-protein coupled receptors (GPCRs) on Ca2+ ion concentration [Ca2+]i desensitise in these cells. Oxytocin, endothelin-1, methacholine, and UTP all caused [Ca2+]i increases which underwent rapid homologous desensitisation in that they were transient and responses to repeat stimuli were attenuated whereas subsequent responses to GnRH were not. To test whether receptor reserve obscures functional desensitisation of GnRH receptors, a photoaffinity antagonist (Pant-1), was used to effect a partial and irreversible receptor blockade. UV crosslinking in medium with 1000 nM Pant-1 reduced GnRH receptor number to 20 +/- 5% and reduced maximal buserelin-stimulated [3H]IP(X) accumulation to 57 +/- 5%, demonstrating removal of receptor reserve. In control alphaT3-1 cells the initial rate of GnRH-stimulated [3H]IP(X) accumulation was maintained for at least 5 min and GnRH caused a sustained increase in Ins(1,4,5)P3 mass (confirming the resistance of GnRH receptors to desensitisation) and Pant-1 pre-treatment reduced the magnitude of these responses without altering their temporal profiles. In alphaT3-1 cells stably transfected with recombinant human muscarinic receptors (alphaT3-1/M3), responses to methacholine were characteristic of desensitising GPCRs (transient Ins(1,4,5)P3 and curvilinear [3H]IP(X) responses) and were unaltered by Pant-1. To test the relevance of phospholipid pool size, alphaT3-1/M3 cells were pre-treated with GnRH or methacholine in medium with LiCl (to deplete PtdIns(4,5)P2 pools). These pre-treatments reduced subsequent responses to methacholine and GnRH comparably, indicating access to a

  12. Atypical nuclear localization of VIP receptors in glioma cell lines and patients. (United States)

    Barbarin, Alice; Séité, Paule; Godet, Julie; Bensalma, Souheyla; Muller, Jean-Marc; Chadéneau, Corinne


    An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

  13. Cell surface receptors for signal transduction and ligand transport: a design principles study.

    Directory of Open Access Journals (Sweden)

    Harish Shankaran


    Full Text Available Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor-ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.

  14. Evaluation of drug-muscarinic receptor affinities using cell membrane chromatography and radioligand binding assay in guinea pig jejunum membrane

    Institute of Scientific and Technical Information of China (English)

    Bing-xiang YUAN; Jin HOU; Lang-chong HE; Guang-de YANG


    Aim: To study if cell membrane chromatography (CMC) could reflect drug-receptor interaction and evaluate the affinity and competitive binding to muscarinic acetylcholine receptor (mAChR). Methods: The cell membrane stationary phase(CMSP) was prepared by immobilizing guinea pig jejunum cell membrane on the surface of a silica carrier, and was used for the rapid on-line chromatographic evaluation of ligand binding affinities to mAChR. The affinity to mAChR was also evaluated from radioligand binding assays (RBA) using the same jejunum membrane preparation. Results: The capacity factor (k') profiles in guinea pig jejunum CMSP were: (-)QNB (15.4)>(+)QNB (11.5)>atropine (5.35)>pirenzepine(5.26)>4-DAMP (4.45)>AF-DX 116 (4.18)>pilocarpine (3.93)>acetylcholine(1.31). These results compared with the affinity rank orders obtained from radioligand binding assays indicated that there wasa positive correlation (r2=0.8525, P<0.0001) between both data sets. Conclusion: The CMC method can be used to evaluate drug-receptor affinities for drug candidates.

  15. The serotonin receptor 5-HT₇R regulates the morphology and migratory properties of dendritic cells. (United States)

    Holst, Katrin; Guseva, Daria; Schindler, Susann; Sixt, Michael; Braun, Armin; Chopra, Himpriya; Pabst, Oliver; Ponimaskin, Evgeni


    Dendritic cells are potent antigen-presenting cells endowed with the unique ability to initiate adaptive immune responses upon inflammation. Inflammatory processes are often associated with an increased production of serotonin, which operates by activating specific receptors. However, the functional role of serotonin receptors in regulation of dendritic cell functions is poorly understood. Here, we demonstrate that expression of serotonin receptor 5-HT7 (5-HT7R) as well as its downstream effector Cdc42 is upregulated in dendritic cells upon maturation. Although dendritic cell maturation was independent of 5-HT7R, receptor stimulation affected dendritic cell morphology through Cdc42-mediated signaling. In addition, basal activity of 5-HT7R was required for the proper expression of the chemokine receptor CCR7, which is a key factor that controls dendritic cell migration. Consistent with this, we observed that 5-HT7R enhances chemotactic motility of dendritic cells in vitro by modulating their directionality and migration velocity. Accordingly, migration of dendritic cells in murine colon explants was abolished after pharmacological receptor inhibition. Our results indicate that there is a crucial role for 5-HT7R-Cdc42-mediated signaling in the regulation of dendritic cell morphology and motility, suggesting that 5-HT7R could be a new target for treatment of a variety of inflammatory and immune disorders.

  16. Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells.

    Directory of Open Access Journals (Sweden)

    Sahil Talwar

    Full Text Available Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αβ heteromeric glycine receptor (GlyR chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems

  17. Expression pattern of mda-7/IL-24 receptors in liver cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Hong Zhu; Zhi-Bin Yang


    BACKGROUND: The mda-7/IL-24 receptor belongs to the typeⅡ cytokine receptor family, and its two heterodimeric receptors are IL-22R1/IL-20R2 and IL-20R1/IL-20R2. Mda-7/IL-24 receptor expression in liver cancer cell lines has not yet been described. This information may be helpful for further clinical gene therapy. METHODS: With normal skin total RNA as template, the cDNA sequences of IL-20R1, IL-20R2 and IL-22R were ampliifed by RT-PCR. Total RNA was extracted from cultured liver cancer cell lines and a normal liver cell line, then detected by northern blotting, and the expression of mda-7/IL-24 receptors was analyzed. RESULTS: PLC/PRF/5 and SMMC-7721 expressed IL-20R1;BEL-7402, Hep3B, HepG2, and PLC/PRF/5 expressed IL-20R2; and HepG2 and PLC/PRF/5 expressed IL-22R. Only HepG2 expressed the IL-22R/IL-20R2 receptor complex. PLC/PRF/5 completely expressed both heterodimeric receptors. Huh-7, QGY-7701 and WRL-68 did not express the IL-24 receptor. CONCLUSION: Complete mda-7/IL-24 receptors are seldom expressed in liver cancer cell lines.

  18. Expression of neuropeptide receptor mRNA during osteoblastic differentiation of mouse iPS cells. (United States)

    Nagao, Satomi; Goto, Tetsuya; Kataoka, Shinji; Toyono, Takashi; Joujima, Takaaki; Egusa, Hiroshi; Yatani, Hirofumi; Kobayashi, Shigeru; Maki, Kenshi


    Various studies have shown a relationship between nerves and bones. Recent evidence suggests that both sensory and sympathetic nerves affect bone metabolism; however, little is known about how neuropeptides are involved in the differentiation of pluripotent stem cells into osteoblastic (OB) cells. To evaluate the putative effects of neuropeptides during the differentiation of mouse induced pluripotent stem (iPS) cells into calcified tissue-forming OB cells, we investigated the expression patterns of neuropeptide receptors at each differentiation stage. Mouse iPS cells were seeded onto feeder cells and then transferred to low-attachment culture dishes to form embryoid bodies (EBs). EBs were cultured for 4 weeks in osteoblastic differentiation medium. The expression of α1-adrenergic receptor (AR), α2-AR, β2-AR, neuropeptide Y1 receptor (NPY1-R), neuropeptide Y2 receptor (NPY2-R), calcitonin gene-related protein receptor (CGRP-R), and neurokinin 1-R (NK1-R) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Among these neuropeptide receptors, CGRP-R and β2-AR were expressed at all stages of cell differentiation, including the iPS cell stage, with peak expression occurring at the early osteoblastic differentiation stage. Another sensory nervous system receptor, NK1-R, was expressed mainly in the late osteoblastic differentiation stage. Furthermore, CGRP-R mRNA showed an additional small peak corresponding to EBs cultured for 3 days, suggesting that EBs may be affected by serum CGRP. These data suggest that the sensory nervous system receptor CGRP-R and the sympathetic nervous system receptor β2-AR may be involved in the differentiation of iPS cells into the osteoblastic lineage. It follows from these findings that CGRP and β2-AR may regulate cell differentiation in the iPS and EB stages, and that each neuropeptide has an optimal period of influence during the differentiation process.

  19. Profiling two indole-2-carboxamides for allosteric modulation of the CB1 receptor. (United States)

    Ahn, Kwang H; Mahmoud, Mariam M; Samala, Sushma; Lu, Dai; Kendall, Debra A


    Allosteric modulation of G-protein coupled receptors (GPCRs) represents a novel approach for fine-tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole-2-carboxamides:5-chloro-3-ethyl-1-methyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-a) and 5-chloro-3-pentyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-b). Although both ICAM-a and ICAM-b enhanced CP55, 940 binding, ICAM-b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G-protein coupling to CB1, ICAM-b induced β-arrestin-mediated downstream activation of extracellular signal-regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.

  20. Gene expression profiling of the androgen receptor antagonists flutamide and vinclozolin in zebrafish (Danio rerio) gonads

    Energy Technology Data Exchange (ETDEWEB)

    Martinovic-Weigelt, Dalma, E-mail: [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804 (United States); University of St. Thomas, 2115 Summit Ave, Saint Paul, MN 55105 (United States); Wang Ronglin [US Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Ecological Exposure Research Division, 26W. Martin Luther King Dr., Cincinnati, OH 45268 (United States); Villeneuve, Daniel L. [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804 (United States); Bencic, David C.; Lazorchak, Jim [US Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Ecological Exposure Research Division, 26W. Martin Luther King Dr., Cincinnati, OH 45268 (United States); Ankley, Gerald T. [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804 (United States)


    The studies presented in this manuscript focus on characterization of transcriptomic responses to anti-androgens in zebrafish (Danio rerio). Research on the effects of anti-androgens in fish has been characterized by a heavy reliance on apical endpoints, and molecular mechanisms of action (MOA) of anti-androgens remain poorly elucidated. In the present study, we examined effects of a short term exposure (24-96 h) to the androgen receptor antagonists flutamide (FLU) and vinclozolin (VZ) on gene expression in gonads of sexually mature zebrafish, using commercially available zebrafish oligonucleotide microarrays (4 x 44 K platform). We found that VZ and FLU potentially impact reproductive processes via multiple pathways related to steroidogenesis, spermatogenesis, and fertilization. Observed changes in gene expression often were shared by VZ and FLU, as demonstrated by overlap in differentially-expressed genes and enrichment of several common key pathways including: (1) integrin and actin signaling, (2) nuclear receptor 5A1 signaling, (3) fibroblast growth factor receptor signaling, (4) polyamine synthesis, and (5) androgen synthesis. This information should prove useful to elucidating specific mechanisms of reproductive effects of anti-androgens in fish, as well as developing biomarkers for this important class of endocrine-active chemicals.

  1. Pharmacological profiles of alpha 2 adrenergic receptor agonists identified using genetically altered mice and isobolographic analysis. (United States)

    Fairbanks, Carolyn A; Stone, Laura S; Wilcox, George L


    Endogenous, descending noradrenergic fibers impose analgesic control over spinal afferent circuitry mediating the rostrad transmission of pain signals. These fibers target alpha 2 adrenergic receptors (alpha(2)ARs) on both primary afferent terminals and secondary neurons, and their activation mediates substantial inhibitory control over this transmission, rivaling that of opioid receptors which share a similar pattern of distribution. The terminals of primary afferent nociceptive neurons and secondary spinal dorsal horn neurons express alpha(2A)AR and alpha(2C)AR subtypes, respectively. Spinal delivery of these agents serves to reduce their side effects, which are mediated largely at supraspinal sites, by concentrating the drugs at the spinal level. Targeting these spinal alpha(2)ARs with one of five selective therapeutic agonists, clonidine, dexmedetomidine, brimonidine, ST91 and moxonidine, produces significant antinociception that can work in concert with opioid agonists to yield synergistic antinociception. Application of several genetically altered mouse lines had facilitated identification of the primary receptor subtypes that likely mediate the antinociceptive effects of these agents. This review provides first an anatomical description of the localization of the three subtypes in the central nervous system, second a detailed account of the pharmacological history of each of the six primary agonists, and finally a comprehensive report of the specific interactions of other GPCR agonists with each of the six principal alpha(2)AR agonists featured.

  2. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation (United States)

    Au-Yeung, Byron B.; Zikherman, Julie; Mueller, James L.; Ashouri, Judith F.; Matloubian, Mehrdad; Cheng, Debra A.; Chen, Yiling; Shokat, Kevan M.; Weiss, Arthur


    T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77–eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery. PMID:25136127

  3. Rac activation by the T-cell receptor inhibits T cell migration.

    Directory of Open Access Journals (Sweden)

    Eva Cernuda-Morollón

    Full Text Available BACKGROUND: T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known. METHODOLOGY/PRINCIPAL FINDINGS: Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin. CONCLUSIONS/SIGNIFICANCE: We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.

  4. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells. (United States)

    Farber, D L; Acuto, O; Bottomly, K


    Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3zeta subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3zeta in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4 T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.

  5. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer


    Lynch, Jennifer R.; Jenny Yingzi Wang


    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in ...

  6. 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


    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...

  7. FcγReceptors and the complement system in T cell activation

    NARCIS (Netherlands)

    Jong, Judith Maria Hendrika de


    Dendritic Cells (DC) are the major Antigen Presenting Cells (APC) of the immune system that are involved in initiation of CD4+ and CD8+ T cell responses, as DC display many receptors involved in antigen uptake, including several types of FcgammaR. However, other APC, like B cells and macrophages als

  8. The stress phenotype makes cancer cells addicted to CDT2, a substrate receptor of the CRL4 ubiquitin ligase. (United States)

    Olivero, Martina; Dettori, Daniela; Arena, Sabrina; Zecchin, Davide; Lantelme, Erica; Di Renzo, Maria Flavia


    CDT2/L2DTL/RAMP is one of the substrate receptors of the Cullin Ring Ubiquitin Ligase 4 that targets for ubiquitin mediated degradation a number of substrates, such as CDT1, p21 and CHK1, involved in the regulation of cell cycle and survival. Here we show that CDT2 depletion was alone able to induce the apoptotic death in 12/12 human cancer cell lines from different tissues, regardless of the mutation profile and CDT2 expression level. Cell death was associated to rereplication and to loss of CDT1 degradation. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to a single specific pathway. CDT2 likely is a non-oncogene to which transformed cells become addicted because of their enhanced cellular stress, such as replicative stress and DNA damage.


    Institute of Scientific and Technical Information of China (English)

    Du Jianqing(杜剑青); Sun Chengwen(孙成文); Tang Jingshi (唐敬师); Colin Sumners; Mohan K Raizada


    Objective To study the action of brain angiotensin Ⅱ(Ang Ⅱ) receptors and underlying intracellular mechanism in the catecholaminergic system(CATH) Methods Action potentials (APs) of the primary co-cultured catecholaminergic tumor (CATH.a) cells were recorded with the whole-cell patch clamp configuration in current clamp mode. Expression of Ang Ⅱ receptors subtypes (AT1 and AT2) was detected by RT-PCR technique. Results The differentiated CATH.a cells represented a neuron-like characterization. All CATH.a cells expressed mRNA encoding both Ang Ⅱ AT1 and AT2 receptor subtypes. Ang Ⅱ increased the firing rate in the CATH.a cells, which was inhibited completely by addition administration of the AT1 but not AT2 receptor antagonist, and partially by using the inhibitors of signal molecules, U73122 (10 μmol*L-1), or KN-93 (10 μmol*L-1), or calphostin C (10 μmol*L-1). Conclusion Ang Ⅱ increases firing rate in CATH.a cells via AT1 receptor. The CATH.a cells expressing functional AT1 and AT2 receptor subtypes may be of general utility for the study of the Ang Ⅱ receptor-induced modulation of brain catecholaminergic system.

  10. Toll-Like Receptor-Dependent Immune Complex Activation of B Cells and Dendritic Cells. (United States)

    Moody, Krishna L; Uccellini, Melissa B; Avalos, Ana M; Marshak-Rothstein, Ann; Viglianti, Gregory A


    High titers of autoantibodies reactive with DNA/RNA molecular complexes are characteristic of autoimmune disorders such as systemic lupus erythematosus (SLE). In vitro and in vivo studies have implicated the endosomal Toll-like receptor 9 (TLR9) and Toll-like receptor 7 (TLR7) in the activation of the corresponding autoantibody producing B cells. Importantly, TLR9/TLR7-deficiency results in the inability of autoreactive B cells to proliferate in response to DNA/RNA-associated autoantigens in vitro, and in marked changes in the autoantibody repertoire of autoimmune-prone mice. Uptake of DNA/RNA-associated autoantigen immune complexes (ICs) also leads to activation of dendritic cells (DCs) through TLR9 and TLR7. The initial studies from our lab involved ICs formed by a mixture of autoantibodies and cell debris released from dying cells in culture. To better understand the nature of the mammalian ligands that can effectively activate TLR7 and TLR9, we have developed a methodology for preparing ICs containing defined DNA fragments that recapitulate the immunostimulatory activity of the previous "black box" ICs. As the endosomal TLR7 and TLR9 function optimally from intracellular acidic compartments, we developed a facile methodology to monitor the trafficking of defined DNA ICs by flow cytometry and confocal microscopy. These reagents reveal an important role for nucleic acid sequence, even when the ligand is mammalian DNA and will help illuminate the role of IC trafficking in the response.

  11. Regulation of hematopoietic cell function by inhibitory immunoglobulin G receptors and their inositol lipid phosphatase effectors. (United States)

    Cady, Carol T; Rice, Jeffrey S; Ott, Vanessa L; Cambier, John C


    Numerous autoimmune and inflammatory disorders stem from the dysregulation of hematopoietic cell activation. The activity of inositol lipid and protein tyrosine phosphatases, and the receptors that recruit them, is critical for prevention of these disorders. Balanced signaling by inhibitory and activating receptors is now recognized to be an important factor in tuning cell function and inflammatory potential. In this review, we provide an overview of current knowledge of membrane proximal events in signaling by inhibitory/regulatory receptors focusing on structural and functional characteristics of receptors and their effectors Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 and SH2 domain-containing inositol 5-phosphatase-1. We review use of new strategies to identify novel regulatory receptors and effectors. Finally, we discuss complementary actions of paired inhibitory and activating receptors, using Fc gammaRIIA and Fc gammaRIIB regulation human basophil activation as a prototype.

  12. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells. (United States)

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng


    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  13. Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer

    DEFF Research Database (Denmark)

    Mereiter, Stefan; Magalhães, Ana; Adamczyk, Barbara


    gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry...... known to be key players in malignancy. Further analysis of RON confirmed its modification with SLe(X) and the concomitant activation. SLe(X) and RON co-expression was validated in gastric tumors. CONCLUSION: The overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells...... affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. GENERAL SIGNIFICANCE: This study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer...

  14. Inhibition of tryptase release from human colon mast cells by histamine receptor antagonists. (United States)

    He, Shao-Heng; Xie, Hua; Fu, Yi-Ling


    The main objective of this study was to investigate the ability of histamine receptor antagonists to modulate tryptase release from human colon mast cells induced by histamine. Enzymatically dispersed cells from human colon were challenged with histamine in the absence or presence of the histamine receptor antagonists, and the tryptase release was determined. It was found that histamine induced tryptase release from colon mast cells was inhibited by up to approximately 61.5% and 24% by the H1 histamine receptor antagonist terfenadine and the H2 histamine receptor antagonist cimetidine, respectively, when histamine and its antagonists were added to cells at the same time. The H3 histamine receptor antagonist clobenpropit had no effect on histamine induced tryptase release from colon mast cells at all concentrations tested. Preincubation of terfenadine, cimetidine or clobenpropit with cells for 20 minutes before challenging with histamine did not enhance the ability of these antihistamines to inhibit histamine induced tryptase release. Apart from terfenadine at 100 microg/ml, the antagonists themselves did not stimulate tryptase release from colon mast cells following both 15 minutes and 35 minutes incubation periods. It was concluded that H1 and H2 histamine receptor antagonists were able to inhibit histamine induced tryptase release from colon mast cells. This not only added some new data to our hypothesis of self-amplification mechanisms of mast cell degranulation, but also suggested that combining these two types of antihistamine drugs could be useful for the treatment of inflammatory bowel disease (IBD).

  15. Involvement of LPA Receptor 3 in LPA-induced BGC- 803 Cell Migration

    Directory of Open Access Journals (Sweden)

    Erdene Oyungerel


    Full Text Available Lysophosphatidic acid ˄ LPA ˅ is a bioactive phospholipid mediator, which elicits a variety of biological functions mainly through G-protein coupled receptors. Although LPA is shown to stimulate proliferation and motility via LPA receptors, LPAR1 and LPAR3 in several cancer cell lines, but the role of LPA receptors in gastric cancer cells is still being unknown. However, several researches reported that LPAR2 play an important role in the carcinogenesis of gastric cancer, but there is no report to show the LPAR3 involvement in the carcinogenesis. For this reason, we examined LPA receptors (LPAR1, LPAR2 and LPAR3 in BGC-803 cells along with real time PCR method. Real-time PCR analyses were used to evaluate the expression of LPA receptors in BGC-803 cells. Among these receptors, LPAR3 was shown to be highly expressed in BGC-803 cells, a human gastric cancer cell line. Transient transfection with LPAR3 siRNA was observed to reduce LPAR3 mRNA in BGC-803 cells and eliminate the LPA-induced cell migration. The results suggest that the LPAR3 regulates LPA-induced BGC-803 cell migration.

  16. Receptors for T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication



    T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1- B20 (in vitro line) (a murine chronic B cell leukemic cell line previou...

  17. Binding of /sup 125/I-labeled reovirus to cell surface receptors

    Energy Technology Data Exchange (ETDEWEB)

    Epstein, R.L.; Powers, M.L.; Rogart, R.B.; Weiner, H.L.


    Quantitative studies of /sup 125/I-labeled reovirus binding at equilibrium to several cell types was studied, including (1) murine L cell fibroblasts; (2) murine splenic T lymphocytes; (3) YAC cells, a murine lymphoma cell line; and (4) R1.1 cells, a murine thymoma cell line. Competition and saturation studies demonstrated (1) specific, saturable, high-affinity binding of reovirus types 1 and 3 to nonidentical receptors on L cell fibroblasts; (2) high-affinity binding of type 3 reovirus to murine splenic lymphocytes and R1.1 cells; (3) low-affinity binding of reovirus type 1 to lymphocytes and R1.1 cells; and (4) no significant binding of either serotype to YAC cells. Differences in the binding characteristics of the two reovirus serotypes to L cell fibroblasts were found to be a property of the viral hemagglutinin, as demonstrated using a recombinant viral clone. The equilibrium dissociation constant (Kd) for viral binding was of extremely high affinity (Kd in the range of 0.5 nM), and was slowly reversible. Experiments demonstrated temperature and pH dependence of reovirus binding and receptor modification studies using pronase, neuraminidase, and various sugars confirmed previous studies that reovirus receptors are predominantly protein in structure. The reovirus receptor site density was in the range of 2-8 X 10(4) sites/cell. These studies demonstrate that the pseudo-first-order kinetic model for ligand-receptor interactions provides a useful model for studying interactions of viral particles with membrane viral receptors. They also suggest that one cell may have distinct receptor sites for two serotypes of the same virus, and that one viral serotype may bind with different kinetics depending on the cell type.

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

    Directory of Open Access Journals (Sweden)

    Callihan Phillip


    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.

  19. Characterization of virus-primed CD8+ T cells with a type 1 cytokine profile

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Stenvang, J P; Marker, O


    parameters of cell-mediated immunity, splenic T cells appear which are able to release high amounts of IFN- gamma, but not IL-5, IL-10 or tumor necrosis factor-alpha upon short-term stimulation with anti-CD3 in vitro. A similar profile is observed analyzing T cells taken from an inflammatory site...

  20. Characterization of a novel, brain-penetrating CB1 receptor inverse agonist: metabolic profile in diet-induced obese models and aspects of central activity. (United States)

    Jacobson, Laura H; Commerford, S Renee; Gerber, Sarah P; Chen, Yu Alice; Dardik, Beatriz; Chaperon, Frederique; Schwartzkopf, Chad; Nguyen-Tran, Van; Hollenbeck, Thomas; McNamara, Peter; He, Xiaohui; Liu, Hong; Seidel, H Martin; Jaton, Anne-Liese; Gromada, Jesper; Teixeira, Sandra


    Pharmacologic antagonism of cannabinoid 1 receptors (CB1 receptors) in the central nervous system (CNS) suppresses food intake, promotes weight loss, and improves the metabolic profile. Since the CB1 receptor is expressed both in the CNS and in peripheral tissues, therapeutic value may be gained with CB1 receptor inverse agonists acting on receptors in both domains. The present report examines the metabolic and CNS actions of a novel CB1 receptor inverse agonist, compound 64, a 1,5,6-trisubstituted pyrazolopyrimidinone. Compound 64 showed similar or superior binding affinity, in vitro potency, and pharmacokinetic profile compared to rimonabant. Both compounds improved the metabolic profile in diet-induced obese (DIO) rats and obese cynomolgus monkeys. Weight loss tended to be greater in compound 64-treated DIO rats compared to pair-fed counterparts, suggesting that compound 64 may have metabolic effects beyond those elicited by weight loss alone. In the CNS, reversal of agonist-induced hypothermia and hypolocomotion indicated that compound 64 possessed an antagonist activity in vivo. Dosed alone, compound 64 suppressed extinction of conditioned freezing (10 mg/kg) and rapid eye movement (REM) sleep (30 mg/kg), consistent with previous reports for rimonabant, although for REM sleep, compound 64 was greater than threefold less potent than for metabolic effects. Together, these data suggested that (1) impairment of extinction learning and REM sleep suppression are classic, centrally mediated responses to CB1 receptor inverse agonists, and (2) some separation may be achievable between central and peripheral effects with brain-penetrating CB1 receptor inverse agonists while maintaining metabolic efficacy. Furthermore, chronic treatment with compound 64 contributes to evidence that peripheral CB1 receptor blockade may yield beneficial outcomes that exceed those elicited by weight loss alone.

  1. Inducible T-cell receptor expression in precursor T-cells for leukemia control (United States)

    Hoseini, Shahabuddin S; Hapke, Martin; Herbst, Jessica; Wedekind, Dirk; Baumann, Rolf; Heinz, Niels; Schiedlmeier, Bernhard; Vignali, Dario AA; van den Brink, Marcel R.M.; Schambach, Axel; Blazar, Bruce R.; Sauer, Martin G.


    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

  2. Fluorescently activated cell sorting followed by microarray profiling of helper T cell subtypes from human peripheral blood.

    Directory of Open Access Journals (Sweden)

    Chiaki Ono

    Full Text Available BACKGROUND: Peripheral blood samples have been subjected to comprehensive gene expression profiling to identify biomarkers for a wide range of diseases. However, blood samples include red blood cells, white blood cells, and platelets. White blood cells comprise polymorphonuclear leukocytes, monocytes, and various types of lymphocytes. Blood is not distinguishable, irrespective of whether the expression profiles reflect alterations in (a gene expression patterns in each cell type or (b the proportion of cell types in blood. CD4+ Th cells are classified into two functionally distinct subclasses, namely Th1 and Th2 cells, on the basis of the unique characteristics of their secreted cytokines and their roles in the immune system. Th1 and Th2 cells play an important role not only in the pathogenesis of human inflammatory, allergic, and autoimmune diseases, but also in diseases that are not considered to be immune or inflammatory disorders. However, analyses of minor cellular components such as CD4+ cell subpopulations have not been performed, partly because of the limited number of these cells in collected samples. METHODOLOGY/PRINCIPAL FINDINGS: We describe fluorescently activated cell sorting followed by microarray (FACS-array technology as a useful experimental strategy for characterizing the expression profiles of specific immune cells in the circulation. We performed reproducible gene expression profiling of Th1 and Th2, respectively. Our data suggest that this procedure provides reliable information on the gene expression profiles of certain small immune cell populations. Moreover, our data suggest that GZMK, GZMH, EOMES, IGFBP3, and STOM may be novel markers for distinguishing Th1 cells from Th2 cells, whereas IL17RB and CNTNAP1 can be Th2-specific markers. CONCLUSIONS/SIGNIFICANCE: Our approach may help in identifying aberrations and novel therapeutic or diagnostic targets for diseases that affect Th1 or Th2 responses and elucidating the

  3. Infection of Polarized MDCK Cells with Herpes Simplex Virus 1: Two Asymmetrically Distributed Cell Receptors Interact with Different Viral Proteins (United States)

    Sears, Amy E.; McGwire, Bradford S.; Roizman, Bernard


    Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.

  4. Authentication of newly established human esophageal squamous cell carcinoma cell line (YM-1) using short tandem repeat (STR) profiling method. (United States)

    Ayyoob, Khosravi; Masoud, Khoshnia; Vahideh, Kazeminejad; Jahanbakhsh, Asadi


    Cross-contamination during or early after establishment of a new cell line could result in the worldwide spread of a misidentified cell line. Therefore, newly established cell lines need to be authenticated by a reference standard method. This study was conducted to investigate the authenticity of a newly established epithelial cell line of human esophageal squamous cell carcinoma (ESCC) called YM-1 using short tandem repeat (STR) DNA profiling method. Primary human ESCC epithelial cells were cultured from the fresh tumor tissue of an adult female patient. Growth characteristics and epithelial originality of YM-1 cells were studied. Genomic DNA was isolated from YM-1 cells harvested at passage 22 and ESCC donor tumor sample on two different days to prevent probable DNA contamination. STR profiling was performed using AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. To address whether YM-1 cells undergo genetic alteration as the passage number increases, STR profiling was performed again on harvested cells at passage 51. YM-1 cells grew as a monolayer with a population doubling time of 40.66 h. Epithelial originality of YM-1 cells was confirmed using ICC/IF staining of cytokeratins AE1/AE3. The STR profile of the ESCC donor tumor sample was the same with YM-1 cells at passage 22. However, STR profile of the donor tumor sample showed an off-ladder (OL) allele in their D7S820 locus. Also, re-profiling of YM-1 cells at passage 51 showed a loss of heterozygosity (LOH) at D18S51 locus. This suggests that long-term culture of cell lines may alter their DNA profile. Comparison of the DNA fingerprinting results in DSMZ, and ATCC STR profiling databases confirmed unique identity of YM-1 cell line. This study provides an easy, fast, and reliable procedure for authentication of newly established cell lines, which helps in preventing the spread of misidentified cells and improving the reproducibility and validity of experiments, consequently.

  5. Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

  6. Relationship between somatostatin receptors and activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 陆良勇; 尤汉宁; 徐芹芳


    Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs.Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation.SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining.Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc.Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.


    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 尤汉宁; 徐芹芳; 陆良勇


    Objective To investigate the relationship between expression of somatostatin receptors (SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1 ~5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1 ~5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1~3 mRNA appeared as HSCs became wholly activated, and SSTR1 ~3 could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc Conclusion The expression of SSTR1~3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

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


    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.

  9. Receptor-mediated gene delivery using polyethylenimine (PEI)coupled with polypeptides targeting FGF receptors on cells surface

    Institute of Scientific and Technical Information of China (English)

    LI Da; WANG Qing-qing; TANG Gu-ping; HUANG Hong-liang; SHEN Fen-ping; LI Jing-zhong; YU Hai


    Objective: To construct a novel kind ofnonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. Methods:The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. Results: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. Conclusion: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.

  10. The potential of single-cell profiling in plants


    Efroni, Idan; Birnbaum, Kenneth D


    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized ce...

  11. 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)


    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.

  12. Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles


    Jouneau, A.; Ciaudo, C.; Sismeiro, O.; Brochard, V.; Jouneau, L.; Vandormael-Pournin, S; Coppee, J.-Y.; Zhou, Q.; Heard, E.; Antoniewski, C.; Cohen-Tannoudji, M.


    Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied. Here, the authors performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC...

  13. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro (United States)


    Bacillus cereus group of bacteria, are attributed to poly- γ-D-glutamate acid (PGA) capsule, lethal toxin (LT) and edema toxin (ET) [10-12]. These toxins...M, Hellman M, Muhie S, et al. (2013) Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in and source are credited. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro Rasha

  14. Improvement of lipid profile by probiotic/protective cultures: study in a non-carcinogenic small intestinal cell model. (United States)

    Gorenjak, Mario; Gradišnik, Lidija; Trapečar, Martin; Pistello, Mauro; Kozmus, Carina Pinto; Škorjanc, Dejan; Skok, Pavel; Langerholc, Tomaž; Cencič, Avrelija


    Plasma lipid levels are important risk factors for the development of atherosclerosis and coronary heart disease. Previous findings have shown that probiotic bacteria exert positive effects on hypercholesterolemia by lowering serum cholesterol and improving lipid profile that, in turn, leads to a reduced risk of coronary heart disease and atherosclerosis. Most of these studies were carried out with tumoral cell lines that have a metabolism quite different from that of normal cells and may thus respond differently to various stimuli. Here, we demonstrate the beneficial effects of some probiotics on cholesterol levels and pathways in normal small intestinal foetal epithelial tissue cells. The results show that Lactobacillus plantarum strain PCS 26 efficiently removes cholesterol from media, exhibits bile salt hydrolase activity, and up-regulates several genes involved in cholesterol metabolism. This study suggests that Lactobacillus plantarum PCS 26 might act as a liver X receptor agonist and help to improve lipid profiles in hypercholesterolemic patients or even dislipidemias in complex diseases such as the metabolic syndrome.

  15. Monte Carlo study of receptor-lipid raft formation on a cell membrane (United States)

    Yu-Yang, Paul; Srinivas Reddy, A.; Raychaudhuri, Subhadip


    Receptors are cell surface molecules that bind with extracellular ligand molecules leading to propagation of downstream signals and cellular activation. Even though ligand binding-induced formation of receptor-lipid rafts has been implicated in such a process, the formation mechanism of such large stable rafts is not understood. We present findings from our Monte Carlo (MC) simulations involving (i) receptor interaction with the membrane lipids and (ii) lipid-lipid interactions between raft forming lipids. We have developed a hybrid MC simulation method that combines a probabilistic MC simulation with an explicit free energy-based MC scheme. Some of the lipid-mediated interactions, such as the cholesterol-lipid interactions, are simulated in an implicit way. We examine the effect of varying attractive interactions between raft forming lipids and ligand-bound receptors and show that strong coupling between receptor-receptor and receptor-sphingolipid molecules generate raft formation similar to that observed in recent biological experiments. We study the effect of variation of receptor affinity for ligands (as happens in adaptive immune cells) on raft formation. Such affinity dependence in receptor-lipid raft formation provides insight into important problems in B cell biology.

  16. A novel system of polymorphic and diverse NK cell receptors in primates.

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    Anne Averdam


    Full Text Available There are two main classes of natural killer (NK cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR and the structurally unrelated killer cell lectin-like receptors (KLR. While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2 rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.

  17. Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines. (United States)

    Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C


    Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

  18. Ligation-free ribosome profiling of cell type-specific translation in the brain. (United States)

    Hornstein, Nicholas; Torres, Daniela; Das Sharma, Sohani; Tang, Guomei; Canoll, Peter; Sims, Peter A


    Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.

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

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    Kellermeier Silvia


    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

  20. The Mannose Receptor Is Involved in the Phagocytosis of Mycobacteria-Induced Apoptotic Cells

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    Teresa Garcia-Aguilar


    Full Text Available Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-β and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect.

  1. Relationship between internalization and calcitonin-induced receptor loss in T 47D cells

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    Findlay, D.M.; Martin, T.J.


    Exposure of T 47D human breast cancer cells to salmon calcitonin (sCT) resulted in a reduction of binding capacity for (/sup 125/I)iodo-sCT in washed cells. The reduction was both time and concentration dependent. Recovery of binding capacity in CT-pretreated T 47D cells occurred in the absence of CT, but was prevented by inhibitors of protein synthesis. Studies were carried out to determine the mechanism of CT-induced reduction of binding capacity. It was found that at 37/sup 0/ C sCT induced a time-dependent loss of cell surface receptors, so that initially the lost binding capacity was largely reclaimable by acid treatment, whereas after longer exposure to sCT, acid treatment was much less effective in regenerating binding capacity. The CT-induced reduction in binding capacity was not observed when cells were pretreated with sCT at 4 C or in the presence of inhibitors of cellular metabolic energy. These results are consistent with the view that initially CT-induced loss of CT receptors in T 47D cells is primarily due to occupancy of cell-surface receptors and later to a reduction in the concentration of cell-surface receptors mediated by an energy requiring internalization process involving the CT-receptor complex; reappearance of receptors requires new protein synthesis.

  2. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

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    Anne-Christine Field

    Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has bee